@article {pmid39242595, year = {2024}, author = {Dvorakova, T and Finisguerra, V and Formenti, M and Loriot, A and Boudhan, L and Zhu, J and Van den Eynde, BJ}, title = {Enhanced tumor response to adoptive T cell therapy with PHD2/3-deficient CD8 T cells.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7789}, pmid = {39242595}, issn = {2041-1723}, support = {T.0091.18//Fonds De La Recherche Scientifique - FNRS (Belgian National Fund for Scientific Research)/ ; WELBIO-CR-2019C-05//Fonds De La Recherche Scientifique - FNRS (Belgian National Fund for Scientific Research)/ ; 2019-094//Stichting Tegen Kanker (Belgian Foundation Against Cancer)/ ; }, mesh = {Animals ; *CD8-Positive T-Lymphocytes/immunology/metabolism ; *Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism/genetics ; *Immunotherapy, Adoptive/methods ; Mice ; *Tumor Microenvironment/immunology ; *Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; Mice, Inbred C57BL ; Cell Line, Tumor ; Lymphocyte Activation/immunology ; CRISPR-Cas Systems ; Humans ; Neoplasms/immunology/therapy/genetics ; Female ; Procollagen-Proline Dioxygenase ; }, abstract = {While adoptive cell therapy has shown success in hematological malignancies, its potential against solid tumors is hindered by an immunosuppressive tumor microenvironment (TME). In recent years, members of the hypoxia-inducible factor (HIF) family have gained recognition as important regulators of T-cell metabolism and function. The role of HIF signalling in activated CD8 T cell function in the context of adoptive cell transfer, however, has not been explored in full depth. Here we utilize CRISPR-Cas9 technology to delete prolyl hydroxylase domain-containing enzymes (PHD) 2 and 3, thereby stabilizing HIF-1 signalling, in CD8 T cells that have already undergone differentiation and activation, modelling the T cell phenotype utilized in clinical settings. We observe a significant boost in T-cell activation and effector functions following PHD2/3 deletion, which is dependent on HIF-1α, and is accompanied by an increased glycolytic flux. This improvement in CD8 T cell performance translates into an enhancement in tumor response to adoptive T cell therapy in mice, across various tumor models, even including those reported to be extremely resistant to immunotherapeutic interventions. These findings hold promise for advancing CD8 T-cell based therapies and overcoming the immune suppression barriers within challenging tumor microenvironments.}, }
@article {pmid39241751, year = {2024}, author = {Birkholz, N and Fineran, PC}, title = {Anti-CRISPRs deconstruct bacterial defense.}, journal = {Molecular cell}, volume = {84}, number = {17}, pages = {3172-3174}, doi = {10.1016/j.molcel.2024.08.008}, pmid = {39241751}, issn = {1097-4164}, mesh = {*CRISPR-Cas Systems ; *Bacteriophages/genetics ; *Bacteria/genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; }, abstract = {Deploying anti-CRISPR proteins is a potent strategy used by phages to inhibit bacterial CRISPR-Cas defense. In a new Nature paper, Trost et al.[1] discover and characterize an exciting anti-CRISPR mechanism with possible implications beyond this microscopic arms race.}, }
@article {pmid39178617, year = {2024}, author = {Punetha, M and Saini, S and Choudhary, S and Sharma, S and Bala, R and Kumar, P and Sharma, RK and Yadav, PS and Datta, TK and Kumar, D}, title = {Establishment of CRISPR-Cas9 ribonucleoprotein mediated MSTN gene edited pregnancy in buffalo: Compare cells transfection and zygotes electroporation.}, journal = {Theriogenology}, volume = {229}, number = {}, pages = {158-168}, doi = {10.1016/j.theriogenology.2024.08.027}, pmid = {39178617}, issn = {1879-3231}, mesh = {Animals ; *Buffaloes/genetics ; *Electroporation/veterinary/methods ; Female ; Pregnancy ; *CRISPR-Cas Systems ; *Gene Editing/methods/veterinary ; *Transfection/veterinary/methods ; *Nuclear Transfer Techniques/veterinary ; Myostatin/genetics ; Zygote/metabolism ; }, abstract = {Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.}, }
@article {pmid39239522, year = {2024}, author = {Wu, Y and Chen, S and Huang, G and Zhang, L and Zhong, L and Feng, Y and Wen, P and Liu, J}, title = {Transcriptome analysis reveals EBF1 ablation-induced injuries in cardiac system.}, journal = {Theranostics}, volume = {14}, number = {12}, pages = {4894-4915}, pmid = {39239522}, issn = {1838-7640}, mesh = {Animals ; Humans ; Mice ; *Trans-Activators/genetics/metabolism ; *Myocytes, Cardiac/metabolism ; *Gene Expression Profiling ; Cell Differentiation/genetics ; Heart/physiopathology ; Mice, Knockout ; Pluripotent Stem Cells/metabolism ; Transcriptome/genetics ; CRISPR-Cas Systems/genetics ; }, abstract = {Rationale: Regulatory processes of transcription factors (TFs) shape heart development and influence the adult heart's response to stress, contributing to cardiac disorders. Despite their significance, the precise mechanisms underpinning TF-mediated regulation remain elusive. Here, we identify that EBF1, as a TF, is highly expressed in human heart tissues. EBF1 is reported to be associated with human cardiovascular disease, but its roles are unclear in heart. In this study, we investigated EBF1 function in cardiac system. Methods: RNA-seq was utilized to profile EBF1 expression patterns. CRISPR/Cas9 was utilized to knock out EBF1 to investigate its effects. Human pluripotent stem cells (hPSCs) differentiated into cardiac lineages were used to mimic cardiac development. Cardiac function was evaluated on mouse model with Ebf1 knockout by using techniques such as echocardiography. RNA-seq was conducted to analyze transcriptional perturbations. ChIP-seq was employed to elucidate EBF1-bound genes and the underlying regulatory mechanisms. Results: EBF1 was expressed in some human and mouse cardiomyocyte. Knockout of EBF1 inhibited cardiac development. ChIP-seq indicated EBF1's binding on promoters of cardiogenic TFs pivotal to cardiac development, facilitating their transcriptional expression and promoting cardiac development. In mouse, Ebf1 depletion triggered transcriptional perturbations of genes, resulting in cardiac remodeling. Mechanistically, we found that EBF1 directly bound to upstream chromatin regions of cardiac hypertrophy-inducing genes, contributing to cardiac hypertrophy. Conclusions: We uncover the mechanisms underlying EBF1-mediated regulatory processes, shedding light on cardiac development, and the pathogenesis of cardiac remodeling. These findings emphasize EBF1's critical role in orchestrating diverse aspects of cardiac processes and provide a promising therapeutic intervention for cardiomyopathy.}, }
@article {pmid39239521, year = {2024}, author = {Ryu, JY and Cerecedo-Lopez, C and Yang, H and Ryu, I and Du, R}, title = {Brain-targeted intranasal delivery of protein-based gene therapy for treatment of ischemic stroke.}, journal = {Theranostics}, volume = {14}, number = {12}, pages = {4773-4786}, pmid = {39239521}, issn = {1838-7640}, mesh = {Animals ; *Administration, Intranasal ; Mice ; Humans ; *Ischemic Stroke/therapy/genetics ; *Nanoparticles/administration & dosage ; *Genetic Therapy/methods ; *Sirtuin 1/genetics/metabolism ; *Brain/metabolism ; *Disease Models, Animal ; Male ; Calcium Phosphates ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; Endothelial Cells/metabolism ; Brain Ischemia/therapy/genetics ; Infarction, Middle Cerebral Artery/therapy/genetics ; Epithelial Cells/metabolism ; }, abstract = {Gene therapy using a protein-based CRISPR system in the brain has practical limitations due to current delivery systems, especially in the presence of arterial occlusion. To overcome these obstacles and improve stability, we designed a system for intranasal administration of gene therapy for the treatment of ischemic stroke. Methods: Nanoparticles containing the protein-based CRISPR/dCas9 system targeting Sirt1 were delivered intranasally to the brain in a mouse model of ischemic stroke. The CRISPR/dCas9 system was encapsulated with calcium phosphate (CaP) nanoparticles to prevent them from being degraded. They were then conjugated with β-hydroxybutyrates (bHb) to target monocarboxylic acid transporter 1 (MCT1) in nasal epithelial cells to facilitate their transfer into the brain. Results: Human nasal epithelial cells were shown to uptake and transfer nanoparticles to human brain endothelial cells with high efficiency in vitro. The intranasal administration of the dCas9/CaP/PEI-PEG-bHb nanoparticles in mice effectively upregulated the target gene, Sirt1, in the brain, decreased cerebral edema and increased survival after permanent middle cerebral artery occlusion. Additionally, we observed no significant in vivo toxicity associated with intranasal administration of the nanoparticles, highlighting the safety of this approach. Conclusion: This study demonstrates that the proposed protein-based CRISPR-dCas9 system targeting neuroprotective genes in general, and SIRT1 in particular, can be a potential novel therapy for acute ischemic stroke.}, }
@article {pmid38816517, year = {2024}, author = {Wang, B and Xu, Y and Wan, AH and Wan, G and Wang, QP}, title = {Integrating genome-wide CRISPR screens and in silico drug profiling for targeted antidote development.}, journal = {Nature protocols}, volume = {19}, number = {9}, pages = {2739-2770}, pmid = {38816517}, issn = {1750-2799}, mesh = {*Antidotes/pharmacology ; Humans ; *CRISPR-Cas Systems ; *Computer Simulation ; Drug Evaluation, Preclinical/methods ; Animals ; }, abstract = {Numerous toxins threaten humans, but specific antidotes are unavailable for most of them. Although CRISPR screening has aided the discovery of the mechanisms of some toxins, developing targeted antidotes remains a significant challenge. Recently, we established a systematic framework to develop antidotes by combining the identification of novel drug targets by using a genome-wide CRISPR screen with a virtual screen of drugs approved by the US Food and Drug Administration. This approach allows for a comprehensive understanding of toxin mechanisms at the whole-genome level and facilitates the identification of promising antidote drugs targeting specific molecules. Here, we present step-by-step instructions for executing genome-scale CRISPR-Cas9 knockout screens of toxins in HAP1 cells. We also provide detailed guidance for conducting an in silico drug screen and an in vivo drug validation. By using this protocol, it takes ~4 weeks to perform the genome-scale screen, 4 weeks for sequencing and data analysis, 4 weeks to validate candidate genes, 1 week for the virtual screen and 2 weeks for in vitro drug validation. This framework has the potential to accelerate the development of antidotes for a wide range of toxins and can rapidly identify promising drug candidates that are already known to be safe and effective. This could lead to the development of new antidotes much more quickly than traditional methods, protecting lives from diverse toxins and advancing human health.}, }
@article {pmid39238167, year = {2024}, author = {Sánchez-León, S and Marín-Sanz, M and Guzmán-López, MH and Gavilán-Camacho, M and Simón, E and Barro, F}, title = {CRISPR/Cas9-mediated multiplex gene editing of gamma and omega gliadins, paving the way for gliadin-free wheat.}, journal = {Journal of experimental botany}, volume = {}, number = {}, pages = {}, doi = {10.1093/jxb/erae376}, pmid = {39238167}, issn = {1460-2431}, abstract = {Wheat is a staple cereal in the human diet. Despite its significance, an increasing percentage of the population suffers adverse reactions to wheat, which are triggered by wheat gluten, particularly the gliadin fractions. In this study, we employed CRISPR/Cas multiplexing to introduce targeted mutations into γ- and ω-gliadin genes of wheat, to produce lines deficient in one or both immunogenic gliadin fractions simultaneously. For this work, eight single guide RNAs (sgRNAs) were designed and combined into four plasmids to produce 59 modified wheat lines, of which 20 exhibited mutations in the target genes. Characterization of these lines through Sanger or NGS sequencing revealed a complex pattern of InDels, including deletions spanning multiple sgRNAs. The mutations were transmitted to the offspring, and the analysis of homozygous derived lines by RP-HPLC and monoclonal antibodies showed a 97.7% reduction in gluten content. Crossing these lines with other CRISPR/Cas lines deficient in the α-gliadins allowed multiple mutations to be combined. This work represents an important step forward in the use of CRISPR/Cas to develop gluten-free wheat.}, }
@article {pmid39237013, year = {2024}, author = {Gattani, A and Mandal, S and Agrawal, A and Patel, P and Jain, AK and Singh, P and Garg, A and Mishra, A}, title = {CRISPR-Based Electrochemical Biosensors For Animal Health: Recent Advances.}, journal = {Progress in biophysics and molecular biology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.pbiomolbio.2024.09.001}, pmid = {39237013}, issn = {1873-1732}, abstract = {Animal diseases are a major concern to animal welfare, human health and the global economy. Early detection, prevention and control of these animal diseases are crucial to ensure sustainability of livestock sector, to reduce farm losses and protecting public health. Points of care (POC) devices are small, portable instruments that provide rapid results thus reduce the risk of disease transmission and enable early intervention. CRISPR based diagnostics offer more accurate and efficient solution for monitoring animal health due to their quick response, can detect very low level of pathogenic organism or disease markers and specificity. These diagnostics are particularly useful in the in area with limited resources or access to common diagnostic methods, especially in developing countries. The ability of electrochemical sensors to detect accurately very low analyte concentration makes them suitable for POC diagnostics and field application. CRISPR base electrochemical biosensors show great potential in revolutionizing disease detection and diagnosis including animal health. However, challenges, such as achieving selectivity and sensitivity, need to be addressed to enhance the competitiveness of these biosensors. Currently, most CRISPR based bioassay research focuses on nucleic acid target detection, but researchers exploring to monitor small organic/ inorganic non-nucleic acid molecules like toxins and proteins. Emerging diagnostics would be centered on CRISPR-Cas system will offer great potential as an accurate, specific and effective means to identify microorganism, virus, toxins, small molecules, peptides and nucleic acid related to various animal health disorders particularly when integrated into electrochemical biosensing platform.}, }
@article {pmid39235647, year = {2024}, author = {Byiringiro, I and Pan, C and Qi, Y}, title = {Orthogonal genome editing and transcriptional activation in tomato using CRISPR-Combo systems.}, journal = {Plant cell reports}, volume = {43}, number = {9}, pages = {227}, pmid = {39235647}, issn = {1432-203X}, support = {IOS-2029889//Directorate for Biological Sciences/ ; IOS-2132693//Directorate for Biological Sciences/ ; 21010111//Foundation for Food and Agriculture Research/ ; }, mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Transcriptional Activation/genetics ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; }, abstract = {The CRISPR-Combo systems (Cas9-Combo and CBE-Combo) are designed for comprehensive genetic manipulation, enabling Cas9-based targeted mutagenesis or cytosine base editing with simultaneous gene activation in tomato stable lines.}, }
@article {pmid39009674, year = {2024}, author = {Pauzaite, T and Wit, N and Seear, RV and Nathan, JA}, title = {Deubiquitinating enzyme mutagenesis screens identify a USP43-dependent HIF-1 transcriptional response.}, journal = {The EMBO journal}, volume = {43}, number = {17}, pages = {3677-3709}, pmid = {39009674}, issn = {1460-2075}, support = {215477/Z/19/Z//Wellcome Trust (WT)/ ; }, mesh = {Humans ; *Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; *Deubiquitinating Enzymes/metabolism/genetics ; Mutagenesis ; CRISPR-Cas Systems ; HEK293 Cells ; Signal Transduction ; Ubiquitination ; }, abstract = {The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.}, }
@article {pmid38884692, year = {2024}, author = {Han, U and Han, N and Park, B and Jeon, TJ}, title = {RapB Regulates Cell Adhesion and Migration in Dictyostelium, Similar to RapA.}, journal = {Journal of microbiology (Seoul, Korea)}, volume = {62}, number = {8}, pages = {627-637}, pmid = {38884692}, issn = {1976-3794}, mesh = {*Dictyostelium/genetics/physiology/metabolism/growth & development/cytology ; *Cell Adhesion ; *Chemotaxis ; *Protozoan Proteins/metabolism/genetics ; *Cell Movement ; CRISPR-Cas Systems ; ras Proteins/metabolism/genetics ; Gene Editing ; Signal Transduction ; }, abstract = {Ras small GTPases act as molecular switches in various cellular signaling pathways, including cell migration, proliferation, and differentiation. Three Rap proteins are present in Dictyostelium; RapA, RapB, and RapC. RapA and RapC have been reported to have opposing functions in the control of cell adhesion and migration. Here, we investigated the role of RapB, a member of the Ras GTPase subfamily in Dictyostelium, focusing on its involvement in cell adhesion, migration, and developmental processes. This study revealed that RapB, similar to RapA, played a crucial role in regulating cell morphology, adhesion, and migration. rapB null cells, which were generated by CRISPR/Cas9 gene editing, displayed altered cell size, reduced cell-substrate adhesion, and increased migration speed during chemotaxis. These phenotypes of rapB null cells were restored by the expression of RapB and RapA, but not RapC. Consistent with these results, RapB, similar to RapA, failed to rescue the phenotypes of rapC null cells, spread morphology, increased cell adhesion, and decreased migration speed during chemotaxis. Multicellular development of rapB null cells remained unaffected. These results suggest that RapB is involved in controlling cell morphology and cell adhesion. Importantly, RapB appears to play an inhibitory role in regulating the migration speed during chemotaxis, possibly by controlling cell-substrate adhesion, resembling the functions of RapA. These findings contribute to the understanding of the functional relationships among Ras subfamily proteins.}, }
@article {pmid39233906, year = {2024}, author = {Gan, T and Yu, J and Deng, Z and He, J}, title = {ERA-CRISPR/Cas12a system: a rapid, highly sensitive and specific assay for Mycobacterium tuberculosis.}, journal = {Frontiers in cellular and infection microbiology}, volume = {14}, number = {}, pages = {1454076}, pmid = {39233906}, issn = {2235-2988}, mesh = {*Mycobacterium tuberculosis/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Tuberculosis/diagnosis/microbiology ; Recombinases/metabolism/genetics ; Molecular Diagnostic Techniques/methods ; Bacterial Proteins/genetics ; DNA, Bacterial/genetics ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases ; }, abstract = {INTRODUCTION: Mycobacterium tuberculosis, the causative agent of human tuberculosis, poses a significant threat to global public health and imposes a considerable burden on the economy. However, existing laboratory diagnostic methods for M. tuberculosis are time-consuming and have limited sensitivity levels.<br><br>METHODS: The CRISPR/Cas system, commonly known as the "gene scissors", demonstrates remarkable specificity and efficient signal amplification capabilities. Enzymatic recombinase amplification (ERA) was utilized to rapidly amplify trace DNA fragments at a consistent temperature without relying on thermal cyclers. By integrating of CRISPR/Cas12a with ERA, we successfully developed an ERA-CRISPR/Cas12a detection system that enables rapid identification of M. tuberculosis.<br><br>RESULTS: The sensitivity of the ERA-CRISPR/Cas12a fluorescence and lateral flow systems was 9 copies/&#x3bc;L and 90 copies/&#x3bc;L, respectively. Simultaneously, the detection system exhibited no cross-reactivity with various of respiratory pathogens and non-tuberculosis mycobacteria, demonstrating a specificity of 100%. The positive concordance rate between the ERA-CRISPR/Cas12a fluorescence system and commercial qPCR was 100% in 60 clinical samples. Meanwhile, the lateral flow system showed a positive concordance rate of 93.8% when compared to commercial qPCR. Both methods demonstrated a negative concordance rate of 100%, and the test results can be obtained in 50 min at the earliest.<br><br>DISCUSSION: The ERA-CRISPR/Cas12a system offers a rapid, sensitive, and specific method that presents a novel approach to laboratory diagnosis of M. tuberculosis.}, }
@article {pmid39232173, year = {2024}, author = {Katz, MA and Sawyer, EM and Oriolt, L and Kozlova, A and Williams, MC and Margolis, SR and Johnson, M and Bondy-Denomy, J and Meeske, AJ}, title = {Diverse viral cas genes antagonize CRISPR immunity.}, journal = {Nature}, volume = {}, number = {}, pages = {}, pmid = {39232173}, issn = {1476-4687}, abstract = {Prokaryotic CRISPR-Cas immunity is subverted by anti-CRISPRs (Acrs), which inhibit Cas protein activities when expressed during the phage lytic cycle or from resident prophages or plasmids[1]. Acrs often bind to specific cognate Cas proteins, and hence inhibition is typically limited to a single CRISPR-Cas subtype[2]. Furthermore, although acr genes are frequently organized together in phage-associated gene clusters[3], how such inhibitors initially evolve has remained unclear. Here we investigated the Acr content and inhibition specificity of diverse Listeria isolates, which naturally harbour four CRISPR-Cas systems (types I-B, II-A, II-C and VI-A). We observed widespread antagonism of CRISPR, which we traced to 11 previously unknown and 4 known acr gene families encoded by endogenous mobile elements. Among these were two Acrs that possess sequence homology to type I-B Cas proteins, one of which assembles into a defective interference complex. Surprisingly, an additional type I-B Cas homologue did not affect type I immunity, but instead inhibited the RNA-targeting type VI CRISPR system by means of CRISPR RNA (crRNA) degradation. By probing viral sequence databases, we detected abundant orphan cas genes located within putative anti-defence gene clusters. Among them, we verified the activity of a particularly broad-spectrum cas3 homologue that inhibits type I-B, II-A and VI-A CRISPR immunity. Our observations provide direct evidence of Acr evolution by cas gene co-option, and new genes with potential for broad-spectrum control of genome editing technologies.}, }
@article {pmid39232160, year = {2024}, author = {Chen, J and Jia, Y and Sun, Y and Liu, K and Zhou, C and Liu, C and Li, D and Liu, G and Zhang, C and Yang, T and Huang, L and Zhuang, Y and Wang, D and Xu, D and Zhong, Q and Guo, Y and Li, A and Seim, I and Jiang, L and Wang, L and Lee, SMY and Liu, Y and Wang, D and Zhang, G and Liu, S and Wei, X and Yue, Z and Zheng, S and Shen, X and Wang, S and Qi, C and Chen, J and Ye, C and Zhao, F and Wang, J and Fan, J and Li, B and Sun, J and Jia, X and Xia, Z and Zhang, H and Liu, J and Zheng, Y and Liu, X and Wang, J and Yang, H and Kristiansen, K and Xu, X and Mock, T and Li, S and Zhang, W and Fan, G}, title = {Global marine microbial diversity and its potential in bioprospecting.}, journal = {Nature}, volume = {}, number = {}, pages = {}, pmid = {39232160}, issn = {1476-4687}, abstract = {The past two decades has witnessed a remarkable increase in the number of microbial genomes retrieved from marine systems[1,2]. However, it has remained challenging to translate this marine genomic diversity into biotechnological and biomedical applications[3,4]. Here we recovered 43,191 bacterial and archaeal genomes from publicly available marine metagenomes, encompassing a wide range of diversity with 138 distinct phyla, redefining the upper limit of marine bacterial genome size and revealing complex trade-offs between the occurrence of CRISPR-Cas systems and antibiotic resistance genes. In silico bioprospecting of these marine genomes led to the discovery of a novel CRISPR-Cas9 system, ten antimicrobial peptides, and three enzymes that degrade polyethylene terephthalate. In vitro experiments confirmed their effectiveness and efficacy. This work provides evidence that global-scale sequencing initiatives advance our understanding of how microbial diversity has evolved in the oceans and is maintained, and demonstrates how such initiatives can be sustainably exploited to advance biotechnology and biomedicine.}, }
@article {pmid39190773, year = {2024}, author = {Kumar, A and Daripa, P and Rasool, K and Chakraborty, D and Jain, N and Maiti, S}, title = {Deciphering the Thermodynamic Landscape of CRISPR/Cas9: Insights into Enhancing Gene Editing Precision and Efficiency.}, journal = {The journal of physical chemistry. B}, volume = {128}, number = {35}, pages = {8409-8422}, doi = {10.1021/acs.jpcb.4c04044}, pmid = {39190773}, issn = {1520-5207}, mesh = {*CRISPR-Cas Systems ; *Thermodynamics ; *Gene Editing/methods ; DNA/chemistry/metabolism/genetics ; Calorimetry ; CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; }, abstract = {The thermodynamic landscape of the CRISPR/Cas9 system plays a crucial role in understanding and optimizing the performance of this revolutionary genome-editing technology. In this research, we utilized isothermal titration calorimetry and microscale thermophoresis techniques to thoroughly investigate the thermodynamic properties governing CRISPR/Cas9 interactions. Our findings revealed that the binding between sgRNA and Cas9 is primarily governed by entropy, which compensates for an unfavorable enthalpy change. Conversely, the interaction between the CRISPR RNP complex and the target DNA is characterized by a favorable enthalpy change, offsetting an unfavorable entropy change. Notably, both interactions displayed negative heat capacity changes, indicative of potential hydration, ionization, or structural rearrangements. However, we noted that the involvement of water molecules and counterions in the interactions is minimal, suggesting that structural rearrangements play a significant role in influencing the binding thermodynamics. These results offer a nuanced understanding of the energetic contributions and structural dynamics underlying CRISPR-mediated gene editing. Such insights are invaluable for optimizing the efficiency and specificity of CRISPR-based genome editing applications, ultimately advancing our ability to precisely manipulate genetic material in various organisms for research, therapeutic, and biotechnological purposes.}, }
@article {pmid39169620, year = {2024}, author = {Capelletti, S and García Soto, SC and Gonçalves, MAFV}, title = {On RNA-programmable gene modulation as a versatile set of principles targeting muscular dystrophies.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.ymthe.2024.08.016}, pmid = {39169620}, issn = {1525-0024}, abstract = {The repurposing of RNA-programmable CRISPR systems from genome editing into epigenome editing tools is gaining pace, including in research and development efforts directed at tackling human disorders. This momentum stems from the increasing knowledge regarding the epigenetic factors and networks underlying cell physiology and disease etiology and from the growing realization that genome editing principles involving chromosomal breaks generated by programmable nucleases are prone to unpredictable genetic changes and outcomes. Hence, engineered CRISPR systems are serving as versatile DNA-targeting scaffolds for heterologous and synthetic effector domains that, via locally recruiting transcription factors and chromatin remodeling complexes, seek interfering with loss-of-function and gain-of-function processes underlying recessive and dominant disorders, respectively. Here, after providing an overview about epigenetic drugs and CRISPR-Cas-based activation and interference platforms, we cover the testing of these platforms in the context of molecular therapies for muscular dystrophies. Finally, we examine attributes, obstacles, and deployment opportunities for CRISPR-based epigenetic modulating technologies.}, }
@article {pmid39163859, year = {2024}, author = {Tran, NT and Han, R}, title = {Rapidly evolving genome and epigenome editing technologies.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {32}, number = {9}, pages = {2803-2806}, doi = {10.1016/j.ymthe.2024.08.011}, pmid = {39163859}, issn = {1525-0024}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Epigenome ; Animals ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; DNA Breaks, Double-Stranded ; Genetic Therapy/methods ; }, abstract = {Genome editing technologies are rapidly evolving, from the early zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9 (Figure 1, initial genome editing technologies), which generate double-strand breaks (DSBs), to base editing, which makes precise nucleobase conversion without inducing DSBs, and prime editing, which can carry out all types of edits without DSBs or donor DNA templates. The emergence of these revolutionary technologies offers us unprecedented opportunities for biomedical research and therapy development.}, }
@article {pmid39127296, year = {2025}, author = {Li, G and Zhou, J and Gao, N and Liu, R and Shen, J}, title = {Establishment of a rapid detection method for Mycoplasma pneumoniae based on RPA-CRISPR-Cas12a technology.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {564}, number = {}, pages = {119906}, doi = {10.1016/j.cca.2024.119906}, pmid = {39127296}, issn = {1873-3492}, mesh = {*Mycoplasma pneumoniae/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Pneumonia, Mycoplasma/diagnosis/microbiology ; }, abstract = {Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 10[2] copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.}, }
@article {pmid39122070, year = {2024}, author = {Wang, Q and Ma, C and Mao, H and Wang, J}, title = {Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates the ZNF334 gene to inhibit the growth of colorectal cancer.}, journal = {International journal of biological macromolecules}, volume = {277}, number = {Pt 4}, pages = {134580}, doi = {10.1016/j.ijbiomac.2024.134580}, pmid = {39122070}, issn = {1879-0003}, mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Expression Regulation, Neoplastic ; Acetylation ; *Epigenesis, Genetic ; Cell Proliferation/genetics ; Promoter Regions, Genetic/genetics ; Animals ; Cell Line, Tumor ; Histones/metabolism ; Mice ; Acetyltransferases/genetics/metabolism ; }, abstract = {Although therapeutic targets for colorectal cancer (CRC) treatment have been developed, the treatment outcomes are not ideal and survival rates for CRC patients remain low. It is critical to identify a specific target and develop an effective CRC treatment system. The ZNF334 gene is a newly identified member of Zinc-finger proteins (ZNFs), which is essential for key biological processes associated with tumorigenesis. Abnormal epigenetic reprogramming of the ZNF334 gene promoter region decreases its expression in CRC and further induces the occurrence of CRC. Here, we clarified that P300 in CRC can regulate the H3K9/27 ac in the ZNF334 promoter. Furthermore, histone acetylation of the ZNF334 promoter region was increased by dCas9-P300 to normalize the deficiency of ZNF334 expression, thereby inhibiting the growth of CRC. Collectively, our findings enable a facile way to affect gene expression using CRISPR/Cas9-based epigenome editing and further determine the causal link between histone acetylation and gene activation, providing a promising gene therapy strategy for the CRC treatment.}, }
@article {pmid39039289, year = {2024}, author = {Yang, Q and Abebe, JS and Mai, M and Rudy, G and Kim, SY and Devinsky, O and Long, C}, title = {T4 DNA polymerase prevents deleterious on-target DNA damage and enhances precise CRISPR editing.}, journal = {The EMBO journal}, volume = {43}, number = {17}, pages = {3733-3751}, pmid = {39039289}, issn = {1460-2075}, support = {Departmental Start-Up Grant//NYU Langone Health/ ; }, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; Mice ; *DNA Damage ; DNA-Directed DNA Polymerase/metabolism/genetics ; Myocytes, Cardiac/metabolism ; Dystrophin/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; }, abstract = {Unintended on-target chromosomal alterations induced by CRISPR/Cas9 in mammalian cells are common, particularly large deletions and chromosomal translocations, and present a safety challenge for genome editing. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified a T4 DNA polymerase derived from phage T4 that strongly prevents undesired on-target damage while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency of correcting common frameshift mutations in DMD and restored higher level of dystrophin expression than Cas9-alone in human cardiomyocytes. Moreover, CasPlus greatly reduced the frequency of on-target large deletions during mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus repressed chromosomal translocations while maintaining gene disruption efficiency that was higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications.}, }
@article {pmid39231976, year = {2024}, author = {Huang, Y and Mei, H and Deng, C and Wang, W and Yuan, C and Nie, Y and Li, JD and Liu, J}, title = {EXTL3 and NPC1 are mammalian host factors for Autographa californica multiple nucleopolyhedrovirus infection.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7711}, pmid = {39231976}, issn = {2041-1723}, mesh = {Animals ; *Nucleopolyhedroviruses/genetics/physiology ; Humans ; Mice ; *Niemann-Pick C1 Protein ; *N-Acetylglucosaminyltransferases/metabolism/genetics ; HEK293 Cells ; Endosomes/metabolism ; Heparitin Sulfate/metabolism ; Virus Internalization ; Transduction, Genetic ; Sf9 Cells ; Liver/metabolism/virology ; CRISPR-Cas Systems ; }, abstract = {Baculovirus is an obligate parasitic virus of the phylum Arthropoda. Baculovirus including Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used in the laboratory and industrial preparation of proteins or protein complexes. Due to its large packaging capacity and non-replicative and non-integrative natures in mammals, baculovirus has been proposed as a gene therapy vector for transgene delivery. However, the mechanism of baculovirus transduction in mammalian cells has not been fully illustrated. Here, we employed a cell surface protein-focused CRISPR screen to identify host dependency factors for baculovirus transduction in mammalian cells. The screening experiment uncovered a series of baculovirus host factors in human cells, including exostosin-like glycosyltransferase 3 (EXTL3) and NPC intracellular cholesterol transporter 1 (NPC1). Further investigation illustrated that EXTL3 affected baculovirus attachment and entry by participating in heparan sulfate biosynthesis. In addition, NPC1 promoted baculovirus transduction by mediating membrane fusion and endosomal escape. Moreover, in vivo, baculovirus transduction in Npc1[-/+] mice showed that disruption of Npc1 gene significantly reduced baculovirus transduction in mouse liver. In summary, our study revealed the functions of EXTL3 and NPC1 in baculovirus attachment, entry, and endosomal escape in mammalian cells, which is useful for understanding baculovirus transduction in human cells.}, }
@article {pmid39231962, year = {2024}, author = {Makar, AN and Boraman, A and Mosen, P and Simpson, JE and Marques, J and Michelberger, T and Aitken, S and Wheeler, AP and Winter, D and von Kriegsheim, A and Gammoh, N}, title = {The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7743}, pmid = {39231962}, issn = {2041-1723}, support = {C52370/A21586//Cancer Research UK (CRUK)/ ; }, mesh = {*Lysosomes/metabolism ; Humans ; *Autophagosomes/metabolism ; *Autophagy ; *Endosomal Sorting Complexes Required for Transport/metabolism/genetics ; CRISPR-Cas Systems ; Vacuolar Proton-Translocating ATPases/metabolism/genetics ; Hydrolases/metabolism/genetics ; HeLa Cells ; HEK293 Cells ; }, abstract = {Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic components. The final degradation step is essential for clearing autophagic cargo and recycling macromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conserved transmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.}, }
@article {pmid39231901, year = {2024}, author = {Fan, X and Lei, Y and Wang, L and Wu, X and Li, D}, title = {Advancing CRISPR base editing technology through innovative strategies and ideas.}, journal = {Science China. Life sciences}, volume = {}, number = {}, pages = {}, pmid = {39231901}, issn = {1869-1889}, abstract = {The innovation of CRISPR/Cas gene editing technology has developed rapidly in recent years. It is widely used in the fields of disease animal model construction, biological breeding, disease diagnosis and screening, gene therapy, cell localization, cell lineage tracking, synthetic biology, information storage, etc. However, developing idealized editors in various fields is still a goal for future development. This article focuses on the development and innovation of non-DSB editors BE and PE in the platform-based CRISPR system. It first explains the application of ideas for improvement such as "substitution", "combination", "adaptation", and "adjustment" in BE and PE development and then catalogues the ingenious inversions and leaps of thought reflected in the innovations made to CRISPR technology. It will then elaborate on the efforts currently being made to develop small editors to solve the problem of AAV overload and summarize the current application status of editors for in vivo gene modification using AAV as a delivery system. Finally, it summarizes the inspiration brought by CRISPR/Cas innovation and assesses future prospects for development of an idealized editor.}, }
@article {pmid39231196, year = {2024}, author = {Pozhydaieva, N and Billau, FA and Wolfram-Schauerte, M and Ramírez Rojas, AA and Paczia, N and Schindler, D and Höfer, K}, title = {Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications.}, journal = {PLoS genetics}, volume = {20}, number = {9}, pages = {e1011384}, doi = {10.1371/journal.pgen.1011384}, pmid = {39231196}, issn = {1553-7404}, abstract = {Lytic bacteriophages hold substantial promise in medical and biotechnological applications. Therefore a comprehensive understanding of phage infection mechanisms is crucial. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.}, }
@article {pmid39231187, year = {2024}, author = {Ghieh, F and Passet, B and Poumerol, E and Castille, J and Calvel, P and Vilotte, JL and Sellem, E and Jouneau, L and Mambu-Mambueni, H and Garchon, HJ and Pailhoux, E and Vialard, F and Mandon-Pépin, B}, title = {A partial deletion within the meiosis-specific sporulation domain SPO22 of Tex11 is not associated with infertility in mice.}, journal = {PloS one}, volume = {19}, number = {9}, pages = {e0309974}, doi = {10.1371/journal.pone.0309974}, pmid = {39231187}, issn = {1932-6203}, mesh = {Animals ; Male ; Mice ; *Spermatogenesis/genetics ; *Meiosis/genetics ; *Azoospermia/genetics/pathology ; Infertility, Male/genetics ; Sequence Deletion ; Humans ; Testis/metabolism/pathology ; CRISPR-Cas Systems ; }, abstract = {Azoospermia (the complete absence of spermatozoa in the semen) is a common cause of male infertility. The etiology of azoospermia is poorly understood. Whole-genome analysis of azoospermic men has identified a number of candidate genes, such as the X-linked testis-expressed 11 (TEX11) gene. Using a comparative genomic hybridization array, an exonic deletion (exons 10-12) of TEX11 had previously been identified in two non-apparent azoospermic patients. However, the putative impact of this genetic alteration on spermatogenesis and the azoospermia phenotype had not been validated functionally. We therefore used a CRISPR/Cas9 system to generate a mouse model (Tex11Ex9-11del/Y) with a partial TEX11 deletion that mimicked the human mutation. Surprisingly, the mutant male Tex11Ex9-11del/Y mice were fertile. The sperm concentration, motility, and morphology were normal. Similarly, the mutant mouse line's testis transcriptome was normal, and the expression of spermatogenesis genes was not altered. These results suggest that the mouse equivalent of the partial deletion observed in two infertile male with azoospermia has no impact on spermatogenesis or fertility in mice, at least of a FVB/N genetic background and until 10 months of age. Mimicking a human mutation does not necessarily lead to the same human phenotype in mice, highlighting significant differences species.}, }
@article {pmid39230778, year = {2024}, author = {Jesudoss, D and Ponnurangan, V and Kumar, MPR and Kumar, KK and Mannu, J and Sankarasubramanian, H and Duraialagaraja, S and Eswaran, K and Loganathan, A and Shanmugam, V}, title = {Advances in breeding, biotechnology, and nanotechnological approaches to combat sheath blight disease in rice.}, journal = {Molecular biology reports}, volume = {51}, number = {1}, pages = {958}, pmid = {39230778}, issn = {1573-4978}, support = {BT/PR40456/AGIII/103/1248/2020//Department of Biotechnology, New Delhi, India/ ; }, mesh = {*Oryza/genetics/microbiology ; *Plant Diseases/microbiology/genetics ; *Disease Resistance/genetics ; *Rhizoctonia/pathogenicity ; *Plant Breeding/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Biotechnology/methods ; Plants, Genetically Modified/genetics ; Nanotechnology/methods ; }, abstract = {Sheath blight, caused by the fungus Rhizoctonia solani, is a major problem that significantly impacts rice production and can lead to substantial yield losses. The disease has become increasingly problematic in recent years due to the widespread use of high-yielding semi-dwarf rice cultivars, dense planting, and heavy application of nitrogenous fertilizers. The disease has become more challenging to manage due to its diverse host range and the lack of resistant cultivars. Despite utilizing traditional methods, the problem persists without a satisfactory solution. Therefore, modern approaches, including advanced breeding, transgenic methods, genome editing using CRISPR/Cas9 technology, and nanotechnological interventions, are being explored to develop rice plants resistant to sheath blight disease. This review primarily focuses on these recent advancements in combating the sheath blight disease.}, }
@article {pmid39228326, year = {2024}, author = {Jansen, PR and van Haelst, M and Wolthuis, RMF}, title = {[Genetic therapy by CRISPR-Cas: paving the way to clinical practice].}, journal = {Nederlands tijdschrift voor geneeskunde}, volume = {168}, number = {}, pages = {}, pmid = {39228326}, issn = {1876-8784}, mesh = {Humans ; *Genetic Therapy/methods/ethics ; *CRISPR-Cas Systems ; Genetic Diseases, Inborn/therapy ; }, abstract = {CRISPR-Cas technology is a revolutionary technology to modify DNA sequences. Owing to its effectiveness and accuracy, CRISPR-Cas holds important promises for 'genetic therapy' for various hereditary disorders. CRISPR-Cas enables researchers to modify specific parts of the genome with unprecedented precision, which, in specific cases, can be applied to correct disease-causing DNA variants. However, several important barriers are complicating the clinical implementation trajectory. There are for example concerns regarding the safety, effectiveness and ethical justification. Nevertheless, as CRISPR-Cas becomes more widely known, doctors and healthcare providers are expected to be well aware of the developments surrounding CRISPR-Cas therapy. Professional expertise and clear communication about the possibilities and limitations to patients with genetic disorders are essential, partly to avoid making promises that are unrealistic in the short term. Altogether, geneticists, medical centers and regulators are now facing the challenges to start translating CRISPR-Cas technology into clinical practice, in an effective and ethical manner.}, }
@article {pmid39228116, year = {2024}, author = {Chokwassanasakulkit, T and McMillan, NAJ}, title = {Merkel Cell Polyomavirus-Pathophysiology and Treatment in the Era of Gene-Targeted Therapies.}, journal = {Reviews in medical virology}, volume = {34}, number = {5}, pages = {e2580}, doi = {10.1002/rmv.2580}, pmid = {39228116}, issn = {1099-1654}, support = {//Griffith University/ ; 2027649//National Health and Medical Research Council/ ; 2027569//National Health and Medical Research Council/ ; }, mesh = {Humans ; *Merkel cell polyomavirus/genetics ; *Carcinoma, Merkel Cell/virology/therapy/genetics ; *Polyomavirus Infections/virology/therapy ; *Genetic Therapy/methods ; Skin Neoplasms/therapy/virology/genetics ; Animals ; Tumor Virus Infections/virology/therapy ; RNA, Small Interfering/genetics ; }, abstract = {Merkel cell polyomavirus (MCPyV) is a significant contributor to the development of Merkel cell carcinoma (MCC), an aggressive skin cancer with high recurrence and a low survival rate. In fact, it is the deadliest skin cancer. The precise routes of transmission for MCPyV-positive MCC remain unclear, but several factors may trigger its development. Conventional treatments for MCC are not highly effective, especially in patients with metastasis, with a clear need for new treatment options. Gene-targeted therapies hold great promise for the treatment of MCC, including the use of siRNA and CRISPR/Cas (C/Cas) but critically none have yet been translated into clinical trials. Validating this approach is the fact that several siRNA products are already FDA licenced, while C/Cas has entered clinical trial, albeit for conditions other than MCC. There are many challenges that must be overcome to move from preclinical research to the clinic. In this review, we provide a comprehensive summary of the current understanding of MCC, with a particular focus on MCPyV-positive MCC, and the status of gene-targeted therapies. Additionally, we discuss the major obstacles that impede MCC research and explore future prospects.}, }
@article {pmid39227830, year = {2024}, author = {Du, Q and Wei, Y and Zhang, L and Ren, D and Gao, J and Dong, X and Bai, L and Li, J}, title = {An improved CRISPR and CRISPR interference (CRISPRi) toolkit for engineering the model methanogenic archaeon Methanococcus maripaludis.}, journal = {Microbial cell factories}, volume = {23}, number = {1}, pages = {239}, pmid = {39227830}, issn = {1475-2859}, support = {32393974, 32393972 and 32270054//National Natural Science Foundation of China/ ; 2020YFA0906800 and 2019YFA0905500//National Key Research and Development Program of China/ ; }, mesh = {*Methanococcus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Archaeal ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; }, abstract = {BACKGROUND: The type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2-based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory.<br><br>RESULTS: This study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5'end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes.<br><br>CONCLUSIONS: Therefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.}, }
@article {pmid39227671, year = {2024}, author = {Grüttner, S and Kempken, F}, title = {A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {20469}, pmid = {39227671}, issn = {2045-2322}, mesh = {*Neurospora crassa/genetics ; *CRISPR-Cas Systems ; *Mutagenesis ; *Gene Editing/methods ; Fungal Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Fungal ; }, abstract = {As a widely used eukaryotic model organism, Neurospora crassa offers advantages in genetic studies due to its diverse biology and rapid growth. Traditional genetic manipulation methods, such as homologous recombination, require a considerable amount of time and effort. In this study, we present an easy-to-use CRIPSR/Cas9 system for N. crassa, in which the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. Our approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process. Using cyclosporin-resistant-1 (csr-1) as a selectable marker gene, we achieved 100% editing efficiency under selection conditions. Furthermore, we successfully edited the non-selectable gene N-acylethanolamine amidohydrolase-2 (naa-2), demonstrating the versatility of the system. Combining gRNAs targeting csr-1 and naa-2 simultaneously increased the probability of finding mutants carrying the non-selectable mutation. The system is not only user-friendly but also effective, providing a rapid and efficient method for generating loss-of-function mutants in N. crassa compared to traditional methods.}, }
@article {pmid39185736, year = {2024}, author = {Tong, S and Hong, R and Chen, W and Chai, M and Zhang, Y and Sun, Y and Wang, Q and Li, D}, title = {Synchronous Bioproduction of Betanin and Mycoprotein in the Engineered Edible Fungus Fusarium venenatum.}, journal = {Journal of agricultural and food chemistry}, volume = {72}, number = {35}, pages = {19462-19469}, doi = {10.1021/acs.jafc.4c06071}, pmid = {39185736}, issn = {1520-5118}, mesh = {*Fusarium/metabolism/genetics ; *Betacyanins/metabolism/chemistry ; *Fungal Proteins/genetics/metabolism ; *Fermentation ; Metabolic Engineering ; CRISPR-Cas Systems ; Food Coloring Agents/metabolism/chemistry ; }, abstract = {Sustainable production of edible microbial proteins and red food colorants is an important demand for future food. Therefore, creation of a chassis strain that can efficiently synthesize both products is extremely necessary and meaningful. To realize this envision, a CRISPR/Cas9-based visual multicopy integration system was successfully developed in Fusarium venenatum. Subsequently, the de novo synthesis of the red food colorant betanin was achieved in the engineered F. venenatum using the above system. After fermentation optimization, the final yields of betanin and mycoprotein reached 1.91 and 9.53 g/L, respectively, when the constant pH naturally decreased from 6 to 4 without the addition of acid after 48 h of fermentation. These results determine a highly suitable chassis strain for the microbial biomanufacturing of betanin, and the obtained engineered strain here is expected to expand the application prospect and improve economic returns of F. venenatum in the field of future food.}, }
@article {pmid39039029, year = {2024}, author = {Crain, AT and Nevil, M and Leatham-Jensen, MP and Reeves, KB and Matera, AG and McKay, DJ and Duronio, RJ}, title = {Redesigning the Drosophila histone gene cluster: an improved genetic platform for spatiotemporal manipulation of histone function.}, journal = {Genetics}, volume = {228}, number = {1}, pages = {}, doi = {10.1093/genetics/iyae117}, pmid = {39039029}, issn = {1943-2631}, support = {R35 GM136435/GM/NIGMS NIH HHS/United States ; T32-GM007092/GF/NIH HHS/United States ; //A.T.C./ ; //M.N./ ; R35-GM136435//R.J.D./ ; R35-GM128851//A.G.M./ ; //D.J.M/ ; }, mesh = {Animals ; *Histones/metabolism/genetics ; *Drosophila melanogaster/genetics ; *Drosophila Proteins/genetics/metabolism ; Gene Editing/methods ; Multigene Family ; CRISPR-Cas Systems ; }, abstract = {Mutating replication-dependent (RD) histone genes is an important tool for understanding chromatin-based epigenetic regulation. Deploying this tool in metazoans is particularly challenging because RD histones in these organisms are typically encoded by many genes, often located at multiple loci. Such gene arrangements make the ability to generate homogenous histone mutant genotypes by site-specific gene editing quite difficult. Drosophila melanogaster provides a solution to this problem because the RD histone genes are organized into a single large tandem array that can be deleted and replaced with transgenes containing mutant histone genes. In the last ∼15 years several different RD histone gene replacement platforms were developed using this simple strategy. However, each platform contains weaknesses that preclude full use of the powerful developmental genetic capabilities available to Drosophila researchers. Here we describe the development of a newly engineered platform that rectifies many of these weaknesses. We used CRISPR to precisely delete the RD histone gene array (HisC), replacing it with a multifunctional cassette that permits site-specific insertion of either one or two synthetic gene arrays using selectable markers. We designed this cassette with the ability to selectively delete each of the integrated gene arrays in specific tissues using site-specific recombinases. We also present a method for rapidly synthesizing histone gene arrays of any genotype using Golden Gate cloning technologies. These improvements facilitate the generation of histone mutant cells in various tissues at different stages of Drosophila development and provide an opportunity to apply forward genetic strategies to interrogate chromatin structure and gene regulation.}, }
@article {pmid39030344, year = {2024}, author = {Jana, B and Liu, X and Dénéréaz, J and Park, H and Leshchiner, D and Liu, B and Gallay, C and Zhu, J and Veening, JW and van Opijnen, T}, title = {CRISPRi-TnSeq maps genome-wide interactions between essential and non-essential genes in bacteria.}, journal = {Nature microbiology}, volume = {9}, number = {9}, pages = {2395-2409}, pmid = {39030344}, issn = {2058-5276}, support = {AI135737//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI156203//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI124302//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI158076//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI124402//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 82270012//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Streptococcus pneumoniae/genetics/metabolism ; *Genes, Essential/genetics ; *Genome, Bacterial/genetics ; *DNA Transposable Elements/genetics ; *CRISPR-Cas Systems ; Gene Regulatory Networks ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genes, Bacterial/genetics ; Bacterial Proteins/genetics/metabolism ; Gene Knockout Techniques ; }, abstract = {Genetic interactions identify functional connections between genes and pathways, establishing gene functions or druggable targets. Here we use CRISPRi-TnSeq, CRISPRi-mediated knockdown of essential genes alongside TnSeq-mediated knockout of non-essential genes, to map genome-wide interactions between essential and non-essential genes in Streptococcus pneumoniae. Transposon-mutant libraries constructed in 13 CRISPRi strains enabled screening of ~24,000 gene pairs. This identified 1,334 genetic interactions, including 754 negative and 580 positive interactions. Network analyses show that 17 non-essential genes pleiotropically interact with more than half the essential genes tested. Validation experiments confirmed that a 7-gene subset protects against perturbations. Furthermore, we reveal hidden redundancies that compensate for essential gene loss, relationships between cell wall synthesis, integrity and cell division, and show that CRISPRi-TnSeq identifies synthetic and suppressor-type relationships between both functionally linked and disparate genes and pathways. Importantly, in species where CRISPRi and Tn-Seq are established, CRISPRi-TnSeq should be straightforward to implement.}, }
@article {pmid39025450, year = {2024}, author = {Wu, CJ and Livak, F and Ashwell, JD}, title = {The histone methyltransferase KMT2D maintains cellular glucocorticoid responsiveness by shielding the glucocorticoid receptor from degradation.}, journal = {The Journal of biological chemistry}, volume = {300}, number = {8}, pages = {107581}, pmid = {39025450}, issn = {1083-351X}, mesh = {Humans ; *Receptors, Glucocorticoid/metabolism/genetics ; Neoplasm Proteins/metabolism/genetics ; Glucocorticoids/metabolism/pharmacology ; DNA-Binding Proteins/metabolism/genetics ; Jurkat Cells ; Proteolysis ; Histone-Lysine N-Methyltransferase/metabolism/genetics ; Apoptosis ; CRISPR-Cas Systems ; Cell Line, Tumor ; }, abstract = {Because of their ability to induce lymphocyte apoptosis, glucocorticoids (GC) are widely used to treat hematological malignancies such as lymphomas and multiple myeloma. Their effectiveness is often limited, however, due to the development of glucocorticoid resistance by a variety of molecular mechanisms. Here we performed an unbiased genome-wide CRISPR screen with the human T-cell leukemia cell line Jurkat to find previously unidentified genes required for GC-induced apoptosis. One such gene was KMT2D (also known as MLL2 or MLL4), which encodes a histone lysine methyltransferase whose mutations are associated with a variety of cancers, blood malignancies in particular, and are considered markers of poor prognosis. Knockout of KMT2D by CRISPR/Cas9 gene editing in Jurkat and several multiple myeloma cell lines downregulated GR protein expression. Surprisingly, this was not due to a reduction in GR transcripts, but rather to a decrease in the protein's half-life, primarily due to proteasomal degradation. Reconstitution of KMT2D expression restored GR levels. In contrast to the known ability of KMT2D to control gene transcription through covalent histone methylation, KMT2D-mediated upregulation of GR levels did not require its methyltransferase activity. Co-immunoprecipitation and proximity ligation assays found constitutive binding of KMT2D to the GR, which was enhanced in the presence of GC. These observations reveal KMT2D to be essential for the stabilization of cellular GR levels, and suggest a possible mechanism by which KMT2D mutations may lead to GC resistance in some malignancies.}, }
@article {pmid38820091, year = {2024}, author = {Petrosky, SJ and Williams, TM and Rebeiz, M}, title = {A genetic screen of transcription factors in the Drosophila melanogaster abdomen identifies novel pigmentation genes.}, journal = {G3 (Bethesda, Md.)}, volume = {14}, number = {9}, pages = {}, doi = {10.1093/g3journal/jkae097}, pmid = {38820091}, issn = {2160-1836}, support = {R35GM14196/GF/NIH HHS/United States ; }, mesh = {Animals ; *Drosophila melanogaster/genetics ; *Pigmentation/genetics ; *Transcription Factors/genetics/metabolism ; *Drosophila Proteins/genetics ; Abdomen ; Phenotype ; CRISPR-Cas Systems ; Genetic Testing ; Animals, Genetically Modified ; Gene Regulatory Networks ; }, abstract = {Gene regulatory networks specify the gene expression patterns needed for traits to develop. Differences in these networks can result in phenotypic differences between organisms. Although loss-of-function genetic screens can identify genes necessary for trait formation, gain-of-function screens can overcome genetic redundancy and identify loci whose expression is sufficient to alter trait formation. Here, we leveraged transgenic lines from the Transgenic RNAi Project at Harvard Medical School to perform both gain- and loss-of-function CRISPR/Cas9 screens for abdominal pigmentation phenotypes. We identified measurable effects on pigmentation patterns in the Drosophila melanogaster abdomen for 21 of 55 transcription factors in gain-of-function experiments and 7 of 16 tested by loss-of-function experiments. These included well-characterized pigmentation genes, such as bab1 and dsx, and transcription factors that had no known role in pigmentation, such as slp2. Finally, this screen was partially conducted by undergraduate students in a Genetics Laboratory course during the spring semesters of 2021 and 2022. We found this screen to be a successful model for student engagement in research in an undergraduate laboratory course that can be readily adapted to evaluate the effect of hundreds of genes on many different Drosophila traits, with minimal resources.}, }
@article {pmid39227603, year = {2024}, author = {Bergman, S and Tuller, T}, title = {Codon usage and expression-based features significantly improve prediction of CRISPR efficiency.}, journal = {NPJ systems biology and applications}, volume = {10}, number = {1}, pages = {100}, pmid = {39227603}, issn = {2056-7189}, mesh = {*Codon Usage/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computational Biology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Codon/genetics ; Gene Expression/genetics ; }, abstract = {CRISPR is a precise and effective genome editing technology; but despite several advancements during the last decade, our ability to computationally design gRNAs remains limited. Most predictive models have relatively low predictive power and utilize only the sequence of the target site as input. Here we suggest a new category of features, which incorporate the target site genomic position and the presence of genes close to it. We calculate four features based on gene expression and codon usage bias indices. We show, on CRISPR datasets taken from 3 different cell types, that such features perform comparably with 425 state-of-the-art predictive features, ranking in the top 2-12% of features. We trained new predictive models, showing that adding expression features to them significantly improves their r[2] by up to 0.04 (relative increase of 39%), achieving average correlations of up to 0.38 on their validation sets; and that these features are deemed important by different feature importance metrics. We believe that incorporating the target site's position, in addition to its sequence, in features such as we have generated here will improve our ability to predict, design and understand CRISPR experiments going forward.}, }
@article {pmid39227468, year = {2024}, author = {Akhtar, N and Shadab, M and Bhatti, N and Sajid Ansarì, M and Siddiqui, MB}, title = {Biotechnological frontiers in harnessing allelopathy for sustainable crop production.}, journal = {Functional &amp; integrative genomics}, volume = {24}, number = {5}, pages = {155}, pmid = {39227468}, issn = {1438-7948}, mesh = {*Allelopathy ; *Crops, Agricultural/genetics/metabolism ; Biotechnology ; Crop Production/methods ; CRISPR-Cas Systems ; Weed Control/methods ; }, abstract = {Allelopathy, the phenomenon in which plants release biochemical compounds that influence the growth and development of neighbouring plants, presents promising opportunities for revolutionizing agriculture towards sustainability. This abstract explores the role of biotechnological advancements in unlocking the potential of allelopathy for sustainable crop production and its applications in agriculture, ecology, and natural resource management. By combining molecular, genetic, biochemical, and bioinformatic tools, researchers can unravel the complexities of allelopathic interactions and their potential for sustainable crop production and environmental stewardship. The development of novel management methods for weed control is getting a lot of attention with the introduction of new genetic technologies such as Gene drive, Transgene technologies, Gene silencing, Marker-assisted selection (MAS), and Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). By strengthening competitive characteristics these tools hold great promise for boosting crops' ability to compete with weeds. Considering recent literature, this review highlights the genetic, transcriptomics, and metabolomics approaches to allelopathy. Employing allelopathic properties in agriculture offer sustainable benefits like natural weed management, pest management, and reduced chemical pollution, but challenges include environmental factors, toxicity, regulatory hurdles, and limited resources. Effective integration requires continued research, regulatory support, and farmer education . Also, we aimed to identify the biotechnological domains requiring more investigation and to provide the basis for future advances through this assessment.}, }
@article {pmid39227390, year = {2024}, author = {Brands, J and Bravo, S and Jürgenliemke, L and Grätz, L and Schihada, H and Frechen, F and Alenfelder, J and Pfeil, C and Ohse, PG and Hiratsuka, S and Kawakami, K and Schmacke, LC and Heycke, N and Inoue, A and König, G and Pfeifer, A and Wachten, D and Schulte, G and Steinmetzer, T and Watts, VJ and Gomeza, J and Simon, K and Kostenis, E}, title = {A molecular mechanism to diversify Ca[2+] signaling downstream of Gs protein-coupled receptors.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7684}, pmid = {39227390}, issn = {2041-1723}, support = {214362475/GRK1873/2//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 494832089 /GRK2873//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 290847012/FOR2372//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 214362475/GRK1873/2//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 494832089 /GRK2873//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; JP21H04791//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP21H05113//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP21H05037//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPJSBP120213501//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22ama121038//Japan Agency for Medical Research and Development (AMED)/ ; JP22zf0127007//Japan Agency for Medical Research and Development (AMED)/ ; JPMJFR215T//MEXT | Japan Science and Technology Agency (JST)/ ; JPMJMS2023//MEXT | Japan Science and Technology Agency (JST)/ ; 22714181//MEXT | Japan Science and Technology Agency (JST)/ ; }, mesh = {Humans ; *Calcium Signaling ; *Phospholipase C beta/metabolism/genetics ; HEK293 Cells ; *Receptors, G-Protein-Coupled/metabolism/genetics ; *Calcium/metabolism ; GTP-Binding Protein alpha Subunits, Gq-G11/metabolism/genetics ; CRISPR-Cas Systems ; GTP-Binding Protein alpha Subunits, Gs/metabolism/genetics ; Cyclic AMP/metabolism ; Animals ; Gene Editing ; Cytosol/metabolism ; GTP-Binding Protein beta Subunits/metabolism/genetics ; Adenylyl Cyclases/metabolism/genetics ; }, abstract = {A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca[2+] in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca[2+]. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCβ1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gβγ as driver of a PLCβ2/3-mediated cytosolic Ca[2+] release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCβ3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCβ3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.}, }
@article {pmid39227100, year = {2024}, author = {Harris, HC and Warren, FJ}, title = {The impact of Cas9-mediated mutagenesis of genes encoding potato starch-branching enzymes on starch structural properties and in vitro digestibility.}, journal = {Carbohydrate polymers}, volume = {345}, number = {}, pages = {122561}, doi = {10.1016/j.carbpol.2024.122561}, pmid = {39227100}, issn = {1879-1344}, mesh = {*Solanum tuberosum/genetics/chemistry ; *1,4-alpha-Glucan Branching Enzyme/genetics/metabolism/chemistry ; *Starch/chemistry/metabolism ; *Mutagenesis ; Digestion ; CRISPR-Cas Systems/genetics ; Amylopectin/chemistry/metabolism ; Amylose/chemistry/metabolism ; Plant Proteins/genetics/chemistry/metabolism ; }, abstract = {The digestibility of starch is affected by amylose content, and increasing amylopectin chain length which can be manipulated by alterations to genes encoding starch-branching enzymes (SBEs). We investigated the impact of Cas9-mediated mutagenesis of SBEs in potato on starch structural properties and digestibility. Four potato starches with edited SBE genes were tested. One lacked SBE1 and SBE2, two lacked SBE2 and had reduced SBE1, and one had reduced SBE2 only. Starch structure and thermal properties were characterised by DSC and XRD. The impact of different thermal treatments on digestibility was studied using an in vitro digestion protocol. All native potato starches were resistant to digestion, and all gelatinised starches were highly digestible. SBE modified starches had higher gelatinisation temperatures than wild type potatoes and retrograded more rapidly. Gelatinisation and 18 h of retrogradation, increased gelatinisation enthalpy, but this did not translate to differences in digestion. Following 7 days of retrogradation, starch from three modified SBE starch lines was less digestible than starch from wild-type potatoes, likely due to the recrystallisation of the long amylopectin chains. Our results indicate that reductions in SBE in potato may be beneficial to health by increasing the amount of fibre reaching the colon after retrogradation.}, }
@article {pmid39226667, year = {2024}, author = {Yang, S and Zhang, Y and Li, C and Tan, X}, title = {Repurposing endogenous Type I-D CRISPR-Cas system for genome editing in Synechococcus sp. PCC7002.}, journal = {Microbiological research}, volume = {288}, number = {}, pages = {127884}, doi = {10.1016/j.micres.2024.127884}, pmid = {39226667}, issn = {1618-0623}, abstract = {Synechococcus sp. PCC7002 has been considered as a photosynthetic chassis for the conversion of CO2 into biochemicals through genetic modification. However, conventional genetic manipulation techniques prove inadequate for comprehensive genetic modifications in this strain. Here, we present the development of a genome editing tool tailored for S. PCC7002, leveraging its endogenous type I-D CRISPR-Cas system. Utilizing this novel tool, we successfully deleted the glgA1 gene and iteratively edited the genome to obtain a double mutant of glgA1 and glgA2 genes. Additionally, large DNA fragments encompassing the entire type I-A (∼14 kb) or III-B CRISPR-Cas (∼21 kb) systems were completely knocked-out in S. PCC7002 using our tool. Furthermore, the endogenous pAQ5 plasmid, approximately 38 kb in length, was successfully cured from S. PCC7002. Our work demonstrates the feasibility of harnessing the endogenous CRISPR-Cas system for genome editing in S. PCC7002, thereby enriching the genetic toolkit for this species and providing a foundation for future enhancements in its biosynthetic efficiency.}, }
@article {pmid39225329, year = {2024}, author = {Feng, S and Zhang, Y and Wang, Y and Gao, Y and Song, Y}, title = {Harnessing Gene Editing Technology for Tumor Microenvironment Modulation: An Emerging Anticancer Strategy.}, journal = {Chemistry (Weinheim an der Bergstrasse, Germany)}, volume = {}, number = {}, pages = {e202402485}, doi = {10.1002/chem.202402485}, pmid = {39225329}, issn = {1521-3765}, abstract = {Cancer is a multifaceted disease influenced by both intrinsic cellular traits and extrinsic factors, with the tumor microenvironment (TME) being crucial in its progression. To satisfy their high proliferation and aggressiveness, cancer cell always plunders large amounts of nutrition and releases various signals to the surrounding, forming a dynamic TME with special metabolic, immune, microbial and physical characteristics. Due to the neglect of interactions between tumor cell and TME, traditional cancer therapies often struggle with challenges such as drug resistance, low efficacy, and recurrence. Importantly, with the development of gene editing technologies, particularly the CRISPR-Cas system, offers promising new strategies for cancer treatment. Combined with nanomaterials strategies, CRISPR-Cas technology exhibits precision, affordability, and user-friendliness with reduced side effects, which holds great promise for profoundly altering the TME at a genetic level, potentially leading to lasting anticancer outcomes. This review will delve into how CRISPR-Cas can be leveraged to manipulate the TME, examining its potential as a transformative anticancer therapy.}, }
@article {pmid39225039, year = {2024}, author = {Ma, J and Zhang, W and Rahimialiabadi, S and Ganesh, NU and Sun, Z and Parvez, S and Peterson, RT and Yeh, JJ}, title = {Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase.}, journal = {Biology open}, volume = {13}, number = {9}, pages = {}, doi = {10.1242/bio.061666}, pmid = {39225039}, issn = {2046-6390}, support = {R01 GM134069/GF/NIH HHS/United States ; 201808210354//China Scholarship Council/ ; //Harvard Medical School; University of Utah/ ; }, mesh = {*CRISPR-Cas Systems ; *Integrases/genetics/metabolism ; Animals ; *Zebrafish/genetics ; *Genotype ; Gene Transfer Techniques ; Genotyping Techniques ; Transgenes ; Genes, Reporter ; Gene Editing/methods ; Animals, Genetically Modified ; }, abstract = {Here, we introduce 'TICIT', targeted integration by CRISPR-Cas9 and integrase technologies, which utilizes the site-specific DNA recombinase - phiC31 integrase - to insert large DNA fragments into CRISPR-Cas9 target loci. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to repeatedly perform site-specific transgenesis at the exact genomic location with high precision and efficiency. We applied this approach to devise a method for the instantaneous determination of a zebrafish's genotype simply by examining its color. When a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, employing this method allows facile identification of a population of homozygous mutant embryos even before the mutant phenotypes manifest. Thus, it should facilitate various downstream applications, such as large-scale chemical screens. We demonstrated that TICIT could also create reporter fish driven by an endogenous promoter. Further, we identified a landing site in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types. In sum, TICIT enables site-specific DNA integration without requiring complex donor DNA construction. It can yield consistent transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms.}, }
@article {pmid39224035, year = {2024}, author = {Chopra, A and Bhuvanagiri, G and Natu, K and Chopra, A}, title = {Role of CRISPR-Cas systems in periodontal disease pathogenesis and potential for periodontal therapy: A review.}, journal = {Molecular oral microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1111/omi.12483}, pmid = {39224035}, issn = {2041-1014}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPRs) are DNA sequences capable of editing a host genome sequence. CRISPR and its specific CRISPR-associated (Cas) protein complexes have been adapted for various applications. These include activating or inhibiting specific genetic sequences or acting as molecular scissors to cut and modify the host DNA precisely. CRISPR-Cas systems are also naturally present in many oral bacteria, where they aid in nutrition, biofilm formation, inter- and intraspecies communication (quorum sensing), horizontal gene transfer, virulence, inflammation modulation, coinfection, and immune response evasion. It even functions as an adaptive immune system, defending microbes against invading viruses and foreign genetic elements from other bacteria by targeting and degrading their DNA. Recently, CRISPR-Cas systems have been tested as molecular editing tools to manipulate specific genes linked with periodontal disease (such as periodontitis) and as novel methods of delivering antimicrobial agents to overcome antimicrobial resistance. With the rapidly increasing role of CRISPR in treating inflammatory diseases, its application in periodontal disease is also becoming popular. Therefore, this review aims to discuss the different types of CRISPR-Cas in oral microbes and their role in periodontal disease pathogenesis and precision periodontal therapy.}, }
@article {pmid39223923, year = {2024}, author = {Shi, S and Ge, Y and Yan, Q and Wan, S and Li, M and Li, M}, title = {Activating UCHL1 through the CRISPR activation system promotes cartilage differentiation mediated by HIF-1α/SOX9.}, journal = {Journal of cellular and molecular medicine}, volume = {28}, number = {17}, pages = {e70051}, doi = {10.1111/jcmm.70051}, pmid = {39223923}, issn = {1582-4934}, support = {2023A04J0427//Basic and Applied Basic Research Foundation of Guangzhou/ ; PY2021028//Science Research Cultivation Program of Stomatological Hospital, Southern Medical University/ ; B2023405//Medical Science and Technology Research Fund of Guangdong Province/ ; }, mesh = {*Ubiquitin Thiolesterase/metabolism/genetics ; *SOX9 Transcription Factor/metabolism/genetics ; *Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; *Cell Differentiation/genetics ; *Chondrogenesis/genetics ; Animals ; Humans ; Cartilage/metabolism ; Chondrocytes/metabolism/cytology ; Mesenchymal Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Mice ; }, abstract = {Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.}, }
@article {pmid39223394, year = {2024}, author = {Razzaq, MK and Babur, MN and Awan, MJA and Raza, G and Mobeen, M and Aslam, A and Siddique, KHM}, title = {Revolutionizing soybean genomics: How CRISPR and advanced sequencing are unlocking new potential.}, journal = {Functional &amp; integrative genomics}, volume = {24}, number = {5}, pages = {153}, pmid = {39223394}, issn = {1438-7948}, mesh = {*Glycine max/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genome, Plant ; Genomics/methods ; Plant Breeding/methods ; Polymorphism, Single Nucleotide ; Genome-Wide Association Study ; }, abstract = {Soybean Glycine max L., paleopolyploid genome, poses challenges to its genetic improvement. However, the development of reference genome assemblies and genome sequencing has completely changed the field of soybean genomics, allowing for more accurate and successful breeding techniques as well as research. During the single-cell revolution, one of the most advanced sequencing tools for examining the transcriptome landscape is single-cell RNA sequencing (scRNA-seq). Comprehensive resources for genetic improvement of soybeans may be found in the SoyBase and other genomics databases. CRISPR-Cas9 genome editing technology provides promising prospects for precise genetic modifications in soybean. This method has enhanced several soybean traits, including as yield, nutritional value, and resistance to both biotic and abiotic stresses. With base editing techniques that allow for precise DNA modifications, the use of CRISPR-Cas9 is further increased. With the availability of the reference genome for soybeans and the following assembly of wild and cultivated soybeans, significant chromosomal rearrangements and gene duplication events have been identified, offering new perspectives on the complex genomic structure of soybeans. Furthermore, major single nucleotide polymorphisms (SNPs) linked to stachyose and sucrose content have been found through genome-wide association studies (GWAS), providing important tools for enhancing soybean carbohydrate profiles. In order to open up new avenues for soybean genetic improvement, future research approaches include investigating transcriptional divergence processes, enhancing genetic resources, and incorporating CRISPR-Cas9 technologies.}, }
@article {pmid39222468, year = {2024}, author = {Inam, S and Muhammad, A and Irum, S and Rehman, N and Riaz, A and Uzair, M and Khan, MR}, title = {Genome editing for improvement of biotic and abiotic stress tolerance in cereals.}, journal = {Functional plant biology : FPB}, volume = {51}, number = {}, pages = {}, doi = {10.1071/FP24092}, pmid = {39222468}, issn = {1445-4416}, mesh = {*Gene Editing ; *Edible Grain/genetics ; *Stress, Physiological/genetics ; *CRISPR-Cas Systems ; Climate Change ; Genome, Plant ; }, abstract = {Global agricultural production must quadruple by 2050 to fulfil the needs of a growing global population, but climate change exacerbates the difficulty. Cereals are a very important source of food for the world population. Improved cultivars are needed, with better resistance to abiotic stresses like drought, salt, and increasing temperatures, and resilience to biotic stressors like bacterial and fungal infections, and pest infestation. A popular, versatile, and helpful method for functional genomics and crop improvement is genome editing. Rapidly developing genome editing techniques including clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) are very important. This review focuses on how CRISPR/Cas9 genome editing might enhance cereals' agronomic qualities in the face of climate change, providing important insights for future applications. Genome editing efforts should focus on improving characteristics that confer tolerance to conditions exacerbated by climate change (e.g. drought, salt, rising temperatures). Improved water usage efficiency, salt tolerance, and heat stress resilience are all desirable characteristics. Cultivars that are more resilient to insect infestations and a wide range of biotic stressors, such as bacterial and fungal diseases, should be created. Genome editing can precisely target genes linked to disease resistance pathways to strengthen cereals' natural defensive systems.}, }
@article {pmid39171996, year = {2024}, author = {Wang, Q and Xia, J and Yang, C and Chen, X and Chen, B and Li, Y and Huang, H and Lin, B and Guo, L and Xu, J}, title = {Cross-Priming-Linked Hierarchical Isothermal Amplification Programming Progressive Activating Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a in miRNA Signaling.}, journal = {Analytical chemistry}, volume = {96}, number = {35}, pages = {14205-14214}, doi = {10.1021/acs.analchem.4c02795}, pmid = {39171996}, issn = {1520-6882}, mesh = {*Nucleic Acid Amplification Techniques/methods ; *MicroRNAs/genetics/analysis ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; Signal Transduction ; Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; CRISPR-Associated Proteins ; }, abstract = {Cascade isothermal nucleic acid amplification, which integrates several different amplification protocols to enhance the assay performance, is widely utilized in biosensing, particularly for detecting microRNAs (miRNAs), crucial biomarkers associated with tumor initiation and progression. However, striking a balance between a high amplification efficiency and simplicity in design remains a challenge. Therefore, methods achieving high amplification efficiency without significantly increasing complexity are highly favored. In this study, we propose a novel approach for miRNA detection, employing cross-priming-linked hierarchical isothermal amplification (CP-HIA) to progressively activate the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. The CP-HIA method strategically combines nicking-rolling circle amplification (n-RCA) and palindrome-aided circular strand displacement amplification (p-CSDA) for miRNA detection. Remarkably, this method utilizes only two main probes. Its key innovation lies in the interactive cross-priming strategy, wherein the amplification product from n-RCA is recycled to further drive p-CSDA, and vice versa. This interactive process establishes a hierarchical amplification, significantly enriching the activation probes for progressive CRISPR/Cas12a activation and subsequent target signal amplification. Consequently, the method exhibits greatly enhanced analytical performance, including high sensitivity and specificity in detecting low concentrations of miRNA. As low as 1.06 fM miRNA can thus be quantitatively detected, and the linear response of the miRNA is from 10 fM to 10 nM. These features demonstrate its potential for early disease diagnosis and monitoring. We anticipate that the CP-HIA method will serve as a promising platform for developing advanced molecular diagnostic tools for biomedical research.}, }
@article {pmid39120142, year = {2024}, author = {Wang, T and Li, A and Zhao, H and Wu, Q and Guo, J and Tian, H and Wang, J and Que, Y and Xu, L}, title = {A novel system with robust compatibility and stability for detecting Sugarcane yellow leaf virus based on CRISPR-Cas12a.}, journal = {Microbiology spectrum}, volume = {12}, number = {9}, pages = {e0114924}, doi = {10.1128/spectrum.01149-24}, pmid = {39120142}, issn = {2165-0497}, mesh = {*Saccharum/virology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/virology ; *Nucleic Acid Amplification Techniques/methods ; *Luteoviridae/genetics/isolation &amp; purification ; *Plant Leaves/virology ; Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Sugarcane yellow leaf virus (SCYLV) can reduce sugarcane productivity. A novel detection system based on reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA) combined with CRISPR-Cas12a, named RT-MIRA-CRISPR-Cas12a, was developed. This innovative approach employs crude leaf extract directly as the reaction template, streamlining the extraction process for simplicity and speed. Combining RT-MIRA and CRISPR-Cas12a in one reaction tube increases the ease of operation while reducing the risk of aerosol contamination. In addition, it exhibits sensitivity equivalent to qPCR, boasting a lower detection limit of 25 copies. Remarkably, the entire process, from sample extraction to reaction completion, requires only 52-57 minutes, just a thermostat water bath. The result can be observed and judged by the naked eye.IMPORTANCESugarcane yellow leaf disease (SCYLD) is an important viral disease that affects sugarcane yield. There is an urgent need for rapid, sensitive, and stable detection methods. The reverse transcription-multienzyme isothermal rapid amplification combined with CRISPR-Cas12a (RT-MIRA-CRISPR-Cas12a) method established in this study has good specificity and high sensitivity. In addition, the system showed good compatibility and stability with the crude leaf extract, as shown by the fact that the crude extract of the positive sample could still be stably detected after 1 week when placed at 4°C. RT-MIRA-CRISPR-Cas12a, reverse transcription polymerase chain reaction (RT-PCR), and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect SCYLV on 33 sugarcane leaf samples collected from the field, and it was found that the three methods reached consistent conclusions. This Cas12a-based detection method proves highly suitable for the rapid on-site detection of the SCYLV.}, }
@article {pmid39018097, year = {2024}, author = {Jadlowsky, JK and Chang, JF and Spencer, DH and Warrington, JM and Levine, BL and June, CH and Fraietta, JA and Singh, N}, title = {Regulatory Considerations for Genome-Edited T-cell Therapies.}, journal = {Cancer immunology research}, volume = {12}, number = {9}, pages = {1132-1135}, doi = {10.1158/2326-6066.CIR-24-0482}, pmid = {39018097}, issn = {2326-6074}, support = {//Damon Runyon Cancer Research Foundation (DRCRF)/ ; R37CA285572/BC/NCI NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing ; *T-Lymphocytes/immunology/metabolism ; Genetic Therapy/methods ; Immunotherapy, Adoptive/methods ; Animals ; Receptors, Chimeric Antigen/genetics/immunology ; CRISPR-Cas Systems ; Receptors, Antigen, T-Cell/genetics ; Cell- and Tissue-Based Therapy/methods ; }, abstract = {Methods to engineer the genomes of human cells for therapeutic intervention continue to advance at a remarkable pace. Chimeric antigen receptor-engineered T lymphocytes have pioneered the way for these therapies, initially beginning with insertions of chimeric antigen receptor transgenes into T-cell genomes using classical gene therapy vectors. The broad use of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies to edit endogenous genes has now opened the door to a new era of precision medicine. To add complexity, many engineered cellular therapies under development integrate gene therapy with genome editing to introduce novel biological functions and enhance therapeutic efficacy. Here, we review the current state of scientific, translational, and regulatory oversight of gene-edited cell products.}, }
@article {pmid38991878, year = {2024}, author = {Cerdà, P and Aguilera, C and Riera-Mestre, A}, title = {Pioneering the future: CRISPR-Cas9 gene therapy for hereditary hemorrhagic telangiectasia. Author's reply.}, journal = {European journal of internal medicine}, volume = {127}, number = {}, pages = {142-143}, doi = {10.1016/j.ejim.2024.07.003}, pmid = {38991878}, issn = {1879-0828}, mesh = {*Telangiectasia, Hereditary Hemorrhagic/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; }, }
@article {pmid38977907, year = {2024}, author = {Giuliano, CJ and Wei, KJ and Harling, FM and Waldman, BS and Farringer, MA and Boydston, EA and Lan, TCT and Thomas, RW and Herneisen, AL and Sanderlin, AG and Coppens, I and Dvorin, JD and Lourido, S}, title = {CRISPR-based functional profiling of the Toxoplasma gondii genome during acute murine infection.}, journal = {Nature microbiology}, volume = {9}, number = {9}, pages = {2323-2343}, pmid = {38977907}, issn = {2058-5276}, support = {R01 AI158501/AI/NIAID NIH HHS/United States ; R01 AI144369/AI/NIAID NIH HHS/United States ; R01 AI145941/AI/NIAID NIH HHS/United States ; R01 AI158501/AI/NIAID NIH HHS/United States ; R01 AI144369/AI/NIAID NIH HHS/United States ; R01 AI145941/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; *Toxoplasma/genetics ; Mice ; *Genome, Protozoan ; *Host-Parasite Interactions/genetics ; CRISPR-Cas Systems ; Protozoan Proteins/genetics/metabolism ; Toxoplasmosis, Animal/parasitology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Toxoplasmosis/parasitology ; Antiprotozoal Agents/pharmacology ; Disease Models, Animal ; Pyrimethamine/pharmacology ; }, abstract = {Examining host-pathogen interactions in animals can capture aspects of infection that are obscured in cell culture. Using CRISPR-based screens, we functionally profile the entire genome of the apicomplexan parasite Toxoplasma gondii during murine infection. Barcoded gRNAs enabled bottleneck detection and mapping of population structures within parasite lineages. Over 300 genes with previously unknown roles in infection were found to modulate parasite fitness in mice. Candidates span multiple axes of host-parasite interaction. Rhoptry Apical Surface Protein 1 was characterized as a mediator of host-cell tropism that facilitates repeated invasion attempts. GTP cyclohydrolase I was also required for fitness in mice and druggable through a repurposed compound, 2,4-diamino-6-hydroxypyrimidine. This compound synergized with pyrimethamine against T. gondii and malaria-causing Plasmodium falciparum parasites. This work represents a complete survey of an apicomplexan genome during infection of an animal host and points to novel interfaces of host-parasite interaction.}, }
@article {pmid38962826, year = {2024}, author = {Xu, J and Ren, J and Xu, K and Fang, M and Ka, M and Xu, F and Wang, X and Wang, J and Han, Z and Feng, G and Zhang, Y and Hai, T and Li, W and Hu, Z}, title = {Elimination of GGTA1, CMAH, β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions.}, journal = {Animal models and experimental medicine}, volume = {7}, number = {4}, pages = {584-590}, doi = {10.1002/ame2.12461}, pmid = {38962826}, issn = {2576-2095}, support = {2019YFA0903800//National Key Research and Development Program/ ; 2021YFA0805701//National Key Research and Development Program/ ; 2021YFA0805905//National Key Research and Development Program/ ; 2022YFA1103603//National Key Research and Development Program/ ; YSBR-012//CAS Project for Young Scientists in Basic Research/ ; 2023ZD0407503//STI 2030-Major Project/ ; 32071456//National Natural Science Foundation of China/ ; 82241224//National Natural Science Foundation of China/ ; XDA16030000//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; }, mesh = {Animals ; Humans ; Swine ; *Transplantation, Heterologous ; *N-Acetylgalactosaminyltransferases/genetics ; *Graft Rejection/genetics/immunology ; Galactosyltransferases/genetics ; Nuclear Proteins/genetics ; Mixed Function Oxygenases/genetics ; Animals, Genetically Modified ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Gene Editing ; Trans-Activators ; }, abstract = {BACKGROUND: Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic, while immunogenic genes need to be eliminated to improve the immune compatibility between humans and pigs. Current knockout strategies are mainly aimed at the genes causing hyperacute immune rejection (HAR) that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure, in which process the MHC II molecule plays critical roles.<br><br>METHODS: Thus, we generate a 4-gene (GGTA1, CMAH, &#x3b2;4GalNT2, and CIITA) knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously.<br><br>RESULTS: We successfully obtained 4KO piglets with deficiency in all alleles of genes, and at cellular and tissue levels. Additionally, the safety of our animals after gene editing was verified by using whole-genome sequencing and karyotyping. Piglets have survived for more than one year in the barrier, and also survived for more than 3&#x2009;months in the conventional environment, suggesting that the piglets without MHC II can be raised in the barrier and then gradually mated in the conventional environment.<br><br>CONCLUSIONS: 4KO piglets have lower immunogenicity, are safe in genomic level, and are easier to breed than the model with both MHC I and II deletion.}, }
@article {pmid38879351, year = {2024}, author = {James, M and Sehgal, VS}, title = {Pioneering the future: CRISPR-Cas9 gene therapy for hereditary hemorrhagic telangiectasia.}, journal = {European journal of internal medicine}, volume = {127}, number = {}, pages = {140-141}, doi = {10.1016/j.ejim.2024.06.012}, pmid = {38879351}, issn = {1879-0828}, mesh = {*Telangiectasia, Hereditary Hemorrhagic/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; Gene Editing/methods ; }, }
@article {pmid38874582, year = {2024}, author = {De Munter, S and Buhl, JL and De Cock, L and Van Parys, A and Daneels, W and Pascal, E and Deseins, L and Ingels, J and Goetgeluk, G and Jansen, H and Billiet, L and Pille, M and Van Duyse, J and Bonte, S and Vandamme, N and Van Dorpe, J and Offner, F and Leclercq, G and Taghon, T and Depla, E and Tavernier, J and Kerre, T and Drost, J and Vandekerckhove, B}, title = {Knocking Out CD70 Rescues CD70-Specific NanoCAR T Cells from Antigen-Induced Exhaustion.}, journal = {Cancer immunology research}, volume = {12}, number = {9}, pages = {1236-1251}, doi = {10.1158/2326-6066.CIR-23-0677}, pmid = {38874582}, issn = {2326-6074}, support = {KW/2196/REG/001/001//Universitair Ziekenhuis Gent (UZ Gent)/ ; 12AP724N//Fonds Wetenschappelijk Onderzoek (FWO)/ ; G028220N//Fonds Wetenschappelijk Onderzoek (FWO)/ ; F2021/IOF-StarTT/107//Ghent University IOF/ ; }, mesh = {Humans ; *CD27 Ligand ; Animals ; Mice ; *Immunotherapy, Adoptive/methods ; *Xenograft Model Antitumor Assays ; *Receptors, Chimeric Antigen/immunology/genetics ; T-Lymphocytes/immunology/metabolism ; Lymphoma, Large B-Cell, Diffuse/immunology/therapy/genetics ; Gene Knockout Techniques ; Cell Line, Tumor ; CRISPR-Cas Systems ; }, abstract = {CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo, using a diffuse large B-cell lymphoma patient-derived xenograft (PDX) model. Although the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. We determined that fratricide was not causing this loss in efficacy but rather CD70 interaction in cis with the nanoCAR-induced exhaustion. Knocking out CD70 in nanoCAR T cells using CRISPR/Cas9 resulted in dramatically enhanced functionality in the diffuse large B-cell lymphoma PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knockout CD70-specific nanoCAR T cells were protected from antigen-induced exhaustion. In addition, we demonstrated that wild-type CD70-specific nanoCAR T cells already exhibited signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T cells. Conversely, the gene signature of knockout CD70-specific nanoCAR T cells overlapped with the gene signature of CAR T-cell infusion products leading to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target.}, }
@article {pmid38869428, year = {2024}, author = {Chang, JF and Wellhausen, N and Engel, NW and Landmann, JH and Hopkins, CR and Salas-McKee, J and Bear, AS and Selli, ME and Agarwal, S and Jadlowsky, JK and Linette, GP and Gill, S and June, CH and Fraietta, JA and Singh, N}, title = {Identification of Core Techniques That Enhance Genome Editing of Human T Cells Expressing Synthetic Antigen Receptors.}, journal = {Cancer immunology research}, volume = {12}, number = {9}, pages = {1136-1146}, doi = {10.1158/2326-6066.CIR-24-0251}, pmid = {38869428}, issn = {2326-6074}, mesh = {Humans ; *Gene Editing/methods ; *T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems ; Receptors, Antigen, T-Cell/genetics/immunology ; }, abstract = {Genome editing technologies have seen remarkable progress in recent years, enabling precise regulation of exogenous and endogenous genes. These advances have been extensively applied to the engineering of human T lymphocytes, leading to the development of practice changing therapies for patients with cancer and the promise of synthetic immune cell therapies for a variety of nonmalignant diseases. Many distinct conceptual and technical approaches have been used to edit T-cell genomes, however targeted assessments of which techniques are most effective for manufacturing, gene editing, and transgene expression are rarely reported. Through extensive comparative evaluation, we identified methods that most effectively enhance engineering of research-scale and preclinical T-cell products at critical stages of manufacturing.}, }
@article {pmid37816604, year = {2024}, author = {Lo, IHY and Matthews, BJ}, title = {Validating Single-Guide RNA for Aedes aegypti Gene Editing.}, journal = {Cold Spring Harbor protocols}, volume = {2024}, number = {9}, pages = {pdb.prot108340}, doi = {10.1101/pdb.prot108340}, pmid = {37816604}, issn = {1559-6095}, mesh = {*Aedes/genetics ; Animals ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Animals, Genetically Modified/genetics ; }, abstract = {Creating transgenic mosquitoes allows for mechanistic studies of basic mosquito biology and the development of novel vector control strategies. CRISPR-Cas9 gene editing has revolutionized gene editing, including in mosquitoes. This protocol details part of the gene editing process of Aedes aegypti mosquitoes via CRISPR-Cas9, through testing and validating single-guide RNAs (sgRNAs). Gene editing activity varies depending on the sequence of sgRNAs used, so validation of sgRNA activity should be done before large-scale generation of mutants or transgenics. sgRNA is designed using online tools and synthesized in <1 h. Once mutants or transgenics are generated via embryo microinjection, sgRNA activity is validated by quick genotyping polymerase chain reaction (PCR) and DNA sequencing.}, }
@article {pmid37816601, year = {2024}, author = {Lo, IHY and Matthews, BJ}, title = {Design and Validation of Guide RNAs for CRISPR-Cas9 Genome Editing in Mosquitoes.}, journal = {Cold Spring Harbor protocols}, volume = {2024}, number = {9}, pages = {pdb.top107688}, doi = {10.1101/pdb.top107688}, pmid = {37816601}, issn = {1559-6095}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Culicidae/genetics ; }, abstract = {CRISPR-Cas9 has revolutionized gene editing for traditional and nontraditional model organisms alike. This tool has opened the door to new mechanistic studies of basic mosquito biology as well as the development of novel vector control strategies based on CRISPR-Cas9, including gene drives that spread genetic elements in the population. Although the promise of the specificity, flexibility, and ease of deployment CRISPR is real, its implementation still requires empirical optimization for each new species of interest, as well as to each genomic target within a given species. Here, we provide an overview of designing and testing single-guide RNAs for the use of CRISPR-based gene editing tools.}, }
@article {pmid39221932, year = {2024}, author = {Kongkaew, R and Uttamapinant, C and Patchsung, M}, title = {Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {210}, pages = {}, doi = {10.3791/66703}, pmid = {39221932}, issn = {1940-087X}, mesh = {*Freeze Drying/methods ; *Point-of-Care Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; Indicators and Reagents/chemistry ; Molecular Diagnostic Techniques/methods ; }, abstract = {Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.}, }
@article {pmid39219618, year = {2024}, author = {Tripathi, L and Ntui, VO and Tripathi, JN}, title = {Application of CRISPR/Cas-based gene-editing for developing better banana.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {12}, number = {}, pages = {1395772}, pmid = {39219618}, issn = {2296-4185}, abstract = {Banana (Musa spp.), including plantain, is one of the major staple food and cash crops grown in over 140 countries in the subtropics and tropics, with around 153 million tons annual global production, feeding about 400 million people. Despite its widespread cultivation and adaptability to diverse environments, banana production faces significant challenges from pathogens and pests that often coexist within agricultural landscapes. Recent advancements in CRISPR/Cas-based gene editing offer transformative solutions to enhance banana resilience and productivity. Researchers at IITA, Kenya, have successfully employed gene editing to confer resistance to diseases such as banana Xanthomonas wilt (BXW) by targeting susceptibility genes and banana streak virus (BSV) by disrupting viral sequences. Other breakthroughs include the development of semi-dwarf plants, and increased β-carotene content. Additionally, non-browning banana have been developed to reduce food waste, with regulatory approval in the Philippines. The future prospects of gene editing in banana looks promising with CRISPR-based gene activation (CRISPRa) and inhibition (CRISPRi) techniques offering potential for improved disease resistance. The Cas-CLOVER system provides a precise alternative to CRISPR/Cas9, demonstrating success in generating gene-edited banana mutants. Integration of precision genetics with traditional breeding, and adopting transgene-free editing strategies, will be pivotal in harnessing the full potential of gene-edited banana. The future of crop gene editing holds exciting prospects for producing banana that thrives across diverse agroecological zones and offers superior nutritional value, ultimately benefiting farmers and consumers. This article highlights the pivotal role of CRISPR/Cas technology in advancing banana resilience, yield and nutritional quality, with significant implications for global food security.}, }
@article {pmid39218584, year = {2024}, author = {Luo, S and Wu, J and Zhong, M and Sun, J and Ao, H and Cao, X and Liu, J and Ju, H}, title = {An electrochemiluminescent imaging strategy based on CRISPR/Cas12a for ultrasensitive detection of nucleic acid.}, journal = {Analytica chimica acta}, volume = {1324}, number = {}, pages = {343040}, doi = {10.1016/j.aca.2024.343040}, pmid = {39218584}, issn = {1873-4324}, mesh = {*Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Biosensing Techniques/methods ; DNA, Viral/analysis/genetics ; Human papillomavirus 18/genetics ; Limit of Detection ; Gold/chemistry ; CRISPR-Associated Proteins ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {BACKGROUND: Persistent infection with human papillomavirus (HPV) significantly contributes to the development of cervical cancer. Thus, it is urgent to develop rapid and accurate methods for HPV detection. Herein, we present an ultrasensitive CRISPR/Cas12a-based electrochemiluminescent (ECL) imaging technique for the detection of HPV-18 DNA.<br><br>RESULT: The ECL DNA sensor array is constructed by applying black hole quencher (BHQ) and polymer dots (Pdots) co-labeled hairpin DNA (hpDNA) onto a gold-coated indium tin oxide slide (Au-ITO). The ECL imaging method involves an incubation process of target HPV-18 with a mixture of crRNA and Cas12a to activate Cas12a, followed by an incubation of the active Cas12a with the ECL sensor. This interaction causes the indiscriminate cleavage of BHQ from Pdots by digesting hpDNA on the sensor surface, leading to the restoration of the ECL signal of Pdots. The ECL brightness readout demonstrates superior performance of the ECL imaging technique, with a linear detection range of 10&#xa0;fM-500 pM and a limit-of-detection (LOD) of 5.3&#xa0;fM.<br><br>SIGNIFICANCE: The Cas12a-based ECL imaging approach offers high sensitivity and a broad detection range, making it highly promising for nucleic acid detection applications.}, }
@article {pmid39218375, year = {2024}, author = {Shidan, Z and Song, L and Yumin, Z and Rong, C and Siteng, W and Meirong, L and Guangjin, L}, title = {First Report of Streptococcus agalactiae isolated from a healthy Captive Sichuan golden snub-nosed monkey (Rhinopithecus roxellana) in China.}, journal = {Microbial pathogenesis}, volume = {}, number = {}, pages = {106907}, doi = {10.1016/j.micpath.2024.106907}, pmid = {39218375}, issn = {1096-1208}, abstract = {Streptococcus agalactiae (S. agalactiae) is an opportunistic pathogen, and to date, studies have mainly focused on S. agalactiae strains isolated from humans, dairy cows, and fish. We reported one S. agalactiae strain, named CFFB, which was isolated from a healthy Sichuan golden snub-nosed monkey. Classical bacteriological approaches, as well as, next-generation sequencing, comparative genomics, and mice challenge test were used to characterize this strain. CFFB was identified as serotype III, ST19 combination which is a common type found in human strains. Phylogenetic analysis showed that the genome of CFFB was closely related to human clinical isolates, rather far away from animal strains. In total, CFFB contained fewer virulence-associated genes and antibiotic resistance genes than human isolates that were close to CFFB in evolutionary relationships. In the mice challenge test, CFFB had a relative weak virulence that just caused death in 33% of ICR mice at a dose of 10[8] CFU by intraperitoneal injection, and CFFB was reisolated from the cardiac blood of the dead mice. Meanwhile, two intact prophages (prophage 1 and 2) were identified in the CFFB genome and shared high similarities with phage Javan52 and Javan29 which from human S. agalactiae isolate Gottschalk 1002A and RBH03, respectively. Moreover, the type II-A CRISPR-Cas system was detected in the CFFB genome, and the spacers from CFFB were the same to the streptococci isolates from human. These results suggest that CFFB isolated from healthy Sichuan golden snub-nosed monkeys may have its origin in human S. agalactiae. Our results suggested some genomic similarities between the S. agalactiae colonized in Sichuan golden snub-nosed monkey and those in infected humans.}, }
@article {pmid39218175, year = {2024}, author = {Hosseini, SA and Elahian, F and Mirzaei, SA}, title = {Innovative genetic scissor strategies and their applications in cancer treatment and prevention: CRISPR modules and challenges.}, journal = {International journal of biological macromolecules}, volume = {}, number = {}, pages = {135239}, doi = {10.1016/j.ijbiomac.2024.135239}, pmid = {39218175}, issn = {1879-0003}, abstract = {There are lots of gene editing tools for targeting genome sequences. Some are almost known, and most are a complete mystery and undiscovered. CRISPR/Cas editing tools have brought about a major revolution in medicine. Researchers have shown that CRISPR can modify DNA much more accurately, economically and easily than previous methods. CRISPR has proven itself effective for the deletion, replacement and insertion of DNA fragments into cell types, tissues and organisms. Recently, combining CRISPR/Cas with factors (transcription factors/repressors, exonucleases, endonucleases, transposons, caspase, fluorescent proteins, oxidoreductive enzymes, DNA/RNA polymerases), and elements (aptamers, barcodes, fluorescent probes, Trigger) have provided genome, transcriptome, proteome and epigenome modification. These modules are being investigated for cancer prevention and therapy and this review focuses on such innovative combinations that hopefully will become a clinical reality in the near future.}, }
@article {pmid39217145, year = {2024}, author = {Niu, H and Maruoka, M and Noguchi, Y and Kosako, H and Suzuki, J}, title = {Phospholipid scrambling induced by an ion channel/metabolite transporter complex.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7566}, pmid = {39217145}, issn = {2041-1723}, support = {21K19261//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 22H02572//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 1199566//MEXT | JST | Core Research for Evolutional Science and Technology (CREST)/ ; }, mesh = {Humans ; *Calcium/metabolism ; *Phospholipid Transfer Proteins/metabolism/genetics ; *Phospholipids/metabolism ; HEK293 Cells ; Ion Channels/metabolism/genetics ; Animals ; CRISPR-Cas Systems ; }, abstract = {Cells establish the asymmetrical distribution of phospholipids and alter their distribution by phospholipid scrambling (PLS) to adapt to environmental changes. Here, we demonstrate that a protein complex, consisting of the ion channel Tmem63b and the thiamine transporter Slc19a2, induces PLS upon calcium (Ca[2+]) stimulation. Through revival screening using a CRISPR sgRNA library on high PLS cells, we identify Tmem63b as a PLS-inducing factor. Ca[2+] stimulation-mediated PLS is suppressed by deletion of Tmem63b, while human disease-related Tmem63b mutants induce constitutive PLS. To search for a molecular link between Ca[2+] stimulation and PLS, we perform revival screening on Tmem63b-overexpressing cells, and identify Slc19a2 and the Ca[2+]-activated K[+] channel Kcnn4 as PLS-regulating factors. Deletion of either of these genes decreases PLS activity. Biochemical screening indicates that Tmem63b and Slc19a2 form a heterodimer. These results demonstrate that a Tmem63b/Slc19a2 heterodimer induces PLS upon Ca[2+] stimulation, along with Kcnn4 activation.}, }
@article {pmid39213744, year = {2024}, author = {Araújo, MRB and Prates, FD and Viana, MVC and Santos, LS and Mattos-Guaraldi, AL and Camargo, CH and Sacchi, CT and Campos, KR and Vieira, VV and Santos, MBN and Bokermann, S and Ramos, JN and Azevedo, V}, title = {Genomic analysis of two penicillin- and rifampin-resistant Corynebacterium rouxii strains isolated from cutaneous infections in dogs.}, journal = {Research in veterinary science}, volume = {179}, number = {}, pages = {105396}, doi = {10.1016/j.rvsc.2024.105396}, pmid = {39213744}, issn = {1532-2661}, abstract = {Although diphtheria is a vaccine-preventable disease, numerous cases are still reported around the world, as well as outbreaks in countries, including European ones. Species of the Corynebacterium diphtheriae complex are potentially toxigenic and, therefore, must be considered given the possible consequences, such as the circulation of clones and transmission of antimicrobial resistance and virulence genes. Recently, Corynebacterium rouxii was characterized and included among the valid species of the complex. Therefore, two cases of C. rouxii infection arising from infections in domestic animals are presented here. We provide molecular characterization, phylogenetic analyses, genome sequencing, and CRISPR-Cas analyses to contribute to a better understanding of the molecular bases, pathogenesis, and epidemiological monitoring of this species, which is still little studied. We confirmed its taxonomic position with genome sequencing and in silico analysis and identified the ST-918 for both strains. The clinical isolates were sensitive resistance to benzylpenicillin and rifampin. Antimicrobial resistance genes, including tetB, rpoB2, and rbpA genes, were predicted. The bla and ampC genes were not found. Several virulence factors were also detected, including adhesion, iron uptake systems, gene regulation (dtxR), and post-translational modification (MdbA). Finally, one prophage and the Type I-E CRISPR-Cas system were identified.}, }
@article {pmid39038660, year = {2024}, author = {Du, Y and Liu, X and Gao, H and Liu, X and Huang, M and Chai, Q and Xing, Z and Zhang, T and Ma, D}, title = {Rapid and one-tube detection of human metapneumovirus using the RT-RPA and CRISPR/Cas12a.}, journal = {Journal of virological methods}, volume = {329}, number = {}, pages = {115001}, doi = {10.1016/j.jviromet.2024.115001}, pmid = {39038660}, issn = {1879-0984}, mesh = {*Metapneumovirus/genetics/isolation &amp; purification ; Humans ; *Paramyxoviridae Infections/diagnosis/virology ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; RNA, Viral/genetics ; Respiratory Tract Infections/virology/diagnosis ; Nucleic Acid Amplification Techniques/methods ; }, abstract = {Human metapneumovirus (HMPV) is a common pathogen that can cause acute respiratory tract infections and is prevalent worldwide. There is yet no effective vaccine or specific treatment for HMPV. Early, rapid, and accurate detection is essential to treat the disease and control the spread of infection. In this study, we created the One-tube assay by combining Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA) with the CRISPR/Cas12a system. By targeting the nucleoprotein (N) gene of HMPV to design specific primers and CRISPR RNAs (crRNAs), combining RT-RPA and CRISPR/Cas12a, established the One-tube assay. Meanwhile, the reaction conditions of the One-tube assay were optimized to achieve rapid and visual detection of HMPV. This assay could detect HMPV at 1 copy/μL in 30 min, without cross-reactivity with nine other respiratory pathogens. We validated the detection performance using clinical specimens and showed that the coincidence rate was 98.53 %，compared to the quantitative reverse-transcription polymerase chain reaction. The One-tube assay reduced the detection time and simplified the manual operation, while maintaining the detection performance and providing a new platform for HMPV detection.}, }
@article {pmid38683016, year = {2024}, author = {Kachwala, MJ and Hamdard, F and Cicek, D and Dagci, H and Smith, CW and Kalla, N and Yigit, MV}, title = {Universal CRISPR-Cas12a and Toehold RNA Cascade Reaction on Paper Substrate for Visual Salmonella Genome Detection.}, journal = {Advanced healthcare materials}, volume = {13}, number = {22}, pages = {e2400508}, doi = {10.1002/adhm.202400508}, pmid = {38683016}, issn = {2192-2659}, support = {2022-08596//National Institute of Food and Agriculture/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Genome, Bacterial/genetics ; *Salmonella/genetics ; Paper ; Lactuca/microbiology/genetics ; Animals ; Milk/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Salmonella, the most prevalent food-borne pathogen, poses significant medical and economic threats. Swift and accurate on-site identification and serotyping of Salmonella is crucial to curb its spread and contamination. Here, a synthetic biology cascade reaction is presented on a paper substrate using CRISPR-Cas12a and recombinase polymerase amplification (RPA), enabling the programming of a standard toehold RNA switch for a genome of choice. This approach employs just one toehold RNA switch design to differentiate between two different Salmonella serotypes, i.e., S. Typhimurium and S. Enteritidis, without the need for reengineering the toehold RNA switch. The sensor exhibits high sensitivity, capable of visually detecting as few as 100 copies of the whole genome from a model Salmonella pathogen on a paper substrate. Furthermore, this robust assay is successfully applied to detect whole genomes in contaminated milk and lettuce samples, demonstrating its potential in real sample analysis. Due to its versatility and practical features, genomes from different organisms can be detected by merely changing a single RNA element in this universal cell-free cascade reaction.}, }
@article {pmid39213392, year = {2024}, author = {Du, Q and Zhang, Z and Yang, W and Zhou, X and Zhou, N and Wu, C and Bao, J}, title = {CBGDA: a manually curated resource for gene-disease associations based on genome-wide CRISPR.}, journal = {Database : the journal of biological databases and curation}, volume = {2024}, number = {}, pages = {}, doi = {10.1093/database/baae077}, pmid = {39213392}, issn = {1758-0463}, support = {31971162 32071275 U20A20410//National Nature Science Foundation of China/ ; 31971162 32071275 U20A20410//National Nature Science Foundation of China/ ; }, mesh = {Humans ; *Databases, Genetic ; *Data Curation/methods ; Genome-Wide Association Study/methods ; Genetic Predisposition to Disease ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Disease/genetics ; }, abstract = {The field of understanding the association between genes and diseases is rapidly expanding, making it challenging for researchers to keep up with the influx of new publications and genetic datasets. Fortunately, there are now several regularly updated databases available that focus on cataloging gene-disease relationships. The development of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system has revolutionized the field of gene editing, providing a highly efficient, accurate, and reliable method for exploring gene-disease associations. However, currently, there is no resource specifically dedicated to collecting and integrating the latest experimentally supported gene-disease association data derived from genome-wide CRISPR screening. To address this gap, we have developed the CRISPR-Based Gene-Disease Associations (CBGDA) database, which includes over 200 manually curated gene-disease association data derived from genome-wide CRISPR screening studies. Through CBGDA, users can explore gene-disease association data derived from genome-wide CRISPR screening, gaining insights into the expression patterns of genes in different diseases, associated chemical data, and variant information. This provides a novel perspective on understanding the associations between genes and diseases. What is more, CBGDA integrates data from several other databases and resources, enhancing its comprehensiveness and utility. In summary, CBGDA offers a fresh perspective and comprehensive insights into the research on gene-disease associations. It fills the gap by providing a dedicated resource for accessing up-to-date, experimentally supported gene-disease association data derived from genome-wide CRISPR screening. Database URL: http://cbgda.zhounan.org/main.}, }
@article {pmid39212859, year = {2024}, author = {Wu, B and Luo, H and Chen, Z and Amin, B and Yang, M and Li, Z and Wu, S and Salmen, SH and Alharbi, SA and Fang, Z}, title = {Rice Promoter Editing: An Efficient Genetic Improvement Strategy.}, journal = {Rice (New York, N.Y.)}, volume = {17}, number = {1}, pages = {55}, pmid = {39212859}, issn = {1939-8425}, support = {32060064/32260498//National Natural Science Foundation of China/ ; Qiankehepingtairencai-YQK (2023) 002//Guizhou Provincial Excellent Young Talents Project of Science and Technology/ ; Qiankehechengguo (2024) General 116 and Qiankehejichu-ZK (2022) Key 008//Guizhou Provincial Science and Technology Projects/ ; Qiankehezhongyindi (2023) 008//Key Laboratory of Molecular Breeding for Grain and Oil Crops in Guizhou Province/ ; Qianjiaoji (2023) 007//Key Laboratory of Functional Agriculture of Guizhou Provincial Department of Education/ ; Qiandongnan Kehe Support (2023) 06//Qiandongnan Science and Technology Support Project/ ; RSP2024R385//Researchers supporting project, King Saud University, Riyadh, Saudi Arabia/ ; }, abstract = {Gene expression levels in rice (Oryza sativa L.) and other plant species are determined by the promoters, which directly control phenotypic characteristics. As essential components of genes, promoters regulate the intensity, location, and timing of gene expression. They contain numerous regulatory elements and serve as binding sites for proteins that modulate transcription, including transcription factors and RNA polymerases. Genome editing can alter promoter sequences, thereby precisely modifying the expression patterns of specific genes, and ultimately affecting the morphology, quality, and resistance of rice. This paper summarizes research on rice promoter editing conducted in recent years, focusing on improvements in yield, heading date, quality, and disease resistance. It is expected to inform the application of promoter editing and encourage further research and development in crop genetic improvement with promote.}, }
@article {pmid39212458, year = {2024}, author = {Lorenzo, CD and Blasco-Escámez, D and Beauchet, A and Wytynck, P and Sanches, M and Garcia Del Campo, JR and Inzé, D and Nelissen, H}, title = {Maize mutant screens: from classical methods to new CRISPR-based approaches.}, journal = {The New phytologist}, volume = {}, number = {}, pages = {}, doi = {10.1111/nph.20084}, pmid = {39212458}, issn = {1469-8137}, support = {833866//H2020 European Research Council/ ; }, abstract = {Mutations play a pivotal role in shaping the trajectory and outcomes of a species evolution and domestication. Maize (Zea mays) has been a major staple crop and model for genetic research for more than 100 yr. With the arrival of site-directed mutagenesis and genome editing (GE) driven by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), maize mutational research is once again in the spotlight. If we combine the powerful physiological and genetic characteristics of maize with the already available and ever increasing toolbox of CRISPR-Cas, prospects for its future trait engineering are very promising. This review aimed to give an overview of the progression and learnings of maize screening studies analyzing forward genetics, natural variation and reverse genetics to focus on recent GE approaches. We will highlight how each strategy and resource has contributed to our understanding of maize natural and induced trait variability and how this information could be used to design the next generation of mutational screenings.}, }
@article {pmid39211481, year = {2024}, author = {Anbazhagan, P and Parameswari, B and Anitha, K and Chaitra, GV and Bajaru, B and Rajashree, A and Mangrauthia, SK and Yousuf, F and Chalam, VC and Singh, GP}, title = {Advances in plant pathogen detection: integrating recombinase polymerase amplification with CRISPR/Cas systems.}, journal = {3 Biotech}, volume = {14}, number = {9}, pages = {214}, doi = {10.1007/s13205-024-04055-x}, pmid = {39211481}, issn = {2190-572X}, abstract = {Plant pathogens are causing substantial economic losses and thus became a significant threat to global agriculture. Effective and timely detection methods are prerequisite for combating the damages caused by the plant pathogens. In the realm of plant pathogen detection, the isothermal amplification techniques, e.g., recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), have emerged as a fast, precise, and most sensitive alternative to conventional PCR but they often comprise high rates of non-specific amplification and operational complexity. In recent advancements, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems, particularly Cas12, have emerged as powerful tools for highly sensitive, specific, and rapid pathogen detection. Exploiting the collateral activities of Cas12, which selectively cleaves single-stranded DNA (ssDNA), novel detection platforms have been developed. The mechanism employs the formation of a triple complex molecule comprising guide RNA, Cas12 enzyme, and the substrate target nucleotide sequence. Upon recognition of the target, Cas12 indiscriminately cleaves the DNA strand, leading to the release of fluorescence from the cleaved ssDNA reporter. Integration of isothermal amplification methods with CRISPR/Cas12 enables one-step detection assays, facilitating rapid pathogen identification within 30 min at a single temperature. This integrated RPA-CRISPR/Cas12a approach eliminates the need for RNA extraction and cDNA conversion, allowing direct use of crude plant sap as a template. With an affordable fluorescence visualization system, this portable method achieves 100-fold greater sensitivity than conventional techniques. This review summarizes recent advances in RPA-CRISPR/Cas12a for detecting plant pathogens, covering primer design, field-level portability, and enhanced sensitivity.}, }
@article {pmid39211201, year = {2024}, author = {Zhang, H and Zhou, C and Mohammad, Z and Zhao, J}, title = {Structural basis of human 20S proteasome biogenesis.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.08.08.607236}, pmid = {39211201}, issn = {2692-8205}, abstract = {New proteasomes are produced to accommodate increases in cellular catabolic demand and prevent the accumulation of cytotoxic proteins. Formation of the proteasomal 20S core complex relies on the function of the five chaperones PAC1-4 and POMP. To understand how these chaperones facilitate proteasome assembly, we tagged the endogenous chaperones using CRISPR/Cas gene editing and examined the chaperone-bound complexes by cryo-EM. We observed an early α-ring intermediate subcomplex that is stabilized by PAC1-4, which transitions to β-ring assembly upon dissociation of PAC3/PAC4 and rearrangement of the PAC1 N-terminal tail. Completion of the β-ring and dimerization of half-proteasomes repositions critical lysine K33 to trigger cleavage of the β pro-peptides, leading to the concerted dissociation of POMP and PAC1/PAC2 to yield mature 20S proteasomes. This study reveals structural insights into critical points along the assembly pathway of the human proteasome and provides a molecular blueprint for 20S biogenesis.}, }
@article {pmid39211128, year = {2024}, author = {Lin, CP and Li, H and Brogan, DJ and Wang, T and Akbari, OS and Komives, EA}, title = {CRISPR-RNA binding drives structural ordering that primes Cas7-11 for target cleavage.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.08.01.606276}, pmid = {39211128}, issn = {2692-8205}, abstract = {Type III-E CRISPR-Cas effectors, of which Cas7-11 is the first, are single proteins that cleave target RNAs without nonspecific collateral cleavage, opening new possibilities for RNA editing. Biochemical experiments combined with amide hydrogen-deuterium exchange (HDX-MS) experiments provide a first glimpse of the conformational dynamics of apo Cas7-11. HDX-MS revealed the backbone comprised of the four Cas7 zinc-binding RRM folds are well-folded but insertion sequences are highly dynamic and fold upon binding crRNA. The crRNA causes folding of disordered catalytic loops and β-hairpins, stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. Target RNA binding causes only minor ordering around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot fully process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces target RNA cleavage. The insertion domain shows the most ordering upon binding of mature crRNA. Finally, we show a crRNA-induced conformational change in one of the TPR-CHAT binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding. The results provide the first glimpse of the apo state of Cas7-11 and reveal how its structure and function are regulated by crRNA binding.}, }
@article {pmid39210508, year = {2024}, author = {Raddaoui, A and Chebbi, Y and Frigui, S and Latorre, J and Ammeri, RW and Abdejlil, NB and Torres, C and Mohamed Salah, A and Achour, W}, title = {Genetic characterization of vancomycin-resistant Enterococcus faecium isolates from neutropenic patients in Tunisia: Spread of the pandemic CC17 clone associated with high genetic diversity in Tn1546-like structures.}, journal = {Journal of applied microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1093/jambio/lxae225}, pmid = {39210508}, issn = {1365-2672}, abstract = {AIMS: In Tunisia, limited research has focused on characterizing clinical vancomycin resistant Enterococcus faecium (VREfm). This study aimed to bridge this knowledge gap by molecular characterisation of antimicrobial resistance, determining the genetic elements mediating vancomycin-resistance, and whole-genome sequencing of one representative VREfm isolate.<br><br>METHODS AND RESULTS: Over six years (2011-2016), a total of eighty VREfm isolates responsible for infection or colonization were identified from hospitalized patients, with the incidence rate increasing from 2% in 2011 to 27% in 2016. All of these strains harbored the vanA gene. The screening for antimicrobial resistance genes revealed the predominance of ermB, tetM, and aac(6')-Ie-aph(2'')-Ia genes and 81.2% of strains harboured the Tn1545. PFGE identified seven clusters, with two major clusters (belonging to ST117 and ST80) persisting throughout the study period. Seven Tn1546 types were detected, with type VI (truncated transposon) being the most prevalent (57.5%). Whole-genome sequencing revealed a 3,028,373 bp chromosome and five plasmids. Mobile genetic elements and a type I CRISPR-cas locus were identified. Notably, the vanA gene was carried by the classic Tn1546 transposon with ISL3 insertion on a rep17pRUM plasmid.<br><br>CONCLUSION: A concerning trend in the prevalence of VREfm essentially attributed to CC17 persistence and to horizontal transfer of multiple genetic variants of truncated vanA-Tn1546.}, }
@article {pmid39210507, year = {2024}, author = {Fan, K and Gökbağ, B and Tang, S and Li, S and Huang, Y and Wang, L and Cheng, L and Li, L}, title = {Synthetic lethal connectivity and graph transformer improve synthetic lethality prediction.}, journal = {Briefings in bioinformatics}, volume = {25}, number = {5}, pages = {}, doi = {10.1093/bib/bbae425}, pmid = {39210507}, issn = {1477-4054}, mesh = {Humans ; *Synthetic Lethal Mutations ; K562 Cells ; Computational Biology/methods ; CRISPR-Cas Systems ; Algorithms ; Jurkat Cells ; Gene Knockout Techniques ; Neoplasms/genetics ; }, abstract = {Synthetic lethality (SL) has shown great promise for the discovery of novel targets in cancer. CRISPR double-knockout (CDKO) technologies can only screen several hundred genes and their combinations, but not genome-wide. Therefore, good SL prediction models are highly needed for genes and gene pairs selection in CDKO experiments. However, lack of scalable SL properties prevents generalizability of SL interactions to out-of-sample data, thereby hindering modeling efforts. In this paper, we recognize that SL connectivity is a scalable and generalizable SL property. We develop a novel two-step multilayer encoder for individual sample-specific SL prediction model (MLEC-iSL), which predicts SL connectivity first and SL interactions subsequently. MLEC-iSL has three encoders, namely, gene, graph, and transformer encoders. MLEC-iSL achieves high SL prediction performance in K562 (AUPR, 0.73; AUC, 0.72) and Jurkat (AUPR, 0.73; AUC, 0.71) cells, while no existing methods exceed 0.62 AUPR and AUC. The prediction performance of MLEC-iSL is validated in a CDKO experiment in 22Rv1 cells, yielding a 46.8% SL rate among 987 selected gene pairs. The screen also reveals SL dependency between apoptosis and mitosis cell death pathways.}, }
@article {pmid39209994, year = {2024}, author = {Zhao, Y and Qin, J and Yu, D and Liu, Y and Song, D and Tian, K and Chen, H and Ye, Q and Wang, X and Xu, T and Xuan, H and Sun, N and Ma, W and Zhong, J and Sun, P and Song, Y and Hu, J and Zhao, Y and Hou, X and Meng, X and Jiang, C and Cai, J}, title = {Polymer-locking fusogenic liposomes for glioblastoma-targeted siRNA delivery and CRISPR-Cas gene editing.}, journal = {Nature nanotechnology}, volume = {}, number = {}, pages = {}, pmid = {39209994}, issn = {1748-3395}, support = {82372901//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82073298//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22007051//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82003022//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82272988//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82230086//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2019M660074//China Postdoctoral Science Foundation/ ; LBH-Z19029//Heilongjiang Postdoctoral Science Foundation/ ; LBH-TZ2218//Heilongjiang Postdoctoral Science Foundation/ ; }, abstract = {In patients with glioblastoma (GBM), upregulated midkine (MDK) limits the survival benefits conferred by temozolomide (TMZ). RNA interference (RNAi) and CRISPR-Cas9 gene editing technology are attractive approaches for regulating MDK expression. However, delivering these biologics to GBM tissue is challenging. Here we demonstrate a polymer-locking fusogenic liposome (Plofsome) that can be transported across the blood-brain barrier (BBB) and deliver short interfering RNA or CRISPR-Cas9 ribonucleoprotein complexes into the cytoplasm of GBM cells. Plofsome is designed by integrating a 'lock' into the fusogenic liposome using a traceless reactive oxygen species (ROS)-cleavable linker so that fusion occurs only after crossing the BBB and entering the GBM tissue with high ROS levels. Our results showed that MDK suppression by Plofsomes significantly reduced TMZ resistance and inhibited GBM growth in orthotopic brain tumour models. Importantly, Plofsomes are effective only at tumour sites and not in normal tissues, which improves the safety of combined RNAi and CRISPR-Cas9 therapeutics.}, }
@article {pmid39209602, year = {2024}, author = {Binan, G and Yalun, W and Xinyan, W and Yongfu, Y and Peng, Z and Yunhaon, C and Xuan, Z and Chenguang, L and Fengwu, B and Ping, X and Qiaoning, H and Shihui, Y}, title = {Efficient genome-editing tools to engineer the recalcitrant non-model industrial microorganism Zymomonas mobilis.}, journal = {Trends in biotechnology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tibtech.2024.05.005}, pmid = {39209602}, issn = {1879-3096}, abstract = {Current biotechnology relies on a few well-studied model organisms, such as Escherichia coli and Saccharomyces cerevisiae, for which abundant information and efficient toolkits are available for genetic manipulation, but which lack industrially favorable characteristics. Non-model industrial microorganisms usually do not have effective and/or efficient genome-engineering toolkits, which hampers the development of microbial cell factories to meet the fast-growing bioeconomy. In this study, using the non-model ethanologenic bacterium Zymomonas mobilis as an example, we developed a workflow to mine and temper the elements of restriction-modification (R-M), CRISPR/Cas, toxin-antitoxin (T-A) systems, and native plasmids, which are hidden within industrial microorganisms themselves, as efficient genome-editing toolkits, and established a genome-wide iterative and continuous editing (GW-ICE) system for continuous genome editing with high efficiency. This research not only provides tools and pipelines for engineering the non-model polyploid industrial microorganism Z. mobilis efficiently, but also sets a paradigm to overcome biotechnological limitations in other genetically recalcitrant non-model industrial microorganisms.}, }
@article {pmid39209209, year = {2024}, author = {Ufret-Vincenty, RL and Kirman, DC and Ulker-Yilmazer, G and Aredo, B and Shrestha, S and Turpin, E and Yuksel, S and Zegeye, Y and Ludwig, S and Moresco, EMY and He, YG and Beutler, B}, title = {Non-redundant role of leishmanolysin-like (Lmln) zinc-metallopeptidase in retinal homeostasis.}, journal = {American journal of ophthalmology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.ajo.2024.08.016}, pmid = {39209209}, issn = {1879-1891}, abstract = {PURPOSE: To determine if Lmln, a Zn-metallopeptidase, is important for retinal homeostasis.<br><br>METHODS: Combining an unbiased N-ethyl-N-nitrosourea mutagenesis pipeline in mice with optical coherence tomography (OCT) screening and automated meiotic mapping, we identified an allele (nemeth) that seemed to be associated with outer nuclear layer (ONL) thinning. Since nemeth was predicted to lead to a nonsense mutation of the Lmln gene, we targeted Lmln using CRISPR/Cas-9 technology and characterized the impact on retinal anatomy and function.<br><br>RESULTS: OCT imaging demonstrated an outer retinal degeneration in Lmln[-/-] mice (P = 7.3&#x202f;&#xd7;&#x202f;10[-9] for ONL at 2 m) that progressed over the first 6 months of life and then stabilized. Light microscopy showed loss of ONL nuclei (P ranged between .00033 and .0097 for posterior measurements), and a TUNEL assay revealed a small but significant increase in apoptosis (P = .034). Lmln[-/-] mice accumulated fundus spots (P = .0030 by 2 m of age) and activated subretinal microglia (p ranged from .0007 to 8&#x202f;&#xd7;&#x202f;10[-13] for Gal3[+] cells). Scotopic electroretinography demonstrated a decrease in retinal function in Lmln[-/-] mice both at 6 m (only a-wave, P < .01 for all stimuli) and at 10 m of age (P < .01 for both a-wave and b-wave with all stimuli).<br><br>CONCLUSIONS: Our work revealed a previously unknown essential requirement for Lmln in maintaining retinal anatomy and function. Further studies using this new model will be aimed at determining the cellular expression of Lmln and its mechanisms of action within the retina.}, }
@article {pmid39203355, year = {2024}, author = {Salinas-Virgen, LI and de la Torre-Hernández, ME and Aguirre-Garrido, JF and Martínez-Abarca, F and Ramírez-Saad, HC}, title = {Genotypic and Phenotypic Characterization of Pseudomonas atacamensis EMP42 a PGPR Strain Obtained from the Rhizosphere of Echinocactus platyacanthus (Sweet Barrel).}, journal = {Microorganisms}, volume = {12}, number = {8}, pages = {}, doi = {10.3390/microorganisms12081512}, pmid = {39203355}, issn = {2076-2607}, abstract = {Plant growth-promoting rhizobacteria (PGPR) are a group of bacteria that associate with the rhizosphere of plants; one of the most abundant bacterial genera in this ecological niche is Pseudomonas, which is constantly expanding due to the emergence of new species such as Pseudomonas atacamensis, whose discovery in 2019 has led to the characterization of several strains from different environments but taxonomically related. The objective of this work was to phenotypically and molecularly characterize P. atacamensis strain EMP42, isolated from the rhizosphere of Echinocactus platyacanthus. The strain EMP42 is able to use different substrates and reduce oxidative stress in plants. It is capable of improving growth parameters such as the number of inflorescences and the height of the aerial body of Arabidopsis thaliana, as well as the germination and seedling survival of the cacti Echinocactus platyacanthus and Astrophytum capricorne. The genetic structure of P. atacamensis EMP42 consists of a closed chromosome of 6.14 Mbp, and 61.1% GC content. It has 5572 genes, including those associated with PGPR activities, such as the trpABCDE, SAP, phoABPRU and acsABC genes, among others, and three ncRNA loci, nine regulatory regions, five complete rRNA operons and three CRISPR-Cas loci, showing phylogenomic similarities with the reference strain P. atacamensis B21-026. Therefore, this study contributes to the understanding of genomic diversity within P. atacamensis and, particularly, highlights the potential application of strain EMP42 as a PGPR.}, }
@article {pmid39202374, year = {2024}, author = {Polidoros, A and Nianiou-Obeidat, I and Tsakirpaloglou, N and Petrou, N and Deligiannidou, E and Makri, NM}, title = {Genome-Editing Products Line up for the Market: Will Europe Harvest the Benefits from Science and Innovation?.}, journal = {Genes}, volume = {15}, number = {8}, pages = {}, doi = {10.3390/genes15081014}, pmid = {39202374}, issn = {2073-4425}, mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/growth &amp; development ; Europe ; *Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems ; Genome, Plant ; Humans ; }, abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies have revolutionized genome editing, significantly advancing the improvement of cultivated crop species. This review provides an overview of genome-edited crops that have either reached the market or received the necessary approvals but are not yet available to consumers. We analyze various genome-editing studies to understand the distribution of different genome-editing systems, the types of site-directed nucleases employed, and the geographical spread of these studies, with a specific focus on global and European contexts. Additionally, we examine the target crops involved. The review also outlines the multiple steps required for the legal acceptance of genome-edited crops within European jurisdictions. We conclude with suggestions for the future prospects of genome-editing research in Europe, aiming to streamline the approval process and enhance the development and adoption of genome-edited crops.}, }
@article {pmid39202365, year = {2024}, author = {Chen, X and Yang, G and Ji, P and Liu, G and Zhang, L}, title = {Identification of Site in the UTY Gene as Safe Harbor Locus on the Y Chromosome of Pig.}, journal = {Genes}, volume = {15}, number = {8}, pages = {}, doi = {10.3390/genes15081005}, pmid = {39202365}, issn = {2073-4425}, support = {32372880//National Natural Science Foundation of China/ ; 2024SKLAB 1-6//Innovative Project of State Key Laboratory of Animal Biotech Breeding and the 2115 Talent Development Program of China Agricultural University/ ; }, mesh = {Animals ; *Y Chromosome/genetics ; Swine/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Male ; }, abstract = {Genomic Safe Harbors (GSH) are loci used for the insertion of exogenous genetic elements, enabling exogenous gene expressing predictably without alterations of the host genome. These sites are becoming increasingly important as the gene editing technologies advance rapidly. Currently, only a few GSHs have been identified in the pig genome. In this study, a novel strategy was demonstrated for the efficient insertion of exogenous genetic material into the third exon of the UTY gene on the Y chromosome using CRISPR/Cas9-mediated homology arm-mediated end joining. The safety of the locus was verified according to the proper expression of the inserted EGFP gene without altering the expression of UTY. This approach enables the integration and expression of the exogenous gene at this locus, indicating that the UTY locus serves as a genomic safe harbor site for gene editing in the pig genome. Located on the Y chromosome, this site can be utilized for sex-biased pig breeding and developing biomedical models.}, }
@article {pmid39201772, year = {2024}, author = {Abounar, SA and El-Nikhely, NA and Turkowski, K and Savai, R and Saeed, H}, title = {CRISPR/Cas-Mediated Knockdown of PD-L1 and KRAS in Lung Cancer Cells.}, journal = {International journal of molecular sciences}, volume = {25}, number = {16}, pages = {}, doi = {10.3390/ijms25169086}, pmid = {39201772}, issn = {1422-0067}, support = {44612//Science and Technology Development Fund/ ; }, mesh = {Humans ; *B7-H1 Antigen/metabolism/genetics ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *Lung Neoplasms/genetics/metabolism/pathology ; *CRISPR-Cas Systems ; *Apoptosis/genetics ; *Gene Knockdown Techniques ; Cell Line, Tumor ; A549 Cells ; Cell Movement/genetics ; Gene Expression Regulation, Neoplastic ; Cell Cycle/genetics ; }, abstract = {Cancer cells can escape death and surveillance by the host immune system in various ways. Programmed cell death ligand 1 (PD-L1) is a transmembrane protein that is expressed by most cell types, including cancer cells, and can provide an inhibitory signal to its receptor PD-1, which is expressed on the surface of activated T cells, impairing the immune response. PD-L1/PD-1-mediated immune evasion is observed in several KRAS-mutated cancers. In the current study, we used the CRISPR/Cas9 system to knock down PD-L1 and KRAS in adenocarcinoma lung cells (A549 and H1975). Knockdown of PD-L1 was validated by qPCR and coculture with lymphocytes. The cells were functionally analyzed for cell cycle, migration and apoptosis. In addition, the effects of PD-L1 and KRAS downregulation on chemotherapy sensitivity and expression of inflammatory markers were investigated. Suppression of PD-L1 and KRAS led to a slowdown of the cell cycle in the G0/G1 phase and reduced migration, increased sensitivity to chemotherapy and triggered apoptosis of cancer cells. In addition, the conditioned medium of the modulated cells significantly affected the native cancer cells and reduced their viability and drug resistance. Our study suggests that dual silencing of PD-L1 and KRAS by CRISPR/Cas9 may be a promising therapeutic approach for the treatment of lung cancer.}, }
@article {pmid39201620, year = {2024}, author = {Khan, N and Choi, SH and Lee, CH and Qu, M and Jeon, JS}, title = {Photosynthesis: Genetic Strategies Adopted to Gain Higher Efficiency.}, journal = {International journal of molecular sciences}, volume = {25}, number = {16}, pages = {}, doi = {10.3390/ijms25168933}, pmid = {39201620}, issn = {1422-0067}, support = {2023R1A2C1003142//National Research Foundation/ ; NRF-2020R1I1A3069080//National Research Foundation/ ; RS-2022-RD010269//Rural Development Administration/ ; RS-2024-00322378//Rural Development Administration/ ; }, mesh = {*Photosynthesis/genetics ; *Gene Editing/methods ; *Crops, Agricultural/genetics/growth &amp; development ; CRISPR-Cas Systems ; Genetic Engineering ; }, abstract = {The global challenge of feeding an ever-increasing population to maintain food security requires novel approaches to increase crop yields. Photosynthesis, the fundamental energy and material basis for plant life on Earth, is highly responsive to environmental conditions. Evaluating the operational status of the photosynthetic mechanism provides insights into plants' capacity to adapt to their surroundings. Despite immense effort, photosynthesis still falls short of its theoretical maximum efficiency, indicating significant potential for improvement. In this review, we provide background information on the various genetic aspects of photosynthesis, explain its complexity, and survey relevant genetic engineering approaches employed to improve the efficiency of photosynthesis. We discuss the latest success stories of gene-editing tools like CRISPR-Cas9 and synthetic biology in achieving precise refinements in targeted photosynthesis pathways, such as the Calvin-Benson cycle, electron transport chain, and photorespiration. We also discuss the genetic markers crucial for mitigating the impact of rapidly changing environmental conditions, such as extreme temperatures or drought, on photosynthesis and growth. This review aims to pinpoint optimization opportunities for photosynthesis, discuss recent advancements, and address the challenges in improving this critical process, fostering a globally food-secure future through sustainable food crop production.}, }
@article {pmid39201545, year = {2024}, author = {Ghenciu, LA and Hațegan, OA and Stoicescu, ER and Iacob, R and Șișu, AM}, title = {Emerging Therapeutic Approaches and Genetic Insights in Stargardt Disease: A Comprehensive Review.}, journal = {International journal of molecular sciences}, volume = {25}, number = {16}, pages = {}, doi = {10.3390/ijms25168859}, pmid = {39201545}, issn = {1422-0067}, mesh = {*Stargardt Disease/genetics ; Humans ; *Genetic Therapy/methods ; *ATP-Binding Cassette Transporters/genetics ; Mutation ; Stem Cell Transplantation/methods ; Animals ; Macular Degeneration/therapy/genetics/congenital ; Retinal Pigment Epithelium/metabolism ; CRISPR-Cas Systems ; }, abstract = {Stargardt disease, one of the most common forms of inherited retinal diseases, affects individuals worldwide. The primary cause is mutations in the ABCA4 gene, leading to the accumulation of toxic byproducts in the retinal pigment epithelium (RPE) and subsequent photoreceptor cell degeneration. Over the past few years, research on Stargardt disease has advanced significantly, focusing on clinical and molecular genetics. Recent studies have explored various innovative therapeutic approaches, including gene therapy, stem cell therapy, and pharmacological interventions. Gene therapy has shown promise, particularly with adeno-associated viral (AAV) vectors capable of delivering the ABCA4 gene to retinal cells. However, challenges remain due to the gene's large size. Stem cell therapy aims to replace degenerated RPE and photoreceptor cells, with several clinical trials demonstrating safety and preliminary efficacy. Pharmacological approaches focus on reducing toxic byproduct accumulation and modulating the visual cycle. Precision medicine, targeting specific genetic mutations and pathways, is becoming increasingly important. Novel techniques such as clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 offer potential for directly correcting genetic defects. This review aims to synthesize recent advancements in understanding and treating Stargardt disease. By highlighting breakthroughs in genetic therapies, stem cell treatments, and novel pharmacological strategies, it provides a comprehensive overview of emerging therapeutic options.}, }
@article {pmid39201470, year = {2024}, author = {Nakahara, T and Tabata, H and Kato, Y and Fuse, R and Nakamura, M and Yamaji, M and Hattori, N and Kiyono, T and Saito, I and Nakanishi, T}, title = {Construction and Stability of All-in-One Adenovirus Vectors Simultaneously Expressing Four and Eight Multiplex Guide RNAs and Cas9 Nickase.}, journal = {International journal of molecular sciences}, volume = {25}, number = {16}, pages = {}, doi = {10.3390/ijms25168783}, pmid = {39201470}, issn = {1422-0067}, support = {23K27095//Japan Society for the Promotion of Science/ ; }, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Vectors/genetics ; *Adenoviridae/genetics ; *Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; Genome, Viral ; CRISPR-Associated Protein 9/metabolism/genetics ; Deoxyribonuclease I/metabolism/genetics ; }, abstract = {CRISPR/Cas9 technology is expected to offer novel genome editing-related therapies for various diseases. We previously showed that an adenovirus vector (AdV) possessing eight expression units of multiplex guide RNAs (gRNAs) was obtained with no deletion of these units. Here, we attempted to construct "all-in-one" AdVs possessing expression units of four and eight gRNAs with Cas9 nickase, although we expected obstacles to obtain complete all-in-one AdVs. The first expected obstacle was that extremely high copies of viral genomes during replication may cause severe off-target cleavages of host cells and induce homologous recombination. However, surprisingly, four units in the all-in-one AdV genome were maintained completely intact. Second, for the all-in-one AdV containing eight gRNA units, we enlarged the E3 deletion in the vector backbone and shortened the U6 promoter of the gRNA expression units to shorten the AdV genome within the adenovirus packaging limits. The final size of the all-in-one AdV genome containing eight gRNA units still slightly exceeded the reported upper limit. Nevertheless, approximately one-third of the eight units remained intact, even upon preparation for in vivo experiments. Third, the genome editing efficiency unexpectedly decreased upon enlarging the E3 deletion. Our results suggested that complete all-in-one AdVs containing four gRNA units could be obtained if the problem of the low genome editing efficiency is solved, and those containing even eight gRNA units could be obtained if the obstacle of the vector size is also removed.}, }
@article {pmid39201401, year = {2024}, author = {Qiao, A and Wei, Y and Liu, Y and Kahn-Krell, A and Ye, L and Nguyen, T and Zhang, J}, title = {Doxycycline-Mediated Control of Cyclin D2 Overexpression in Human-Induced Pluripotent Stem Cells.}, journal = {International journal of molecular sciences}, volume = {25}, number = {16}, pages = {}, doi = {10.3390/ijms25168714}, pmid = {39201401}, issn = {1422-0067}, support = {NIH RO1s HL138990, HL114120, HL 131017, HL149137, P01 HL160476, and UO1 HL134764/NH/NIH HHS/United States ; }, mesh = {*Doxycycline/pharmacology ; *Cyclin D2/metabolism/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology/drug effects ; *Promoter Regions, Genetic ; Myocytes, Cardiac/metabolism/drug effects ; Gene Expression Regulation/drug effects ; Cell Differentiation/drug effects ; RNA, Guide, CRISPR-Cas Systems ; }, abstract = {Previous studies have demonstrated that when the cyclin D2 (CCND2), a cell-cycle regulatory protein, is overexpressed in human-induced pluripotent stem cells (hiPSCs), cardiomyocytes (CMs) differentiated from these CCND2-overexpressing hiPSCs can proliferate after transplantation into infarcted hearts, which significantly improves the cells' potency for myocardial regeneration. However, persistent CM proliferation could lead to tumor growth or the development of arrhythmogenic complications; thus, the goal of the current study was to generate a line of hiPSCs in which CCND2 overexpression could be tightly controlled. First, we transfected hiPSCs with vectors coding for a doxycycline-inducible Tet-On transactivator and S. pyogenes dCas9 fused to the VPR activation domain; then, the same hiPSCs were engineered to express guide RNAs targeting the CCND2 promotor. Thus, treatment with doxycycline (dox) activated dCas9-VPR expression, and the guide RNAs directed dCas9-VPR to the CCND2 promoter, which activated CCND2 expression. Subsequent experiments confirmed that CCND2 expression was dox-dependent in this newly engineered line of hiPSCs ([dox]CCND2-hiPSCs): CCND2 protein was abundantly expressed after 48 h of treatment with dox and declined to near baseline level ~96 h after dox treatment was discontinued.}, }
@article {pmid39201387, year = {2024}, author = {Qin, C and Jiang, S and Xu, K and Zhu, J and Wang, L and Yang, W and Xiao, F and Yang, K and Huang, Q and Meng, H}, title = {One-Step Genetic Modification by Embryonic Doral Aorta Injection of Adenoviral CRISPR/Cas9 Vector in Chicken.}, journal = {International journal of molecular sciences}, volume = {25}, number = {16}, pages = {}, doi = {10.3390/ijms25168692}, pmid = {39201387}, issn = {1422-0067}, mesh = {Animals ; *CRISPR-Cas Systems ; Chick Embryo ; *Genetic Vectors/genetics ; *Chickens/genetics ; *Adenoviridae/genetics ; *Aorta/metabolism ; Gene Editing/methods ; Male ; }, abstract = {In the avian species, genetic modification by cell nuclear transfer is infeasible due to its unique reproductive system. The in vitro primordial germ cell modification approach is difficult and cumbersome, although it is the main method of genetic modification in chickens. In the present study, the adenoviral CRISPR/Cas9 vector was directly microinjected into the dorsal aorta of chicken embryos to achieve in vivo genetic modification. The results demonstrated that keratin 75-like 4 (KRT75L4), a candidate gene crucial for feather development, was widely knocked out, and an 8bp deletion was the predominant mutation that occurred in multiple tissues in chimeras, particularly in the gonad (2.63-11.57%). As we expected, significant modification was detected in the sperm of G0 (0.16-4.85%), confirming the potential to generate homozygous chickens and establishing this vector as a simple and effective method for genetic modification in avian species.}, }
@article {pmid39201382, year = {2024}, author = {Pavlova, SV and Shulgina, AE and Zakian, SM and Dementyeva, EV}, title = {Studying Pathogenetic Contribution of a Variant of Unknown Significance, p.M659I (c.1977G &gt; A) in MYH7, to the Development of Hypertrophic Cardiomyopathy Using CRISPR/Cas9-Engineered Isogenic Induced Pluripotent Stem Cells.}, journal = {International journal of molecular sciences}, volume = {25}, number = {16}, pages = {}, doi = {10.3390/ijms25168695}, pmid = {39201382}, issn = {1422-0067}, support = {22-15-00271//Russian Science Foundation/ ; }, mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; *Cardiac Myosins/genetics/metabolism ; *Myosin Heavy Chains/genetics/metabolism ; Humans ; *Cardiomyopathy, Hypertrophic/genetics/pathology ; *Myocytes, Cardiac/metabolism/pathology/cytology ; Cell Differentiation/genetics ; Gene Editing/methods ; Mutation ; Cell Line ; }, abstract = {Hypertrophic cardiomyopathy (HCM) is a cardiovascular pathology that is caused by variants in genes encoding sarcomere-associated proteins. However, the clinical significance of numerous variants in HCM-associated genes is still unknown. CRISPR/Cas9 is a tool of nucleotide sequence editing that allows for the unraveling of different biological tasks. In this study, introducing a mutation with CRISPR/Cas9 into induced pluripotent stem cells (iPSCs) of a healthy donor and the directed differentiation of the isogenic iPSC lines into cardiomyocytes were used to assess the pathogenicity of a variant of unknown significance, p.M659I (c.1977G > A) in MYH7, which was found previously in an HCM patient. Using two single-stranded donor oligonucleotides with and without the p.M659I (c.1977G > A) mutation, together with CRISPR/Cas9, an iPSC line heterozygous at the p.M659I (c.1977G > A) variant in MYH7 was generated. No CRISPR/Cas9 off-target activity was observed. The iPSC line with the introduced p.M659I (c.1977G > A) mutation in MYH7 retained its pluripotent state and normal karyotype. Compared to the isogenic control, cardiomyocytes derived from the iPSCs with the introduced p.M659I (c.1977G > A) mutation in MYH7 recapitulated known HCM features: enlarged size, elevated diastolic calcium level, changes in the expression of HCM-related genes, and disrupted energy metabolism. These findings indicate the pathogenicity of the variant.}, }
@article {pmid39199403, year = {2024}, author = {Protacio, RU and Dixon, S and Davidson, MK and Wahls, WP}, title = {Creating Meiotic Recombination-Regulating DNA Sites by SpEDIT in Fission Yeast Reveals Inefficiencies, Target-Site Duplications, and Ectopic Insertions.}, journal = {Biomolecules}, volume = {14}, number = {8}, pages = {}, doi = {10.3390/biom14081016}, pmid = {39199403}, issn = {2218-273X}, support = {GM145834/GM/NIGMS NIH HHS/United States ; }, mesh = {*Schizosaccharomyces/genetics/metabolism ; *Meiosis/genetics ; *CRISPR-Cas Systems/genetics ; *Schizosaccharomyces pombe Proteins/genetics/metabolism ; Recombination, Genetic ; DNA, Fungal/genetics/metabolism ; Gene Editing/methods ; }, abstract = {Recombination hotspot-activating DNA sites (e.g., M26, CCAAT, Oligo-C) and their binding proteins (e.g., Atf1-Pcr1 heterodimer; Php2-Php3-Php5 complex, Rst2, Prdm9) regulate the distribution of Spo11 (Rec12)-initiated meiotic recombination. We sought to create 14 different candidate regulatory DNA sites via bp substitutions in the ade6 gene of Schizosaccharomyces pombe. We used a fission yeast-optimized CRISPR-Cas9 system (SpEDIT) and 196 bp-long dsDNA templates with centrally located bp substitutions designed to ablate the genomic PAM site, create specific 15 bp-long DNA sequences, and introduce a stop codon. After co-transformation with a plasmid that encoded both the guide RNA and Cas9 enzyme, about one-third of colonies had a phenotype diagnostic for DNA sequence changes at ade6. PCR diagnostics and DNA sequencing revealed a diverse collection of alterations at the target locus, including: (A) complete or (B) partial template-directed substitutions; (C) non-homologous end joinings; (D) duplications; (E) bp mutations, and (F) insertions of ectopic DNA. We concluded that SpEDIT can be used successfully to generate a diverse collection of DNA sequence elements within a reporter gene of interest. However, its utility is complicated by low efficiency, incomplete template-directed repair events, and undesired alterations to the target locus.}, }
@article {pmid39199291, year = {2024}, author = {Liu, Y and Zong, X and Cao, W and Zhang, W and Zhang, N and Yang, N}, title = {Gene Therapy for Retinitis Pigmentosa: Current Challenges and New Progress.}, journal = {Biomolecules}, volume = {14}, number = {8}, pages = {}, doi = {10.3390/biom14080903}, pmid = {39199291}, issn = {2218-273X}, support = {2023AFC019//Natural Science Foundation of Hubei Province/ ; 2023KFH019//Hubei Key Laboratories Opening Project/ ; 2021KFY055//Hubei Key Laboratories Opening Project/ ; 2020CFB240//Natural Science Foundation of Hubei Province/ ; 2042020kf0065//Fundamental Research Funds for Central Universities/ ; }, mesh = {*Retinitis Pigmentosa/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *Gene Editing/methods ; Animals ; Mutation ; Genetic Vectors/genetics ; Eye Proteins/genetics ; }, abstract = {Retinitis pigmentosa (RP) poses a significant threat to eye health worldwide, with prevalence rates of 1 in 5000 worldwide. This genetically diverse retinopathy is characterized by the loss of photoreceptor cells and atrophy of the retinal pigment epithelium. Despite the involvement of more than 3000 mutations across approximately 90 genes in its onset, finding an effective treatment has been challenging for a considerable time. However, advancements in scientific research, especially in gene therapy, are significantly expanding treatment options for this most prevalent inherited eye disease, with the discovery of new compounds, gene-editing techniques, and gene loci offering hope for more effective treatments. Gene therapy, a promising technology, utilizes viral or non-viral vectors to correct genetic defects by either replacing or silencing disease-causing genes, potentially leading to complete recovery. In this review, we primarily focus on the latest applications of gene editing research in RP. We delve into the most prevalent genes associated with RP and discuss advancements in genome-editing strategies currently employed to correct various disease-causing mutations.}, }
@article {pmid39198401, year = {2024}, author = {Truong, P and Shen, S and Joshi, S and Islam, MI and Zhong, L and Raftery, MJ and Afrasiabi, A and Alinejad-Rokny, H and Nguyen, M and Zou, X and Bhuyan, GS and Sarowar, CH and Ghodousi, ES and Stonehouse, O and Mohamed, S and Toscan, CE and Connerty, P and Kakadia, PM and Bohlander, SK and Michie, KA and Larsson, J and Lock, RB and Walkley, CR and Thoms, JAI and Jolly, CJ and Pimanda, JE}, title = {TOPORS E3 ligase mediates resistance to hypomethylating agent cytotoxicity in acute myeloid leukemia cells.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7360}, pmid = {39198401}, issn = {2041-1723}, support = {GNT2011627//Department of Health | National Health and Medical Research Council (NHMRC)/ ; TPG2152//Cancer Institute NSW (Cancer Institute New South Wales)/ ; }, mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/drug therapy/pathology/metabolism ; Animals ; *Drug Resistance, Neoplasm/genetics/drug effects ; Cell Line, Tumor ; Mice ; *Myelodysplastic Syndromes/drug therapy/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; Sumoylation/drug effects ; Ubiquitin-Protein Ligases/metabolism/genetics ; DNA Damage/drug effects ; DNA Methylation/drug effects ; Xenograft Model Antitumor Assays ; DNA (Cytosine-5-)-Methyltransferase 1/metabolism/genetics/antagonists &amp; inhibitors ; Female ; }, abstract = {Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However, acquired resistance and treatment failure are commonplace. To address this, we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line, MDS-L, and identify TOPORS as a loss-of-function target that synergizes with HMAs, reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable, we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall, our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML.}, }
@article {pmid39198387, year = {2024}, author = {Kaito, S and Aoyama, K and Oshima, M and Tsuchiya, A and Miyota, M and Yamashita, M and Koide, S and Nakajima-Takagi, Y and Kozuka-Hata, H and Oyama, M and Yogo, T and Yabushita, T and Ito, R and Ueno, M and Hirao, A and Tohyama, K and Li, C and Kawabata, KC and Yamaguchi, K and Furukawa, Y and Kosako, H and Yoshimi, A and Goyama, S and Nannya, Y and Ogawa, S and Agger, K and Helin, K and Yamazaki, S and Koseki, H and Doki, N and Harada, Y and Harada, H and Nishiyama, A and Nakanishi, M and Iwama, A}, title = {Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7359}, pmid = {39198387}, issn = {2041-1723}, support = {19H05653//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 19H05746//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21zf0127003h0001//Japan Agency for Medical Research and Development (AMED)/ ; }, mesh = {*DNA (Cytosine-5-)-Methyltransferase 1/metabolism/antagonists &amp; inhibitors/genetics ; Humans ; *Ubiquitination/drug effects ; *Sumoylation/drug effects ; *DNA Methylation/drug effects ; Animals ; Ubiquitin-Protein Ligases/metabolism/genetics ; Cell Line, Tumor ; Mice ; CRISPR-Cas Systems ; HEK293 Cells ; }, abstract = {DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks.}, }
@article {pmid39196449, year = {2024}, author = {Peer, LA and Bhat, MY and Lone, AA and Dar, ZA and Mir, BA}, title = {Genetic, molecular and physiological crosstalk during drought tolerance in maize (Zea mays): pathways to resilient agriculture.}, journal = {Planta}, volume = {260}, number = {4}, pages = {81}, pmid = {39196449}, issn = {1432-2048}, mesh = {*Zea mays/genetics/physiology ; *Droughts ; *Quantitative Trait Loci/genetics ; Stress, Physiological/genetics ; Genome-Wide Association Study ; Gene Expression Regulation, Plant ; Agriculture/methods ; CRISPR-Cas Systems ; Crops, Agricultural/genetics/physiology ; Gene Regulatory Networks ; Drought Resistance ; }, abstract = {This review comprehensively elucidates maize drought tolerance mechanisms, vital for global food security. It highlights genetic networks, key genes, CRISPR-Cas applications, and physiological responses, guiding resilient variety development. Maize, a globally significant crop, confronts the pervasive challenge of drought stress, impacting its growth and yield significantly. Drought, an important abiotic stress, triggers a spectrum of alterations encompassing maize's morphological, biochemical, and physiological dimensions. Unraveling and understanding these mechanisms assumes paramount importance for ensuring global food security. Approaches like developing drought-tolerant varieties and harnessing genomic and molecular applications emerge as effective measures to mitigate the negative effects of drought. The multifaceted nature of drought tolerance in maize has been unfolded through complex genetic networks. Additionally, quantitative trait loci mapping and genome-wide association studies pinpoint key genes associated with drought tolerance, influencing morphophysiological traits and yield. Furthermore, transcription factors like ZmHsf28, ZmNAC20, and ZmNF-YA1 play pivotal roles in drought response through hormone signaling, stomatal regulation, and gene expression. Genes, such as ZmSAG39, ZmRAFS, and ZmBSK1, have been reported to be pivotal in enhancing drought tolerance through diverse mechanisms. Integration of CRISPR-Cas9 technology, targeting genes like gl2 and ZmHDT103, emerges as crucial for precise genetic enhancement, highlighting its role in safeguarding global food security amid pervasive drought challenges. Thus, decoding the genetic and molecular underpinnings of drought tolerance in maize sheds light on its resilience and paves the way for cultivating robust and climate-smart varieties, thus safeguarding global food security amid climate challenges. This comprehensive review covers quantitative trait loci mapping, genome-wide association studies, key genes and functions, CRISPR-Cas applications, transcription factors, physiological responses, signaling pathways, offering a nuanced understanding of intricate mechanisms involved in maize drought tolerance.}, }
@article {pmid39196424, year = {2024}, author = {Araújo, MRB and Prates, FD and Ramos, JN and Sousa, EG and Bokermann, S and Sacchi, CT and de Mattos-Guaraldi, AL and Campos, KR and Sousa, M&#xc2;B and Vieira, VV and Santos, MBN and Camargo, CH and de Oliveira Sant'Anna, L and Dos Santos, LS and Azevedo, V}, title = {Infection by a multidrug-resistant Corynebacterium diphtheriae strain: prediction of virulence factors, CRISPR-Cas system analysis, and structural implications of mutations conferring rifampin resistance.}, journal = {Functional &amp; integrative genomics}, volume = {24}, number = {5}, pages = {145}, pmid = {39196424}, issn = {1438-7948}, mesh = {*Corynebacterium diphtheriae/genetics/pathogenicity/drug effects ; Humans ; *Virulence Factors/genetics ; *CRISPR-Cas Systems ; *Rifampin/pharmacology ; Mutation ; Phylogeny ; Diphtheria/microbiology ; Genome, Bacterial ; Bacterial Proteins/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; }, abstract = {Cases of diphtheria, even in immunized individuals, are still reported in several parts of the world, including in Brazil. New outbreaks occur in Europe and other continents. In this context, studies on Corynebacterium diphtheriae infections are highly relevant, both for a better understanding of the pathogenesis of the disease and for controlling the circulation of clones and antimicrobial resistance genes. Here we present a case of cutaneous infection by multidrug-resistant Corynebacterium diphtheriae and provide its whole-genome sequencing. Genomic analysis revealed resistance genes, including tet(W), sul1, cmx, rpoB2, rbpA and mutation in rpoB. We performed phylogenetic analyzes and used the BRIG to compare the predicted resistance genes with those found in genomes from other significant isolates, including those associated with some outbreaks. Virulence factors such as spaD, srtBC, spaH, srtDE, surface-anchored pilus proteins (sapD), nonfimbrial adhesins (DIP0733, DIP1281, and DIP1621), embC and mptC (putatively involved in CdiLAM), sigA, dtxR and MdbA (putatively involved) in post-translational modification, were detected. We identified the CRISPR-Cas system in our isolate, which was classified as Type II-U based on the database and contains 15 spacers. This system functions as an adaptive immune mechanism. The strain was attributed to a new sequence type ST-928, and phylogenetic analysis confirmed that it was related to ST-634 of C. diphtheriae strains isolated in French Guiana and Brazil. In addition, since infections are not always reported, studies with the sequence data might be a way to complement and inform C. diphtheriae surveillance.}, }
@article {pmid39179418, year = {2024}, author = {Strucko, T and Gadar-Lopez, AE and Frøhling, FB and Frost, ET and Iversen, EF and Olsson, H and Jarczynska, ZD and Mortensen, UH}, title = {Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering of Komagataella phaffii.}, journal = {FEMS yeast research}, volume = {24}, number = {}, pages = {}, doi = {10.1093/femsyr/foae026}, pmid = {39179418}, issn = {1567-1364}, support = {6150-00031B//Innovation Fund Denmark/ ; }, mesh = {*CRISPR-Cas Systems ; *Saccharomycetales/genetics ; Oligonucleotides/genetics ; Genetic Engineering/methods ; Gene Deletion ; Genetic Vectors/genetics ; DNA End-Joining Repair ; Gene Editing/methods ; }, abstract = {Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bioproduction of valuable compounds cannot be fully exploited as genetic tools are lagging behind those that are available for baker's yeast. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of K. phaffii, but improvements in gene-editing methods are desirable to further accelerate engineering of this yeast. In this study, we have developed a versatile vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences including single-stranded oligonucleotides. Notably, we performed site-specific point mutations and full gene deletions using short (90 nt) single-stranded oligonucleotides at very high efficiencies. Lastly, we present a strategy for transient inactivation of nonhomologous end-joining (NHEJ) pathway, where KU70 gene is disrupted by a visual marker (uidA gene). This system enables precise CRISPR-Cas9-based editing (including multiplexing) and facilitates simple reversion to NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering of K. phaffii strains and are compatible with high-throughput automated workflows.}, }
@article {pmid39177875, year = {2024}, author = {Beraza-Millor, M and Rodríguez-Castejón, J and Del Pozo-Rodríguez, A and Rodríguez-Gascón, A and Solinís, M&#xc1;}, title = {Systematic Review of Genetic Substrate Reduction Therapy in Lysosomal Storage Diseases: Opportunities, Challenges and Delivery Systems.}, journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy}, volume = {38}, number = {5}, pages = {657-680}, pmid = {39177875}, issn = {1179-190X}, support = {RTI2018-098672-B-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; IT1587-22//Eusko Jaurlaritza/ ; GIC21/34//Eusko Jaurlaritza/ ; PRE_2020_2_01164//Eusko Jaurlaritza/ ; PIF21/61//Euskal Herriko Unibertsitatea/ ; }, mesh = {Humans ; *Lysosomal Storage Diseases/therapy/genetics ; *Genetic Therapy/methods ; Animals ; *Nanoparticles ; RNA, Small Interfering/administration &amp; dosage/therapeutic use ; CRISPR-Cas Systems ; Gene Silencing ; Drug Delivery Systems ; Liposomes ; }, abstract = {BACKGROUND: Genetic substrate reduction therapy (gSRT), which involves the use of nucleic acids to downregulate the genes involved in the biosynthesis of storage substances, has been investigated in the treatment of lysosomal storage diseases (LSDs).<br><br>OBJECTIVE: To analyze the application of gSRT to the treatment of LSDs, identifying the silencing tools and delivery systems used, and the main challenges for its development and clinical translation, highlighting the contribution of nanotechnology to overcome them.<br><br>METHODS: A systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines was performed. PubMed, Scopus, and Web of Science databases were used for searching terms related to LSDs and gene-silencing strategies and tools.<br><br>RESULTS: Fabry, Gaucher, and Pompe diseases and mucopolysaccharidoses I and III are the only LSDs for which gSRT has been studied, siRNA and lipid nanoparticles being the silencing strategy and the delivery system most frequently employed, respectively. Only in&#xa0;one recently published study&#xa0;was CRISPR/Cas9 applied to treat&#xa0;Fabry disease. Specific tissue targeting, availability of relevant&#xa0;cell and animal LSD models, and the rare disease condition are the main challenges with gSRT for the treatment of these diseases. Out of the 11 studies identified, only two gSRT studies were evaluated in animal models.<br><br>CONCLUSIONS: Nucleic acid therapies are expanding the clinical tools and therapies currently available for LSDs. Recent advances in CRISPR/Cas9 technology and the growing impact of nanotechnology are expected to boost the clinical translation of gSRT in the near future, and not only for LSDs.}, }
@article {pmid39163202, year = {2024}, author = {Kaplan, SJ and Wong, W and Yan, J and Pulecio, J and Cho, HS and Li, Q and Zhao, J and Leslie-Iyer, J and Kazakov, J and Murphy, D and Luo, R and Dey, KK and Apostolou, E and Leslie, CS and Huangfu, D}, title = {CRISPR screening uncovers a long-range enhancer for ONECUT1 in pancreatic differentiation and links a diabetes risk variant.}, journal = {Cell reports}, volume = {43}, number = {8}, pages = {114640}, doi = {10.1016/j.celrep.2024.114640}, pmid = {39163202}, issn = {2211-1247}, mesh = {Humans ; *Enhancer Elements, Genetic/genetics ; *Cell Differentiation/genetics ; *Pancreas/metabolism/pathology ; Diabetes Mellitus, Type 2/genetics/metabolism/pathology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; GATA6 Transcription Factor/metabolism/genetics ; }, abstract = {Functional enhancer annotation is critical for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants. However, unbiased enhancer discovery in disease-relevant contexts remains challenging. To identify enhancers pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers identified, we focused on an enhancer we named ONECUT1e-664kb, ∼664 kb from the ONECUT1 promoter. Previous studies have linked ONECUT1 coding mutations to pancreatic hypoplasia and neonatal diabetes. We found that homozygous deletion of ONECUT1e-664kb in hPSCs leads to a near-complete loss of ONECUT1 expression and impaired pancreatic differentiation. ONECUT1e-664kb contains a type 2 diabetes-associated variant (rs528350911) disrupting a GATA motif. Introducing the risk variant into hPSCs reduced binding of key pancreatic transcription factors (GATA4, GATA6, and FOXA2), supporting its causal role in diabetes. This work highlights the utility of unbiased enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.}, }
@article {pmid39154508, year = {2024}, author = {Xu, Y and Ma, J and Dai, C and Mao, Z and Zhou, Y}, title = {CRISPR/Cas12a-drived electrochemiluminescence and fluorescence dual-mode magnetic biosensor for sensitive detection of Pseudomonas aeruginosa based on iridium(III) complex as luminophore.}, journal = {Biosensors &amp; bioelectronics}, volume = {264}, number = {}, pages = {116678}, doi = {10.1016/j.bios.2024.116678}, pmid = {39154508}, issn = {1873-4235}, mesh = {*Pseudomonas aeruginosa/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Iridium/chemistry ; Humans ; *Limit of Detection ; *Luminescent Measurements ; *CRISPR-Cas Systems ; Electrochemical Techniques/methods ; Pseudomonas Infections/diagnosis/microbiology ; Magnetite Nanoparticles/chemistry ; Fluorescence ; Coordination Complexes/chemistry ; }, abstract = {The opportunistic human pathogen Pseudomonas aeruginosa (P. aeruginosa) poses a significant threat to human health, causing sepsis, inflammation, and pneumonia, so it is crucial to devise an expeditious detection platform for the P. aeruginosa. In this work, bis (2- (3, 5- dimethylphenyl) quinoline- C2, N') (acetylacetonato) iridium (III) Ir (dmpq)2 (acac) with excellent electrochemiluminescence (ECL) and fluorescence (FL) and magnetic nanoparticles were encapsulated in silica spheres. The luminescent units exhibited equal ECL and FL properties compared with single iridium complexes, and enabled rapid separation, which was of vital significance for the establishment of biosensors with effective detection. In addition, the luminescent units were further reacted with the DNA with quenching units to obtain the signal units, and the ECL/FL dual-mode biosensor was employed with the CRISPR/Cas12a system to further improve its specific recognition ability. The ECL detection linear range of as-proposed biosensor in this work was 100 fM-10 nM with the detection limit of 73 fM (S/N = 3), and FL detection linear range was 1 pM-10 nM with the detection limit of 0.126 pM (S/N = 3). Importantly, the proposed dual-mode biosensor exhibited excellent repeatability and stability in the detection of P. aeruginosa in real samples, underscoring its potential as an alternative strategy for infection prevention and safeguarding public health and safety in the future.}, }
@article {pmid39151261, year = {2024}, author = {Shen, X and Lin, Z and Jiang, X and Zhu, X and Zeng, S and Cai, S and Liu, H}, title = {Dumbbell probe initiated multi-rolling circle amplification assisted CRISPR/Cas12a for highly sensitive detection of clinical microRNA.}, journal = {Biosensors &amp; bioelectronics}, volume = {264}, number = {}, pages = {116676}, doi = {10.1016/j.bios.2024.116676}, pmid = {39151261}, issn = {1873-4235}, mesh = {Humans ; *MicroRNAs/genetics/analysis ; *CRISPR-Cas Systems ; *Lung Neoplasms/genetics/diagnosis ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; }, abstract = {A novel miRNA detection technique named Dumbbell probe initiated multi-Rolling Circle Amplification assisted CRISPR/Cas12a (DBmRCA) was developed relying on the ligation-free dumbbell probe and the high-sensitivity CRISPR/Cas12a signal out strategy. This DBmRCA assay streamlines miRNA quantification within a mere 30-min timeframe and with exceptional analytical precision. The efficacy of this method was validated by assessing miRNA levels in clinical samples, revealing distinct expression panel of miR-200a and miR-126 in lung cancer/adjacent/normal tissue specimens. Moreover, a predictive model was established to classify benign and malignant tumor. Due to its time efficiency, enhanced sensitivity, and streamlined workflow, this assay would be a reliable tool for miRNA analysis in clinical settings, offering potential guidance for early diagnosis and treatment of lung cancer.}, }
@article {pmid39137521, year = {2024}, author = {Pian, H and Wang, H and Wang, H and Tang, F and Li, Z}, title = {Capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for highly sensitive visual detection of HPV DNA.}, journal = {Biosensors &amp; bioelectronics}, volume = {264}, number = {}, pages = {116657}, doi = {10.1016/j.bios.2024.116657}, pmid = {39137521}, issn = {1873-4235}, mesh = {*Biosensing Techniques/instrumentation/methods ; Humans ; *DNA, Viral/analysis/genetics ; *CRISPR-Cas Systems ; *Papillomavirus Infections/virology/diagnosis ; *Hydrogels/chemistry ; Endodeoxyribonucleases/chemistry ; Limit of Detection ; Papillomaviridae/genetics ; CRISPR-Associated Proteins/chemistry ; Female ; Bacterial Proteins/chemistry ; }, abstract = {The rapid and specific identification and sensitive detection of human papillomavirus (HPV) infection is critical for preventing cervical cancer, particularly in resource-limited regions. In this work, we hope to propose a capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for point-of-care (POC) DNA testing. Using the thermal reversibility of DNA hydrogel and capillarity, the novel DNA hydrogel distance sensor can be rapidly and simply constructed by loading an ultra-thin CRISPR/Cas12a-responsive DNA-crosslinked hydrogel film at the end of the capillary tube. The target DNA-specific recombinase polymerase reaction (RPA) amplicons activate the trans-cleavage activity of the Cas12a enzyme, cleaving the crosslinked DNA in hydrogel film, and causing an increase of hydrogel's permeability. As a result, a sample solution containing target DNA travels into the capillary tube at a longer distance compared to the negative samples. Reading the solution traveling distance in capillary tubes, the novel sensor realizes target DNA detection without any special equipment. Benefiting from the exponential target amplification of RPA and multiple turnover response of trans-cleavage of CRISPR/Cas12a, the developed sensor can visually and specifically detect as low as 1 aM HPV 16 DNA within 30 min. These outstanding features, including exceptional sensitivity and specificity, simple and portable design, mild measurement conditions, quick turnaround time, and user-friendly read-out, make the novel distance sensor a promising option for POC diagnostic applications.}, }
@article {pmid39126914, year = {2024}, author = {Mizushima, S and Ogawa, Y and Kuroiwa, A}, title = {Initial formation of and sex differences in primordial germ cells in Japanese quail.}, journal = {Reproductive biology}, volume = {24}, number = {3}, pages = {100922}, doi = {10.1016/j.repbio.2024.100922}, pmid = {39126914}, issn = {2300-732X}, mesh = {Animals ; *Coturnix ; Male ; Female ; *Germ Cells/physiology/cytology ; *DEAD-box RNA Helicases/metabolism/genetics ; Sex Characteristics ; Cell Proliferation/physiology ; CRISPR-Cas Systems ; }, abstract = {DEAD-box RNA helicase 4 (DDX4) is posited to be a key maternal germ cell factor regulating avian germ cell formation. We herein showed that the DDX4 gene product of zygotic genome activation associated with the nuclear localization of the cyclin D1 protein in presumptive primordial germ cells (PGCs) plays an essential role in the proliferation of PGCs using a CRISPR/Cas9 system approach combined with in vitro fertilization techniques in Japanese quail. A proteome analysis also revealed molecular-based differences in the features of early male and female PGCs.}, }
@article {pmid39126653, year = {2024}, author = {Chuang, YM and Dong, Y and Stone, H and Abouneameh, S and Tang, XD and Raduwan, H and Dimopoulos, G and Fikrig, E}, title = {Anopheles gambiae lacking AgTRIO probe inefficiently on a mammalian host.}, journal = {Cell reports}, volume = {43}, number = {8}, pages = {114600}, doi = {10.1016/j.celrep.2024.114600}, pmid = {39126653}, issn = {2211-1247}, mesh = {Animals ; *Anopheles/parasitology/genetics ; Malaria/transmission/parasitology ; Insect Proteins/genetics/metabolism ; Mice ; CRISPR-Cas Systems/genetics ; Female ; Mosquito Vectors/parasitology/genetics ; }, abstract = {Malaria is initiated as Plasmodium sporozoites are injected into the dermis when an infected mosquito probes on a vertebrate host for a blood meal. Factors in the mosquito saliva, such as AgTRIO, can alter the ability of Anopheles gambiae to transmit Plasmodium. We therefore used CRISPR-Cas9-mediated genome editing to generate AgTRIO knockout (KO) A. gambiae and examined the ability of these mosquitoes to probe on a vertebrate host. AgTRIO KO mosquitoes showed a diminished host probing capacity and required repetitive probing to locate a blood resource to complete a blood meal. This increased probing resulted in enhanced Plasmodium transmission to the vertebrate host. Our data demonstrate the importance of the A. gambiae saliva protein AgTRIO in probing and its influence on the ability of mosquitoes to transmit malaria.}, }
@article {pmid39116728, year = {2024}, author = {Zhang, H and Gao, H and Liu, S and Ren, X and Que, L and Gu, X and Rong, S and Ma, H and Ruan, J and Miao, M and Qi, X and Chang, D and Pan, H}, title = {Dual electrochemical signal "signal-on-off" sensor based on CHA-Td-HCR and CRISPR-Cas12a for MUC1 detection.}, journal = {Talanta}, volume = {279}, number = {}, pages = {126665}, doi = {10.1016/j.talanta.2024.126665}, pmid = {39116728}, issn = {1873-3573}, mesh = {*Mucin-1/analysis/genetics ; *Electrochemical Techniques/methods ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Hybridization ; Methylene Blue/chemistry ; Magnetite Nanoparticles/chemistry ; DNA, Single-Stranded/chemistry/genetics ; Electrodes ; Limit of Detection ; Gold/chemistry ; }, abstract = {Mucin 1 (MUC1) is frequently overexpressed in various cancers and is essential for early cancer detection. Current methods to detect MUC1 are expensive, time-consuming, and require skilled personnel. Therefore, developing a simple, sensitive, highly selective MUC1 detection sensor is necessary. In this study, we proposed a novel "signal-on-off" strategy that, in the presence of MUC1, synergistically integrates catalytic hairpin assembly (CHA) with DNA tetrahedron (Td)-based nonlinear hybridization chain reaction (HCR) to enhance the immobilization of electrochemically active methylene blue (MB) on magnetic nanoparticles (MNP), marking the MB signal "on". Concurrently, the activation of CRISPR-Cas12a by isothermal amplification products triggers the cleavage of single-stranded DNA (ssDNA) at the electrode surface, resulting in a reduction of MgAl-LDH@Fc-AuFe-MIL-101 (containing ferrocene, Fc) on the electrode, presenting the "signal-off" state. Both MB and MgAl-LDH@Fc-AuFe-MIL-101 electrochemical signals were measured and analyzed. Assay parameters were optimized, and sensitivity, stability, and linear range were assessed. Across a concentration spectrum of MUC1 spanning from 10 fg/mL to 100 ng/mL, the MB and MgAl-LDH@Fc-AuFe-MIL-101 signals were calibrated with each other, demonstrating a "signal-on-off" dual electrochemical signaling pattern. This allows for the precise and quantitative detection of MUC1 in clinical samples, offering significant potential for medical diagnosis.}, }
@article {pmid39116491, year = {2024}, author = {Wang, Y and Li, J and Zhu, J and Ma, H and Zhuang, B and Zhao, J and Zhang, F and Yu, L}, title = {TgMIC6 inhibition of autophagy is partially responsible for the phenotypic differences between Chinese 1 Toxoplasma gondii strains.}, journal = {International immunopharmacology}, volume = {140}, number = {}, pages = {112857}, doi = {10.1016/j.intimp.2024.112857}, pmid = {39116491}, issn = {1878-1705}, mesh = {*Toxoplasma/pathogenicity/immunology/genetics ; Animals ; *Autophagy ; *Protozoan Proteins/genetics/metabolism ; Humans ; Mice ; Virulence ; Female ; Cytokines/metabolism ; Cell Line ; ErbB Receptors/metabolism/genetics ; Phenotype ; China ; Toxoplasmosis/immunology/parasitology ; Toxoplasmosis, Animal/immunology ; CRISPR-Cas Systems ; East Asian People ; }, abstract = {Chinese1 is the predominant Toxoplasma gondii lineage in China, and significant phenotypic differences are observed within the lineage. WH3 and WH6 are two representative strains of Chinese 1, which exhibit divergent virulence and pathogenicity in mice. However, virulence determinants and their modulating mechanisms remain elusive. A global genome expression analysis of the WH3 and WH6 transcriptional profiles identified microneme secretory protein 6 (MIC6), which may be associated with the phenotypic difference observed in WH3. In the present study, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing technique was used to generate a T. gondii microneme secretory protein (TgMIC6) knockout in WH3. Wild-type mice and different mouse and human cell lines were infected with the WH3, WH3-Δmic6, and WH6 strains. The survival rate of mice, related cytokine levels in serum, and the proliferation of parasites were observed. These results suggested that TgMIC6 is an important effector molecule that determines the differential virulence of WH3 in vivo and in vitro. Furthermore, MIC6 may enhance WH3 virulence via inhibition of host cell autophagy and activation of key molecules in the epidermal growth factor receptor (EGFR)-Akt-mammalian target of rapamycin (mTOR) classical autophagy pathway. CD40L was cleared in vivo by i.p injection of CD40L monoclonal antibody, and it was found that the virulence of WH3-Δmic6 to mice was restored to a certain extent in the absence of CD40L. This study elucidates the virulence determinants and immune escape strategies of Toxoplasma gondii in China. Moreover, these data will aid the development of effective strategies for the prevention and control of toxoplasmosis.}, }
@article {pmid39112518, year = {2024}, author = {Cui, M and Liu, Z and Wang, S and Bae, S and Guo, H and Zhou, J and Liu, R and Wang, L}, title = {CRISPR-based dissection of microRNA-23a ~ 27a ~ 24-2 cluster functionality in hepatocellular carcinoma.}, journal = {Oncogene}, volume = {43}, number = {36}, pages = {2708-2721}, pmid = {39112518}, issn = {1476-5594}, mesh = {*Carcinoma, Hepatocellular/genetics/pathology ; Humans ; *MicroRNAs/genetics ; *Liver Neoplasms/genetics/pathology ; *Gene Expression Regulation, Neoplastic ; Animals ; Mice ; Cell Line, Tumor ; Cell Proliferation/genetics ; Multigene Family ; Epithelial-Mesenchymal Transition/genetics ; CRISPR-Cas Systems ; Cell Movement/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {The miR-23a ~ 27a ~ 24-2 cluster, commonly upregulated in diverse cancers, including hepatocellular carcinoma (HCC), raises questions about the specific functions of its three mature miRNAs and their integrated function. Utilizing CRISPR knockout (KO), CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) technologies, we established controlled endogenous miR-23a ~ 27 ~ a24-2 cell models to unravel their roles and signaling pathways in HCC. Both miR-23a KO and miR-27a KO displayed reduced cell growth in vitro and in vivo, revealing an integrated oncogenic function. Functional analysis indicated cell cycle arrest, particularly at the G2/M phase, through the downregulation of CDK1/cyclin B activation. High-throughput RNA-seq, combined with miRNA target prediction, unveiled the miR-23a/miR-27a-regulated gene network, validated through diverse technologies. While miR-23a and miR-27a exhibited opposing roles in cell migration and mesenchymal-epithelial transition, an integrated CRISPRi/a analysis suggested an oncogenic role of the miR-23a ~ 27a ~ 24-2 cluster in cell migration. This involvement potentially encompasses two signaling axes: miR-23a-BMPR2 and miR-27a-TMEM170B in HCC cells. In conclusion, our CRISPRi/a study provides a valuable tool for comprehending the integrated roles and underlying mechanisms of endogenous miRNA clusters, paving the way for promising directions in miRNA-targeted therapy interventions.}, }
@article {pmid39083378, year = {2024}, author = {Rafnsdottir, S and Jang, K and Halldorsdottir, ST and Vinod, M and Tomasdottir, A and Möller, K and Halldorsdottir, K and Reynisdottir, T and Atladottir, LH and Allison, KE and Ostacolo, K and He, J and Zhang, L and Northington, FJ and Magnusdottir, E and Chavez-Valdez, R and Anderson, KJ and Bjornsson, HT}, title = {SMYD5 is a regulator of the mild hypothermia response.}, journal = {Cell reports}, volume = {43}, number = {8}, pages = {114554}, doi = {10.1016/j.celrep.2024.114554}, pmid = {39083378}, issn = {2211-1247}, mesh = {Animals ; *Histone-Lysine N-Methyltransferase/metabolism/genetics ; *Histones/metabolism ; Humans ; Mice ; Hypothermia/metabolism/genetics ; Methylation ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; HEK293 Cells ; }, abstract = {The mild hypothermia response (MHR) maintains organismal homeostasis during cold exposure and is thought to be critical for the neuroprotection documented with therapeutic hypothermia. To date, little is known about the transcriptional regulation of the MHR. We utilize a forward CRISPR-Cas9 mutagenesis screen to identify the histone lysine methyltransferase SMYD5 as a regulator of the MHR. SMYD5 represses the key MHR gene SP1 at euthermia. This repression correlates with temperature-dependent levels of histone H3 lysine 26 trimethylation (H3K36me3) at the SP1 locus and globally, indicating that the mammalian MHR is regulated at the level of histone modifications. We have identified 37 additional SMYD5-regulated temperature-dependent genes, suggesting a broader MHR-related role for SMYD5. Our study provides an example of how histone modifications integrate environmental cues into the genetic circuitry of mammalian cells and provides insights that may yield therapeutic avenues for neuroprotection after catastrophic events.}, }
@article {pmid39078737, year = {2024}, author = {Casey, MJ and Chan, PP and Li, Q and Zu, JF and Jette, CA and Kohler, M and Myers, BR and Stewart, RA}, title = {A simple and scalable zebrafish model of Sonic hedgehog medulloblastoma.}, journal = {Cell reports}, volume = {43}, number = {8}, pages = {114559}, doi = {10.1016/j.celrep.2024.114559}, pmid = {39078737}, issn = {2211-1247}, support = {R01 NS106527/NS/NINDS NIH HHS/United States ; }, mesh = {*Medulloblastoma/genetics/pathology/metabolism ; Animals ; *Zebrafish ; *Hedgehog Proteins/metabolism/genetics ; *Disease Models, Animal ; *Zebrafish Proteins/metabolism/genetics ; *Patched-1 Receptor/genetics/metabolism ; Cerebellar Neoplasms/genetics/pathology/metabolism ; Humans ; Tumor Suppressor Protein p53/metabolism/genetics ; CRISPR-Cas Systems/genetics ; }, abstract = {Medulloblastoma (MB) is the most common malignant brain tumor in children and is stratified into three major subgroups. The Sonic hedgehog (SHH) subgroup represents ∼30% of all MB cases and has significant survival disparity depending upon TP53 status. Here, we describe a zebrafish model of SHH MB using CRISPR to create mutant ptch1, the primary genetic driver of human SHH MB. In these animals, tumors rapidly arise in the cerebellum and resemble human SHH MB by histology and comparative onco-genomics. Similar to human patients, MB tumors with loss of both ptch1 and tp53 have aggressive tumor histology and significantly worse survival outcomes. The simplicity and scalability of the ptch1-crispant MB model makes it highly amenable to CRISPR-based genome-editing screens to identify genes required for SHH MB tumor formation in vivo, and here we identify the gene encoding Grk3 kinase as one such target.}, }
@article {pmid39029887, year = {2024}, author = {Li, B and Zhu, X and Zhao, D and Li, Y and Yang, Y and Li, J and Bi, C and Zhang, X}, title = {igRNA Prediction and Selection AI Models (igRNA-PS) for Bystander-less ABE Base Editing.}, journal = {Journal of molecular biology}, volume = {436}, number = {18}, pages = {168714}, doi = {10.1016/j.jmb.2024.168714}, pmid = {39029887}, issn = {1089-8638}, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; }, abstract = {CRISPR derived base editing techniques tend to edit multiple bases in the targeted region, which impedes precise reversion of disease-associated single nucleotide variations (SNVs). We designed an imperfect gRNA (igRNA) editing strategy to achieve bystander-less single-base editing. To predict the performance and provide ready-to-use igRNAs, we employed a high-throughput method to edit 5000 loci, each with approximate 19 systematically designed ABE igRNAs. Through deep learning of the relationship of editing efficiency, original gRNA sequence and igRNA sequence, AI models were constructed and tested, designated igRNA Prediction and Selection AI models (igRNA-PS). The models have three functions, First, they can identify the major editing site from the bystanders on a gRNA protospacer with a near 90% accuracy. second, a modified single-base editing efficiency (SBE), considering both single-base editing efficiency and product purity, can be predicted for any given igRNAs. Third, for an editing locus, a set of 64 igRNAs derived from a gRNA can be generated, evaluated through igRNA-PS to select for the best performer, and provided to the user. In this work, we overcome one of the most significant obstacles of base editors, and provide a convenient and efficient approach for single-base bystander-less ABE base editing.}, }
@article {pmid39029179, year = {2024}, author = {Zheng, W and Tang, H and Ye, B and Lin, J and Wang, H and Liu, Y and Wang, D and Wu, Z and Xie, W and Dong, WF and Zan, M}, title = {Fast, portable and sensitive detection of group B streptococcus DNA using one-pot MIRA-CRISPR system with suboptimal PAM.}, journal = {Talanta}, volume = {279}, number = {}, pages = {126574}, doi = {10.1016/j.talanta.2024.126574}, pmid = {39029179}, issn = {1873-3573}, mesh = {*Streptococcus agalactiae/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *DNA, Bacterial/analysis/genetics ; Limit of Detection ; Streptococcal Infections/diagnosis ; Female ; Pregnancy ; }, abstract = {The group B Streptococcus (GBS) can generate vertical transmission to infants during delivery, has been seriously threatening the health of infants. Rapid and accurate prenatal GBS diagnosis for pregnant women is a deterministic blueprint to avoid infant viruses. Here, we developed an extraction-free nucleic acid isothermal amplification/CRISPR-Cas12a cutting one-pot system for GBS diagnostic assay by using suboptimal protospacer adjacent motifs, effectively avoiding multiple handling steps and uncapping contamination. The GBS diagnosis assay based on a one-pot system was validated by using fluorescent technique and lateral flow assay strips, exhibited fantastic specificity, accuracy and sensitivity with a limit of detection of 32 copies per reaction (0.64 copies/μL). Moreover, a portable device was constructed and integrated with the one-pot system to realize the GBS detection without professional and scene restrictions, it showed excellent performance in clinical sample detection, which achieved optical and portable GBS detection for point-of-care testing or home-self testing.}, }
@article {pmid39020177, year = {2024}, author = {Chen, W and Choi, J and Li, X and Nathans, JF and Martin, B and Yang, W and Hamazaki, N and Qiu, C and Lalanne, JB and Regalado, S and Kim, H and Agarwal, V and Nichols, E and Leith, A and Lee, C and Shendure, J}, title = {Symbolic recording of signalling and cis-regulatory element activity to DNA.}, journal = {Nature}, volume = {632}, number = {8027}, pages = {1073-1081}, pmid = {39020177}, issn = {1476-4687}, mesh = {Animals ; Mice ; *DNA/genetics/metabolism ; *Gene Editing ; *Signal Transduction/genetics ; *NF-kappa B/metabolism ; Enhancer Elements, Genetic/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Transcription Factors/metabolism/genetics ; Cell Differentiation/genetics ; Reproducibility of Results ; Mouse Embryonic Stem Cells/metabolism/cytology ; Genomics ; Time Factors ; Regulatory Sequences, Nucleic Acid/genetics ; Wnt Signaling Pathway/genetics ; Transcription, Genetic ; }, abstract = {Measurements of gene expression or signal transduction activity are conventionally performed using methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm in which such biological activities are stably recorded to the genome. Enhancer-driven genomic recording of transcriptional activity in multiplex (ENGRAM) is based on the signal-dependent production of prime editing guide RNAs that mediate the insertion of signal-specific barcodes (symbols) into a genomically encoded recording unit. We show how this strategy can be used for multiplex recording of the cell-type-specific activities of dozens to hundreds of cis-regulatory elements with high fidelity, sensitivity and reproducibility. Leveraging signal transduction pathway-responsive cis-regulatory elements, we also demonstrate time- and concentration-dependent genomic recording of WNT, NF-κB and Tet-On activities. By coupling ENGRAM to sequential genome editing via DNA Typewriter[1], we stably record information about the temporal dynamics of two orthogonal signalling pathways to genomic DNA. Finally we apply ENGRAM to integratively record the transient activity of nearly 100 transcription factor consensus motifs across daily windows spanning the differentiation of mouse embryonic stem cells into gastruloids, an in vitro model of early mammalian development. Although these are proof-of-concept experiments and much work remains to fully realize the possibilities, the symbolic recording of biological signals or states within cells, to the genome and over time, has broad potential to complement contemporary paradigms for how we make measurements in biological systems.}, }
@article {pmid38553609, year = {2024}, author = {Yang, L and Huo, Y and Wang, M and Zhang, D and Zhang, T and Wu, H and Rao, X and Meng, H and Yin, S and Mei, J and Zhang, D and Chen, X and Lv, J and Liu, M and Cheng, Y and Guan, Y and Feng, B and Song, G and Yi, C and Liu, M and Zeng, F and Wang, L and Li, D}, title = {Engineering APOBEC3A deaminase for highly accurate and efficient base editing.}, journal = {Nature chemical biology}, volume = {20}, number = {9}, pages = {1176-1187}, pmid = {38553609}, issn = {1552-4469}, mesh = {*Gene Editing/methods ; Humans ; Animals ; *Cytidine Deaminase/genetics/metabolism ; Mice ; HEK293 Cells ; Protein Engineering/methods ; Proteins/genetics/metabolism/chemistry ; CRISPR-Cas Systems ; Dependovirus/genetics ; Cytosine/metabolism/chemistry ; }, abstract = {Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.}, }
@article {pmid39196112, year = {2024}, author = {Padmanabhan, A and de Soysa, TY and Pelonero, A and Sapp, V and Shah, PP and Wang, Q and Li, L and Lee, CY and Sadagopan, N and Nishino, T and Ye, L and Yang, R and Karnay, A and Poleshko, A and Bolar, N and Linares-Saldana, R and Ranade, SS and Alexanian, M and Morton, SU and Jain, M and Haldar, SM and Srivastava, D and Jain, R}, title = {A genome-wide CRISPR screen identifies BRD4 as a regulator of cardiomyocyte differentiation.}, journal = {Nature cardiovascular research}, volume = {3}, number = {3}, pages = {317-331}, pmid = {39196112}, issn = {2731-0590}, support = {K08HL157700//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; P01 HL146366/HL/NHLBI NIH HHS/United States ; R01 HL057181/HL/NHLBI NIH HHS/United States ; R01 HL127240/HL/NHLBI NIH HHS/United States ; R01 HL015100//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; }, mesh = {*Myocytes, Cardiac/metabolism/cytology ; *Transcription Factors/genetics/metabolism ; Animals ; *Cell Differentiation/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/genetics/metabolism ; Mice ; Mouse Embryonic Stem Cells/metabolism/cytology ; Nuclear Proteins/genetics/metabolism ; Gene Expression Regulation, Developmental ; Cell Lineage/genetics ; Cells, Cultured ; Single-Cell Analysis ; Bromodomain Containing Proteins ; }, abstract = {Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs.}, }
@article {pmid39194596, year = {2024}, author = {Hu, K and Yin, W and Bai, Y and Zhang, J and Yin, J and Zhu, Q and Mu, Y}, title = {CRISPR-Based Biosensors for Medical Diagnosis: Readout from Detector-Dependence Detection Toward Naked Eye Detection.}, journal = {Biosensors}, volume = {14}, number = {8}, pages = {}, doi = {10.3390/bios14080367}, pmid = {39194596}, issn = {2079-6374}, support = {No.32071481//National Natural Science Foundation of China/ ; No. LQ22F010005//Zhejiang Provincial Natural Science Foundation of China under Grant/ ; }, mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; Humans ; Biomarkers ; }, abstract = {The detection of biomarkers (such as DNA, RNA, and protein) plays a vital role in medical diagnosis. The CRISPR-based biosensors utilize the CRISPR/Cas system for biometric recognition of targets and use biosensor strategy to read out biological signals without the employment of professional operations. Consequently, the CRISPR-based biosensors demonstrate great potential for the detection of biomarkers with high sensitivity and specificity. However, the signal readout still relies on specialized detectors, limiting its application in on-site detection for medical diagnosis. In this review, we summarize the principles and advances of the CRISPR-based biosensors with a focus on medical diagnosis. Then, we review the advantages and progress of CRISPR-based naked eye biosensors, which can realize diagnosis without additional detectors for signal readout. Finally, we discuss the challenges and further prospects for the development of CRISPR-based biosensors.}, }
@article {pmid39194140, year = {2024}, author = {Domingues, W and Folgosi, V&#xc2; and Sanabani, SS and Leite Junior, PD and Assone, T and Casseb, J}, title = {Novel approaches for HTLV-1 therapy: innovative applications of CRISPR-Cas9.}, journal = {Revista do Instituto de Medicina Tropical de Sao Paulo}, volume = {66}, number = {}, pages = {e48}, doi = {10.1590/S1678-9946202466048}, pmid = {39194140}, issn = {1678-9946}, mesh = {Humans ; *CRISPR-Cas Systems ; *Human T-lymphotropic virus 1/genetics ; *Gene Editing/methods ; *HTLV-I Infections/therapy/genetics/virology ; Genetic Therapy ; Paraparesis, Tropical Spastic/therapy/genetics/virology ; }, abstract = {The human T-cell lymphotropic virus type 1 (HTLV-1) is a single-stranded positive-sense RNA virus that belongs to the Retroviridae family, genus Deltaretro, and infects approximately five to 10 million people worldwide. Although a significant number of individuals living with HTLV-1 remain asymptomatic throughout their lives, some develop one or more severe clinical conditions, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive and debilitating disease, and/or a subtype of non-Hodgkin's lymphoma with a more threatening course known as adult T-cell leukemia/lymphoma (ATLL). Moreover, current therapeutic options are limited and focus primarily on treating symptoms and controlling viral latency. CRISPR-Cas9 gene editing is proposed as a promising tool to address the intricate links associated with HTLV-1. By targeting or silencing key genes during initial infection and dysregulating immune signaling pathways, CRISPR-Cas9 offers potential intervention opportunities. In this review, we address the therapeutic potential of CRISPR-Cas9 gene editing, as well as examine the primary mechanisms involved in editing potential target genes and discuss the existing evidence in the current scientific literature.}, }
@article {pmid39193213, year = {2024}, author = {Kanapiya, A and Amanbayeva, U and Tulegenova, Z and Abash, A and Zhangazin, S and Dyussembayev, K and Mukiyanova, G}, title = {Recent advances and challenges in plant viral diagnostics.}, journal = {Frontiers in plant science}, volume = {15}, number = {}, pages = {1451790}, doi = {10.3389/fpls.2024.1451790}, pmid = {39193213}, issn = {1664-462X}, abstract = {Accurate and timely diagnosis of plant viral infections plays a key role in effective disease control and maintaining agricultural productivity. Recent advances in the diagnosis of plant viruses have significantly expanded our ability to detect and monitor viral pathogens in agricultural crops. This review discusses the latest advances in diagnostic technologies, including both traditional methods and the latest innovations. Conventional methods such as enzyme-linked immunosorbent assay and DNA amplification-based assays remain widely used due to their reliability and accuracy. However, diagnostics such as next-generation sequencing and CRISPR-based detection offer faster, more sensitive and specific virus detection. The review highlights the main advantages and limitations of detection systems used in plant viral diagnostics including conventional methods, biosensor technologies and advanced sequence-based techniques. In addition, it also discusses the effectiveness of commercially available diagnostic tools and challenges facing modern diagnostic techniques as well as future directions for improving informed disease management strategies. Understanding the main features of available diagnostic methodologies would enable stakeholders to choose optimal management strategies against viral threats and ensure global food security.}, }
@article {pmid39192133, year = {2025}, author = {Wu, XM and Zheng, SY and Chang, MX}, title = {Zebrafish as a Model for Investigating Antiviral Innate Immunity.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2854}, number = {}, pages = {221-236}, pmid = {39192133}, issn = {1940-6029}, mesh = {Animals ; *Zebrafish/immunology/genetics/virology ; *Immunity, Innate/genetics ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Virus Diseases/immunology/genetics ; Gene Knockout Techniques ; Animals, Genetically Modified ; }, abstract = {Zebrafish is a widely used model organism in genetics, developmental biology, pathology, and immunology research. Due to their fast reproduction, large numbers, transparent early embryos, and high genetic conservation with the human genome, zebrafish have been used as a model for studying human and fish viral diseases. In particular, the ability to easily perform forward and reverse genetics and lacking a functional adaptive immune response during the early period of development establish the zebrafish as a favored option to assess the functional implication of specific genes in the antiviral innate immune response and the pathogenesis of viral diseases. In this chapter, we detail protocols for the antiviral innate immunity analysis using the zebrafish model, including the generation of gene-overexpression zebrafish, generation of gene-knockout zebrafish by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, methods of viral infection in zebrafish larvae, analyzing the expression of antiviral genes in zebrafish larvae using qRT-PCR, Western blotting and transcriptome sequencing, and in vivo antiviral assays. These experimental protocols provide effective references for studying the antiviral immune response in the zebrafish model.}, }
@article {pmid39192119, year = {2025}, author = {Shen, Y and Zhao, X and Zheng, C and Chen, Q}, title = {CRISPR-Mediated Construction of Gene-Knockout Mice for Investigating Antiviral Innate Immunity.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2854}, number = {}, pages = {61-74}, pmid = {39192119}, issn = {1940-6029}, mesh = {Animals ; *Immunity, Innate/genetics ; *CRISPR-Cas Systems ; Mice ; *Mice, Knockout ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Gene Knockout Techniques/methods ; Nucleotidyltransferases/genetics/metabolism ; Virus Diseases/immunology/genetics ; }, abstract = {With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.}, }
@article {pmid39192118, year = {2025}, author = {Shen, Y and Feng, Z and Zheng, C and Chen, Q}, title = {CRISPR-Mediated Library Screening of Gene-Knockout Cell Lines for Investigating Antiviral Innate Immunity.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2854}, number = {}, pages = {51-60}, pmid = {39192118}, issn = {1940-6029}, mesh = {*Immunity, Innate/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Gene Knockout Techniques ; *Gene Library ; High-Throughput Nucleotide Sequencing/methods ; Host-Pathogen Interactions/immunology/genetics ; Cell Line ; Lentivirus/genetics ; }, abstract = {The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.}, }
@article {pmid39192117, year = {2025}, author = {Feng, Z and Shen, Y and Zheng, C and Chen, Q}, title = {CRISPR-Mediated Viral Gene Knockout to Investigate Viral Evasion of Antiviral Innate Immunity.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2854}, number = {}, pages = {41-50}, pmid = {39192117}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems/genetics ; *Immunity, Innate/genetics ; *Herpesvirus 1, Human/immunology/genetics ; *Immune Evasion/genetics ; *Gene Knockout Techniques ; Humans ; Gene Editing/methods ; Animals ; Host-Pathogen Interactions/immunology/genetics ; Herpes Simplex/immunology/virology/genetics ; }, abstract = {The innate immune system relies on a variety of pathogen recognition receptors (PRRs) as the first line of defense against pathogenic invasions. Viruses have evolved multiple strategies to evade the host immune system through coevolution with hosts. The CRISPR-Cas system is an adaptive immune system in bacteria or archaea that defends against viral reinvasion by targeting nucleic acids for cleavage. Based on the characteristics of Cas proteins and their variants, the CRISPR-Cas system has been developed into a versatile gene-editing tool capable of gene knockout or knock-in operations to achieve genetic variations in organisms. It is now widely used in the study of viral immune evasion mechanisms. This chapter will introduce the use of the CRISPR-Cas9 system for editing herpes simplex virus 1 (HSV-1) genes to explore the mechanisms by which HSV-1 evades host innate immunity and the experimental procedures involved.}, }
@article {pmid39191721, year = {2024}, author = {Zhang, L and Zhou, X and Aryal, S and Veasey, V and Zhang, P and Li, FJ and Luan, Y and Bhatia, R and Zhou, Y and Lu, R}, title = {CRISPR screen of venetoclax response-associated genes identifies transcription factor ZNF740 as a key functional regulator.}, journal = {Cell death &amp; disease}, volume = {15}, number = {8}, pages = {627}, pmid = {39191721}, issn = {2041-4889}, support = {CA259480//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; RSG-22-036-01-DMC//American Cancer Society (American Cancer Society, Inc.)/ ; }, mesh = {*Sulfonamides/pharmacology ; Humans ; *Bridged Bicyclo Compounds, Heterocyclic/pharmacology ; *Leukemia, Myeloid, Acute/genetics/drug therapy/metabolism/pathology ; Animals ; Cell Line, Tumor ; Proto-Oncogene Proteins c-bcl-2/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice ; Drug Resistance, Neoplasm/genetics ; Antineoplastic Agents/pharmacology/therapeutic use ; Transcription Factors/metabolism/genetics ; CRISPR-Cas Systems/genetics ; }, abstract = {BCL-2 inhibitors such as venetoclax offer therapeutic promise in acute myeloid leukemia (AML) and other cancers, but drug resistance poses a significant challenge. It is crucial to understand the mechanisms that regulate venetoclax response. While correlative studies have identified numerous genes linked to venetoclax sensitivity, their direct impact on the drug response remains unclear. In this study, we targeted around 1400 genes upregulated in venetoclax-sensitive primary AML samples and carried out a CRISPR knockout screen to evaluate their direct effects on venetoclax response. Our screen identified the transcription factor ZNF740 as a critical regulator, with its expression consistently predicting venetoclax sensitivity across subtypes of the FAB classification. ZNF740 depletion leads to increased resistance to ventoclax, while its overexpression enhances sensitivity to the drug. Mechanistically, our integrative transcriptomic and genomic analysis identifies NOXA as a direct target of ZNF740, which negatively regulates MCL-1 protein stability. Loss of ZNF740 downregulates NOXA and increases the steady state protein levels of MCL-1 in AML cells. Restoring NOXA expression in ZNF740-depleted cells re-sensitizes AML cells to venetoclax treatment. Furthermore, we demonstrated that dual targeting of MCL-1 and BCL-2 effectively treats ZNF740-deficient AML in vivo. Together, our work systematically elucidates the causal relationship between venetoclax response signature genes and establishes ZNF740 as a novel transcription factor regulating venetoclax sensitivity.}, }
@article {pmid39191394, year = {2024}, author = {Gupta, V and Chitranshi, N and Gupta, VB}, title = {Genetic Risk, Inflammation, and Therapeutics: An Editorial Overview of Recent Advances in Aging Brains and Neurodegeneration.}, journal = {Aging and disease}, volume = {15}, number = {5}, pages = {1989-1993}, doi = {10.14336/AD.2024.0986}, pmid = {39191394}, issn = {2152-5250}, mesh = {Humans ; *Neurodegenerative Diseases/therapy/genetics/metabolism/pathology ; *Aging/pathology ; *Brain/pathology/metabolism ; Inflammation/genetics/therapy ; Oxidative Stress ; Neuroinflammatory Diseases/metabolism/genetics ; }, abstract = {Neurodegenerative disorders, including Dementia, Parkinson's disease, various Vision disorders, Multiple sclerosis, and transsynaptic degenerative changes represent a significant challenge in aging populations. This editorial synthesizes and discusses recent advancements in understanding the genetic and environmental factors contributing to these diseases. Central to these advancements is the role of neuroinflammation and oxidative stress, which exacerbate neuronal damage and accelerate disease progression. Emerging research underscores the significance of mitochondrial dysfunction and protein aggregation in neurodegenerative pathology, highlighting shared mechanisms across various disorders. Innovative therapeutic strategies, including gene therapy, CRISPR-Cas technology, and the use of naturally occurring antioxidant molecules, are being investigated to target and manage these conditions. Additionally, lifestyle interventions such as exercise and healthy diet have shown promise in enhancing brain plasticity and reducing neuroinflammation. Advances in neuroimaging and biomarker discovery are necessary to improve early diagnosis, while clinical and preclinical studies are essential for the translation of these novel treatments. This edition aims to bridge the gap between molecular mechanisms and therapeutic applications, offering insights into potential interventions to mitigate the impact of neurodegenerative diseases. By establishing a deeper understanding of these complex processes, we aim to move closer to effective prevention and treatment strategies, ultimately improving the quality of life for those affected by neurodegenerative disorders.}, }
@article {pmid39191105, year = {2024}, author = {Song, SF and Zhang, XW and Chen, S and Shu, Y and Yu, YL and Wang, JH}, title = {CRISPR-based dual-aptamer proximity ligation coupled hybridization chain reaction for precise detection of tumor extracellular vesicles and cancer diagnosis.}, journal = {Talanta}, volume = {280}, number = {}, pages = {126780}, doi = {10.1016/j.talanta.2024.126780}, pmid = {39191105}, issn = {1873-3573}, abstract = {Tumor cell-derived extracellular vesicles (TEVs) contain numerous cellular molecules and are considered potential biomarkers for non-invasive liquid biopsy. However, due to the low abundance of TEVs secreted by tumor cells and their phenotypic heterogeneity, there is a lack of sensitive and specific methods to quantify TEVs. Here, we developed a dual-aptamer proximity ligation-coupled hybridization chain reaction (HCR) method for tracing TEVs, exploiting CRISPR to achieve highly sensitive detection. Taking advantage of the high binding affinity of aptamers, the two aptamers (AptEpCAM, AptHER2) exhibited the high selectivity for TEVs recognition. HCR generated long-repeated sequence containing multiple crRNA targetable barcodes, and the signals were further amplified by CRISPR upon recognizing the HCR sequences, thereby enhancing the sensitivity. Under optimal conditions, the developed method demonstrated a favorable linear relationship in the range of 2 × 10[3]-10[7] particles/μL, with a limit of detection (LOD) of 3.3 × 10[2] particles/μL. We directly applied our assay to clinical plasma analysis, achieving 100 % accuracy in cancer diagnosis, thus demonstrating the potential clinical applications of TEVs. Due to its simplicity and rapidity, excellent sensitivity and specificity, this method has broad applications in clinical medicine.}, }
@article {pmid39056144, year = {2024}, author = {Stockhammer, A and Spalt, C and Klemt, A and Benz, LS and Harel, S and Natalia, V and Wiench, L and Freund, C and Kuropka, B and Bottanelli, F}, title = {When less is more - a fast TurboID knock-in approach for high-sensitivity endogenous interactome mapping.}, journal = {Journal of cell science}, volume = {137}, number = {16}, pages = {}, doi = {10.1242/jcs.261952}, pmid = {39056144}, issn = {1477-9137}, support = {278001972//Deutsche Forschungsgemeinschaft/ ; //Freie Universitat Berlin/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; Gene Knock-In Techniques ; Protein Interaction Mapping/methods ; HEK293 Cells ; }, abstract = {In recent years, proximity labeling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. Although physiological expression of labeling enzymes is beneficial for the mapping of interactors, generation of the desired cell lines remains time-consuming and challenging. Using our established pipeline for rapid generation of C- and N-terminal CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labeling enzymes when endogenously expressed. Endogenous tagging of the µ subunit of the adaptor protein (AP)-1 complex with TurboID allowed identification of known interactors and cargo proteins that simple overexpression of a labeling enzyme fusion protein could not reveal. We used the KI strategy to compare the interactome of the different AP complexes and clathrin and were able to assemble lists of potential interactors and cargo proteins that are specific for each sorting pathway. Our approach greatly simplifies the execution of proximity labeling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry analysis and interactome data in just over a month.}, }
@article {pmid38973166, year = {2024}, author = {Li, M and Guan, M and Lin, J and Zhu, K and Zhu, J and Guo, M and Li, Y and Chen, Y and Chen, Y and Zou, Y and Wu, D and Xu, J and Yi, W and Fan, Y and Ma, S and Chen, Y and Xu, J and Yang, L and Dai, J and Ye, T and Lu, Z and Chen, Y}, title = {Early blood immune molecular alterations in cynomolgus monkeys with a PSEN1 mutation causing familial Alzheimer's disease.}, journal = {Alzheimer's &amp; dementia : the journal of the Alzheimer's Association}, volume = {20}, number = {8}, pages = {5492-5510}, doi = {10.1002/alz.14046}, pmid = {38973166}, issn = {1552-5279}, support = {2021YFE0203000//National Key R&amp;D Program of China/ ; 2018YFA0801404//National Key R&amp;D Program of China/ ; 2021YFC2500103//National Key R&amp;D Program of China/ ; 32061160472//National Natural Science Foundation of China (NSFC)/RGC Joint Research Scheme/ ; 32100778//National Natural Science Foundation of China/ ; 81961128019//National Natural Science Foundation of China/ ; 82071187//National Natural Science Foundation of China/ ; 2019B1515130004//Guangdong Provincial Fund for Basic and Applied Basic Research/ ; 2022A1515011639//Guangdong Provincial Fund for Basic and Applied Basic Research/ ; 2018B030336001//Guangdong Provincial Key S&amp;T Program/ ; F-2021-Z99-504979//Development and Reform Commission of Shenzhen/ ; JCYJ20220818100800001//Technology Innovation Commission of Shenzhen/ ; JCYJ20210324102006018//Technology Innovation Commission of Shenzhen/ ; JCYJ20220531100217038//Technology Innovation Commission of Shenzhen/ ; ZDSYS20200828154800001//Technology Innovation Commission of Shenzhen/ ; KQTD20221101093608028//Technology Innovation Commission of Shenzhen/ ; INNOHK18SC01//Innovation and Technology Commission of HKSAR/ ; }, mesh = {Animals ; *Macaca fascicularis ; *Alzheimer Disease/genetics/blood ; *Presenilin-1/genetics ; *Disease Models, Animal ; *Mutation/genetics ; Amyloid beta-Peptides/cerebrospinal fluid ; tau Proteins/genetics ; CRISPR-Cas Systems ; Exons/genetics ; Male ; Transcriptome ; Biomarkers/blood/cerebrospinal fluid ; }, abstract = {INTRODUCTION: More robust non-human primate models of Alzheimer's disease (AD) will provide new opportunities to better understand the pathogenesis and progression of AD.<br><br>METHODS: We designed a CRISPR/Cas9 system to achieve precise genomic deletion of exon 9 in cynomolgus monkeys using two guide RNAs targeting the 3' and 5' intron sequences of PSEN1 exon 9. We performed biochemical, transcriptome, proteome, and biomarker analyses to characterize the cellular and molecular dysregulations of this non-human primate model.<br><br>RESULTS: We observed early changes of AD-related pathological proteins (cerebrospinal fluid A&#x3b2;42 and phosphorylated tau) in PSEN1 mutant (ie, PSEN1-&#x394;E9) monkeys. Blood transcriptome and proteome profiling revealed early changes in inflammatory and immune molecules in juvenile PSEN1-&#x394;E9 cynomolgus monkeys.<br><br>DISCUSSION: PSEN1 mutant cynomolgus monkeys recapitulate AD-related pathological protein changes, and reveal early alterations in blood immune signaling. Thus, this model might mimic AD-associated pathogenesis and has potential utility for developing early diagnostic and therapeutic interventions.<br><br>HIGHLIGHTS: A dual-guide CRISPR/Cas9 system successfully mimics AD PSEN1-&#x394;E9 mutation by genomic excision of exon 9. PSEN1 mutant cynomolgus monkey-derived fibroblasts exhibit disrupted PSEN1 endoproteolysis and increased A&#x3b2; secretion. Blood transcriptome and proteome profiling implicate early inflammatory and immune molecular dysregulation in juvenile PSEN1 mutant cynomolgus monkeys. Cerebrospinal fluid from juvenile PSEN1 mutant monkeys recapitulates early changes of AD-related pathological proteins (increased A&#x3b2;42 and phosphorylated tau).}, }
@article {pmid39190135, year = {2024}, author = {Li, J and Wang, D and Zhao, P and Chen, T and Ma, L and Zhou, Y}, title = {Engineering broad-spectrum resistance to rice bacterial blight by editing the OsETR susceptible haplotype using CRISPR/Cas9.}, journal = {Plant cell reports}, volume = {43}, number = {9}, pages = {222}, pmid = {39190135}, issn = {1432-203X}, support = {ZDYF2022XDNY180//the Key Research and Development Program of Hainan Province/ ; 31661143009//Natural Science Foundation for Young Scientists of Shanxi Province/ ; 31571632//Innovative Research Group Project of the National Natural Science Foundation of China/ ; }, mesh = {*Oryza/genetics/microbiology ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Gene Editing/methods ; *Haplotypes/genetics ; Plant Proteins/genetics/metabolism ; Xanthomonas/pathogenicity/physiology ; Plants, Genetically Modified/genetics ; }, abstract = {CRISPR/Cas9-mediated knockout of the susceptible haplotype of OsETR, encoding an embryogenesis transmembrane protein, confers broad-spectrum resistance to bacterial leaf blight in a susceptible rice cultivar without yield penalty.}, }
@article {pmid39189452, year = {2024}, author = {Rahimi, S and Balusamy, SR and Perumalsamy, H and Ståhlberg, A and Mijakovic, I}, title = {CRISPR-Cas target recognition for sensing viral and cancer biomarkers.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae736}, pmid = {39189452}, issn = {1362-4962}, support = {NNF20OC0064547//Novo Nordisk Foundation/ ; C 2021-1705//Kristina Stenborgs foundation for scientific research/ ; 2020-04733//Vinnova-Swelifes, Medtech4Healths/ ; 2023-01315//FORMAS/ ; 23-299//&#xc5;Forsk/ ; 22-2080//Swedish Cancer Society/ ; 2022-0030//Swedish Childhood Cancer Foundation/ ; 2021-01008//Swedish Research Council/ ; //Swedish state/ ; 965065//ALF/ ; 2020-04141//Sweden's Innovation Agency/ ; //Sj&#xf6;berg Foundation/ ; //Chalmers library/ ; }, abstract = {Nucleic acid-based diagnostics is a promising venue for detection of pathogens causing infectious diseases and mutations related to cancer. However, this type of diagnostics still faces certain challenges, and there is a need for more robust, simple and cost-effective methods. Clustered regularly interspaced short palindromic repeats (CRISPRs), the adaptive immune systems present in the prokaryotes, has recently been developed for specific detection of nucleic acids. In this review, structural and functional differences of CRISPR-Cas proteins Cas9, Cas12 and Cas13 are outlined. Thereafter, recent reports about applications of these Cas proteins for detection of viral genomes and cancer biomarkers are discussed. Further, we highlight the challenges associated with using these technologies to replace the current diagnostic approaches and outline the points that need to be considered for designing an ideal Cas-based detection system for nucleic acids.}, }
@article {pmid39185796, year = {2024}, author = {Yang, C and Wei, Y and Li, X and Xu, K and Huo, X and Chen, G and Zhao, H and Wang, J and Wei, T and Qing, Y and Guo, J and Zhao, H and Zhang, X and Jiao, D and Xiong, Z and Jamal, MA and Zhao, HY and Wei, HJ}, title = {Production of Four-Gene (GTKO/hCD55/hTBM/hCD39)-Edited Donor Pigs and Kidney Xenotransplantation.}, journal = {Xenotransplantation}, volume = {31}, number = {4}, pages = {e12881}, doi = {10.1111/xen.12881}, pmid = {39185796}, issn = {1399-3089}, support = {2019YFA0110700//National Key R&amp;D Program of China/ ; 202102AA310047//Major Science and Technology Project of Yunnan Province/ ; }, mesh = {Animals ; *Transplantation, Heterologous/methods ; *Kidney Transplantation/methods ; Swine ; *Gene Editing/methods ; *Animals, Genetically Modified ; *Galactosyltransferases/genetics ; CRISPR-Cas Systems ; Macaca mulatta ; Nuclear Transfer Techniques ; Heterografts ; Humans ; Graft Survival/immunology ; Graft Rejection/immunology ; Apyrase ; Antigens, CD ; }, abstract = {BACKGROUND: The number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene-editing technologies. However, it remains unclear which gene combination is suitable for specific organ transplantation.<br><br>METHODS: In this study, we utilized CRISPR/Cas9 gene editing technology, piggyBac transposon system, and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs.<br><br>RESULTS: First, 107 cell colonies were obtained through drug selection, of which seven were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer (SCNT), resulting in seven fetuses, of which four were GGTA1 biallelic knockout. Out of these four, two fetuses had higher expression of hCD55, hTBM, and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM, and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation.<br><br>CONCLUSION: These results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey's life.}, }
@article {pmid39184070, year = {2024}, author = {Maxim, DS and Wu, DW and Johnson, NS and Charu, V and Carter, JN and Anand, S and Church, GM and Bhalla, V}, title = {EditABLE: A Simple Web Application for Designing Genome Editing Experiments.}, journal = {Research square}, volume = {}, number = {}, pages = {}, doi = {10.21203/rs.3.rs-4775705/v1}, pmid = {39184070}, issn = {2693-5015}, abstract = {CRISPR-Cas genome editing is transformative; however, there is no simple tool available for determining the optimal genome editing technology to create specific mutations for experimentation or to correct mutations as a curative therapy for specific diseases. We developed editABLE, an online resource (editable-app.stanford.edu) to provide computationally validated CRISPR editors and guide RNAs based on user provided sequence data. We demonstrate the utility of editABLE by applying it to one of the most common monogenic disorders, autosomal dominant polycystic kidney disease (ADPKD), identifying specific editing tools across the landscape of ADPKD mutations.}, }
@article {pmid39146367, year = {2024}, author = {Matoušková, M and Plachý, J and Kučerová, D and Pecnová, &#x13d; and Reinišová, M and Geryk, J and Karafiát, V and Hron, T and Hejnar, J}, title = {Rapid adaptive evolution of avian leukosis virus subgroup J in response to biotechnologically induced host resistance.}, journal = {PLoS pathogens}, volume = {20}, number = {8}, pages = {e1012468}, doi = {10.1371/journal.ppat.1012468}, pmid = {39146367}, issn = {1553-7374}, mesh = {Animals ; *Avian Leukosis Virus/genetics/physiology ; *Chickens/virology ; *Avian Leukosis/virology/genetics ; Poultry Diseases/virology/genetics ; Disease Resistance/genetics ; CRISPR-Cas Systems ; Gene Editing ; Chick Embryo ; Evolution, Molecular ; Viral Envelope Proteins/genetics/metabolism ; Fibroblasts/virology/metabolism ; }, abstract = {Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.}, }
@article {pmid39043964, year = {2024}, author = {Tran, NT and Graf, R and Acevedo-Ochoa, E and Trombke, J and Weber, T and Sommermann, T and Salomon, C and Kühn, R and Rajewsky, K and Chu, VT}, title = {In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis.}, journal = {Leukemia}, volume = {38}, number = {9}, pages = {2003-2015}, pmid = {39043964}, issn = {1476-5551}, support = {CH 1968/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; ZT-0027//Helmholtz Association/ ; }, mesh = {*Hematopoiesis/genetics ; Animals ; *CRISPR-Cas Systems ; *Hematopoietic Stem Cells/metabolism/cytology ; Mice ; *E2F4 Transcription Factor/genetics/metabolism ; *Transcription Factor DP1/genetics/metabolism ; Cell Proliferation ; Humans ; Cell Cycle/genetics ; Cell Differentiation/genetics ; Mice, Inbred C57BL ; }, abstract = {Hematopoiesis is a continuous process of blood cell production driven by hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Proliferation and differentiation of HSPCs are regulated by complex transcriptional networks. In order to identify transcription factors with key roles in HSPC-mediated hematopoietic reconstitution, we developed an efficient and robust CRISPR/Cas9-based in vivo genetic screen. Using this experimental system, we identified the TFDP1 transcription factor to be essential for HSPC proliferation and post-transplant hematopoiesis. We further discovered that E2F4, an E2F transcription factor, serves as a binding partner of TFDP1 and is required for HSPC proliferation. Deletion of TFDP1 caused downregulation of genes associated with the cell cycle, with around 50% of these genes being identified as direct targets of TFDP1 and E2F4. Thus, our study expands the transcriptional network governing hematopoietic development through an in vivo CRISPR/Cas9-based genetic screen and identifies TFDP1/E2F4 as positive regulators of cell cycle genes in HSPCs.}, }
@article {pmid39011887, year = {2024}, author = {Yang, Y and Rocamonde-Lago, I and Shen, B and Berzina, I and Zipf, J and Högberg, B}, title = {Re-engineered guide RNA enables DNA loops and contacts modulating repression in E. coli.}, journal = {Nucleic acids research}, volume = {52}, number = {15}, pages = {9328-9339}, doi = {10.1093/nar/gkae591}, pmid = {39011887}, issn = {1362-4962}, support = {KAW 2017.0114//Knut and Alice Wallenberg Foundation/ ; //G&#xf6;ran Gustafsson Foundation for Research in Natural Sciences and Medicine/ ; 2019-01474//Swedish Research Council/ ; 341&#x2009;908//Academy of Finland/ ; 724&#x2009;872/ERC_/European Research Council/International ; }, mesh = {*Escherichia coli/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Nucleic Acid Conformation ; CRISPR-Cas Systems ; DNA/metabolism/chemistry/genetics ; DNA, Bacterial/metabolism/genetics ; Aptamers, Nucleotide/chemistry/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; }, abstract = {RNA serves as information media as well as molecular scaffold in nature and synthetic systems. The single guide RNA (sgRNA) widely applied in CRISPR techniques exemplifies both functions, with a guide region bearing DNA base-pairing information, and a structural motif for Cas9 protein scaffolding. The scaffold region has been modified by fusing RNA aptamers to the tetra-stem loop. The guide region is typically not regarded as a pluggable module as it encodes the essential function of DNA sequence recognition. Here, we investigate a chimera of two sgRNAs, with distinct guide sequences joined by an RNA linker (dgRNA), regarding its DNA binding function and loop induction capability. First, we studied the sequence bi-specificity of the dgRNA and discovered that the RNA linker allows distal parts of double-stranded DNA to be brought into proximity. To test the activity of the dgRNA in organisms, we used the LacZ gene as a reporter and recapitulated the loop-mediated gene inhibition by LacI in E. coli. We found that the dgRNA can be applied to target distal genomic regions with comparable levels of inhibition. The capability of dgRNA to induce DNA contacts solely requires dCas9 and RNA, making it a minimal system to remodel chromosomal conformation in various organisms.}, }
@article {pmid38995679, year = {2024}, author = {Wang, J and Liao, Z and Jin, X and Liao, L and Zhang, Y and Zhang, R and Zhao, X and Qin, H and Chen, J and He, Y and Zhuang, C and Tang, J and Huang, S}, title = {Xanthomonas oryzae pv. oryzicola effector Tal10a directly activates rice OsHXK5 expression to facilitate pathogenesis.}, journal = {The Plant journal : for cell and molecular biology}, volume = {119}, number = {5}, pages = {2423-2436}, doi = {10.1111/tpj.16929}, pmid = {38995679}, issn = {1365-313X}, support = {2020GXNSFDA297026//Guangxi Natural Science Foundation/ ; SKLCUSA-a202009//Independent project of State Key Laboratory State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/ ; 31860032//National Natural Science Foundation of China/ ; }, mesh = {*Oryza/microbiology/genetics ; *Xanthomonas/pathogenicity/physiology/genetics ; *Plant Diseases/microbiology/genetics ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; *Bacterial Proteins/genetics/metabolism ; Transcription Activator-Like Effectors/genetics/metabolism ; Virulence/genetics ; Promoter Regions, Genetic/genetics ; Disease Resistance/genetics ; CRISPR-Cas Systems ; Gene Editing ; Plants, Genetically Modified ; }, abstract = {Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc), is a major bacterial disease in rice. Transcription activator-like effectors (TALEs) from Xanthomonas can induce host susceptibility (S) genes and facilitate infection. However, knowledge of the function of Xoc TALEs in promoting bacterial virulence is limited. In this study, we demonstrated the importance of Tal10a for the full virulence of Xoc. Through computational prediction and gene expression analysis, we identified the hexokinase gene OsHXK5 as a host target of Tal10a. Tal10a directly binds to the gene promoter region and activates the expression of OsHXK5. CRISPR/Cas9-mediated gene editing in the effector binding element (EBE) of OsHXK5 significantly increases rice resistance to Xoc, while OsHXK5 overexpression enhances the susceptibility of rice plants and impairs rice defense responses. Moreover, simultaneous editing of the promoters of OsSULTR3;6 and OsHXK5 confers robust resistance to Xoc in rice. Taken together, our findings highlight the role of Tal10a in targeting OsHXK5 to promote infection and suggest that OsHXK5 represents a potential target for engineering rice resistance to Xoc.}, }
@article {pmid38953163, year = {2024}, author = {Schep, R and Trauernicht, M and Vergara, X and Friskes, A and Morris, B and Gregoricchio, S and Manzo, SG and Zwart, W and Beijersbergen, RL and Medema, RH and van Steensel, B}, title = {Chromatin context-dependent effects of epigenetic drugs on CRISPR-Cas9 editing.}, journal = {Nucleic acids research}, volume = {52}, number = {15}, pages = {8815-8832}, doi = {10.1093/nar/gkae570}, pmid = {38953163}, issn = {1362-4962}, support = {91&#xa0;215&#xa0;067//ZonMW TOP/ ; //The Dutch Cancer Society/ ; //EU/MUR MSCA Young Researcher Fellowship/ ; 694466/ERC_/European Research Council/International ; //KWF Dutch Cancer Society/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Epigenesis, Genetic/drug effects ; *Chromatin/metabolism/genetics ; *Histone Deacetylase Inhibitors/pharmacology ; DNA Repair ; CRISPR-Associated Protein 9/metabolism/genetics ; Heterochromatin/metabolism/genetics ; Cell Line ; Histones/metabolism ; Euchromatin/genetics ; DNA Breaks, Double-Stranded/drug effects ; }, abstract = {The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break in different chromatin environments. We then measured Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 58 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g. PCI-24781), while others were only effective in euchromatin and H3K27me3-marked regions (e.g. apicidin). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a resource to improve Cas9 editing more selectively at the desired location.}, }
@article {pmid38924638, year = {2024}, author = {Chen, M and Huang, X and Shi, Y and Wang, W and Huang, Z and Tong, Y and Zou, X and Xu, Y and Dai, Z}, title = {CRISPR/Pepper-tDeg: A Live Imaging System Enables Non-Repetitive Genomic Locus Analysis with One Single-Guide RNA.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {11}, number = {32}, pages = {e2402534}, doi = {10.1002/advs.202402534}, pmid = {38924638}, issn = {2198-3844}, support = {22274169//National Natural Science Foundation of China/ ; 2023A1515012027//Guangdong Basic and Applied Basic Research Foundation/ ; A2303001//Shenzhen Medical Research Fund/ ; JCYJ20210324124003008//Shenzhen Fundamental Research Program/ ; 2023B03J1380//Science and Technology Program of Guangzhou City/ ; 202103000003//Science and Technology Program of Guangzhou City/ ; 2020B1212060077//Guangdong Science and Technology Plan Project Grant/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genomics/methods ; Telomere/genetics ; }, abstract = {CRISPR-based genomic-imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal-to-noise ratio (SNR) at a non-repetitive genomic locus. Here, an efficient genomic-imaging system is proposed, termed CRISPR/Pepper-tDeg, by engineering the CRISPR sgRNA scaffolds with the degron-binding Pepper aptamers for binding fluorogenic proteins fused with Tat peptide derived degron domain (tDeg). The target-dependent stability switches of both sgRNA and fluorogenic protein allow this system to image repetitive telomeres sensitively with a 5-fold higher SNR than conventional CRISPR/MS2-MCP system using "always-on" fluorescent protein tag. Subsequently, CRISPR/Pepper-tDeg is applied to simultaneously label and track two different genomic loci, telomeres and centromeres, in living cells by combining two systems. Given a further improved SNR by the split fluorescent protein design, CRISPR/Pepper-tDeg system is extended to non-repetitive sequence imaging using only one sgRNA with two aptamer insertions. Neither complex sgRNA design nor difficult plasmid construction is required, greatly reducing the technical barriers to define spatiotemporal organization and dynamics of both repetitive and non-repetitive genomic loci in living cells, and thus demonstrating the large application potential of this genomic-imaging system in biological research, clinical diagnosis and therapy.}, }
@article {pmid38923275, year = {2024}, author = {Zhang, H and Jiang, W and Song, T and Song, M and Liu, S and Zhou, J and Cheng, H and Ding, Y}, title = {Lipid-Polymer Nanoparticles Mediate Compartmentalized Delivery of Cas9 and sgRNA for Glioblastoma Vasculature and Immune Reprogramming.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {11}, number = {32}, pages = {e2309314}, doi = {10.1002/advs.202309314}, pmid = {38923275}, issn = {2198-3844}, support = {82073401//National Natural Science Foundation of China/ ; 82104097//National Natural Science Foundation of China/ ; 82372113//National Natural Science Foundation of China/ ; BK20231018//Natural Science Foundation of Jiangsu Province/ ; 2632023TD05//Fundamental Research Funds for the Central Universities/ ; 2632023GR18//Fundamental Research Funds for the Central Universities/ ; //National Ten Thousand Talents Program for Young Top-notch Talents/ ; TJ-2021-002//Young Talent Support Project of Jiangsu Association for Science and Technology/ ; [2022]3-16-190//Jiangsu 333 High-level Talent Training Project/ ; CPU2022QZ18//Double First-Rate Construction Plan of China Pharmaceutical University/ ; QJHKY[2022]387//Project of the State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University/ ; }, mesh = {*Glioblastoma/metabolism/genetics/drug therapy ; *Nanoparticles/chemistry ; Mice ; Animals ; *Polymers/chemistry ; Humans ; Lipids/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Brain Neoplasms/genetics/drug therapy/metabolism ; Cell Line, Tumor ; Blood-Brain Barrier/metabolism ; STAT3 Transcription Factor/metabolism/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; }, abstract = {Hypervascularized glioblastoma is naturally sensitive to anti-angiogenesis but suffers from low efficacy of transient vasculature normalization. In this study, a lipid-polymer nanoparticle is synthesized to execute compartmentalized Cas9 and sgRNA delivery for a permanent vasculature editing strategy by knocking out the signal transducer and activator of transcription 3 (STAT3). The phenylboronic acid branched cationic polymer is designed to condense sgRNA electrostatically (inner compartment) and patch Cas9 coordinatively (outer compartment), followed by liposomal hybridization with angiopep-2 decoration for blood-brain barrier (BBB) penetration. The lipid-polymer nanoparticles can reach glioblastoma within 2 h post intravenous administration, and hypoxia in tumor cells triggers charge-elimination and degradation of the cationic polymer for burst release of Cas9 and sgRNA, accompanied by instant Cas9 RNP assembly, yielding ≈50% STAT3 knockout. The downregulation of downstream vascular endothelial growth factor (VEGF) reprograms vasculature normalization to improve immune infiltration, collaborating with interleukin-6 (IL-6) and interleukin-10 (IL-10) reduction to develop anti-glioblastoma responses. Collectively, the combinational assembly for compartmentalized Cas9/sgRNA delivery provides a potential solution in glioblastoma therapy.}, }
@article {pmid38908026, year = {2024}, author = {Wu, P and Yao, M and Wang, W}, title = {Differential impact of quiescent non-coding loci on chromatin entropy.}, journal = {Nucleic acids research}, volume = {52}, number = {15}, pages = {8778-8799}, doi = {10.1093/nar/gkae535}, pmid = {38908026}, issn = {1362-4962}, support = {RB5-07012//California Institute for Regenerative Medicine/ ; R01HG009626/NH/NIH HHS/United States ; R01HG009626/NH/NIH HHS/United States ; }, mesh = {Humans ; *Chromatin/metabolism/genetics ; *Entropy ; *Genome, Human ; Machine Learning ; Epigenesis, Genetic ; Gene Regulatory Networks ; CRISPR-Cas Systems ; Genetic Loci ; }, abstract = {Non-coding regions of the human genome are important for functional regulations, but their mechanisms remain elusive. We used machine learning to guide a CRISPR screening on hubs (i.e. non-coding loci forming many 3D contacts) and significantly increased the discovery rate of hubs essential for cell growth. We found no clear genetic or epigenetic differences between essential and nonessential hubs, but we observed that some neighboring hubs in the linear genome have distinct spatial contacts and opposite effects on cell growth. One such pair in an epigenetically quiescent region showed different impacts on gene expression, chromatin accessibility and chromatin organization. We also found that deleting the essential hub altered the genetic network activity and increased the entropy of chromatin accessibility, more severe than that caused by deletion of the nonessential hub, suggesting that they are critical for maintaining an ordered chromatin structure. Our study reveals new insights into the system-level roles of non-coding regions in the human genome.}, }
@article {pmid38692586, year = {2024}, author = {Noguchi, Y and Matsui, R and Suh, J and Dou, Y and Suzuki, J}, title = {Genome-Wide Screening Approaches for Biochemical Reactions Independent of Cell Growth.}, journal = {Annual review of genomics and human genetics}, volume = {25}, number = {1}, pages = {51-76}, doi = {10.1146/annurev-genom-121222-115958}, pmid = {38692586}, issn = {1545-293X}, mesh = {Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Flow Cytometry/methods ; Animals ; Genome-Wide Association Study ; Gene Editing/methods ; Gene Library ; }, abstract = {Genome-wide screening is a potent approach for comprehensively understanding the molecular mechanisms of biological phenomena. However, despite its widespread use in the past decades across various biological targets, its application to biochemical reactions with temporal and reversible biological outputs remains a formidable challenge. To uncover the molecular machinery underlying various biochemical reactions, we have recently developed the revival screening method, which combines flow cytometry-based cell sorting with library reconstruction from collected cells. Our refinements to the traditional genome-wide screening technique have proven successful in revealing the molecular machinery of biochemical reactions of interest. In this article, we elucidate the technical basis of revival screening, focusing on its application to CRISPR-Cas9 single guide RNA (sgRNA) library screening. Finally, we also discuss the future of genome-wide screening while describing recent achievements from in vitro and in vivo screening.}, }
@article {pmid39181868, year = {2024}, author = {Mittal, P and Myers, JA and Carter, RD and Radko-Juettner, S and Malone, HA and Rosikiewicz, W and Robertson, AN and Zhu, Z and Narayanan, IV and Hansen, BS and Parrish, M and Bhanu, NV and Mobley, RJ and Rehg, JE and Xu, B and Drosos, Y and Pruett-Miller, SM and Ljungman, M and Garcia, BA and Wu, G and Partridge, JF and Roberts, CWM}, title = {PHF6 cooperates with SWI/SNF complexes to facilitate transcriptional progression.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7303}, pmid = {39181868}, issn = {2041-1723}, support = {R01 CA113794/CA/NCI NIH HHS/United States ; R01 CA172152/CA/NCI NIH HHS/United States ; R01 CA273455/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *SMARCB1 Protein/metabolism/genetics ; *Rhabdoid Tumor/genetics/metabolism/pathology ; *Micrognathism/genetics/metabolism ; Cell Line, Tumor ; *Transcription Factors/metabolism/genetics ; *Repressor Proteins/metabolism/genetics ; *Promoter Regions, Genetic/genetics ; Face/abnormalities ; Chromatin/metabolism ; Intellectual Disability/genetics/metabolism ; Chromatin Assembly and Disassembly ; Mutation ; Foot Deformities, Congenital/genetics/metabolism ; Chromosomal Proteins, Non-Histone/metabolism/genetics ; CRISPR-Cas Systems ; Transcription, Genetic ; Animals ; Mice ; Abnormalities, Multiple ; Hand Deformities, Congenital ; Neck/abnormalities ; }, abstract = {Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are mutated in nearly 25% of cancers. To gain insight into the mechanisms by which SWI/SNF mutations drive cancer, we contributed ten rhabdoid tumor (RT) cell lines mutant for SWI/SNF subunit SMARCB1 to a genome-scale CRISPR-Cas9 depletion screen performed across 896 cell lines. We identify PHF6 as specifically essential for RT cell survival and demonstrate that dependency on Phf6 extends to Smarcb1-deficient cancers in vivo. As mutations in either SWI/SNF or PHF6 can cause the neurodevelopmental disorder Coffin-Siris syndrome, our findings of a dependency suggest a previously unrecognized functional link. We demonstrate that PHF6 co-localizes with SWI/SNF complexes at promoters, where it is essential for maintenance of an active chromatin state. We show that in the absence of SMARCB1, PHF6 loss disrupts the recruitment and stability of residual SWI/SNF complex members, collectively resulting in the loss of active chromatin at promoters and stalling of RNA Polymerase II progression. Our work establishes a mechanistic basis for the shared syndromic features of SWI/SNF and PHF6 mutations in CSS and the basis for selective dependency on PHF6 in SMARCB1-mutant cancers.}, }
@article {pmid39180405, year = {2024}, author = {Kim, DY and Lee, SY and Ha, HJ and Park, HH}, title = {Structural basis of Cas3 activation in type I-C CRISPR-Cas system.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae723}, pmid = {39180405}, issn = {1362-4962}, support = {NRF-2021R1A2C3003331//National Research Foundation of Korea/ ; }, abstract = {CRISPR-Cas systems function as adaptive immune mechanisms in bacteria and archaea and offer protection against phages and other mobile genetic elements. Among many types of CRISPR-Cas systems, Type I CRISPR-Cas systems are most abundant, with target interference depending on a multi-subunit, RNA-guided complex known as Cascade that recruits a transacting helicase nuclease, Cas3, to degrade the target. While structural studies on several other types of Cas3 have been conducted long ago, it was only recently that the structural study of Type I-C Cas3 in complex with Cascade was revealed, shedding light on how Cas3 achieve its activity in the Cascade complex. In the present study, we elucidated the first structure of standalone Type I-C Cas3 from Neisseria lactamica (NlaCas3). Structural analysis revealed that the histidine-aspartate (HD) nuclease active site of NlaCas3 was bound to two Fe2+ ions that inhibited its activity. Moreover, NlaCas3 could cleave both single-stranded and double-stranded DNA in the presence of Ni2+ or Co2+, showing the highest activity in the presence of both Ni2+ and Mg2+ ions. By comparing the structural studies of various Cas3 proteins, we determined that our NlaCas3 stays in an inactive conformation, allowing us to understand the structural changes associated with its activation and their implication.}, }
@article {pmid39067682, year = {2024}, author = {Yu, J and Ji, S and Tao, H and Shan, X and Yan, Y and Sun, X and Tu, X and Li, L and Deng, C}, title = {Cell-specific expression of Cre recombinase in rat noradrenergic neurons via CRISPR-Cas9 system.}, journal = {Neuroscience}, volume = {556}, number = {}, pages = {31-41}, doi = {10.1016/j.neuroscience.2024.07.032}, pmid = {39067682}, issn = {1873-7544}, mesh = {Animals ; *Integrases/genetics/metabolism ; *Adrenergic Neurons/metabolism ; *CRISPR-Cas Systems ; *Rats, Transgenic ; Rats ; *Dopamine beta-Hydroxylase/genetics/metabolism ; Locus Coeruleus/metabolism ; Male ; Rats, Sprague-Dawley ; }, abstract = {Noradrenergic neurons play a crucial role in the functioning of the nervous system. They formed compact small clusters in the central nervous system. To target noradrenergic neurons in combination with viral tracing and achieve cell-type specific functional manipulation using chemogenetic or optogenetic tools, new transgenic animal lines are needed, especially rat models for their advantages in large body size with facilitating easy operation, physiological parameter monitoring, and accommodating complex behavioral and cognitive studies. In this study, we successfully generated a transgenic rat strain capable of expressing Cre recombinase under the control of the dopamine beta-hydroxylase (DBH) gene promoter using the CRISPR-Cas9 system. Our validation process included co-immunostaining with Cre and DBH antibodies, confirming the specific expression of Cre recombinase. Furthermore, stereotaxic injection of a fluorescence-labeled AAV-DIO virus illustrated the precise Cre-loxP-mediated recombination activity in noradrenergic neurons within the locus coeruleus (LC). Through crossbreeding with the LSL-fluorescence reporter rat line, DBH-Cre rats proved instrumental in delineating the position and structure of noradrenergic neuron clusters A1, A2, A6 (LC), and A7 in rats. Additionally, our specific activation of the LC noradrenergic neurons showed effective behavioral readout using chemogenetics of this rat line. Our results underscore the effectiveness and specificity of Cre recombinase in noradrenergic neurons, serving as a robust tool for cell-type specific targeting of small-sized noradrenergic nuclei. This approach enhances our understanding of their anatomical, physiological, and pathological roles, contributing to a more profound comprehension of noradrenergic neuron function in the nervous system.}, }
@article {pmid39029255, year = {2024}, author = {Yu, Z and Shao, Y and Shi, D and Dong, Y and Zhang, Y and Cheng, F and Wang, Z and Tu, J and Qi, K and Song, X}, title = {A rapid, ultrasensitive, and highly specific method for detecting fowl adenovirus serotype 4 based on the LAMP-CRISPR/Cas12a system.}, journal = {Poultry science}, volume = {103}, number = {9}, pages = {104048}, pmid = {39029255}, issn = {1525-3171}, mesh = {*Adenoviridae Infections/veterinary/virology/diagnosis ; *Poultry Diseases/virology/diagnosis ; Animals ; *Chickens ; *Aviadenovirus/genetics/classification/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; Serogroup ; Molecular Diagnostic Techniques/veterinary/methods ; }, abstract = {Fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium hepatitis syndrome in chickens, which causes severe economic impact to the poultry industry. A simple, swift and reliable detection is crucial for timely identification of FAdV-4 infection, promoting effective viral prevention and control measures. Herein, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system detection platform based on loop-mediated isothermal amplification (LAMP) was studied. The CRISPR RNA (crRNA) and LAMP primers were designed and screened based on the highly conserved region of the FAdV-4 hexon gene. The parameters were then optimized individually to achieve the ideal reaction performance. The platform could lead visual detection of FAdV-4 to achieve as low as 1 copy in less than 40 min without the need for specialized instrumentation or complex equipment. Moreover, it was greatly specific, and did not cross-react with other common avian viruses. Following the validation of 30 clinical samples of suspected FAdV-4 infection, the results LAMP-CRISPR/Cas12a method generated showed fully concordance with which of the gold standard quantitative real-time PCR. To summarize, this study presented a novel, swift, expedient and inexpensive detection platform for FAdV-4, which is beneficial to viral inchoate diagnosis and point-of-care testing.}, }
@article {pmid39179961, year = {2024}, author = {Wu, X and Yang, Y and Ru, Y and Hao, R and Zhao, D and Ren, R and Lu, B and Li, Y and Sun, S and Zheng, H and Wang, W}, title = {Knockout of the WD40 domain of ATG16L1 enhances foot and mouth disease virus replication.}, journal = {BMC genomics}, volume = {25}, number = {1}, pages = {796}, pmid = {39179961}, issn = {1471-2164}, support = {2021YFD1800300//National Key Research and Development Program of China/ ; 2022SDZG02//The open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province, China/ ; 21ZD3NA001-5//Key Technologies R&amp;D Program of Gansu Province, China/ ; NCTIP-XD/C03//Project of National Center of Technology Innovation for Pigs, China/ ; }, mesh = {*Virus Replication ; *Foot-and-Mouth Disease Virus/genetics/physiology ; *Autophagy-Related Proteins/genetics/metabolism ; Animals ; *Gene Knockout Techniques ; *Autophagy/genetics ; Cell Line ; WD40 Repeats/genetics ; CRISPR-Cas Systems ; Foot-and-Mouth Disease/virology ; }, abstract = {The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus-host interactions and potential antiviral strategies.}, }
@article {pmid39179646, year = {2024}, author = {Benites-Pariente, JS and Samolski, I and Ludeña, Y and Villena, GK}, title = {CRISPR/Cas9 mediated targeted knock-in of eglA gene to improve endoglucanase activity of Aspergillus fumigatus LMB-35Aa.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {19661}, pmid = {39179646}, issn = {2045-2322}, support = {177-2015-CONCYTEC-FONDECYT-DE//Consejo Nacional de Ciencia, Tecnolog&#xed;a e Innovaci&#xf3;n Tecnol&#xf3;gica -Peru (/ ; Grant No 144-PROINNOVATE-CETF2-2022//PROINNOVATE-Ministry of Production of Peru/ ; }, mesh = {*CRISPR-Cas Systems ; *Aspergillus fumigatus/genetics/enzymology ; *Cellulase/genetics/metabolism ; *Gene Editing/methods ; *Fungal Proteins/genetics/metabolism ; Gene Knock-In Techniques ; Biofilms/growth &amp; development ; Fermentation ; }, abstract = {Bioeconomy goals for using biomass feedstock for biofuels and bio-based production has arisen the demand for fungal strains and enzymes for biomass processing. Despite well-known Trichoderma and Aspergillus commercial strains, continuous bioprospecting has revealed the fungal biodiversity potential for production of biomass degrading enzymes. The strain Aspergillus fumigatus LMB-35Aa has revealed a great potential as source of lignocellulose-degrading enzymes. Nevertheless, genetic improvement should be considered to increase its biotechnological potential. Molecular manipulation based on homologous direct recombination (HDR) in filamentous fungi poses a challenge since its low recombination rate. Currently, CRISPR/Cas9-mediated mutagenesis can enable precise and efficient editing of filamentous fungi genomes. In this study, a CRISPR/Cas9-mediated gene editing strategy for improving endoglucanase activity of A. fumigatus LMB-35Aa strain was successfully used, which constitutes the first report of heterologous cellulase production in filamentous fungi using this technology. For this, eglA gene from A. niger ATCC 10,864 was integrated into conidial melanin pksP gene locus, which facilitated the selection of edited events discerned by the emergence of albino colonies. Heterologous production of the EglA enzyme in a biofilm fermentation system resulted in a 40% improvement in endoglucanase activity of the mutant strain compared to the wild type.}, }
@article {pmid39179566, year = {2024}, author = {Guo, J and Gong, L and Yu, H and Li, M and An, Q and Liu, Z and Fan, S and Yang, C and Zhao, D and Han, J and Xiang, H}, title = {Engineered minimal type I CRISPR-Cas system for transcriptional activation and base editing in human cells.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7277}, pmid = {39179566}, issn = {2041-1723}, support = {32100499//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32150020//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32230061//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32150020//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Transcriptional Activation ; HEK293 Cells ; CRISPR-Associated Protein 9/metabolism/genetics ; Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Type I CRISPR-Cas systems are widespread and have exhibited high versatility and efficiency in genome editing and gene regulation in prokaryotes. However, due to the multi-subunit composition and large size, their application in eukaryotes has not been thoroughly investigated. Here, we demonstrate that the type I-F2 Cascade, the most compact among type I systems, with a total gene size smaller than that of SpCas9, can be developed for transcriptional activation in human cells. The efficiency of the engineered I-F2 tool can match or surpass that of dCas9. Additionally, we create a base editor using the I-F2 Cascade, which induces a considerably wide editing window (~30 nt) with a bimodal distribution. It can expand targetable sites, which is useful for disrupting functional sequences and genetic screening. This research underscores the application of compact type I systems in eukaryotes, particularly in the development of a base editor with a wide editing window.}, }
@article {pmid39179547, year = {2024}, author = {Gholami, A and Mohkam, M and Soleimanian, S and Sadraeian, M and Lauto, A}, title = {Bacterial nanotechnology as a paradigm in targeted cancer therapeutic delivery and immunotherapy.}, journal = {Microsystems &amp; nanoengineering}, volume = {10}, number = {1}, pages = {113}, pmid = {39179547}, issn = {2055-7434}, abstract = {Cancer, a multifaceted and diverse ailment, presents formidable obstacles to traditional treatment modalities. Nanotechnology presents novel prospects for surmounting these challenges through its capacity to facilitate meticulous and regulated administration of therapeutic agents to malignant cells while concurrently modulating the immune system to combat neoplasms. Bacteria and their derivatives have emerged as highly versatile and multifunctional platforms for cancer nanotherapy within the realm of nanomaterials. This comprehensive review delves into the multifaceted and groundbreaking implementations of bacterial nanotechnology within cancer therapy. This review encompasses four primary facets: the utilization of bacteria as living conveyors of medicinal substances, the employment of bacterial components as agents that stimulate the immune system, the deployment of bacterial vectors as tools for delivering genetic material, and the development of bacteria-derived nano-drugs as intelligent nano-medications. Furthermore, we elucidate the merits and modalities of operation pertaining to these bacterial nano-systems, along with their capacity to synergize with other cutting-edge nanotechnologies, such as CRISPR-Cas systems. Additionally, we offer insightful viewpoints regarding the forthcoming trajectories and prospects within this expanding domain. It is our deduction that bacterial nanotechnology embodies a propitious and innovative paradigm in the realm of cancer therapy, which has the potential to provide numerous advantages and synergistic effects in enhancing the outcomes and quality of life for individuals afflicted with cancer.}, }
@article {pmid39179542, year = {2024}, author = {Kantor, B and O'Donovan, B and Rittiner, J and Hodgson, D and Lindner, N and Guerrero, S and Dong, W and Zhang, A and Chiba-Falek, O}, title = {The therapeutic implications of all-in-one AAV-delivered epigenome-editing platform in neurodegenerative disorders.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7259}, pmid = {39179542}, issn = {2041-1723}, support = {R01 AG057522/AG/NIA NIH HHS/United States ; R41 AG077992/AG/NIA NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *Dependovirus/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Genetic Vectors/genetics ; Mice ; Neurodegenerative Diseases/genetics/therapy ; Genetic Therapy/methods ; Epigenome ; HEK293 Cells ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Epigenesis, Genetic ; Alzheimer Disease/genetics/therapy ; Apolipoproteins E/genetics ; Staphylococcus aureus/genetics ; }, abstract = {Safely and efficiently controlling gene expression is a long-standing goal of biomedical research, and CRISPR/Cas system can be harnessed to create powerful tools for epigenetic editing. Adeno-associated-viruses (AAVs) represent the delivery vehicle of choice for therapeutic platform. However, their small packaging capacity isn't suitable for large constructs including most CRISPR/dCas9-effector vectors. Thus, AAV-based CRISPR/Cas systems have been delivered via two separate viral vectors. Here we develop a compact CRISPR/dCas9-based repressor system packaged in AAV as a single optimized vector. The system comprises the small Staphylococcus aureus (Sa)dCas9 and an engineered repressor molecule, a fusion of MeCP2's transcription repression domain (TRD) and KRAB. The dSaCas9-KRAB-MeCP2(TRD) vector platform repressed robustly and sustainably the expression of multiple genes-of-interest, in vitro and in vivo, including ApoE, the strongest genetic risk factor for late onset Alzheimer's disease (LOAD). Our platform broadens the CRISPR/dCas9 toolset available for transcriptional manipulation of gene expression in research and therapeutic settings.}, }
@article {pmid39179185, year = {2024}, author = {Tharp, O and Sansbury, BM and Kmiec, EB}, title = {CRISPR-directed gene-editing induces genetic rearrangement within the human globin gene locus.}, journal = {Gene}, volume = {}, number = {}, pages = {148879}, doi = {10.1016/j.gene.2024.148879}, pmid = {39179185}, issn = {1879-0038}, abstract = {CRISPR-Cas is a revolutionary technology but has already demonstrated significant feasibility for clinical and non-clinical applications. While the efficiency and precision of this remarkable genetic tool is unprecedented, unfortunately, a series of collateral genetic rearrangement have been reported in response to double-stranded DNA breakage. Once these molecular scissions occur, the cascade of DNA repair reactions can lead to genomic rearrangements especially if breakage takes place within a family of sequence related genes. Here, we demonstrate that CRISPR- directed gene editing near the sickle cell mutation site generates a curious genetic outcome; a footprint of the δ globin gene proximal to the CRISPR/Cas cut site(s). This rearrangement is not dependent on the presence of an exogenously added DNA template but is apparently dependent on a double strand break. Our results the highlight recombinational capacity of double strand breaks in human chromosomes where the aim is to edit a human gene.}, }
@article {pmid39178725, year = {2024}, author = {Hu, X and Enbar, T and Tang, L}, title = {Delivery approaches of immunomodulatory nucleic acids for cancer therapy.}, journal = {Current opinion in biotechnology}, volume = {89}, number = {}, pages = {103182}, doi = {10.1016/j.copbio.2024.103182}, pmid = {39178725}, issn = {1879-0429}, abstract = {Messenger RNA (mRNA) vaccines have made remarkable public health contributions during the pandemic and initiated a new era for nucleic acid-based therapeutics. With the unique strength of nucleic acids, including not only mRNA but also DNA, microRNA, small interfering RNA (siRNA), and other nucleic acids, either in tuning off genes or introducing function, nucleic acid therapeutics have been regarded as potential candidates for the treatment of many different diseases, especially for the immunomodulation in cancer. However, the scope of the applications was limited by the challenges in delivery due to intrinsic properties of nucleic acids including low stability, immunogenicity, and toxicity. Bioengineering approaches toward efficient and targeted delivery of therapeutic nucleic acids have gained momentum in clinical applications in the past few decades. Recent advances in the biotechnological approaches for the delivery of mRNA, siRNA, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas for immunomodulatory are promising alternatives in designing future cancer immunotherapy.}, }
@article {pmid39176473, year = {2024}, author = {Zhang, L and Hou, L and Cai, HH and Sun, B and Han, DM and Chen, FZ}, title = {Cascading CRISPR/Cas and Nanozyme for Enhanced Organic Photoelectrochemical Transistor Detection with Triple Signal Amplification.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.4c03220}, pmid = {39176473}, issn = {1520-6882}, abstract = {Innovative signal amplification and transduction play pivotal roles in bioanalysis. Herein, cascading CRISPR/Cas and the nanozyme are integrated with electronic amplification in an organic photoelectrochemical transistor (OPECT) to enable triple signal amplification, which is exemplified by the miRNA-triggered CRISPR/Cas13a system and polyoxometalate nanozyme for OPECT detection of miRNA-21. The CRISPR/Cas13a-enabled release of glucose oxidase could synergize with peroxidase-like SiW12 to induce catalytic precipitation on the photogate, inhibiting the interfacial mass transfer and thus the significant suppression of the channel current. The as-developed OPECT sensor demonstrates good sensitivity and selectivity for miRNA-21 detection, with a linear range from 1 fM to 10 nM and an ultralow detection limit of 0.53 fM. This study features the integration of bio- and nanoenzyme cascade and electronic triple signal amplification for OPECT detection.}, }
@article {pmid39121652, year = {2024}, author = {Schuster, J and Fatima, A and Papadopoulos, N and de Guidi, C and Sobol, M and Dahl, N}, title = {Generation of a ZEB2 deficient human iPSC line (KICRi002A-4).}, journal = {Stem cell research}, volume = {80}, number = {}, pages = {103521}, doi = {10.1016/j.scr.2024.103521}, pmid = {39121652}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Zinc Finger E-box Binding Homeobox 2/metabolism/genetics ; Cell Line ; Cell Differentiation ; CRISPR-Cas Systems ; }, abstract = {The transcription factor ZEB2 is essential for early embryonic development. Using CRISPR/Cas9, we generated a ZEB2 deficient human iPSC cell line (KICRi002A-4), carrying a homozygous 790 bp deletion in ZEB2 that involves part of exon 5, intron 5 and part of exon 6. The deletion leads to markedly reduced levels of a truncated ZEB2 transcript. No ZEB2 protein was detected by immunopreciptation. The iPSC line expressed pluripotency markers and showed a capacity to differentiate into the three germ layers in vitro. Assessment of genomic integrity revealed a normal karyotype without detectable OFF-target editing. The iPSC line KICRi002A-4 thus offers a valuable resource to study the role of ZEB2 for the commitment and differentiation of various human cell lineages.}, }
@article {pmid39121651, year = {2024}, author = {Oh, JY and Yoo, DH and Im, YS and Kim, YO}, title = {Generation and characterization of a human iPSC line expressing EGFP-tagged CDH1, KSCBi002-A-3.}, journal = {Stem cell research}, volume = {80}, number = {}, pages = {103510}, doi = {10.1016/j.scr.2024.103510}, pmid = {39121651}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cadherins/metabolism/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; CRISPR-Cas Systems ; Antigens, CD/metabolism/genetics ; Cell Differentiation ; }, abstract = {E-cadherin, a transmembrane protein, is essential for maintaining the integrity and structure of human pluripotent stem cells (hPSCs) by facilitating strong cell-cell adhesion and communication, which is crucial for their colony formation and pluripotency. Here, we used the CRISPR/Cas9 system to introduce the enhanced green fluorescent protein (EGFP)-tagged CDH1 into the AAVS1 locus, a safe harbour site, of human induced pluripotent stem cells (hiPSCs). The engineered cell line, KSCBi002-A-3, expressed functional CDH1-EGFP fusion protein, exhibited normal cell morphology, maintained a normal karyotype, and retained pluripotent state.}, }
@article {pmid39111000, year = {2024}, author = {Lim, SW and Lee, KI and Cui, S and Fang, X and Shin, YJ and Lee, H and Lee, JY and Chung, BH and Yang, CW}, title = {Generation of green fluorescent protein reporter knock-in iPSC line at the 3'UTR region of the KLOTHO locus.}, journal = {Stem cell research}, volume = {80}, number = {}, pages = {103499}, doi = {10.1016/j.scr.2024.103499}, pmid = {39111000}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Klotho Proteins ; *3' Untranslated Regions ; *Green Fluorescent Proteins/metabolism/genetics ; *Glucuronidase/metabolism/genetics ; Cell Line ; CRISPR-Cas Systems ; Genes, Reporter ; Cell Differentiation ; Gene Knock-In Techniques/methods ; Genetic Loci ; }, abstract = {We generated a human induced pluripotent stem cell (hiPSC) line (CMCi014-A-78) expressing a GFP reporter in the 3'-UTR region of the KLOTHO locus using CRISPR/Cas9-mediated homologous recombination to screen for candidates regulating KLOTHO. The established cell line exhibits a normal karyotype, typical stem cell morphology, expression of pluripotency markers, and the ability to differentiate into the three germ layers. Consequently, this hiPSC line could serve as a valuable resource for screening KLOTHO regulators in hiPSC-derived target cells or organoids.}, }
@article {pmid39098170, year = {2024}, author = {Fatima, N and Dillen, L and Hommersom, MP and Çepni, E and Fatima, F and van Beusekom, E and Albert, S and Ali Khan, A and de Brouwer, APM and van Bokhoven, H}, title = {Generation of induced pluripotent stem cell line (UCSFi001-A-77) carrying a biallelic frameshift variant in exon 4 of SGIP1 through CRISPR/Cas9.}, journal = {Stem cell research}, volume = {80}, number = {}, pages = {103511}, doi = {10.1016/j.scr.2024.103511}, pmid = {39098170}, issn = {1876-7753}, mesh = {*Induced Pluripotent Stem Cells/metabolism ; Humans ; *CRISPR-Cas Systems ; *Frameshift Mutation ; *Exons ; Cell Line ; Carrier Proteins/genetics/metabolism ; Cell Differentiation ; }, abstract = {SGIP1 encodes a protein Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1. It is involved in the regulation of clathrin-mediated endocytosis along with having a role in energy homeostasis in neuronal systems. We generated an isogenic human induced pluripotent stem cell (iPSC) line with a biallelic frameshift variant in SGIP1. This exon has been shown to be subject to alternative splicing, leading to an isoform lacking 24 amino acids that are present in the longest SGIP isoform. The newly generated iPSC line will be helpful to dissect the differential properties of the two SGIP isoforms.}, }
@article {pmid39096898, year = {2024}, author = {Xue, N and Hong, D and Zhang, D and Wang, Q and Zhang, S and Yang, L and Chen, X and Li, Y and Han, H and Hu, C and Liu, M and Song, G and Guan, Y and Wang, L and Zhu, Y and Li, D}, title = {Engineering IscB to develop highly efficient miniature editing tools in mammalian cells and embryos.}, journal = {Molecular cell}, volume = {84}, number = {16}, pages = {3128-3140.e4}, doi = {10.1016/j.molcel.2024.07.007}, pmid = {39096898}, issn = {1097-4164}, mesh = {Animals ; *Gene Editing/methods ; Mice ; Humans ; *CRISPR-Cas Systems ; Embryo, Mammalian/metabolism ; HEK293 Cells ; Protein Engineering/methods ; }, abstract = {The IscB proteins, as the ancestors of Cas9 endonuclease, hold great promise due to their small size and potential for diverse genome editing. However, their activity in mammalian cells is unsatisfactory. By introducing three residual substitutions in IscB, we observed an average 7.5-fold increase in activity. Through fusing a sequence-non-specific DNA-binding protein domain, the eIscB-D variant achieved higher editing efficiency, with a maximum of 91.3%. Moreover, engineered ωRNA was generated with a 20% reduction in length and slightly increased efficiency. The engineered eIscB-D/eωRNA system showed an average 20.2-fold increase in activity compared with the original IscB. Furthermore, we successfully adapted eIscB-D for highly efficient cytosine and adenine base editing. Notably, eIscB-D is highly active in mouse cell lines and embryos, enabling the efficient generation of disease models through mRNA/ωRNA injection. Our study suggests that these miniature genome-editing tools have great potential for diverse applications.}, }
@article {pmid39059534, year = {2024}, author = {You, D and Xu, T and Huang, BZ and Zhu, L and Wu, F and Deng, LS and Liu, ZY and Duan, JQ and Wang, YM and Ge, LP and Liu, ZH and Sun, J and Zeng, X and Lang, LQ and Zhou, YC and Chen, DS and Lai, SY and Ai, YR and Huang, JB and Xu, ZW}, title = {Rapid, sensitive, and visual detection of swine Japanese encephalitis virus with a one-pot RPA-CRISPR/EsCas13d-based dual readout portable platform.}, journal = {International journal of biological macromolecules}, volume = {277}, number = {Pt 1}, pages = {134151}, doi = {10.1016/j.ijbiomac.2024.134151}, pmid = {39059534}, issn = {1879-0003}, mesh = {*Encephalitis Virus, Japanese/isolation &amp; purification/genetics ; Animals ; *Nucleic Acid Amplification Techniques/methods ; Encephalitis, Japanese/diagnosis/virology ; Molecular Diagnostic Techniques/methods ; Swine ; CRISPR-Cas Systems ; Sensitivity and Specificity ; RNA, Viral/genetics/analysis ; }, abstract = {Japanese encephalitis (JE), a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV), poses a serious threat to global public health. The low viremia levels typical in JEV infections make RNA detection challenging, necessitating early and rapid diagnostic methods for effective control and prevention. This study introduces a novel one-pot detection method that combines recombinant enzyme polymerase isothermal amplification (RPA) with CRISPR/EsCas13d targeting, providing visual fluorescence and lateral flow assay (LFA) results. Our portable one-pot RPA-EsCas13d platform can detect as few as two copies of JEV nucleic acid within 1 h, without cross-reactivity with other pathogens. Validation against clinical samples showed 100 % concordance with real-time PCR results, underscoring the method's simplicity, sensitivity, and specificity. This efficacy confirms the platform's suitability as a novel point-of-care testing (POCT) solution for detecting and monitoring the JE virus in clinical and vector samples, especially valuable in remote and resource-limited settings.}, }
@article {pmid39059080, year = {2024}, author = {Jiang, S and Dai, T and Li, Q and Xu, T and Zhang, W and Sun, J and Liu, H}, title = {Generation of ASCL1-mCherry knock-in reporter in human embryonic stem cell line, WAe001-A-2E, using CRISPR/Cas9-based gene targeting.}, journal = {Stem cell research}, volume = {80}, number = {}, pages = {103500}, doi = {10.1016/j.scr.2024.103500}, pmid = {39059080}, issn = {1876-7753}, mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Basic Helix-Loop-Helix Transcription Factors/metabolism/genetics ; Cell Line ; *Gene Targeting ; Gene Knock-In Techniques ; Genes, Reporter ; Cell Differentiation ; Red Fluorescent Protein ; }, abstract = {Achaete-Scute Complex Homolog 1 (ASCL1) is a key regulator in the development and function of the nervous system, particularly in the process of neuronal and neuroendocrine cell differentiation. By employing the CRISPR/Cas9 system, we successfully established an ASCL1-mCherry knock-in human embryonic stem cell (hESC) line by inserting a P2A-mCherry fragment at the ASCL1 locus. The mCherry reporter effectively demonstrated the expression level of endogenous ASCL1 during the process of inducing pulmonary neuroendocrine cells (PNECs) from hESC. This reporter cell line holds significant value as a research tool for investigating the process of lung neuroendocrine cell differentiation, conducting drug screening, and exploring the underlying mechanisms of lung diseases associated with PNECs dysfunction.}, }
@article {pmid38702575, year = {2024}, author = {Pandey, P and Vavilala, SL}, title = {From Gene Editing to Biofilm Busting: CRISPR-CAS9 Against Antibiotic Resistance-A Review.}, journal = {Cell biochemistry and biophysics}, volume = {82}, number = {2}, pages = {549-560}, pmid = {38702575}, issn = {1559-0283}, mesh = {*CRISPR-Cas Systems ; *Biofilms/drug effects ; *Gene Editing ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Microbial/genetics ; Humans ; Bacteria/genetics/drug effects ; Drug Resistance, Bacterial/genetics ; }, abstract = {In recent decades, the development of novel antimicrobials has significantly slowed due to the emergence of antimicrobial resistance (AMR), intensifying the global struggle against infectious diseases. Microbial populations worldwide rapidly develop resistance due to the widespread use of antibiotics, primarily targeting drug-resistant germs. A prominent manifestation of this resistance is the formation of biofilms, where bacteria create protective layers using signaling pathways such as quorum sensing. In response to this challenge, the CRISPR-Cas9 method has emerged as a ground-breaking strategy to counter biofilms. Initially identified as the "adaptive immune system" of bacteria, CRISPR-Cas9 has evolved into a state-of-the-art genetic engineering tool. Its exceptional precision in altering specific genes across diverse microorganisms positions it as a promising alternative for addressing antibiotic resistance by selectively modifying genes in diverse microorganisms. This comprehensive review concentrates on the historical background, discovery, developmental stages, and distinct components of CRISPR Cas9 technology. Emphasizing its role as a widely used genome engineering tool, the review explores how CRISPR Cas9 can significantly contribute to the targeted disruption of genes responsible for biofilm formation, highlighting its pivotal role in reshaping strategies to combat antibiotic resistance and mitigate the challenges posed by biofilm-associated infectious diseases.}, }
@article {pmid39176261, year = {2024}, author = {Liu, T and Liu, Q and Chen, F and Shi, Y and Maimaiti, G and Yang, Z and Zheng, S and Lu, X and Li, H and Chen, Z}, title = {An accurate and convenient method for Mycoplasma pneumoniae via one-step LAMP-CRISPR/Cas12b detection platform.}, journal = {Frontiers in cellular and infection microbiology}, volume = {14}, number = {}, pages = {1409078}, doi = {10.3389/fcimb.2024.1409078}, pmid = {39176261}, issn = {2235-2988}, mesh = {*Mycoplasma pneumoniae/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Sensitivity and Specificity ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; Child ; Limit of Detection ; }, abstract = {INTRODUCTION: Mycoplasma pneumoniae (MP) is the major cause of respiratory infections that threaten the health of children and adolescents worldwide. Therefore, an early, simple, and accurate detection approach for MP is critical to prevent outbreaks of MP-induced community-acquired pneumonia.<br><br>METHODS: Here, we explored a simple and accurate method for MP identification that combines loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12b assay in a one-pot reaction.<br><br>RESULTS: In the current study, the whole reaction was completed within 1 h at a constant temperature of 57&#xb0;C. The limit of detection of this assay was 33.7 copies per reaction. The specificity of the LAMP-CRISPR/Cas12b method was 100%, without any cross-reactivity with other pathogens. Overall, 272 clinical samples were used to evaluate the clinical performance of LAMP-CRISPR/Cas12b. Compared with the gold standard results from real-time PCR, the present method provided a sensitivity of 88.11% (126/143), specificity of 100% (129/129), and consistency of 93.75% (255/272).<br><br>DISCUSSION: Taken together, our preliminary results illustrate that the LAMP-CRISPR/Cas12b method is a simple and reliable tool for MP diagnosis that can be performed in resource-limited regions.}, }
@article {pmid39174599, year = {2024}, author = {Geng, Z and Tai, YT and Wang, Q and Gao, Z}, title = {AUTS2 disruption causes neuronal differentiation defects in human cerebral organoids through hyperactivation of the WNT/β-catenin pathway.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {19522}, pmid = {39174599}, issn = {2045-2322}, support = {R35GM133496/GM/NIGMS NIH HHS/United States ; }, mesh = {Humans ; *Organoids/metabolism ; *Wnt Signaling Pathway ; *Neurons/metabolism ; *Cell Differentiation ; *Transcription Factors/metabolism/genetics ; *Cytoskeletal Proteins/metabolism/genetics ; CRISPR-Cas Systems ; beta Catenin/metabolism/genetics ; Human Embryonic Stem Cells/metabolism ; Brain/metabolism/pathology ; }, abstract = {Individuals with the Autism Susceptibility Candidate 2 (AUTS2) gene disruptions exhibit symptoms such as intellectual disability, microcephaly, growth retardation, and distinct skeletal and facial differences. The role of AUTS2 in neurodevelopment has been investigated using animal and embryonic stem cell models. However, the precise molecular mechanisms of how AUTS2 influences neurodevelopment, particularly in humans, are not thoroughly understood. Our study employed a 3D human cerebral organoid culture system, in combination with genetic, genomic, cellular, and molecular approaches, to investigate how AUTS2 impacts neurodevelopment through cellular signaling pathways. We used CRISPR/Cas9 technology to create AUTS2-deficient human embryonic stem cells and then generated cerebral organoids with these cells. Our transcriptomic analyses revealed that the absence of AUTS2 in cerebral organoids reduces the populations of cells committed to the neuronal lineage, resulting in an overabundance of cells with a transcription profile resembling that of choroid plexus (ChP) cells. Intriguingly, we found that AUTS2 negatively regulates the WNT/β-catenin signaling pathway, evidenced by its overactivation in AUTS2-deficient cerebral organoids and in luciferase reporter cells lacking AUTS2. Importantly, treating the AUTS2-deficient cerebral organoids with a WNT inhibitor reversed the overexpression of ChP genes and increased the downregulated neuronal gene expression. This study offers new insights into the role of AUTS2 in neurodevelopment and suggests potential targeted therapies for neurodevelopmental disorders.}, }
@article {pmid39078660, year = {2024}, author = {Gao, Y and Ang, YS and Yung, LL}, title = {One-Pot Detection of Proteins Using a Two-Way Extension-Based Assay with Cas12a.}, journal = {ACS sensors}, volume = {9}, number = {8}, pages = {3928-3937}, doi = {10.1021/acssensors.4c00370}, pmid = {39078660}, issn = {2379-3694}, mesh = {*CRISPR-Cas Systems ; Immunoassay/methods ; Humans ; Aptamers, Nucleotide/chemistry ; CRISPR-Associated Proteins ; Nucleic Acid Amplification Techniques/methods ; Becaplermin/analysis ; Endodeoxyribonucleases/chemistry ; Bacterial Proteins/chemistry ; }, abstract = {Protein biomarkers are an important class of biomarkers in disease diagnosis and are traditionally detected by enzyme-linked immunosorbent assay and mass spectrometry, which involve multiple steps and a complex workflow. In recent years, many CRISPR-Cas12a-based methods for protein detection have been developed; however, most of them have not overcome the workflow complications observed in traditional assays, limiting their applicability in point-of-care testing. In this work, we designed a single-step, one-pot, and proximity-based isothermal immunoassay integrating CRISPR Cas12a for homogeneous protein target detection with a simplified workflow and high sensitivity. Probes consisting of different binders (small molecule, aptamer, and antibody) conjugated with oligonucleotides undergo two-way extension upon binding to the protein targets, leading to downstream DNA amplification by a pair of nicking enzymes and polymerases to generate target sequences for Cas12a signal generation. We used the streptavidin-biotin model to demonstrate the design of our assay and proved that all three elements of protein detection (target protein binding, DNA amplification, and Cas12a signal generation) could coexist in one pot and proceed isothermally in a single buffer system at a low reaction volume of 10 μL. The plug-and-play applicability of our assay has been successfully demonstrated using four different protein targets, streptavidin, PDGF-BB, antidigoxigenin antibody, and IFNγ, with the limit of detection ranging from fM to pM.}, }
@article {pmid39013507, year = {2024}, author = {Li, P and Wei, Y and Shi, J and Wu, J and Wu, Y and Yan, J and Liu, S and Tan, X and Huang, KJ}, title = {CRISPR/Cas12a-triggered ordered concatemeric DNA probes signal-on/off multifunctional analytical sensing system for ultrasensitive detection of thalassemia.}, journal = {International journal of biological macromolecules}, volume = {276}, number = {Pt 1}, pages = {133884}, doi = {10.1016/j.ijbiomac.2024.133884}, pmid = {39013507}, issn = {1879-0003}, mesh = {Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *DNA Probes/chemistry/genetics ; *Thalassemia/diagnosis/genetics ; Glucose Oxidase/chemistry/metabolism ; Electrochemical Techniques/methods ; Limit of Detection ; Electrodes ; }, abstract = {Based on CRISPR/Cas12a triggered ordered concatemeric DNA probes, a "on/off" self-powered biosensor is developed to achieve highly sensitive detection of thalassemia gene CD142 through open-circuit potential-assisted visual signal output. The ingeniously constructed glucose oxidase (GOD)-functionalized ordered concatemeric DNA probe structure can significantly amplify signal output, while the coupled CRISPR/Cas12a system is served as a "signal switch" with excellent signal-transducing capabilities. When the ordered concatemeric DNA probe structure is anchored on electrode, the response signal of the sensing system is in the "signal on" mode. While, the presence of the target activates the non-specific cleavage activity of the CRISPR/Cas12a system, causing the sensing system to switch to the "signal off" mode. In the detection system, GOD catalyzes the oxidation of glucose to produce hydrogen peroxide, which further catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to form a color product, enabling visual signal of the target through naked-eye color contrast. By employing a multifunctional analytical mode combining electrochemical and visual signal outputs, accurate determination of the target is achieved, with linear ranges of 0.0001-100 pM, and detection limits of 48.1 aM (S/N = 3). This work provides a reference method for sensitive detection of thalassemia genes and holds great diagnostic potential in biomedical applications.}, }
@article {pmid39173952, year = {2024}, author = {Jung, H and Inaba, Y and Banta, S}, title = {CRISPR/dCas12a knock-down of Acidithiobacillus ferrooxidans electron transport chain bc1 complexes enables enhanced metal sulfide bioleaching.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {107703}, doi = {10.1016/j.jbc.2024.107703}, pmid = {39173952}, issn = {1083-351X}, abstract = {Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that plays an important role in biogeochemical iron and sulfur cycling and is a member of the consortia used in industrial hydrometallurgical processing of copper. Metal sulfide bioleaching is catalyzed by the regeneration of ferric iron, however, bioleaching of chalcopyrite, the dominant unmined form of copper on Earth, is inhibited by surface passivation. Here, we report the implementation of CRISPR interference (CRISPRi) using the catalytically inactive Cas12a (dCas12a) in A. ferrooxidans to knockdown the expression of genes in the petI and petII operons. These operons encode bc1 complex proteins and knockdown of these genes enabled the manipulation (enhancement or repression) of iron oxidation. The petB2 gene knockdown strain enhanced iron oxidation, leading to enhanced pyrite and chalcopyrite oxidation, which correlated with reduced biofilm formation and decreased surface passivation of the minerals. These findings highlight the utility of CRISPRi/dCas12a technology for engineering A. ferrooxidans while unveiling a new strategy to manipulate and improve bioleaching efficiency.}, }
@article {pmid39174527, year = {2024}, author = {Chen, R and Shi, X and Yao, X and Gao, T and Huang, G and Ning, D and Cao, Z and Xu, Y and Liang, W and Tian, SZ and Zhu, Q and Fang, L and Zheng, M and Hu, Y and Cui, H and Chen, W}, title = {Specific multivalent molecules boost CRISPR-mediated transcriptional activation.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7222}, pmid = {39174527}, issn = {2041-1723}, mesh = {*CRISPR-Cas Systems ; *Transcriptional Activation ; Humans ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; Binding Sites ; Chromatin/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Enhancer Elements, Genetic ; }, abstract = {CRISPR/Cas-based transcriptional activators can be enhanced by intrinsically disordered regions (IDRs). However, the underlying mechanisms are still debatable. Here, we examine 12 well-known IDRs by fusing them to the dCas9-VP64 activator, of which only seven can augment activation, albeit independently of their phase separation capabilities. Moreover, modular domains (MDs), another class of multivalent molecules, though ineffective in enhancing dCas9-VP64 activity on their own, show substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and fusing dCas9-VP64 with different IDRs/MDs, we uncover that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs yields synergistic effects, which can be further amplified via enhancing chromatin interactions. Overall, our study develops a versatile platform for efficient gene activation and sheds important insights into CRIPSR-based transcriptional activators enhanced with multivalent molecules.}, }
@article {pmid39173188, year = {2024}, author = {Zhang, L and Wang, H and Yang, S and Liu, J and Li, J and Lu, Y and Cheng, J and Xu, Y}, title = {High-Throughput and Integrated CRISPR/Cas12a-Based Molecular Diagnosis Using a Deep Learning Enabled Microfluidic System.}, journal = {ACS nano}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsnano.4c05734}, pmid = {39173188}, issn = {1936-086X}, abstract = {CRISPR/Cas-based molecular diagnosis demonstrates potent potential for sensitive and rapid pathogen detection, notably in SARS-CoV-2 diagnosis and mutation tracking. Yet, a major hurdle hindering widespread practical use is its restricted throughput, limited integration, and complex reagent preparation. Here, a system, microfluidic multiplate-based ultrahigh throughput analysis of SARS-CoV-2 variants of concern using CRISPR/Cas12a and nonextraction RT-LAMP (mutaSCAN), is proposed for rapid detection of SARS-CoV-2 and its variants with limited resource requirements. With the aid of the self-developed reagents and deep-learning enabled prototype device, our mutaSCAN system can detect SARS-CoV-2 in mock swab samples below 30 min as low as 250 copies/mL with the throughput up to 96 per round. Clinical specimens were tested with this system, the accuracy for routine and mutation testing (22 wildtype samples, 26 mutational samples) was 98% and 100%, respectively. No false-positive results were found for negative (n = 24) samples.}, }
@article {pmid39173055, year = {2024}, author = {Gong, M and Peng, C and Yang, C and Wang, Z and Qian, H and Hu, X and Zhou, P and Shan, C and Ding, Q}, title = {Genome-wide CRISPR/Cas9 screen identifies SLC39A9 and PIK3C3 as crucial entry factors for Ebola virus infection.}, journal = {PLoS pathogens}, volume = {20}, number = {8}, pages = {e1012444}, doi = {10.1371/journal.ppat.1012444}, pmid = {39173055}, issn = {1553-7374}, mesh = {*Virus Internalization ; *Ebolavirus/genetics/physiology/metabolism ; Humans ; *CRISPR-Cas Systems ; *Hemorrhagic Fever, Ebola/virology/metabolism/genetics ; Cation Transport Proteins/metabolism/genetics ; Class III Phosphatidylinositol 3-Kinases/metabolism/genetics ; Virus Replication ; Animals ; HEK293 Cells ; }, abstract = {The Ebola virus (EBOV) has emerged as a significant global health concern, notably during the 2013-2016 outbreak in West Africa. Despite the clinical approval of two EBOV antibody drugs, there is an urgent need for more diverse and effective antiviral drugs, along with comprehensive understanding of viral-host interactions. In this study, we harnessed a biologically contained EBOVΔVP30-EGFP cell culture model which could recapitulate the entire viral life cycle, to conduct a genome-wide CRISPR/Cas9 screen. Through this, we identified PIK3C3 (phosphatidylinositide 3-kinase) and SLC39A9 (zinc transporter) as crucial host factors for EBOV infection. Genetic depletion of SLC39A9 and PIK3C3 lead to reduction of EBOV entry, but not impact viral genome replication, suggesting that SLC39A9 and PIK3C3 act as entry factors, facilitating viral entry into host cells. Moreover, PIK3C3 kinase activity is indispensable for the internalization of EBOV virions, presumably through the regulation of endocytic and autophagic membrane traffic, which has been previously recognized as essential for EBOV internalization. Notably, our study demonstrated that PIK3C3 kinase inhibitor could effectively block EBOV infection, underscoring PIK3C3 as a promising drug target. Furthermore, biochemical analysis showed that recombinant SLC39A9 protein could directly bind viral GP protein, which further promotes the interaction of viral GP protein with cellular receptor NPC1. These findings suggests that SLC39A9 plays dual roles in EBOV entry. Initially, it serves as an attachment factor during the early entry phase by engaging with the viral GP protein. Subsequently, SLC39A9 functions an adaptor protein, facilitating the interaction between virions and the NPC1 receptor during the late entry phase, prior to cathepsin cleavage on the viral GP. In summary, this study offers novel insights into virus-host interactions, contributing valuable information for the development of new therapies against EBOV infection.}, }
@article {pmid39172826, year = {2024}, author = {Aguirre Rivera, J and Mao, G and Sabantsev, A and Panfilov, M and Hou, Q and Lindell, M and Chanez, C and Ritort, F and Jinek, M and Deindl, S}, title = {Massively parallel analysis of single-molecule dynamics on next-generation sequencing chips.}, journal = {Science (New York, N.Y.)}, volume = {385}, number = {6711}, pages = {892-898}, doi = {10.1126/science.adn5371}, pmid = {39172826}, issn = {1095-9203}, mesh = {*High-Throughput Nucleotide Sequencing/methods ; *Single Molecule Imaging/methods ; *DNA/chemistry/genetics ; *Microscopy, Fluorescence/methods ; CRISPR-Associated Protein 9 ; Sequence Analysis, DNA/methods ; Gene Library ; CRISPR-Cas Systems ; }, abstract = {Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. Here we describe multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics. We comprehensively profiled the sequence dependence of DNA hairpin properties and Cas9-induced target DNA unwinding-rewinding dynamics. The ability to explore a large sequence space for Cas9 allowed us to identify a number of target sequences with unexpected behaviors. We envision that MUSCLE will enable the mechanistic exploration of many fundamental biological processes.}, }
@article {pmid39172289, year = {2024}, author = {Wang, L and Cui, J and Zhang, N and Wang, X and Su, J and Vallés, MP and Wu, S and Yao, W and Chen, X and Chen, D}, title = {OsIPK1 frameshift mutations disturb phosphorus homeostasis and impair starch synthesis during grain filling in rice.}, journal = {Plant molecular biology}, volume = {114}, number = {5}, pages = {91}, pmid = {39172289}, issn = {1573-5028}, support = {21JCYBJC00010//Natural Science Foundation of Tianjin/ ; 60//Fundamental Research Funds for the Central Universities, Nankai University/ ; 32070349//National Natural Science Foundation of China/ ; ITTRRS20211000-02//Rice Industry Technological System of Tianjin/ ; }, mesh = {*Frameshift Mutation ; *Oryza/genetics/metabolism/growth &amp; development ; *Starch/biosynthesis/metabolism ; *Phosphorus/metabolism ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; *Homeostasis ; Seeds/genetics/metabolism/growth &amp; development ; CRISPR-Cas Systems ; Edible Grain/genetics/metabolism ; Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism ; Plants, Genetically Modified ; Phytic Acid/metabolism/biosynthesis ; }, abstract = {Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) catalyzes the final step in phytic acid (InsP6) synthesis. In this study, the effects of OsIPK1 mutations on InsP6 synthesis, grain filling and their underlying mechanisms were investigated. Seven gRNAs were designed to disrupt the OsIPK1 gene via CRISPR/CAS9 system. Only 4 of them generated 29 individual insertion or deletion T0 plants, in which nine biallelic or heterozygous genotypes were identified. Segregation analysis revealed that OsIPK1 frameshift mutants are homozygous lethality. The biallelic and heterozygous frameshift mutants exhibited significant reduction in yield-related traits, particularly in the seed-setting rate and yield per plant. Despite a notable decline in pollen viability, the male and female gametes had comparable transmission rates to their progenies in the mutants. A significant number of the filling-aborted (FA) grains was observed in mature grains of these heterozygous frameshift mutants. These grains exhibited a nearly complete blockage of InsP6 synthesis, resulting in a pronounced increase in Pi content. In contrast, a slight decline in InsP6 content was observed in the plump grains. During the filling stage, owing to the excessive accumulation of Pi, starch synthesis was significantly impaired, and the endosperm development-specific gene expression was nearly abolished. Consistently, the activity of whereas AGPase, a key enzyme in starch synthesis, was significantly decreased and Pi transporter gene expression was upregulated in the FA grains. Taken together, these results demonstrate that OsIPK1 frameshift mutations result in excessive Pi accumulation, decreased starch synthesis, and ultimately leading to lower yields in rice.}, }
@article {pmid39171866, year = {2024}, author = {Alalmaie, A and Khashan, R}, title = {Mechanistic Insight Into the Conformational Changes of Cas8 Upon Binding to Different PAM Sequences in the Transposon-Encoded Type I-F CRISPR-Cas System.}, journal = {Proteins}, volume = {}, number = {}, pages = {}, doi = {10.1002/prot.26730}, pmid = {39171866}, issn = {1097-0134}, abstract = {The INTEGRATE system is a gene-editing approach that offers advantages over the widely used CRISPR-Cas9 system. It does not introduce double strand breaks in the target DNA but rather integrates the desired DNA sequence directly into it. The first step in the integration process is PAM recognition, which is critical to understanding and optimizing the system. Experimental testing revealed varying integration efficiencies of different PAM mutants, and computational simulations were carried out to gain mechanistic insight into the conformational changes of Cas8 during PAM recognition. Our results showed that the interaction between Arg246 and guanine at position (-1) of the target strand is critical for PAM recognition. We found that unfavorable interactions in the 5'-AC-3' PAM mutant disrupted this interaction and may be responsible for its 0% integration efficiency. Additionally, we discovered that PAM sequences not only initiate the integration process but also regulate it through an allosteric mechanism that connects the N-terminal domain and the helical bundle of Cas8. This allosteric regulation was present in all PAMs tested, even those with lower integration efficiencies, such as 5'-TC-3' and 5'-AC-3'. We identified the Cas8 residues that are involved in this regulation. Our findings provide valuable insights into PAM recognition mechanisms in the INTEGRATE system and can help improve the gene-editing technology.}, }
@article {pmid39168259, year = {2024}, author = {Zanganeh, S and Zahedi, AM and Bardsiri, MS and Bazi, A and Bastanifard, M and Shool, S and Kouhbananinejad, SM and Farsinejad, A and Afgar, A and Shahabi, A and Mirzaei-Parsa, MJ}, title = {Recent advances and applications of the CRISPR-Cas system in the gene therapy of blood disorders.}, journal = {Gene}, volume = {}, number = {}, pages = {148865}, doi = {10.1016/j.gene.2024.148865}, pmid = {39168259}, issn = {1879-0038}, }
@article {pmid39089555, year = {2024}, author = {Chen, X and Moran Torres, JP and Tedjai, SVK and Lugones, LG and Wösten, HAB}, title = {Functional analysis of FlbA-regulated transcription factor genes in Aspergillus niger using a multiplexed CRISPRoff system.}, journal = {International journal of biological macromolecules}, volume = {277}, number = {Pt 2}, pages = {134326}, doi = {10.1016/j.ijbiomac.2024.134326}, pmid = {39089555}, issn = {1879-0003}, mesh = {*Aspergillus niger/genetics ; *Transcription Factors/genetics/metabolism ; *Gene Expression Regulation, Fungal ; *Fungal Proteins/genetics ; CRISPR-Cas Systems/genetics ; Spores, Fungal/genetics ; Phenotype ; Promoter Regions, Genetic/genetics ; }, abstract = {FlbA of Aspergillus niger (indirectly) regulates 36 transcription factor (TF) genes. As a result, it promotes sporulation and represses vegetative growth, protein secretion and lysis. In this study, the functions of part of the FlbA-regulated TF genes were studied by using CRISPRoff. This system was recently introduced as an epigenetic tool for modulating gene expression in A. niger. A plasmid encompassing an optimized CRISPRoff system as well as a library of sgRNA genes that target the promoters of the 36 FlbA-regulated TF genes was introduced in A. niger. Out of 24 transformants that exhibited a sporulation phenotype, 12 and 18 strains also showed a biomass and secretion phenotype, respectively. The transforming sgRNAs, and thus the genes responsible for the phenotypes, were identified from five of the transformants. The results show that the genes dofA, dofB, dofC, dofD, and socA are involved in sporulation and extracellular enzyme activity, while dofA and socA also play roles in biomass formation. Overall, this study shows that the multiplexed CRISPRoff system can be effectively used for functional analysis of genes in a fungus.}, }
@article {pmid39019219, year = {2024}, author = {Yun, S and Noh, M and Yu, J and Kim, HJ and Hui, CC and Lee, H and Son, JE}, title = {Unlocking biological mechanisms with integrative functional genomics approaches.}, journal = {Molecules and cells}, volume = {47}, number = {8}, pages = {100092}, doi = {10.1016/j.mocell.2024.100092}, pmid = {39019219}, issn = {0219-1032}, mesh = {Humans ; *Genomics/methods ; CRISPR-Cas Systems ; Epigenesis, Genetic ; Chromatin/metabolism/genetics ; High-Throughput Nucleotide Sequencing/methods ; }, abstract = {Reverse genetics offers precise functional insights into genes through the targeted manipulation of gene expression followed by phenotypic assessment. While these approaches have proven effective in model organisms such as Saccharomyces cerevisiae, large-scale genetic manipulations in human cells were historically unfeasible due to methodological limitations. However, recent advancements in functional genomics, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-based screening technologies and next-generation sequencing platforms, have enabled pooled screening technologies that allow massively parallel, unbiased assessments of biological phenomena in human cells. This review provides a comprehensive overview of cutting-edge functional genomic screening technologies applicable to human cells, ranging from short hairpin RNA screens to modern CRISPR screens. Additionally, we explore the integration of CRISPR platforms with single-cell approaches to monitor gene expression, chromatin accessibility, epigenetic regulation, and chromatin architecture following genetic perturbations at the omics level. By offering an in-depth understanding of these genomic screening methods, this review aims to provide insights into more targeted and effective strategies for genomic research and personalized medicine.}, }
@article {pmid38878231, year = {2024}, author = {Dalgleish, R}, title = {Re: Identification of a family with van der Hoeve's syndrome harboring a novel COL1A1 mutation and generation of patient-derived iPSC lines and CRISPR/Cas9-corrected isogenic iPSCs.}, journal = {Human cell}, volume = {37}, number = {5}, pages = {1610-1611}, pmid = {38878231}, issn = {1749-0774}, mesh = {Humans ; *Induced Pluripotent Stem Cells ; *CRISPR-Cas Systems/genetics ; *Mutation/genetics ; *Collagen Type I, alpha 1 Chain ; *Collagen Type I/genetics/metabolism ; Cell Line ; }, }
@article {pmid39167979, year = {2024}, author = {Lei, H and Xiong, W and Li, M and Qi, Q and Liu, X and Wang, S and Tian, T and Zhou, X}, title = {Enhanced control of RNA modification and CRISPR-Cas activity through redox-triggered disulfide cleavage.}, journal = {Bioorganic &amp; medicinal chemistry}, volume = {112}, number = {}, pages = {117878}, doi = {10.1016/j.bmc.2024.117878}, pmid = {39167979}, issn = {1464-3391}, abstract = {Chemical RNA modification has emerged as a flexible approach for post-synthetic modifications in chemical biology research. Guide RNA (gRNA) plays a crucial role in the clustered regularly interspaced short palindromic repeats and associated protein system (CRISPR-Cas). Several toolkits have been developed to regulate gene expression and editing through modifications of gRNA. However, conditional regulation strategies to control gene editing in cells as required are still lacking. In this context, we introduce a strategy employing a cyclic disulfide-substituted acylating agent to randomly acylate the 2'-OH group on the gRNA strand. The CRISPR-Cas systems demonstrate off-on transformation activity driven by redox-triggered disulfide cleavage and undergo intramolecular cyclization, which releases the functionalized gRNA. Dithiothreitol (DTT) exhibits superior reductive capabilities in cleaving disulfides compared to glutathione (GSH), requiring fewer reductants. This acylation method with cyclic disulfides enables conditional control of CRISPR-Cas9, CRISPR-Cas13a, RNA hybridization, and aptamer folding. Our strategy facilitates precise in vivo control of gene editing, making it particularly valuable for targeted applications.}, }
@article {pmid39166823, year = {2024}, author = {He, Y and Gao, M and Zhu, X and Peng, W and Zhou, Y and Cheng, J and Bai, L and Bao, J}, title = {Large-Scale Formation and Long-Term Culture of Hepatocyte Organoids From Streamlined In Vivo Genome-Edited GGTA1[-/-] Pigs for Bioartificial Liver Applications.}, journal = {Xenotransplantation}, volume = {31}, number = {4}, pages = {e12878}, doi = {10.1111/xen.12878}, pmid = {39166823}, issn = {1399-3089}, support = {82070640//National Natural Science Foundation of China/ ; ZYIC21014//West China Hospital Sichuan University/ ; }, mesh = {Animals ; *Hepatocytes ; *Galactosyltransferases/genetics ; Swine ; *Transplantation, Heterologous/methods ; *Organoids/metabolism ; *Liver, Artificial ; *Gene Editing/methods ; Humans ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Gene Knockout Techniques/methods ; Coculture Techniques/methods ; }, abstract = {Hepatocyte transplantation and bioartificial liver (BAL) systems hold significant promise as less invasive alternatives to traditional transplantation, providing crucial temporary support for patients with acute and chronic liver failure. Although human hepatocytes are ideal, their use is limited by ethical concerns and donor availability, leading to the use of porcine hepatocytes in BAL systems due to their functional similarities. Recent advancements in gene-editing technology have improved porcine organ xenotransplantation clinical trials by addressing immune rejection issues. Gene-edited pigs, such as alpha-1,3-galactosyltransferase (GGTA1) knockout pigs, offer a secure source of primary cells for BAL systems. Our research focuses on optimizing the safety and functionality of porcine primary hepatocytes during large-scale cultivation. We achieved this by creating GGTA1 knockout pigs through one-step delivery of CRISPR/Cas9 to pig zygotes via oviduct injection of rAAV, and enhancing hepatocyte viability and function by co-culturing hepatocytes with Roof plate-specific spondin 1 overexpressing HUVECs (R-HUVECs). Using a Rocker culture system, approximately 10[10] primary porcine hepatocytes and R-HUVECs rapidly formed organoids with a diameter of 92.1 ± 28.1 µm within 24 h. These organoids not only maintained excellent functionality but also supported partial hepatocyte self-renewal during long-term culture over 28 days. Gene-edited primary porcine hepatocyte organoids will significantly advance the applications of hepatocyte transplantation and BAL systems.}, }
@article {pmid39166476, year = {2024}, author = {Binder, SC and Schneberger, N and Schmitz, M and Engeser, M and Geyer, M and Rouillon, C and Hagelueken, G}, title = {The SAVED domain of the type III CRISPR protease CalpL is a ring nuclease.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae676}, pmid = {39166476}, issn = {1362-4962}, support = {HA-6805/6-1//German Research Foundation/ ; EXC2151-390873048//Germany's Excellence Strategy/ ; //University of Bonn/ ; }, abstract = {Prokaryotic CRISPR-Cas immune systems detect and cleave foreign nucleic acids. In type III CRISPR-Cas systems, the Cas10 subunit of the activated recognition complex synthesizes cyclic oligoadenylates (cOAs), second messengers that activate downstream ancillary effector proteins. Once the viral attack has been weathered, elimination of extant cOA is essential to limit the antiviral response and to allow cellular recovery. Various families of ring nucleases have been identified, specializing in the degradation of cOAs either as standalone enzymes or as domains of effector proteins. Here we describe the ring nuclease activity inherent in the SAVED domain of the cA4-activated CRISPR Lon protease CalpL. We characterize the kinetics of cA4 cleavage and identify key catalytic residues. We demonstrate that cA4-induced oligomerization of CalpL is essential not only for activation of the protease, but is also required for nuclease activity. Further, the nuclease activity of CalpL poses a limitation to the protease reaction, indicating a mechanism for regulation of the CalpL/T/S signaling cascade. This work is the first demonstration of a catalytic SAVED domain and gives new insights into the dynamics of transcriptional adaption in CRISPR defense systems.}, }
@article {pmid39166136, year = {2024}, author = {Gholami, A and Mohkam, M and Soleimanian, S and Sadraeian, M and Lauto, A}, title = {Bacterial nanotechnology as a paradigm in targeted cancer therapeutic delivery and immunotherapy.}, journal = {Microsystems &amp; nanoengineering}, volume = {10}, number = {}, pages = {113}, pmid = {39166136}, issn = {2055-7434}, abstract = {Cancer, a multifaceted and diverse ailment, presents formidable obstacles to traditional treatment modalities. Nanotechnology presents novel prospects for surmounting these challenges through its capacity to facilitate meticulous and regulated administration of therapeutic agents to malignant cells while concurrently modulating the immune system to combat neoplasms. Bacteria and their derivatives have emerged as highly versatile and multifunctional platforms for cancer nanotherapy within the realm of nanomaterials. This comprehensive review delves into the multifaceted and groundbreaking implementations of bacterial nanotechnology within cancer therapy. This review encompasses four primary facets: the utilization of bacteria as living conveyors of medicinal substances, the employment of bacterial components as agents that stimulate the immune system, the deployment of bacterial vectors as tools for delivering genetic material, and the development of bacteria-derived nano-drugs as intelligent nano-medications. Furthermore, we elucidate the merits and modalities of operation pertaining to these bacterial nano-systems, along with their capacity to synergize with other cutting-edge nanotechnologies, such as CRISPR-Cas systems. Additionally, we offer insightful viewpoints regarding the forthcoming trajectories and prospects within this expanding domain. It is our deduction that bacterial nanotechnology embodies a propitious and innovative paradigm in the realm of cancer therapy, which has the potential to provide numerous advantages and synergistic effects in enhancing the outcomes and quality of life for individuals afflicted with cancer.}, }
@article {pmid39165749, year = {2024}, author = {Hwang, S and Lee, W and Lee, Y}, title = {Development of a nucleic acid detection method based on the CRISPR-Cas13 for point-of-care testing of bovine viral diarrhea virus-1b.}, journal = {Journal of animal science and technology}, volume = {66}, number = {4}, pages = {781-791}, pmid = {39165749}, issn = {2055-0391}, abstract = {Bovine viral diarrhea (BVD) is a single-stranded, positive-sense ribonucleic acid (RNA) virus belonging to the genus Pestivirus of the Flaviviridae family. BVD frequently causes economic losses to farmers. Among bovine viral diarrhea virus (BVDV) strains, BVDV-1b is predominant and widespread in Hanwoo calves. Reverse-transcription polymerase chain reaction (RT-PCR) is an essential method for diagnosing BVDV-1b and has become the gold standard for diagnosis in the Republic of Korea. However, this diagnostic method is time-consuming and requires expensive equipment. Therefore, Clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems have been used for point-of-care (POC) testing of viruses. Developing a sensitive and specific method for POC testing of BVDV-1b would be advantageous for controlling the spread of infection. Thus, this study aimed to develop a novel nucleic acid detection method using the CRISPR-Cas13 system for POC testing of BVDV-1b. The sequence of the BVD virus was extracted from National Center for Biotechnology Information (NC_001461.1), and the 5' untranslated region, commonly used for detection, was selected. CRISPR RNA (crRNA) was designed using the Cas13 design program and optimized for the expression and purification of the LwCas13a protein. Madin Darby bovine kidney (MDBK) cells were infected with BVDV-1b, incubated, and the viral RNA was extracted. To enable POC viral detection, the compatibility of the CRISPR-Cas13 system was verified with a paper-based strip through collateral cleavage activity. Finally, a colorimetric assay was used to evaluate the detection of BVDV-1b by combining the previously obtained crRNA and Cas13a protein on a paper strip. In conclusion, the CRISPR-Cas13 system is highly sensitive, specific, and capable of nucleic acid detection, making it an optimal system for the early point-of-care testing of BVDV-1b.}, }
@article {pmid39165171, year = {2024}, author = {Liaghat, A and Yang, J and Whitaker, R and Pascual, M}, title = {Punctuated virus-driven succession generates dynamical alternations in CRISPR-mediated microbe-virus coevolution.}, journal = {Journal of the Royal Society, Interface}, volume = {21}, number = {217}, pages = {20240195}, doi = {10.1098/rsif.2024.0195}, pmid = {39165171}, issn = {1742-5662}, support = {//Division of Biological Infrastructure/ ; //Gordon and Betty Moore Foundation/ ; }, mesh = {*CRISPR-Cas Systems ; Host-Pathogen Interactions/genetics ; Models, Biological ; Viruses/genetics ; Evolution, Molecular ; Biological Evolution ; }, abstract = {The coevolutionary dynamics of lytic viruses and microbes with CRISPR-Cas immunity exhibit alternations between sustained host control of viral proliferation and major viral epidemics in previous computational models. These alternating dynamics have yet to be observed in other host-pathogen systems. Here, we address the breakdown of control and transition to large outbreaks with a stochastic eco-evolutionary model. We establish the role of host density-dependent competition in punctuated virus-driven succession and associated diversity trends that concentrate escape pathways during control phases. Using infection and escape networks, we derive the viral emergence probability whose fluctuations of increasing size and frequency characterize the approach to large outbreaks. We explore alternation probabilities as a function of non-dimensional parameters related to the probability of viral escape and host competition. Our results demonstrate how emergent feedbacks between host competition and viral diversification render the host immune structure fragile, potentiating a dynamical transition to large epidemics.}, }
@article {pmid39163906, year = {2024}, author = {Sabrina Freitas-Alves, N and Moreira-Pinto, CE and Távora, FTPK and Paes-de-Melo, B and Arraes, FBM and Lourenço-Tessutti, IT and Moura, SM and Oliveira, AC and Morgante, CV and Qi, Y and Fatima Grossi-de-Sa, M}, title = {CRISPR/Cas genome editing in soybean: Challenges and new insights to overcome existing bottlenecks.}, journal = {Journal of advanced research}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.jare.2024.08.024}, pmid = {39163906}, issn = {2090-1224}, abstract = {BACKGROUND: Soybean is a worldwide-cultivated crop due to its applications in the food, feed, and biodiesel industries. Genome editing in soybean began with ZFN and TALEN technologies; however, CRISPR/Cas has emerged and shortly became the preferable approach for soybean genome manipulation since it is more precise, easy to handle, and cost-effective. Recent reports have focused on the conventional Cas9 nuclease, Cas9 nickase (nCas9) derived base editors, and Cas12a (formally Cpf1) as the most commonly used genome editors in soybean. Nonetheless, several challenges in the complex plant genetic engineering pipeline need to be overcome to effectively edit the genome of an elite soybean cultivar. These challenges include (1) optimizing CRISPR cassette design (i.e., gRNA and Cas promoters, gRNA design and testing, number of gRNAs, and binary vector), (2) improving transformation frequency, (3) increasing the editing efficiency ratio of targeted plant cells, and (4) improving soybean crop production.<br><br>AIM OF REVIEW: This review provides an overview of soybean genome editing using CRISPR/Cas technology, discusses current challenges, and highlights theoretical (insights) and practical suggestions to overcome the existing bottlenecks.<br><br>The CRISPR/Cas system was discovered as part of the bacterial innate immune system. It has been used as a biotechnological tool for genome editing and efficiently applied in soybean to unveil gene function, improve agronomic traits such as yield and nutritional grain quality, and enhance biotic and abiotic stress tolerance. To date, the editing efficiency has been validated using protoplasts and hairy root assays, while stable plant transformation relies on Agrobacterium-mediated and particle bombardment methods. Nevertheless, most steps of the CRISPR/Cas workflow require optimizations to achieve a more effective genome editing in soybean plants.}, }
@article {pmid39163781, year = {2024}, author = {Shi, X and Zhang, J and Ding, Y and Li, H and Yao, S and Hu, T and Zhao, C and Wang, J}, title = {Ultrasensitive detection platform for Staphylococcus aureus based on DNAzyme tandem blocking CRISPR/Cas12a system.}, journal = {Biosensors &amp; bioelectronics}, volume = {264}, number = {}, pages = {116671}, doi = {10.1016/j.bios.2024.116671}, pmid = {39163781}, issn = {1873-4235}, abstract = {Detection methods based on CRISPR/Cas12a have been widely developed in the application of pathogenic microorganisms to guarantee food safety and public health. For sensitive detection, the CRISPR-based strategies are often in tandem with amplification methods. However, that may increase the detection time and the process may introduce nucleic acid contamination resulting in non-specific amplification. Herein, we established a sensitive S. aureus detection strategy based on the CRISPR/Cas12a system combined with DNAzyme. The activity of Cas12a is blocked by extending the spacer of crRNA (bcrRNA) and can be reactivated by Mn[2+]. NH2-modified S. aureus-specific aptamer was loaded on the surface of Fe3O4 MNPs (apt-Fe3O4 MNPs) and MnO2 NPs (apt-MnO2 NPs) by EDC/NHS chemistry. The S. aureus was captured to form apt-Fe3O4 MNPs/S. aureus/apt-MnO2 NPs complex and then MnO2 NPs were etched to release Mn[2+] to activate DNAzyme. The active DNAzyme can cleave the hairpin structure in bcrRNA to recover the activity of the CRISPR/Cas system. By initiating the whole detection process by generating Mn[2+] through nanoparticle etching, we established a rapid detection assay without nucleic acid extraction and amplification process. The proposed strategy has been applied in the ultrasensitive quantitative detection of S. aureus and has shown good performance with an LOD of 5 CFU/mL in 29 min. Besides, the proposed method can potentially be applied to other targets by simply changing the recognition element and has the prospect of developing a universal detection strategy.}, }
@article {pmid39163294, year = {2024}, author = {Kim, CS and Cairns, J and Quarantotti, V and Kaczkowski, B and Wang, Y and Konings, P and Zhang, X}, title = {A statistical simulation model to guide the choices of analytical methods in arrayed CRISPR screen experiments.}, journal = {PloS one}, volume = {19}, number = {8}, pages = {e0307445}, pmid = {39163294}, issn = {1932-6203}, mesh = {*CRISPR-Cas Systems ; *Models, Statistical ; Gene Editing/methods ; Humans ; Computer Simulation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {An arrayed CRISPR screen is a high-throughput functional genomic screening method, which typically uses 384 well plates and has different gene knockouts in different wells. Despite various computational workflows, there is currently no systematic way to find what is a good workflow for arrayed CRISPR screening data analysis. To guide this choice, we developed a statistical simulation model that mimics the data generating process of arrayed CRISPR screening experiments. Our model is flexible and can simulate effects on phenotypic readouts of various experimental factors, such as the effect size of gene editing, as well as biological and technical variations. With two examples, we showed that the simulation model can assist making principled choice of normalization and hit calling method for the arrayed CRISPR data analysis. This simulation model is implemented in an R package and can be downloaded from Github.}, }
@article {pmid39134876, year = {2024}, author = {Abdallah, K and Huys, I and Claes, KJ and Simoens, S}, title = {Budget Impact of Disease-Modifying Treatments and a CRISPR Gene-Edited Therapy for Sickle Cell Disease.}, journal = {Clinical drug investigation}, volume = {44}, number = {8}, pages = {611-627}, pmid = {39134876}, issn = {1179-1918}, support = {G0B9819N//Fonds Wetenschappelijk Onderzoek/ ; }, mesh = {Humans ; *Anemia, Sickle Cell/genetics/drug therapy/economics/therapy ; *Antibodies, Monoclonal, Humanized/economics/therapeutic use ; *Budgets ; Belgium ; Gene Editing/methods/economics ; CRISPR-Cas Systems ; Genetic Therapy/economics/methods ; Cost-Benefit Analysis ; }, abstract = {BACKGROUND AND OBJECTIVE: Treatment of sickle cell disease (SCD) has traditionally focused on symptomatic and preventative care. Recent advances in novel therapeutic developments, likely to be orphan-designated, are anticipated to carry a substantial price tag. This study assesses the potential budget impact of adopting disease-modifying treatments, crizanlizumab and voxelotor, and pioneering CRISPR gene-edited therapy, CTX001, in the Belgian healthcare system.<br><br>METHODS: The perspective of the Belgian healthcare payer (RIZIV-INAMI including patient copayments), a 5-year horizon with a 2-10% uptake of disease-modifying interventions, and a 2% uptake of CTX001 were considered. Data, encompassing target population, current (chronic and acute management, curative hematopoietic stem cell transplantation) and new (crizanlizumab, voxelotor, and CTX001) interventions, clinical effectiveness, adverse events, healthcare resource utilization, and associated costs, were gathered through a comprehensive literature review (first phase) and two Delphi panels involving hematologists (second phase). The cost difference between a "world with and without crizanlizumab, voxelotor, and CTX001" was calculated to obtain the budget impact.&#xa0;Three scenario analyses were conducted: a 5-13% and 4% uptake analysis, a 10-18% and 8% uptake analysis, respectively for disease-modifying treatments (crizanlizumab and voxelotor) and CTX001, and a 0% crizanlizumab uptake and managed entry agreements analysis .&#xa0;A&#x2009;&#xb1;&#x2009;20% univariate sensitivity analysis was performed to test the robustness of the analysis.<br><br>RESULTS: The total five-year cumulative budget impact was estimated at &#x20ac;30,024,968, with 91% attributed to drug acquisition costs. The largest budget impact share was for CTX001 (&#x20ac;25,575,150), while crizanlizumab (&#x20ac;2,301,095) and voxelotor (&#x20ac;2,148,723) was relatively small. In scenarios one and three, a two-fold increase of the cumulative budget impact to &#x20ac;60,731,772 and a four-fold increase to &#x20ac;120,846,256 from the base case was observed. In scenario three, this budget impact decreased by 63% to &#x20ac;11,212,766. Patient population size, number of treated patients, and drug costs influenced the analysis the most, while discontinuation, acute crisis, and adverse event rates had comparatively minimal impact.<br><br>CONCLUSIONS: Belgian decision-makers may consider alternative financing models, such as outcome-based risk-sharing agreements or annuities, to ensure sustainable coverage of these treatments. This study adheres to recommended practices for assessing budget impact of orphan drugs, distinguishing it from earlier studies with potentially weaker methodologies.}, }
@article {pmid39121862, year = {2024}, author = {Yoshimi, K and Kuno, A and Yamauchi, Y and Hattori, K and Taniguchi, H and Mikamo, K and Iida, R and Ishida, S and Goto, M and Takeshita, K and Ito, R and Takahashi, R and Takahashi, S and Mashimo, T}, title = {Genome editing using type I-E CRISPR-Cas3 in mice and rat zygotes.}, journal = {Cell reports methods}, volume = {4}, number = {8}, pages = {100833}, doi = {10.1016/j.crmeth.2024.100833}, pmid = {39121862}, issn = {2667-2375}, mesh = {Animals ; *Zygote/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Rats ; Mice ; Female ; }, abstract = {The type I CRISPR system has recently emerged as a promising tool, especially for large-scale genomic modification, but its application to generate model animals by editing zygotes had not been established. In this study, we demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system, which efficiently generates deletions of several thousand base pairs at targeted loci in mice with 40%-70% editing efficiency without off-target mutations. To overcome the difficulties associated with detecting the variable deletions, we used a newly long-read sequencing-based multiplex genotyping approach. Demonstrating remarkable versatility, our Cas3-based technique was successfully extended to rats as well as mice, even by zygote electroporation methods. Knockin for SNP exchange and genomic replacement with a donor plasmid were also achieved in mice. This pioneering work with the type I CRISPR zygote editing system offers increased flexibility and broader applications in genetic engineering across different species.}, }
@article {pmid39054057, year = {2024}, author = {Jansen, G and Gebert, D and Kumar, TR and Simmons, E and Murphy, S and Teixeira, FK}, title = {Tolerance thresholds underlie responses to DNA damage during germline development.}, journal = {Genes &amp; development}, volume = {38}, number = {13-14}, pages = {631-654}, doi = {10.1101/gad.351701.124}, pmid = {39054057}, issn = {1549-5477}, mesh = {Animals ; *Germ Cells ; *DNA Breaks, Double-Stranded ; *DNA Transposable Elements/genetics ; CRISPR-Cas Systems/genetics ; DNA Damage/genetics ; Drosophila melanogaster/genetics ; Female ; Oogenesis/genetics ; }, abstract = {Selfish DNA modules like transposable elements (TEs) are particularly active in the germline, the lineage that passes genetic information across generations. New TE insertions can disrupt genes and impair the functionality and viability of germ cells. However, we found that in P-M hybrid dysgenesis in Drosophila, a sterility syndrome triggered by the P-element DNA transposon, germ cells harbor unexpectedly few new TE insertions despite accumulating DNA double-strand breaks (DSBs) and inducing cell cycle arrest. Using an engineered CRISPR-Cas9 system, we show that generating DSBs at silenced P-elements or other noncoding sequences is sufficient to induce germ cell loss independently of gene disruption. Indeed, we demonstrate that both developing and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis regardless of the accumulated genome damage. Our findings establish DNA damage tolerance thresholds as crucial safeguards of genome integrity during germline development.}, }
@article {pmid39048074, year = {2024}, author = {Guan, Q and Wang, Z and Zhang, K and Liu, Z and Zhou, H and Cao, D and Mao, X}, title = {CRISPR/Cas9-mediated neuronal deletion of 5-lipoxygenase alleviates deficits in mouse models of epilepsy.}, journal = {Journal of advanced research}, volume = {63}, number = {}, pages = {73-90}, doi = {10.1016/j.jare.2024.07.018}, pmid = {39048074}, issn = {2090-1224}, mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *Disease Models, Animal ; *Arachidonate 5-Lipoxygenase/metabolism/genetics ; *Neurons/metabolism ; *Hippocampus/metabolism ; *Epilepsy/genetics ; Male ; Mice, Inbred C57BL ; Gene Deletion ; Epilepsy, Temporal Lobe/genetics ; Dependovirus/genetics ; Pilocarpine ; }, abstract = {INTRODUCTION: Our previous work reveals a critical role of activation of neuronal Alox5 in exacerbating brain injury post seizures. However, whether neuronal Alox5 impacts the pathological process of epilepsy remains unknown.<br><br>OBJECTIVES: To prove the feasibility of neuron-specific deletion of Alox5 via CRISPR-Cas9 in the blockade of seizure onset and epileptic progression.<br><br>METHODS: Here, we employed a Clustered regularly interspaced short-palindromic repeat-associated proteins 9 system (CRISPR/Cas9) system delivered by adeno-associated virus (AAV) to specifically delete neuronal Alox5 gene in the hippocampus to explore its therapeutic potential in various epilepsy mouse models and possible mechanisms.<br><br>RESULTS: Neuronal depletion of Alox5 was successfully achieved in the brain. AAV delivery of single guide RNA of Alox5 in hippocampus resulted in reducing seizure severity, delaying epileptic progression and improving epilepsy-associated neuropsychiatric comorbidities especially anxiety, cognitive deficit and autistic-like behaviors in pilocarpine- and kainic acid-induced temporal lobe epilepsy (TLE) models. In addition, neuronal Alox5 deletion also reversed neuron loss, neurodegeneration, astrogliosis and mossy fiber sprouting in TLE model. Moreover, a battery of tests including analysis of routine blood test, hepatic function, renal function, routine urine test and inflammatory factors demonstrated no noticeable toxic effect, suggesting that Alox5 deletion possesses the satisfactory biosafety. Mechanistically, the anti-epileptic effect of Alox5 deletion might be associated with reduction of glutamate level to restore excitatory/inhibitory balance by reducing CAMKII-mediated phosphorylation of Syn I[Ser603].<br><br>CONCLUSION: Our findings showed the translational potential of AAV-mediated delivery of CRISPR-Cas9 system including neuronal Alox5 gene for an alternative promising therapeutic approach to treat epilepsy.}, }
@article {pmid38987595, year = {2024}, author = {Brödel, AK and Charpenay, LH and Galtier, M and Fuche, FJ and Terrasse, R and Poquet, C and Havránek, J and Pignotti, S and Krawczyk, A and Arraou, M and Prevot, G and Spadoni, D and Yarnall, MTN and Hessel, EM and Fernandez-Rodriguez, J and Duportet, X and Bikard, D}, title = {In situ targeted base editing of bacteria in the mouse gut.}, journal = {Nature}, volume = {632}, number = {8026}, pages = {877-884}, pmid = {38987595}, issn = {1476-4687}, mesh = {Animals ; Mice ; *Escherichia coli/genetics ; *Gene Editing/methods ; *Klebsiella pneumoniae/genetics ; *beta-Lactamases/genetics/metabolism ; *Gastrointestinal Microbiome ; Female ; Bacteriophages/genetics ; CRISPR-Cas Systems/genetics ; Male ; Gastrointestinal Tract/microbiology ; Genes, Bacterial/genetics ; Genetic Vectors/genetics ; }, abstract = {Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health[1]. Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells[2], we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle to deliver a base editor and modify Escherichia coli colonizing the mouse gut. Editing of a β-lactamase gene in a model E. coli strain resulted in a median editing efficiency of 93% of the target bacterial population with a single dose. Edited bacteria were stably maintained in the mouse gut for at least 42 days following treatment. This was achieved using a non-replicative DNA vector, preventing maintenance and dissemination of the payload. We then leveraged this approach to edit several genes of therapeutic relevance in E. coli and Klebsiella pneumoniae strains in vitro and demonstrate in situ editing of a gene involved in the production of curli in a pathogenic E. coli strain. Our work demonstrates the feasibility of modifying bacteria directly in the gut, offering a new avenue to investigate the function of bacterial genes and opening the door to the design of new microbiome-targeted therapies.}, }
@article {pmid38894572, year = {2024}, author = {Bai, Y and Nan, Y and Wu, T and Zhu, A and Xie, X and Sun, Y and Deng, Y and Dou, Z and Hu, X and Zhou, R and Xu, S and Zhang, Y and Fan, J and Ju, D}, title = {Lipid Nanoparticle-Mediated Delivery of CRISPR-Cas9 Against Rubicon Ameliorates NAFLD by Modulating CD36 Along with Glycerophospholipid Metabolism.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {11}, number = {31}, pages = {e2400493}, doi = {10.1002/advs.202400493}, pmid = {38894572}, issn = {2198-3844}, support = {2023YFC3404000//National Key Research and Development Program of China/ ; 82371781//National Natural Science Foundation of China/ ; 82073752//National Natural Science Foundation of China/ ; 32070935//National Natural Science Foundation of China/ ; 21QB1401800//Shanghai Rising-Star Program/ ; 20S11904700//Shanghai Science and Technology Development Foundation/ ; }, mesh = {Animals ; *Non-alcoholic Fatty Liver Disease/genetics/metabolism/therapy ; Mice ; *Glycerophospholipids/metabolism ; *Nanoparticles ; *CRISPR-Cas Systems/genetics ; *CD36 Antigens/genetics/metabolism ; *Disease Models, Animal ; Mice, Inbred C57BL ; Male ; Lipids ; Lipid Metabolism/genetics ; Humans ; Liposomes ; }, abstract = {Non-alcoholic fatty liver disease (NAFLD) is a prominent cause of various chronic metabolic hepatic diseases with limited therapeutics. Rubicon, an essential regulator in lysosomal degradation, is reported to exacerbate hepatic steatosis in NAFLD mice and patients, indicating its probability of being a therapeutic target for NAFLD treatment. In this study, the therapeutic potential of Rubicon blockage is investigated. Lipid nanoparticles carrying Rubicon-specific CRISPR-Cas9 components exhibited liver accumulation, cell internalization, and Rubicon knockdown. A single administration of the nanoparticles results in attenuated lipid deposition and hepatic steatosis, with lower circulating lipid levels and decreased adipocyte size in NAFLD mice. Furthermore, the increase of phosphatidylcholine and phosphatidylethanolamine levels can be observed in the NAFLD mice livers after Rubicon silencing, along with regulatory effects on metabolism-related genes such as CD36, Gpcpd1, Chka, and Lpin2. The results indicate that knockdown of Rubicon improves glycerophospholipid metabolism and thereby ameliorates the NAFLD progression, which provides a potential strategy for NAFLD therapy via the restoration of Rubicon.}, }
@article {pmid38881503, year = {2024}, author = {Zhang, G and Song, Z and Huang, S and Wang, Y and Sun, J and Qiao, L and Li, G and Feng, Y and Han, W and Tang, J and Chen, Y and Huang, X and Liu, F and Wang, X and Liu, J}, title = {nCas9 Engineering for Improved Target Interaction Presents an Effective Strategy to Enhance Base Editing.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {11}, number = {31}, pages = {e2405426}, doi = {10.1002/advs.202405426}, pmid = {38881503}, issn = {2198-3844}, support = {2021YFF1000704//National Key R&amp;D Program of China/ ; 2019YFA0802802//National Key R&amp;D Program of China/ ; 2023ZD0405104//Sci-Tech Innovation 2030 Key Program/ ; 32272848//National Natural Science Foundation of China/ ; 2023TQ0329//China Postdoctoral Science Foundation/ ; 2023M743261//China Postdoctoral Science Foundation/ ; 2022ZB708//Excellent Postdoctoral Fellow of Jiangsu Province/ ; ZDBS-ZRKJZ-TLC008//Key Research Program of Chinese Academy of Sciences/ ; 2022GD-TSLD-46//Double chain fusion project of Shaanxi/ ; }, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; HEK293 Cells ; }, abstract = {Base editors (BEs) are a recent generation of genome editing tools that couple a cytidine or adenosine deaminase activity to a catalytically impaired Cas9 moiety (nCas9) to enable specific base conversions at the targeted genomic loci. Given their strong application potential, BEs are under active developments toward greater levels of efficiency and safety. Here, a previously overlooked nCas9-centric strategy is explored for enhancement of BE. Based on a cytosine BE (CBE), 20 point mutations associated with nCas9-target interaction are tested. Subsequently, from the initial positive X-to-arginine hits, combinatorial modifications are applied to establish further enhanced CBE variants (1.1-1.3). Parallel nCas9 modifications in other versions of CBEs including A3A-Y130F-BE4max, YEE-BE4max, CGBE, and split-AncBE4max, as well as in the context of two adenine BEs (ABE), likewise enhance their respective activities. The same strategy also substantially improves the efficiencies of high-fidelity nCas9/BEs. Further evidence confirms that the stabilization of nCas9-substrate interactions underlies the enhanced BE activities. In support of their translational potential, the engineered CBE and ABE variants respectively enable 82% and 25% higher rates of editing than the controls in primary human T-cells. This study thus demonstrates a highly adaptable strategy for enhancing BE, and for optimizing other forms of Cas9-derived tools.}, }
@article {pmid39162876, year = {2024}, author = {Li, G and Li, S and Li, X and He, W and Tan, X and Liang, J and Zhou, Z}, title = {A novel electrochemical aptasensor based on NrGO-H-Mn3O4 NPs integrated CRISPR/Cas12a system for ultrasensitive low-density lipoprotein determination.}, journal = {Mikrochimica acta}, volume = {191}, number = {9}, pages = {547}, pmid = {39162876}, issn = {1436-5073}, support = {Nos. 62161009//National Natural Science Foundation of China/ ; YCSW2023301//Innovation Project of Guangxi Graduate Education/ ; RC-XJ2022000401//Projects of Talents Recruitment of GDUPT/ ; 2024GXNSFAA010440//the Natural Science Foundation of Guangxi Zhuang Autonomous Region/ ; No. GuikeAA 24011005//the Guangxi Science and Technology Major Program/ ; }, mesh = {*Manganese Compounds/chemistry ; *Lipoproteins, LDL/blood/chemistry ; Humans ; *Electrochemical Techniques/methods ; *Oxides/chemistry ; *Graphite/chemistry ; *CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry ; *Hemin/chemistry ; *Biosensing Techniques/methods ; *Limit of Detection ; DNA, Single-Stranded/chemistry ; Nanoparticles/chemistry ; }, abstract = {Atherosclerosis cardiovascular disease (ASCVD) has become one of the leading death causes in humans. Low-density lipoprotein (LDL) is an important biomarker for assessing ASCVD risk level. Thus, monitoring LDL levels can be an important means for early diagnosis of ASCVD. Herein, a novel electrochemical aptasensor for determination LDL was designed based on nitrogen-doped reduced graphene oxide-hemin-manganese oxide nanoparticles (NrGO-H-Mn3O4 NPs) integrated with clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR/Cas12a) system. NrGO-H-Mn3O4 NPs not only have a large surface area and remarkable enhanced electrical conductivity but also the interconversion of different valence states of iron in hemin can provide an electrical signal. Nonspecific single-stranded DNA (ssDNA) was bound to NrGO-H-Mn3O4 NPs to form a signaling probe and was immobilized on the electrode surface. The CRISPR/Cas12a system has excellent trans-cleavage activity, which can be used to cleave ssDNA, thus detaching the NrGO-H-Mn3O4 NPs from the sensing interface and attenuating the electrical signal. Significant signal change triggered by the target was ultimately obtained, thus achieving sensitive detection of the LDL in range from 0.005 to 1000.0 nM with the detection limit of 0.005 nM. The proposed sensor exhibited good stability, selectivity, and stability and achieved reliable detection of LDL in serum samples, demonstrating its promising application prospects for the diagnostic application of LDL.}, }
@article {pmid39162556, year = {2024}, author = {Ma, Z and Qian, C and Yao, Z and Tang, M and Chen, K and Zhao, D and Hu, P and Zhou, T and Cao, J}, title = {Coexistence of plasmid-mediated tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 in Klebsiella quasipneumoniae.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0387423}, doi = {10.1128/spectrum.03874-23}, pmid = {39162556}, issn = {2165-0497}, abstract = {Klebsiella quasipneumoniae is a potential pathogen that has not been studied comprehensively. The emergence of multidrug-resistant (MDR) K. quasipneumoniae, specifically strains resistant to tigecycline and carbapenem, presents a significant challenge to clinical treatment. This investigation aimed to characterize MDR K. quasipneumoniae strain FK8966, co-carrying tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 by plasmids. It was observed that FK8966's MDR was primarily because of the IncHI1B-like plasmid co-carrying tmexCD2-toprJ2 and blaIMP-4, and an IncFIB(K)/IncFII(K) plasmid harboring blaNDM-1. Furthermore, the phylogenetic analysis revealed that IncHI1B-like plasmids carrying tmexCD2-toprJ2 were disseminated among different bacteria, specifically in China. Additionally, according to the comparative genomic analysis, the MDR regions indicated that the tmexCD2-toprJ2 gene cluster was inserted into the umuC gene, while blaIMP-4 was present in transposon TnAs3 linked to the class 1 integron (IntI1). It was also observed that an ΔTn3000 insertion with blaNDM-1 made a novel blaNDM-1 harboring IncFIB(K)/IncFII(K) plasmid. The antimicrobial resistance prevalence and phylogenetic analyses of K. quasipneumoniae strains indicated that FK8966 is a distinct MDR branch of K. quasipneumoniae. Furthermore, CRISPR-Cas system analysis showed that many K. quasipneumoniae CRISPR-Cas systems lacked spacers matching the two aforementioned novel resistance plasmids, suggesting that these resistance plasmids have the potential to disseminate within K. quasipneumoniae. Therefore, the spread of MDR K. quasipneumoniae and plasmids warrants further attention.IMPORTANCEThe emergence of multidrug-resistant K. quasipneumoniae poses a great threat to clinical care, and the situation is exacerbated by the dissemination of tigecycline- and carbapenem-resistant genes. Therefore, monitoring these pathogens and their resistance plasmids is urgent and crucial. This study identified tigecycline- and carbapenem-resistant K. quasipneumoniae strain, FK8966. Furthermore, it is the first study to report the coexistence of tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 in K. quasipneumoniae. Moreover, the CRISPR-Cas system of many K. quasipneumoniae lacks spacers that match the plasmids carried by FK8966, which are crucial for mediating resistance against tigecycline and carbapenems, indicating their potential to disseminate within K. quasipneumoniae.}, }
@article {pmid39162172, year = {2024}, author = {Markley, HC and Helms, KJ and Maar, M and Zentner, GE and Wade, MJ and Zelhof, AC}, title = {Generating and testing the efficacy of reagents for CRISPR/Cas9 homology directed repair-based manipulations in Tribolium.}, journal = {Journal of insect science (Online)}, volume = {24}, number = {4}, pages = {}, doi = {10.1093/jisesa/ieae082}, pmid = {39162172}, issn = {1536-2442}, support = {2019-33522-30064//NIFA/ ; 2019-33522-30064//USDA/ ; IOS-1928781//NSF/ ; }, mesh = {*Tribolium/genetics ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Recombinational DNA Repair ; Gene Drive Technology/methods ; }, abstract = {CRISPR/Cas9 manipulations are possible in many insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of CRISPR-based (clustered regularly interspaced short palindromic repeats) genome-editing tools and methodologies are dependent upon direct experimentation. One useful technique is Cas9-dependent homologous recombination, which is a critical tool for studying gene function but also for developing pest related applications like gene drive. Here, we report our attempts to induce Cas9 homology directed repair (HDR) and subsequent gene drive in Tribolium castaneum (Herbst; Insecta: Coleoptera: Tenebrionidae). Utilizing constructs containing 1 or 2 target gRNAs in combination with Cas9 under 2 different promoters and corresponding homology arms, we found a high incidence of CRISPR/Cas9 induced mutations but no evidence of homologous recombination. Even though the generated constructs provide new resources for CRISPR/Cas9 modification of the Tribolium genome, our results suggest that additional modifications and increased sample sizes will be necessary to increase the potential and detection for HDR of the Tribolium genome.}, }
@article {pmid39161182, year = {2024}, author = {Hu, R and Guo, C and Liu, C and Zhang, Q and Zhang, X and Chen, Y and Liu, Y}, title = {From Lab to Home: Ultrasensitive Rapid Detection of SARS-CoV-2 with a Cascade CRISPR/Cas13a-Cas12a System Based Lateral Flow Assay.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.4c02726}, pmid = {39161182}, issn = {1520-6882}, abstract = {Currently, CRISPR/Cas-based molecular diagnostic techniques usually rely on the introduction of nucleic acid amplification to improve their sensitivity, which is usually more time-consuming, susceptible to aerosol contamination, and therefore not suitable for at-home molecular testing. In this research, we developed an advanced CRISPR/Cas13a-Cas12a-based lateral flow assay that facilitated the ultrasensitive and rapid detection of SARS-CoV-2 RNA directly from samples, without the need for nucleic acid amplification. This method was called CRISPR LFA enabling at-home RNA testing (CLEAR). CLEAR used a novel cascade mechanism with specially designed probes that fold into hairpin structures, enabling visual detection of SARS-CoV-2 sequences down to 1 aM sensitivity levels. More importantly, CLEAR had a positive coincidence rate of 100% and a negative coincidence rate of 100% for clinical nasopharyngeal swabs from 16 patients. CLEAR was particularly suitable for at-home molecular testing, providing a low-cost, user-friendly solution that can efficiently distinguish between different SARS-CoV-2 variants. CLEAR overcame the common limitations of high sensitivity and potential contamination associated with traditional PCR-based systems, making it a promising tool for widespread public health application, especially in environments with limited access to laboratory resources.}, }
@article {pmid39160220, year = {2024}, author = {Liu, F and Zhang, X and Yang, Y}, title = {Simulation of CRISPR-Cas9 editing on evolving barcode and accuracy of lineage tracing.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {19213}, pmid = {39160220}, issn = {2045-2322}, support = {R01 CA251950/CA/NCI NIH HHS/United States ; R01 CA251950/CA/NCI NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Computer Simulation ; Algorithms ; DNA Barcoding, Taxonomic/methods ; Cell Lineage/genetics ; INDEL Mutation ; Mutation ; }, abstract = {We designed a simulation program that mimics the CRISPR-Cas9 editing on evolving barcode and double strand break repair procedure along with cell divisions. Emerging barcode mutations tend to build upon previously existing mutations, occurring sequentially with each generation. This process results in a unique mutation profile in each cell. We sample the barcodes in leaf cells and reconstruct the lineage, comparing it to the original lineage tree to test algorithm accuracy under different parameter settings. Our computational simulations validate the reasonable assumptions deduced from experimental observations, emphasizing that factors such as sampling size, barcode length, multiple barcodes, indel probabilities, and Cas9 activity are critical for accurate and successful lineage tracing. Among the many factors we found that sampling size and indel probabilities are two major ones that affect lineage tracing accuracy. Large segment deletions in early generations could greatly impact lineage accuracy. These simulation results offer insightful recommendations for enhancing the design and analysis of Cas9-mediated molecular barcodes in actual experiments.}, }
@article {pmid39160035, year = {2024}, author = {Gao, R and Liu, X and Xiong, Z and Wang, G and Ai, L}, title = {Research progress on detection of foodborne pathogens: The more rapid and accurate answer to food safety.}, journal = {Food research international (Ottawa, Ont.)}, volume = {193}, number = {}, pages = {114767}, doi = {10.1016/j.foodres.2024.114767}, pmid = {39160035}, issn = {1873-7145}, mesh = {*Foodborne Diseases/microbiology/prevention &amp; control ; *Food Microbiology/methods ; *Food Safety/methods ; Humans ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Food Contamination/analysis ; }, abstract = {In recent years, foodborne diseases have posed a serious threat to human health, and rapid detection of foodborne pathogens is particularly crucial for the prevention and control of such diseases. This article offers a detailed overview of the development of detection techniques for foodborne pathogens, transitioning from traditional microbiological culture methods to the current array of techniques, including immunological, molecular biological, and biosensor-based methods. It summarizes the technical principles, advantages, disadvantages, and research progress of these diverse methods. Furthermore, the article demonstrates that the combination of different methods enhances the efficiency and accuracy of pathogens detection. Specifically, the article focuses on the application and advantages of combining CRISPR/Cas systems with other detection methods in the detection of foodborne pathogens. CRISPR/Cas systems, with their high specificity, sensitivity, and ease of operation, show great potential in the field of foodborne pathogens detection. When integrated with other detection techniques such as immunological detection techniques, molecular biology detection techniques, and biosensors, the accuracy and efficiency of detection can be further improved. By fully utilizing these tools, early detection and control of foodborne diseases can be achieved, enhancing public health and preventing disease outbreaks. This article serves as a valuable reference for exploring more convenient, accurate, and sensitive field detection methods for foodborne pathogens, promoting the application of rapid detection techniques, and ensuring food safety and human health.}, }
@article {pmid39159817, year = {2024}, author = {Madaan, V and Kollara, A and Spaner, D and Brown, TJ}, title = {ISGylation enhances double-stranded RNA-induced interferon response and NFκB signaling in fallopian tube epithelial cells.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {107686}, doi = {10.1016/j.jbc.2024.107686}, pmid = {39159817}, issn = {1083-351X}, abstract = {Heritable mutations in BRCA1 associate with increased risk of high-grade serous tubo-ovarian cancer (HGSTOC). Non-genetic risk factors associated with this cancer, which arises from fallopian tube epithelial (FTE) cells, suggests a role for repetitive ovulation wherein FTE cells are exposed to inflammatory signaling molecules within follicular fluid. We previously reported increased NFκB and EGFR signaling in BRCA1-deficient primary FTE cells, with follicular fluid exposure further increasing abundance of interferon-stimulated gene (ISG) transcripts, including the ubiquitin-like protein ISG15 and other ISGylation pathway members. Both NFκB and type I interferon signaling are upregulated by stimulation of cGAS-STING or MDA5 and RIGI pattern recognition receptors (PRRs). Since some PRRs and their signal transduction pathway members are ISGylated, we tested the impact of ISG15 and ISGylation on IRF3 and NFκB signaling through cGAS-STING or RIGI and MDA5 activation. Expression of ISG15 or UBA7, the E1-like ISG15 activating enzyme, in immortalized FTE cells was disrupted by CRISPR gene editing. Activation of IRF3 by RIGI or MDA5 but not cGAS-STING was attenuated by loss of either ISG15 or UBA7 and this was reflected by a similar effect on NFκB activation and downstream targets. Loss of ISGylation decreased levels of both MDA5 and RIGI, with knock-down of RIGI but not MDA5, decreasing IRF3 and NFκB activation in parental cells. These finding indicate that ISGylation enhances the ability of dsRNA to activate cytokine release and pro-inflammatory signaling. Further work to explore ISGylation as a target for prevention of HGSTOC in BRCA1 mutation carriers is warranted.}, }
@article {pmid39158453, year = {2024}, author = {Ishida, S and Sawai, T}, title = {From CRISPR to Conscience: Ethical Dilemmas in Gene Editing and Genetic Selection.}, journal = {The American journal of bioethics : AJOB}, volume = {24}, number = {8}, pages = {67-70}, doi = {10.1080/15265161.2024.2361900}, pmid = {39158453}, issn = {1536-0075}, mesh = {Humans ; *Gene Editing/ethics ; *Conscience ; Selection, Genetic ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, }
@article {pmid39158272, year = {2024}, author = {Upreti, C and Kumar, P and Durso, LM and Palmer, KL}, title = {CRISPR-Cas inhibits plasmid transfer and immunizes bacteria against antibiotic resistance acquisition in manure.}, journal = {Applied and environmental microbiology}, volume = {}, number = {}, pages = {e0087624}, doi = {10.1128/aem.00876-24}, pmid = {39158272}, issn = {1098-5336}, abstract = {The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system clustered regularly interspaced short palindromic repeats (CRISPR)-Cas acts as a barrier to the spread of antibiotic resistance plasmids, and CRISPR-Cas-based antimicrobials can be effective to selectively deplete antibiotic-resistant bacteria. While significant surveillance efforts monitor the spread of antibiotic-resistant bacteria in the clinical context, a major, often overlooked aspect of the issue is resistance emergence in agriculture. Farm animals are commonly treated with antibiotics, and antibiotic resistance in agriculture is on the rise. Yet, CRISPR-Cas efficacy has not been investigated in this setting. Here, we evaluate the prevalence of CRISPR-Cas in agricultural Enterococcus faecalis strains and its antiplasmid efficacy in an agricultural niche: manure. Analyzing 1,986 E. faecalis genomes from human and animal hosts, we show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural E. faecalis strains. Using plasmid conjugation assays, we found that CRISPR-Cas is a significant barrier against resistance plasmid transfer in manure. Finally, we used a CRISPR-based antimicrobial approach to cure resistant E. faecalis of erythromycin resistance, but this was limited by delivery efficiency of the CRISPR antimicrobial in manure. However, immunization of bacteria against resistance gene acquisition in manure was highly effective. Together, our results show that E. faecalis CRISPR-Cas is prevalent and effective in an agricultural setting and has the potential to be utilized for depleting antibiotic-resistant populations. Our work has broad implications for tackling antibiotic resistance in the increasingly relevant agricultural setting, in line with a One Health approach.IMPORTANCEAntibiotic resistance is a growing global health crisis in human and veterinary medicine. Previous work has shown technologies based on CRISPR-Cas-a bacterial defense system-to be effective in tackling antibiotic resistance. Here we test if CRISPR-Cas is present and effective in agricultural niches, specifically in the ubiquitously present bacterium, Enterococcus faecalis. We show that CRISPR-Cas is both prevalent and functional in manure and has the potential to be used to specifically kill bacteria carrying antibiotic resistance genes. This study demonstrates the utility of CRISPR-Cas-based strategies for control of antibiotic resistance in agricultural settings.}, }
@article {pmid39156766, year = {2024}, author = {Liu, F and Li, R and Zhu, Z and Yang, Y and Lu, F}, title = {Current developments of gene therapy in human diseases.}, journal = {MedComm}, volume = {5}, number = {9}, pages = {e645}, pmid = {39156766}, issn = {2688-2663}, abstract = {Gene therapy has witnessed substantial advancements in recent years, becoming a constructive tactic for treating various human diseases. This review presents a comprehensive overview of these developments, with a focus on their diverse applications in different disease contexts. It explores the evolution of gene delivery systems, encompassing viral (like adeno-associated virus; AAV) and nonviral approaches, and evaluates their inherent strengths and limitations. Moreover, the review delves into the progress made in targeting specific tissues and cell types, spanning the eye, liver, muscles, and central nervous system, among others, using these gene technologies. This targeted approach is crucial in addressing a broad spectrum of genetic disorders, such as inherited lysosomal storage diseases, neurodegenerative disorders, and cardiovascular diseases. Recent clinical trials and successful outcomes in gene therapy, particularly those involving AAV and the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins, are highlighted, illuminating the transformative potentials of this approach in disease treatment. The review summarizes the current status of gene therapy, its prospects, and its capacity to significantly ameliorate patient outcomes and quality of life. By offering comprehensive analysis, this review provides invaluable insights for researchers, clinicians, and stakeholders, enriching the ongoing discourse on the trajectory of disease treatment.}, }
@article {pmid39155782, year = {2024}, author = {Lin, L and Tao, J and Meng, Y and Gan, Y and He, X and Li, S and Zhang, J and Gao, F and Xin, D and Wang, L and Fan, Y and Chen, B and Lu, Z and Xu, Y}, title = {Genome-wide CRISPR screening identifies critical role of phosphatase and tensin homologous (PTEN) in sensitivity of acute myeloid leukemia to chemotherapy.}, journal = {Journal of Zhejiang University. Science. B}, volume = {25}, number = {8}, pages = {700-710}, doi = {10.1631/jzus.B2300555}, pmid = {39155782}, issn = {1862-1783}, support = {LY21H080005//the Zhejiang Provincial Natural Science Foundation of China/ ; 81572920 and 82100171//the National Natural Science Foundation of China/ ; }, mesh = {Humans ; *PTEN Phosphohydrolase/metabolism/genetics ; *Leukemia, Myeloid, Acute/drug therapy/genetics ; *Apoptosis/drug effects ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Proliferation/drug effects ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Antineoplastic Agents/pharmacology/therapeutic use ; Cell Survival/drug effects ; Drug Resistance, Neoplasm ; CRISPR-Cas Systems ; }, abstract = {Although significant progress has been made in the development of novel targeted drugs for the treatment of acute myeloid leukemia (AML) in recent years, chemotherapy still remains the mainstay of treatment and the overall survival is poor in most patients. Here, we demonstrated the antileukemia activity of a novel small molecular compound NL101, which is formed through the modification on bendamustine with a suberanilohydroxamic acid (SAHA) radical. NL101 suppresses the proliferation of myeloid malignancy cells and primary AML cells. It induces DNA damage and caspase 3-mediated apoptosis. A genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) library screen revealed that phosphatase and tensin homologous (PTEN) gene is critical for the regulation of cell survival upon NL101 treatment. The knockout or inhibition of PTEN significantly reduced NL101-induced apoptosis in AML and myelodysplastic syndrome (MDS) cells, accompanied by the activation of protein kinase B (AKT) signaling pathway. The inhibition of mammalian target of rapamycin (mTOR) by rapamycin enhanced the sensitivity of AML cells to NL101-induced cell death. These findings uncover PTEN protein expression as a major determinant of chemosensitivity to NL101 and provide a novel strategy to treat AML with the combination of NL101 and rapamycin.}, }
@article {pmid39155100, year = {2024}, author = {Wang, T and Ding, K and Wang, X and Wang, Z and Liu, G and Zang, Y and Lin, S and Zhou, H and Wang, Q}, title = {Dual amplification dynamic DNA network system for CRISPR/Cas12a based p53 gene detection.}, journal = {Analytica chimica acta}, volume = {1321}, number = {}, pages = {343048}, doi = {10.1016/j.aca.2024.343048}, pmid = {39155100}, issn = {1873-4324}, mesh = {*Tumor Suppressor Protein p53/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques ; Biosensing Techniques/methods ; DNA/chemistry/genetics ; Limit of Detection ; Genes, p53 ; Nucleic Acid Hybridization ; }, abstract = {BACKGROUND: It is estimated that over 50 % of human cancers are caused by mutations in the p53 gene. Early sensitive and accurate detection of the p53 gene is important for diagnosis of cancers in the early stage. However, conventional detection techniques often suffer from strict reaction conditions, or unsatisfied sensitivity, so we need to develop a new strategy for accurate detection of p53 gene with smart designability, multiple signal amplification in mild reaction conditions.<br><br>RESULTS: In this study, CRISPR/Cas system is exploited in entropy-driven catalysis (EDC) and hybridization chain reaction (CHA) dual signal amplification sensing strategies. The products of both reactions can efficiently and separately activate CRISPR/Cas12a which greatly amplifies the fluorescent signal. The method has good linearity in p53 detection with the concentration ranged from 0.1&#xa0;fM to 0.5 pM with ultra-low detection limit of 0.096&#xa0;fM. It also showed good performance in serum, offering potentials for early disease detection.<br><br>SIGNIFICANCE: The designed dual amplification dynamic DNA network system exhibits an ultra-sensitive fluorescence biosensing for p53 gene identification. The method is simple to operate and requires only one buffer for the experiment, and meanwhile shows smart designability which could be used for a wide range of markers. Thus, we believe the present work will provide a potential tool for the construction and development of sensitive fluorescent biosensors for diseases.}, }
@article {pmid39058317, year = {2024}, author = {Chaumont, L and Peruzzi, M and Huetz, F and Raffy, C and Le Hir, J and Minke, J and Boudinot, P and Collet, B}, title = {Salmonid Double-stranded RNA-Dependent Protein Kinase Activates Apoptosis and Inhibits Protein Synthesis.}, journal = {Journal of immunology (Baltimore, Md. : 1950)}, volume = {213}, number = {5}, pages = {700-717}, doi = {10.4049/jimmunol.2400076}, pmid = {39058317}, issn = {1550-6606}, support = {871108//EU/ ; 817923//EU/ ; 301083//RCN/ ; 2020/0646//Association Nationale de la Recherche et de la Technologie (ANRT)/ ; }, mesh = {Animals ; *Apoptosis/immunology ; *eIF-2 Kinase/metabolism/genetics ; *Salmon/immunology ; Cell Line ; *Protein Biosynthesis/immunology ; Immunity, Innate ; Novirhabdovirus/physiology/immunology ; Fish Proteins/genetics/immunology/metabolism ; RNA, Double-Stranded/immunology ; Fish Diseases/immunology ; CRISPR-Cas Systems ; }, abstract = {dsRNA-dependent protein kinase R (PKR) is a key factor of innate immunity. It is involved in translation inhibition, apoptosis, and enhancement of the proinflammatory and IFN responses. However, how these antiviral functions are conserved during evolution remains largely unknown. Overexpression and knockout studies in a Chinook salmon (Oncorhynchus tshawytscha) cell line were conducted to assess the role of salmonid PKR in the antiviral response. Three distinct mRNA isoforms from a unique pkr gene, named pkr-fl (full length), pkr-ml (medium length) and pkr-sl (short length), were cloned and a pkr-/- clonal fish cell line was developed using CRISPR/Cas9 genome editing. PKR-FL includes an N-terminal dsRNA-binding domain and a C-terminal kinase domain, whereas PKR-ML and PKR-SL display a truncated or absent kinase domain, respectively. PKR-FL is induced during IFNA2 stimulation but not during viral hemorrhagic septicemia virus (VHSV) infection. Overexpression experiments showed that only PKR-FL possesses antiviral functions, including activation of apoptosis and inhibition of de novo protein synthesis. Knockout experiments confirmed that PKR is involved in apoptosis activation during the late stage of VHSV infection. Endogenous PKR also plays a critical role in translation inhibition upon poly(I:C) transfection after IFNA2 treatment. It is, however, not involved in translational arrest during VHSV infection. Extra- and intracellular titrations showed that endogenous PKR does not directly inhibit viral replication but apparently favors virion release into the supernatant, likely by triggering late apoptosis. Altogether, our data confirm that salmonid PKR has conserved molecular functions that VHSV appears to bypass with subversion strategies.}, }
@article {pmid39051914, year = {2024}, author = {Liu, Y and Gou, S and Qiu, L and Xu, Z and Yang, H and Yang, S and Zhao, Y}, title = {A CRISPR/Cas12a-powered gold/nickel foam surface-enhanced Raman spectroscopy biosensor for nucleic acid specific detection in foods.}, journal = {The Analyst}, volume = {149}, number = {17}, pages = {4343-4350}, doi = {10.1039/d4an00778f}, pmid = {39051914}, issn = {1364-5528}, mesh = {*Spectrum Analysis, Raman/methods ; *Biosensing Techniques/methods ; *Gold/chemistry ; *CRISPR-Cas Systems ; *Limit of Detection ; *Metal Nanoparticles/chemistry ; Drinking Water/analysis ; Silver/chemistry ; DNA, Single-Stranded/chemistry ; Food Analysis/methods ; Endodeoxyribonucleases/chemistry ; CRISPR-Associated Proteins/genetics ; Food Contamination/analysis ; Bacterial Proteins/genetics ; }, abstract = {Food is a necessary source of energy, but it also serves as a pathway for transmitting infectious pathogens, making food safety a matter of great concern. Rapid, accurate, and specific detection methods for foodborne viruses are crucial. Surface-Enhanced Raman Scattering (SERS), due to its superior sensitivity and characteristic fingerprint spectra, holds enormous potential. However, due to the limitations of SERS, it requires specific conditions to achieve specificity. In order to enhance the specificity and accuracy of nucleic acid detection based on SERS, we have developed a CRISPR-Cas12a-mediated SERS technique to identify target DNA, harnessing the targeting recognition capability of CRISPR-Cas12a and ultra-sensitive SERS tags and successfully addressing SERS' lack of specific detection capability. This system includes a gold/nickel foam substrate (Au-NFs) and a reporter (ssDNA-ROX). The phenomenon of colloidal gold/silver nano-aggregation due to magnesium ions, which is commonly encountered in CRISPR-SERS, was simultaneously solved using AuNFs. The qualitative and quantitative analysis of target DNA in drinking water was performed by monitoring the intensity change of ROX Raman reporter molecules. The results showed that the sensor detected DNA within 30 min and the limit of detection (LOD) was 8.23 fM. This is expected to become one of the alternative methods for nucleic acid detection for its rapid detection and high specificity.}, }
@article {pmid39011640, year = {2024}, author = {Gong, S and Song, K and Pan, W and Li, N and Tang, B}, title = {CRISPR-Cas12a-based ultrasensitive assay for visual detection of SARS-CoV-2 RNA.}, journal = {The Analyst}, volume = {149}, number = {17}, pages = {4418-4424}, doi = {10.1039/d4an00479e}, pmid = {39011640}, issn = {1364-5528}, mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *RNA, Viral/analysis/genetics ; Humans ; *Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; *COVID-19/diagnosis/virology ; COVID-19 Nucleic Acid Testing/methods/instrumentation ; Magnetite Nanoparticles/chemistry ; Endodeoxyribonucleases/chemistry/genetics ; CRISPR-Associated Proteins/genetics ; DNA, Single-Stranded/chemistry/genetics ; Bacterial Proteins ; }, abstract = {The development of ultrasensitive and visual methods is of great significance for molecular diagnosis at the point-of-care. In this study, we have integrated recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system to design an ultrasensitive strategy for visual nucleic acid testing. RPA is utilized to amplify the target nucleic acid, producing amplicons that activate the single-stranded DNase property of CRISPR-Cas12a. The activated CRISPR-Cas12a then degrades the single-stranded DNA on magnetic nanoparticles (MNPs), releasing immobilized GOx from the MNPs which catalyses the chromogenic substrate. The developed method exhibits remarkable sensitivity, successfully detecting as low as 10 aM (∼6 copies per μL) of the target nucleic acid by visual colour changes in solution. The instrumental limit of detection is calculated to be 2.86 aM (∼2 copies per μL), comparable to the sensitivity of polymerase chain reaction (PCR). Importantly, this approach only requires isothermal incubation operation and does not involve costly instruments. The method has been validated by visually detecting the SARS-CoV-2 RNA gene fragment within 50 minutes. With its ultrasensitivity, simplicity of operation, and potential for integration into a point-of-care detection kit, this strategy holds great promise for nucleic acid testing in various settings.}, }
@article {pmid38803114, year = {2024}, author = {Wei, Y and Zhang, H and Fan, J and Cai, Q and Zhang, Z and Wang, J and Zhang, M and Yan, F and Jiang, J and Xie, H and Luo, X and Wei, L and Lin, Y and He, W and Qu, M and Zhang, X and Zhu, Y and Xie, H and Zhang, J}, title = {Multiplex-genome-editing based rapid directional improvement of complex traits in rice.}, journal = {Plant biotechnology journal}, volume = {22}, number = {9}, pages = {2624-2628}, doi = {10.1111/pbi.14375}, pmid = {38803114}, issn = {1467-7652}, support = {2020J011354//The Natural Science Foundation of Fujian Province/ ; CARS-01-20//The National Rice Industry Technology System of Modern Agriculture for China/ ; KJZD202401//Key Science and Technology Projects of Fujian Academy of Agricultural Sciences/ ; XTCXGC2021001//The "5511" Collaborative Innovation project for High-quality Development and Surpasses of Agriculture between Government of Fujian and Chinese Academy of Agricultural Sciences/ ; 2020NZ08016//Key Program of Science and Technology in Fujian Province/ ; GBMUC-2019-002//Open Project Fund of Key Laboratory of Ministry of Education for Genetics, Breeding, and Multiple Utilization of Crops of Fujian Agricultrue and Forestry University/ ; }, mesh = {*Oryza/genetics/growth &amp; development ; *Gene Editing/methods ; *Genome, Plant/genetics ; Plant Breeding/methods ; Phenotype ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; }, abstract = {Although thousands of genes have been identified or cloned in rice (Oryza sativa) in the last two decades, the majority of them have only been separately characterized in specific varieties or single-gene modified backgrounds, thus limiting their practical application. We developed an optimized multiplex genome editing (MGE) toolbox that can efficiently assemble and stably express up to twelve sgRNA targets in a single plant expression vector. In this study, we established the MGE-based Rapid Directional Improvement (MRDI) strategy for directional improvement of complex agronomic traits in one small-scale rice transformation. This approach provides a rapid and practical procedure, encompassing sgRNA assembly, transgene-free screening and the creation of promising germplasm, by combining the precision of gene editing with phenotype-based field breeding. The MRDI strategy was used to generate the full diversity of twelve main agronomic genes in rice cultivar FXZ for the directional improvement of its growth duration and plant architecture. After applying the MRDI to FXZ, ideal plants with the desired traits of early heading date reduced plant height, and more effective panicles were generated without compromising yield, blast resistance and grain quality. Furthermore, the results of whole-genome sequencing (WGS), including the analysis of structural variations (SVs) and single nucleotide variations (SNVs) in the MGE plants, confirmed the high specificity and low frequency of unwanted mutations associated with this strategy. The MRDI breeding strategy would be a robust approach for exploring and applying crucial agronomic genes, as well as for generating novel elite germplasm in the future.}, }
@article {pmid38713743, year = {2024}, author = {He, Y and Han, Y and Ma, Y and Liu, S and Fan, T and Liang, Y and Tang, X and Zheng, X and Wu, Y and Zhang, T and Qi, Y and Zhang, Y}, title = {Expanding plant genome editing scope and profiles with CRISPR-FrCas9 systems targeting palindromic TA sites.}, journal = {Plant biotechnology journal}, volume = {22}, number = {9}, pages = {2488-2503}, doi = {10.1111/pbi.14363}, pmid = {38713743}, issn = {1467-7652}, support = {32072045//National Natural Science Foundation of China/ ; 32101205//National Natural Science Foundation of China/ ; 32270433//National Natural Science Foundation of China/ ; 2023ZD04074//STI 2030-Major Projects/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Genome, Plant/genetics ; *Oryza/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; }, abstract = {CRISPR-Cas9 is widely used for genome editing, but its PAM sequence requirements limit its efficiency. In this study, we explore Faecalibaculum rodentium Cas9 (FrCas9) for plant genome editing, especially in rice. FrCas9 recognizes a concise 5'-NNTA-3' PAM, targeting more abundant palindromic TA sites in plant genomes than the 5'-NGG-3' PAM sites of the most popular SpCas9. FrCas9 shows cleavage activities at all tested 5'-NNTA-3' PAM sites with editing outcomes sharing the same characteristics of a typical CRISPR-Cas9 system. FrCas9 induces high-efficiency targeted mutagenesis in stable rice lines, readily generating biallelic mutants with expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion with the exonuclease, TREX2. TREX2-FrCas9 generates much larger deletions than FrCas9 without compromise in editing efficiency. We demonstrate TREX2-FrCas9 as an efficient tool for genetic knockout of a microRNA gene. Furthermore, FrCas9-derived cytosine base editors (CBEs) and adenine base editors (ABE) are developed to produce targeted C-to-T and A-to-G base edits in rice plants. Whole-genome sequencing-based off-target analysis suggests that FrCas9 is a highly specific nuclease. Expression of TREX2-FrCas9 in plants, however, causes detectable guide RNA-independent off-target mutations, mostly as single nucleotide variants (SNVs). Together, we have established an efficient CRISPR-FrCas9 system for targeted mutagenesis, large deletions, C-to-T base editing, and A-to-G base editing in plants. The simple palindromic TA motif in the PAM makes the CRISPR-FrCas9 system a promising tool for genome editing in plants with an expanded targeting scope.}, }
@article {pmid38709858, year = {2024}, author = {Wang, X and Fang, T and Lu, J and Tripathi, L and Qi, Y}, title = {Broad range plastid genome editing with monomeric TALE-linked cytosine and dual base editors.}, journal = {Plant biotechnology journal}, volume = {22}, number = {9}, pages = {2441-2443}, doi = {10.1111/pbi.14358}, pmid = {38709858}, issn = {1467-7652}, support = {22010332//United States Agency for International Development/ ; MD-PSLA-243089//Maryland Agricultural Experiment Station/ ; IOS-2029889//National Science Fundation/ ; IOS-2132693//National Science Fundation/ ; }, mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; *Genome, Plastid/genetics ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plastids/genetics ; }, }
@article {pmid38643514, year = {2024}, author = {Niu, Q and Xie, H and Cao, X and Song, M and Wang, X and Li, S and Pang, K and Zhang, Y and Zhu, JK and Zhu, J}, title = {Engineering soybean with high levels of herbicide resistance with a Cas12-SF01-based cytosine base editor.}, journal = {Plant biotechnology journal}, volume = {22}, number = {9}, pages = {2435-2437}, doi = {10.1111/pbi.14356}, pmid = {38643514}, issn = {1467-7652}, support = {32270367//National Natural Science Foundation of China/ ; NK2022010301//National Key Research and Development Program of China/ ; 2021LZGC003//Seed-Industrialized Development Program in Shandong Province, China/ ; 2021LZGC012//Key Research &amp; Development Program of Shandong Province, China/ ; }, mesh = {*Glycine max/genetics/metabolism/drug effects ; *Herbicide Resistance/genetics ; *Cytosine/metabolism ; Gene Editing ; CRISPR-Cas Systems/genetics ; Herbicides/pharmacology ; Plants, Genetically Modified/genetics ; }, }
@article {pmid38623687, year = {2024}, author = {Zhang, S and Wu, S and Jia, Z and Zhang, J and Li, Y and Ma, X and Fan, B and Wang, P and Gao, Y and Ye, Z and Wang, W}, title = {Exploring the influence of a single-nucleotide mutation in EIN4 on tomato fruit firmness diversity through fruit pericarp microstructure.}, journal = {Plant biotechnology journal}, volume = {22}, number = {9}, pages = {2379-2394}, doi = {10.1111/pbi.14352}, pmid = {38623687}, issn = {1467-7652}, support = {31902024//National Natural Science Foundation of China/ ; 212102110413//Scientific and Technological Project of Henan Province/ ; 30500495//Research fund for the Doctoral program of Henan Agricultural University/ ; }, mesh = {*Solanum lycopersicum/genetics/growth &amp; development ; *Fruit/genetics/growth &amp; development ; *Plant Proteins/genetics/metabolism ; *Polymorphism, Single Nucleotide/genetics ; *Receptors, Cell Surface/genetics/metabolism ; *Genome-Wide Association Study ; Gene Expression Regulation, Plant ; Gene Editing ; CRISPR-Cas Systems ; }, abstract = {Tomato (Solanum lycopersicum) stands as one of the most valuable vegetable crops globally, and fruit firmness significantly impacts storage and transportation. To identify genes governing tomato firmness, we scrutinized the firmness of 266 accessions from core collections. Our study pinpointed an ethylene receptor gene, SlEIN4, located on chromosome 4 through a genome-wide association study (GWAS) of fruit firmness in the 266 tomato core accessions. A single-nucleotide polymorphism (SNP) (A → G) of SlEIN4 distinguished lower (AA) and higher (GG) fruit firmness genotypes. Through experiments, we observed that overexpression of SlEIN4[AA] significantly delayed tomato fruit ripening and dramatically reduced fruit firmness at the red ripe stage compared with the control. Conversely, gene editing of SlEIN4[AA] with CRISPR/Cas9 notably accelerated fruit ripening and significantly increased fruit firmness at the red ripe stage compared with the control. Further investigations revealed that fruit firmness is associated with alterations in the microstructure of the fruit pericarp. Additionally, SlEIN4[AA] positively regulates pectinase activity. The transient transformation assay verified that the SNP (A → G) on SlEIN4 caused different genetic effects, as overexpression of SlEIN4[GG] increased fruit firmness. Moreover, SlEIN4 exerts a negative regulatory role in tomato ripening by impacting ethylene evolution through the abundant expression of ethylene pathway regulatory genes. This study presents the first evidence of the role of ethylene receptor genes in regulating fruit firmness. These significant findings will facilitate the effective utilization of firmness and ripening traits in tomato improvement, offering promising opportunities for enhancing tomato storage and transportation capabilities.}, }
@article {pmid35271349, year = {2024}, author = {Fu, X and Duan, Z and Lu, X and Zhu, Y and Ren, Y and Zhang, W and Sun, X and Ge, L and Yang, J}, title = {SND1 Promotes Radioresistance in Cervical Cancer Cells by Targeting the DNA Damage Response.}, journal = {Cancer biotherapy &amp; radiopharmaceuticals}, volume = {39}, number = {6}, pages = {425-434}, doi = {10.1089/cbr.2021.0371}, pmid = {35271349}, issn = {1557-8852}, mesh = {Humans ; *Uterine Cervical Neoplasms/genetics/radiotherapy/pathology/metabolism ; *Radiation Tolerance/genetics ; *DNA Damage ; Female ; HeLa Cells ; *Endonucleases/genetics/metabolism ; Apoptosis/genetics ; DNA Repair ; CRISPR-Cas Systems ; Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; }, abstract = {Background: Radiotherapy is one of the most effective therapeutic strategies for cervical cancer patients, although radioresistance-mediated residual and recurrent tumors are the main cause of treatment failure. However, the mechanism of tumor radioresistance is still elusive. DNA damage response pathways are key determinants of radioresistance. The purpose of this study was to investigate the role and mechanism of SND1 in radioresistance of cervical cancer. Methods: A stable HeLa cell line with SND1 knockout (HeLa-KO) was generated through a modified CRISPR/Cas9 double-nicking gene editing system. The stable CaSki cell lines with SND1 knockdown (CaSki-Ctrl, CaSki-SND1-sh-1, CaSki-SND1-sh-2) were constructed through lentivirus transfection with the pSil-SND1-sh-1 and pSil-SND1-sh-2 plasmids. Results: It was observed that SND1 deficiency significantly increased the radiosensitivity of cervical cancer cells. It was also found that silencing SND1 promotes radiation-induced apoptosis. Significantly, the cells with a loss of SND1 function exhibited inefficient ataxia telangiectasia mutated pathway activation, subsequently impairing DNA repair and G2/M checkpoint arrest. In addition, threonine 103 is an important phosphorylation site of SND1 under DNA damaging stress. Conclusion: Collectively, the results of this study reveal a potent radiosensitizing effect of silencing SND1 or T103 mutation on cervical cancer cells, providing novel insights into potential therapeutic strategies for cervical cancer treatment.}, }
@article {pmid39155053, year = {2024}, author = {Koller, U and Bauer, JW}, title = {Emerging DNA &amp; RNA editing strategies for the treatment of epidermolysis bullosa.}, journal = {The Journal of dermatological treatment}, volume = {35}, number = {1}, pages = {2391452}, doi = {10.1080/09546634.2024.2391452}, pmid = {39155053}, issn = {1471-1753}, mesh = {Humans ; *Epidermolysis Bullosa/therapy/genetics ; *Genetic Therapy ; *Gene Editing ; *CRISPR-Cas Systems ; RNA Editing ; Quality of Life ; }, abstract = {Background: Epidermolysis bullosa (EB) is a clinically-heterogeneous genodermatosis with severe manifestations in the skin and other organs. The significant burden this condition places on patients justifies the development of gene therapeutic strategies targeting the genetic cause of the disease. Methods: Emerging RNA and DNA editing tools have shown remarkable advances in efficiency and safety. Applicable both in ex vivo- and in vivo settings, these gene therapeutics based on gene replacement or editing are either at the pre-clinical or clinical stage. Results: The recent landmark FDA approvals for gene editing based on CRISPR/Cas9, along with the first FDA-approved redosable in vivo gene replacement therapy for EB, will invigorate ongoing research efforts, increasing the likelihood of achieving local cure via CRISPR-based technologies in the near future. Conclusions: This review discusses the status quo of current gene therapeutics that act at the level of RNA or DNA, all with the common aim of improving the quality of life for EB patients.}, }
@article {pmid39153653, year = {2024}, author = {Razipour, M and Jamali, Z and Khorsand, M and Zargar, M and Maghsudlu, M and Ghadami, E and Shakoori, A}, title = {Circular RNAs in laryngeal cancer.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {}, number = {}, pages = {119916}, doi = {10.1016/j.cca.2024.119916}, pmid = {39153653}, issn = {1873-3492}, abstract = {Laryngeal cancer remains a significant global health concern, with poor prognosis for advanced-stage disease highlighting the need for novel diagnostic, prognostic, and therapeutic approaches. Circular RNAs (circRNAs), a class of covalently closed non-coding RNAs, have emerged as important regulators of gene expression and cellular processes in various cancers, including laryngeal cancer. This review summarizes the current understanding of circRNAs in laryngeal cancer, covering their biogenesis, regulatory mechanisms, and potential clinical applications. We explore the diverse functions of circRNAs, including their roles as miRNA sponges, protein interactors, and direct mRNA regulators, and their influence on key cellular processes such as proliferation, invasion, and metastasis. The review highlights promising circRNAs as diagnostic and prognostic biomarkers, as well as potential therapeutic targets. We also outline current strategies for circRNA modulation, including suppression techniques like RNA interference and CRISPR/Cas systems, and overexpression methods using vectors and synthetic circRNAs.}, }
@article {pmid39152113, year = {2024}, author = {van de Kooij, B and van der Wal, FJ and Rother, MB and Wiegant, WW and Creixell, P and Stout, M and Joughin, BA and Vornberger, J and Altmeyer, M and van Vugt, MATM and Yaffe, MB and van Attikum, H}, title = {The Fanconi anemia core complex promotes CtIP-dependent end resection to drive homologous recombination at DNA double-strand breaks.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {7076}, pmid = {39152113}, issn = {2041-1723}, support = {ERC-CoG-617485//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; VI.C.182.052//Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research)/ ; BUIT 2015-7546//KWF Kankerbestrijding (Dutch Cancer Society)/ ; C9545/A29580//Cancer Research UK (CRUK)/ ; C9685/A26398//Cancer Research UK (CRUK)/ ; C42454/A28596//Cancer Research UK (CRUK)/ ; 310030_197003//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; P30-CA14051//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01-ES015339//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R35-ES028374//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01-CA226898//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; }, mesh = {Humans ; *DNA Breaks, Double-Stranded ; *Fanconi Anemia Complementation Group D2 Protein/metabolism/genetics ; *Fanconi Anemia Complementation Group L Protein/metabolism/genetics ; *Ubiquitin-Conjugating Enzymes/metabolism/genetics ; *Homologous Recombination ; Nuclear Proteins/metabolism/genetics ; Carrier Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Ubiquitination ; Fanconi Anemia/genetics/metabolism ; Endodeoxyribonucleases/metabolism/genetics ; HEK293 Cells ; Recombinational DNA Repair ; DNA Repair ; DNA End-Joining Repair ; DNA Helicases ; }, abstract = {During the repair of interstrand crosslinks (ICLs) a DNA double-strand break (DSB) is generated. The Fanconi anemia (FA) core complex, which is recruited to ICLs, promotes high-fidelity repair of this DSB by homologous recombination (HR). However, whether the FA core complex also promotes HR at ICL-independent DSBs, for example induced by ionizing irradiation or nucleases, remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen. Using isogenic cell line models, we further demonstrated an HR-promoting function of FANCL and Ube2T, and of their ubiquitination substrate FANCD2. We show that FANCL and Ube2T localize at DSBs in a FANCM-dependent manner, and are required for the DSB accumulation of FANCD2. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of CtIP at DSBs, thereby promoting end resection and Rad51 loading. Together, these data demonstrate a dual genome maintenance function of the FA core complex and FANCD2 in promoting repair of both ICLs and DSBs.}, }
@article {pmid39079650, year = {2024}, author = {Patra, S and Chatterjee, D and Basak, S and Sen, S and Mandal, A}, title = {CRISPR/Cas9 opens new horizon of crop improvement under stress condition.}, journal = {Biochimica et biophysica acta. General subjects}, volume = {1868}, number = {10}, pages = {130685}, doi = {10.1016/j.bbagen.2024.130685}, pmid = {39079650}, issn = {1872-8006}, mesh = {*CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; *Gene Editing/methods ; *Stress, Physiological/genetics ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; Genome, Plant/genetics ; }, abstract = {Plants are exposed to a myriad of stresses, stemming from abiotic and biotic sources, significantly threatening agricultural productivity. The low crop yield, coupled with the global burden of population has resulted in the scarcity of quality food, exacerbating socio-economic issues like poverty, hunger, and malnutrition. Conventional breeding methods for the generation of stress-tolerant plants are time-consuming, limit genetic diversity, and are not sustainable for the consistent production of high-yielding crops. In recent years, the use of high-throughput, genome editing (GE) technique has revolutionized the crop-improvement paradigm, ushering greater prospects for agricultural progress. Among these tools, the Clustered regularly interspaced short palindromic repeat (CRISPR), and its associated nuclease protein Cas9, have appeared as a ground-breaking technology, allowing precise knockout (KO), upregulation, and downregulation of target gene expression. Apart from its high efficacy and speed, this programmable nuclease offers exceptional specificity with minimal off-target effects. Here in, we aim to review the latest findings on the application of the CRISPR/Cas9 genome editing tool for generating resilience in plants against environmental stresses.}, }
@article {pmid39151151, year = {2024}, author = {Philippidis, A}, title = {Roche, Ascidian Launch Up-to-$1.8B RNA Exon Editing Collaboration.}, journal = {Human gene therapy}, volume = {35}, number = {15-16}, pages = {517-519}, doi = {10.1089/hum.2024.378421}, pmid = {39151151}, issn = {1557-7422}, mesh = {Humans ; *Exons/genetics ; RNA Editing ; Genetic Therapy/methods ; CRISPR-Cas Systems ; }, }
@article {pmid39121791, year = {2024}, author = {Mohd Abas, MD and Mohd Asri, MF and Yusafawi, NAS and Rosman, NAZ and Baharudin, NAZ and Taher, M and Susanti, D and Khotib, J}, title = {Advancements of gene therapy in cancer treatment: A comprehensive review.}, journal = {Pathology, research and practice}, volume = {261}, number = {}, pages = {155509}, doi = {10.1016/j.prp.2024.155509}, pmid = {39121791}, issn = {1618-0631}, mesh = {Humans ; *Genetic Therapy/methods ; *Neoplasms/therapy/genetics ; Animals ; Genetic Vectors ; CRISPR-Cas Systems/genetics ; }, abstract = {Cancer is the main contributor for mortality in the world. Conventional therapy that available as the treatment options are chemotherapy, radiotherapy and surgery. However, these treatments are hardly cell-specific most of the time. Nowadays, extensive research and investigations are made to develop cell-specific approaches prior to cancer treatment. Some of them are photodynamic therapy, hyperthermia, immunotherapy, stem cell transplantation and targeted therapy. This review article will be focusing on the development of gene therapy in cancer. The objective of gene therapy is to correct specific mutant genes causing the excessive proliferation of the cell that leads to cancer. There are lots of explorations in the approach to modify the gene. The delivery of this therapy plays a big role in its success. If the inserted gene does not find its way to the target, the therapy is considered a failure. Hence, vectors are needed and the common vectors used are viral, non viral or synthetic, polymer based and lipid based vectors. The advancement of gene therapy in cancer treatment will be focussing on the top three cancer cases in the world which are breast, lung and colon cancer. In breast cancer, the discussed therapy are CRISPR/Cas9, siRNA and gene silencing whereas in colon cancer miRNA and suicide gene therapy and in lung cancer, replacement of tumor suppressor gene, CRISPR/Cas9 and miRNA.}, }
@article {pmid39039305, year = {2024}, author = {Minton, K}, title = {Epigenetic editing works like a CHARM.}, journal = {Nature reviews. Genetics}, volume = {25}, number = {9}, pages = {600}, pmid = {39039305}, issn = {1471-0064}, mesh = {Humans ; *Epigenesis, Genetic ; *Gene Editing/methods ; CRISPR-Cas Systems ; Epigenomics/methods ; Animals ; }, }
@article {pmid38909850, year = {2024}, author = {Thomas, LW and Stephen, JM and Ashcroft, M}, title = {CHCHD4 regulates the expression of mitochondrial genes that are essential for tumour cell growth.}, journal = {Biochimica et biophysica acta. Molecular basis of disease}, volume = {1870}, number = {7}, pages = {167282}, doi = {10.1016/j.bbadis.2024.167282}, pmid = {38909850}, issn = {1879-260X}, mesh = {Humans ; *Neoplasms/genetics/pathology/metabolism ; *Mitochondria/metabolism/genetics ; *Gene Expression Regulation, Neoplastic ; Cell Proliferation/genetics ; Mitochondrial Proteins/genetics/metabolism ; Mitochondrial Precursor Protein Import Complex Proteins ; Genes, Mitochondrial ; Cell Line, Tumor ; Proteomics/methods ; CRISPR-Cas Systems ; Mitochondrial Membrane Transport Proteins/genetics/metabolism ; }, abstract = {CHCHD4 (MIA40) is central to the functions of the mitochondrial disulfide relay system (DRS). CHCHD4 is essential and evolutionarily conserved. Previously, we have shown CHCHD4 to be a critical regulator of tumour cell growth. Here, we use integrated analysis of our genome-wide CRISPR/Cas9 and SILAC proteomic screening data to delineate mechanisms of CHCHD4 essentiality in cancer. We identify a shortlist of common essential genes/proteins regulated by CHCHD4, including subunits of complex I that are known DRS substrates, and genes/proteins involved in key metabolic pathways. Our study highlights a range of CHCHD4-regulated nuclear encoded mitochondrial genes/proteins essential for tumour cell growth.}, }
@article {pmid38716631, year = {2024}, author = {Liu, J and Li, N and Zhang, L and Lu, Y and Shen, M and Zhang, Y and Feng, L and Jing, J and Cheng, J and Xu, Y}, title = {A Wax Interface-Enabled One-Pot Multiplexed Nucleic Acid Testing Platform for Rapid and Sensitive Detection of Viruses and Variants.}, journal = {Small methods}, volume = {8}, number = {8}, pages = {e2400030}, doi = {10.1002/smtd.202400030}, pmid = {38716631}, issn = {2366-9608}, support = {2021YFF0703400//National Key Research and Development Program/ ; 32201246//National Natural Science Foundation of China/ ; 22074078//National Natural Science Foundation of China/ ; //Beijing Nova Program 2020 and Beijing Lab Foundation/ ; }, mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; *COVID-19/diagnosis/virology ; Humans ; *Waxes/chemistry ; Sensitivity and Specificity ; Animals ; COVID-19 Nucleic Acid Testing/methods/instrumentation ; Cats ; Smartphone ; Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; CRISPR-Cas Systems ; Molecular Diagnostic Techniques/methods ; }, abstract = {High-quality, low-cost, and rapid detection is essential for the society to reopen the economy during the critical period of transition from Coronavirus Disease 2019 (COVID-19) pandemic response to pandemic control. In addition to performing sustainable and target-driven tracking of SARS-CoV-2, conducting comprehensive surveillance of variants and multiple respiratory pathogens is also critical due to the frequency of reinfections, mutation immune escape, and the growing prevalence of the cocirculation of multiple viruses. By utilizing a 0.05 cents wax interface, a Stable Interface assisted Multiplex Pathogenesis Locating Estimation in Onepot (SIMPLEone) using nested RPA and CRISPR/Cas12a enzymatic reporting system is successfully developed. This smartphone-based SIMPLEone system achieves highly sensitive one-pot detection of SARS-CoV-2 and its variants, or multiple respiratory viruses, in 40 min. A total of 89 clinical samples, 14 environmental samples, and 20 cat swab samples are analyzed by SIMPLEone, demonstrating its excellent sensitivity (3-6 copies/reaction for non-extraction detection of swab and 100-150 copies/mL for RNA extraction-based assay), accuracy (>97.7%), and specificity (100%). Furthermore, a high percentage (44.2%) of co-infection cases are detected in SARS-CoV-2-infected patients using SIMPLEone's multiplex detection capability.}, }
@article {pmid38658741, year = {2024}, author = {Li, B and Sun, C and Li, J and Gao, C}, title = {Targeted genome-modification tools and their advanced applications in crop breeding.}, journal = {Nature reviews. Genetics}, volume = {25}, number = {9}, pages = {603-622}, pmid = {38658741}, issn = {1471-0064}, mesh = {*Gene Editing/methods ; *Plant Breeding/methods ; *Crops, Agricultural/genetics ; *Genome, Plant ; *CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; }, abstract = {Crop improvement by genome editing involves the targeted alteration of genes to improve plant traits, such as stress tolerance, disease resistance or nutritional content. Techniques for the targeted modification of genomes have evolved from generating random mutations to precise base substitutions, followed by insertions, substitutions and deletions of small DNA fragments, and are finally starting to achieve precision manipulation of large DNA segments. Recent developments in base editing, prime editing and other CRISPR-associated systems have laid a solid technological foundation to enable plant basic research and precise molecular breeding. In this Review, we systematically outline the technological principles underlying precise and targeted genome-modification methods. We also review methods for the delivery of genome-editing reagents in plants and outline emerging crop-breeding strategies based on targeted genome modification. Finally, we consider potential future developments in precise genome-editing technologies, delivery methods and crop-breeding approaches, as well as regulatory policies for genome-editing products.}, }
@article {pmid38164884, year = {2024}, author = {Gu, P and Zhao, J and Zhang, W and Ruan, X and Hu, L and Zeng, Y and Hou, X and Zheng, X and Gao, M and Chi, J}, title = {An Inducible CRISPR-dCas9-Based Transcriptional Repression System for Cancer Therapy.}, journal = {Small methods}, volume = {8}, number = {8}, pages = {e2301310}, doi = {10.1002/smtd.202301310}, pmid = {38164884}, issn = {2366-9608}, support = {82004158//National Natural Science Foundation of China/ ; 82172821//National Natural Science Foundation of China/ ; 82102639//National Natural Science Foundation of China/ ; 19JCYBJC27400//Tianjin Municipal Science and Technology Project/ ; 21JCZDJC00360//Tianjin Municipal Science and Technology Project/ ; 2021ZD033//Science &amp;Technology Development Fund of Tianjin Education Commission for Higher Education/ ; TJYXZDXK-058B//Tianjin Medical Key Discipline (Specialty) Construction Project/ ; TJYXZDXK-009A//Tianjin Medical Key Discipline (Specialty) Construction Project/ ; TJWJ2022XK024//Tianjin Health Research Project/ ; TJWJ2021QN047//Tianjin Health Science and Technology Program/ ; TJWJ2023MS020//Tianjin Health Science and Technology Program/ ; TJSQNYXXR-D2-133//Tianjin Health Science and Technology Program/ ; 22JCYBJC00780//Natural Science Foundation of Tianjin Municipality/ ; 22JCQNJC00850//Natural Science Foundation of Tianjin Municipality/ ; 2023QNRC001//Young Elite Scientists Sponsorship Program by CAST/ ; }, mesh = {Humans ; *Oleanolic Acid/pharmacology/analogs &amp; derivatives ; *CRISPR-Cas Systems ; Animals ; *Aurora Kinase A/genetics/metabolism/antagonists &amp; inhibitors ; Mice ; Lung Neoplasms/genetics/drug therapy/therapy ; Cell Line, Tumor ; Thyroid Neoplasms/genetics/drug therapy/therapy ; Genetic Therapy/methods ; Histone Demethylases/genetics/metabolism ; }, abstract = {Gene therapy has been adapted for improving malignant tumor treatment. However, pharmacotherapies targeting cancer remain limited and are generally inapplicable for rare disease patients. Oleanolic acid (OA) is a plant-derived triterpenoid that is frequently used in Chinese medicine as a safe but slow-acting treatment for many disorders. Here, the congruent pharmacological activities of OA and CRISPR-dCas9 in targeting AURKA or KDM1A and improving disease-specific prognosis and used a synthetic-biology-inspired design principle to engineer a therapeutic gene circuit that enables a concerted action of both drugs are utilized. In particular, the OA-triggered CRISPR-dCas9 transcriptional repression system rapidly and simultaneously attenuated lung and thyroid cancer. Collectively, this work shows that rationally engineered synthetic gene circuits are capable of treating multifactorial diseases in a synergistic manner by multiplexing the targeting efficiencies of single therapeutics.}, }
@article {pmid39150856, year = {2024}, author = {Barrangou, R}, title = {AI and SynBio Meet CRISPR Heralding a New Genome Editing Era.}, journal = {The CRISPR journal}, volume = {7}, number = {4}, pages = {179}, doi = {10.1089/crispr.2024.0063}, pmid = {39150856}, issn = {2573-1602}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Artificial Intelligence ; }, }
@article {pmid39150073, year = {2024}, author = {Sjøgaard-Frich, LM and Henriksen, MS and Lam, SM and Birkbak, FJ and Czaplinska, D and Flinck, M and Pedersen, SF}, title = {NHE1 regulation in NAFLD in vitro contributes to hepatocyte injury and HSC crosstalk.}, journal = {The Journal of endocrinology}, volume = {}, number = {}, pages = {}, doi = {10.1530/JOE-24-0099}, pmid = {39150073}, issn = {1479-6805}, abstract = {Non-alcoholic fatty liver disease (NAFLD) is the fastest growing cause of liver-associated death globally. Whole-body knockout (KO) of Na+/H+ exchanger 1 (NHE1, SLC9A1) was previously proposed to protect against high fat diet-induced liver damage, however, mechanistic insight was lacking. The aim of the present work was to address this question in vitro to determine how NHE1 specifically in hepatocytes impacts lipid overload-induced inflammation, fibrosis, and hepatocyte- hepatic stellate cell (HSC) crosstalk. We induced palmitate (PA)-based steatosis in AML12 and HepG2 hepatocytes, manipulated NHE1 activity pharmacologically and by CRISPR-Cas knockout (KO) and -overexpression, and measured intracellular pH (pHi), steatosis-associated inflammatory and fibrotic mediators and cell death. PA treatment increased NHE1 mRNA levels but modestly reduced NHE1 protein expression and hepatocyte pHi. NHE1 KO in hepatocytes did not alter lipid droplet accumulation but reduced inflammatory signaling (p38 MAPK activity), lipotoxicity (4-HNE accumulation) and apoptosis (PARP cleavage). Conditioned medium from PA-treated hepatocytes increased expression of NHE1 and of the fibrosis regulator tissue inhibitor of matrix metalloproteases-2 (TIMP2) in LX-2 HSCs, in a manner abolished by NHE1 KO in hepatocytes. We conclude that NHE1 is regulated in NAFLD in vitro and contributes to the ensuing damage by aggravating hepatocyte injury and stimulating hepatocyte-HSC crosstalk.}, }
@article {pmid39150015, year = {2024}, author = {Janns, JH and Mikkelsen, JG}, title = {Gene editing by ferrying of CRISPR/Cas ribonucleoprotein complexes in enveloped virus-derived particles.}, journal = {Human gene therapy}, volume = {}, number = {}, pages = {}, doi = {10.1089/hum.2024.105}, pmid = {39150015}, issn = {1557-7422}, abstract = {The invention of next-generation CRISPR/Cas gene editing tools, like base and prime editing, for correction of gene variants causing disease, has created hope for in vivo use in patients leading to wider clinical translation. To realize this potential, delivery vehicles that can ferry gene editing tool kits safely and effectively into specific cell populations or tissues are in great demand. Here, we describe the development of enveloped retrovirus-derived particles as carriers of 'ready-to-work' ribonucleoprotein complexes consisting of Cas9-derived editor proteins and single guide RNAs. We present arguments for adapting viruses for cell-targeted protein delivery and describe the status after a decade-long development period, which has already shown effective editing in primary cells, including T-cells and hematopoietic stem cells, and in tissues targeted in vivo, including mouse retina, liver, and brain. Emerging evidence has demonstrated that engineered virus-derived nanoparticles can accommodate both base and prime editors, and seem to fertilize a sprouting hope that such particles can be further developed and produced on a large scale for therapeutic applications.}, }
@article {pmid39149034, year = {2024}, author = {Kadkhoda, H and Gholizadeh, P and Samadi Kafil, H and Ghotaslou, R and Pirzadeh, T and Ahangarzadeh Rezaee, M and Nabizadeh, E and Feizi, H and Aghazadeh, M}, title = {Role of CRISPR-Cas systems and anti-CRISPR proteins in bacterial antibiotic resistance.}, journal = {Heliyon}, volume = {10}, number = {14}, pages = {e34692}, doi = {10.1016/j.heliyon.2024.e34692}, pmid = {39149034}, issn = {2405-8440}, abstract = {The emergence and development of antibiotic resistance in bacteria is a serious threat to global public health. Antibiotic resistance genes (ARGs) are often located on mobile genetic elements (MGEs). They can be transferred among bacteria by horizontal gene transfer (HGT), leading to the spread of drug-resistant strains and antibiotic treatment failure. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated genes) is one of the many strategies bacteria have developed under long-term selection pressure to restrict the HGT. CRISPR-Cas systems exist in about half of bacterial genomes and play a significant role in limiting the spread of antibiotic resistance. On the other hand, bacteriophages and other MGEs encode a wide range of anti-CRISPR proteins (Acrs) to counteract the immunity of the CRISPR-Cas system. The Acrs could decrease the CRISPR-Cas system's activity against phages and facilitate the acquisition of ARGs and virulence traits for bacteria. This review aimed to assess the relationship between the CRISPR-Cas systems and Acrs with bacterial antibiotic resistance. We also highlighted the CRISPR technology and Acrs to control and prevent antibacterial resistance. The CRISPR-Cas system can target nucleic acid sequences with high accuracy and reliability; therefore, it has become a novel gene editing and gene therapy tool to prevent the spread of antibiotic resistance. CRISPR-based approaches may pave the way for developing smart antibiotics, which could eliminate multidrug-resistant (MDR) bacteria and distinguish between pathogenic and beneficial microorganisms. Additionally, the engineered anti-CRISPR gene-containing phages in combination with antibiotics could be used as a cutting-edge treatment approach to reduce antibiotic resistance.}, }
@article {pmid39147780, year = {2024}, author = {Saraswathy, VM and Zhou, L and Mokalled, MH}, title = {Single-cell analysis of innate spinal cord regeneration identifies intersecting modes of neuronal repair.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6808}, pmid = {39147780}, issn = {2041-1723}, support = {NS113915//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; }, mesh = {Animals ; *Zebrafish ; *Spinal Cord Injuries/metabolism ; *Single-Cell Analysis ; *Spinal Cord Regeneration ; *Neuronal Plasticity/physiology ; *Neurogenesis/genetics ; *Spinal Cord/metabolism ; Neurons/metabolism/physiology ; CRISPR-Cas Systems ; GABAergic Neurons/metabolism ; Recovery of Function ; Disease Models, Animal ; Nerve Regeneration/physiology ; Animals, Genetically Modified ; }, abstract = {Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here, we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory balance after injury. In addition, a transient population of injury-responsive neurons (iNeurons) show elevated plasticity 1 week post-injury. We found iNeurons are injury-surviving neurons that acquire a neuroblast-like gene expression signature after injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity-driven neural repair.}, }
@article {pmid39111828, year = {2024}, author = {Ma, G and Qi, H and Deng, H and Dong, L and Zhang, Q and Ma, J and Yang, Y and Yan, X and Duan, Y and Lei, H}, title = {Prime Editing of Vascular Endothelial Growth Factor Receptor 2 Attenuates Angiogenesis In Vitro.}, journal = {The CRISPR journal}, volume = {7}, number = {4}, pages = {188-196}, doi = {10.1089/crispr.2024.0019}, pmid = {39111828}, issn = {2573-1602}, mesh = {*Vascular Endothelial Growth Factor Receptor-2/metabolism/genetics ; Humans ; *Gene Editing/methods ; *Endothelial Cells/metabolism ; Neovascularization, Physiologic ; CRISPR-Cas Systems ; Phosphorylation ; RNA, Guide, CRISPR-Cas Systems ; Vascular Endothelial Growth Factor A/metabolism/genetics ; Neovascularization, Pathologic/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; Retina/metabolism ; Genetic Vectors ; Angiogenesis ; }, abstract = {Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo.}, }
@article {pmid39111827, year = {2024}, author = {Nakamura, A and Yamamoto, H and Yano, T and Hasegawa, R and Makino, Y and Mitsuda, N and Terakawa, T and Ito, S and Sugano, SS}, title = {Expanding the Genome-Editing Toolbox with Abyssicoccus albus Cas9 Using a Unique Protospacer Adjacent Motif Sequence.}, journal = {The CRISPR journal}, volume = {7}, number = {4}, pages = {197-209}, doi = {10.1089/crispr.2024.0013}, pmid = {39111827}, issn = {2573-1602}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Streptococcus pyogenes/genetics/enzymology ; Nucleotide Motifs ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {The genome-editing efficiency of the CRISPR-Cas9 system hinges on the recognition of the protospacer adjacent motif (PAM) sequence, which is essential for Cas9 binding to DNA. The commonly used Streptococcus pyogenes (SpyCas9) targets the 5'-NGG-3' PAM sequence, which does not cover all the potential genomic-editing sites. To expand the toolbox for genome editing, SpyCas9 has been engineered to recognize flexible PAM sequences and Cas9 orthologs have been used to recognize novel PAM sequences. In this study, Abyssicoccus albus Cas9 (AalCas9, 1059 aa), which is smaller than SpyCas9, was found to recognize a unique 5'-NNACR-3' PAM sequence. Modification of the guide RNA sequence improved the efficiency of AalCas9-mediated genome editing in both plant and human cells. Predicted structure-assisted introduction of a point mutation in the putative PAM recognition site shifted the sequence preference of AalCas9. These results provide insights into Cas9 diversity and novel tools for genome editing.}, }
@article {pmid39051564, year = {2024}, author = {Pandit, B and Fang, L and Kool, ET and Royzen, M}, title = {Reversible RNA Acylation Using Bio-Orthogonal Chemistry Enables Temporal Control of CRISPR-Cas9 Nuclease Activity.}, journal = {ACS chemical biology}, volume = {19}, number = {8}, pages = {1719-1724}, doi = {10.1021/acschembio.4c00117}, pmid = {39051564}, issn = {1554-8937}, mesh = {Humans ; Acylation ; *CRISPR-Cas Systems ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; RNA/metabolism/chemistry ; Green Fluorescent Proteins/genetics/metabolism ; }, abstract = {The CRISPR-Cas9 system is a widely popular tool for genome engineering. There is strong interest in developing tools for temporal control of CRISPR-Cas9 activity to address some of the challenges and to broaden the scope of potential applications. In this work, we describe a bio-orthogonal chemistry-based approach to control nuclease activity with temporal precision. We report a trans-cyclooctene (TCO)-acylimidazole reagent that acylates 2'-OH groups of RNA. Poly acylation ("cloaking") of RNA was optimized in vitro using a model 18-nt oligonucleotide, as well as CRISPR single guide RNA (sgRNA). Two hours of treatment completely inactivated sgRNA for Cas9-assisted DNA cleavage. Nuclease activity was restored upon addition of tetrazine, which removes the TCO moieties via a two-step process ("uncloaking"). The approach was applied to target the GFP gene in live HEK293 cells. GFP expression was analyzed by flow cytometry. In the future, we anticipate that our approach will be useful in the field of developmental biology, by enabling investigation of genes of interest at different stages of an organism's development.}, }
@article {pmid39046112, year = {2024}, author = {Wang, Y and Liu, M and Lin, X and Wang, H and Dong, N and Liu, H and Shao, H and Zhang, W}, title = {Genome Editing of Mammalian Cells Through RNA Transcript-Mediated Homologous Recombination Repair.}, journal = {Human gene therapy}, volume = {35}, number = {15-16}, pages = {555-563}, doi = {10.1089/hum.2024.025}, pmid = {39046112}, issn = {1557-7422}, mesh = {*Gene Editing/methods ; *Recombinational DNA Repair ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; Animals ; HEK293 Cells ; RNA/genetics/metabolism ; DNA Breaks, Double-Stranded ; }, abstract = {Double-stranded break (DSB) repair of eukaryotic DNA is mainly accomplished by nonhomologous end joining and homologous recombination (HR). Providing exogenous templates during HR repair can result in the editing of target genes, which is the central mechanism of the well-established clustered regularly interspaced short palindromic repeats (CRISPR) gene editing system. Currently, exogenous templates are mainly DNA molecules, which can provoke a cellular immune response within the cell. In order to verify the feasibility of RNA molecules as repair templates for HR in mammalian cell genome editing, we fused RNA template molecules to the 3'-end of single guide RNA (sgRNA), so that the sgRNA and the homologous template RNA form a single RNA molecule. The results show this construct can be used as a repair template to achieve target gene editing in mammalian cells. In addition, the factors influencing HR mediated by RNA template molecules were investigated, and it was found that increasing the length of homologous arms and inducing an R-loop near the DSBcan effectively promote HR repair. Furthermore, intracellular homologous chromosomes may compete with exogenous RNA templates. The findings in this article provide a reference for the utilization of RNA template molecules to mediate target gene editing in eukaryotic cells, as well as a basis for the study of the mechanism by which RNA molecules mediate the repair of DSBs.}, }
@article {pmid39033464, year = {2024}, author = {Sanford, PA and Woolston, BM}, title = {Development of a Recombineering System for the Acetogen Eubacterium limosum with Cas9 Counterselection for Markerless Genome Engineering.}, journal = {ACS synthetic biology}, volume = {13}, number = {8}, pages = {2505-2514}, doi = {10.1021/acssynbio.4c00253}, pmid = {39033464}, issn = {2161-5063}, mesh = {*Eubacterium/genetics ; *CRISPR-Cas Systems/genetics ; Metabolic Engineering/methods ; Recombination, Genetic/genetics ; Genome, Bacterial/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Recombinases/genetics/metabolism ; Genetic Engineering/methods ; Gene Editing/methods ; Multigene Family ; }, abstract = {Eubacterium limosum is a Clostridial acetogen that efficiently utilizes a wide range of single-carbon substrates and contributes to metabolism of health-associated compounds in the human gut microbiota. These traits have led to interest in developing it as a platform for sustainable CO2-based biofuel production to combat carbon emissions, and for exploring the importance of the microbiota in human health. However, synthetic biology and metabolic engineering in E. limosum have been hindered by the inability to rapidly make precise genomic modifications. Here, we screened a diverse library of recombinase proteins to develop a highly efficient oligonucleotide-based recombineering system based on the viral recombinase RecT. Following optimization, the system is capable of catalyzing ssDNA recombination at an efficiency of up to 2%. Addition of a Cas9 counterselection system eliminated unrecombined cells, with up to 100% of viable cells encoding the desired mutation, enabling creation of genomic point mutations in a scarless and markerless manner. We deployed this system to create a clean knockout of the extracellular polymeric substance (EPS) gene cluster, generating a strain incapable of biofilm formation. This approach is rapid and simple, not requiring laborious homology arm cloning, and can readily be retargeted to almost any genomic locus. This work overcomes a major bottleneck in E. limosum genetic engineering by enabling precise genomic modifications, and provides both a roadmap and associated recombinase plasmid library for developing similar systems in other Clostridia of interest.}, }
@article {pmid38976494, year = {2024}, author = {Waltz, M and Walker, RL and Flatt, MA and MacKay, D and Conley, JM and Juengst, ET and Cadigan, RJ}, title = {Challenging the Boundaries Between Treatment, Prevention, and Enhancement in Human Genome Editing.}, journal = {The CRISPR journal}, volume = {7}, number = {4}, pages = {180-187}, doi = {10.1089/crispr.2024.0021}, pmid = {38976494}, issn = {2573-1602}, support = {R01 HG010661/HG/NHGRI NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing/methods/ethics ; *Genome, Human ; CRISPR-Cas Systems ; Genetic Therapy/methods/ethics ; }, abstract = {Traditional distinctions between treatment and enhancement goals for human genome editing (HGE) have animated oversight considerations, yet these categories have been complicated by the addition of prevention as a possible target for HGE applications. To assess the role these three categories might play in continued HGE governance efforts, we report on interviews with genome editing scientists and governance group members. While some accepted traditional distinctions between treatment and enhancement and rejected the latter as unacceptable, others argued that the concept of enhancement is largely irrelevant or not as morally problematic as suggested. Others described how preventive goals for HGE create gray zones where prevention and enhancement may be difficult to distinguish, which may stymie uses of HGE. We conclude by discussing the governance implications of these various understandings of treatment, prevention, and enhancement as HGE research moves beyond the treatment of serious disease to embrace longer range preventive goals.}, }
@article {pmid38934772, year = {2024}, author = {Liu, L and Zhang, Z and Wang, C and Yan, F and Sun, W and Zhou, X and Miao, W and Zhou, H}, title = {Developing guanine base editors for G-to-T editing in rice.}, journal = {Journal of integrative plant biology}, volume = {66}, number = {8}, pages = {1557-1560}, doi = {10.1111/jipb.13729}, pmid = {38934772}, issn = {1744-7909}, support = {2023ZD04074//Biological Breeding-Major Projects/ ; YBXM2313//Nanfan special project of the Chinese Academy of Agricultural Sciences/ ; B23CJ0208//Hainan Seed Industry Laboratory/ ; }, mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Guanine/metabolism ; CRISPR-Cas Systems/genetics ; DNA Glycosylases/metabolism/genetics ; Thymine/metabolism ; }, abstract = {Two guanine base editors created using an engineered N-methylpurine DNA glycosylase with CRISPR systems achieved targeted G-to-T editing with 4.94-12.50% efficiency in rice (Oryza sativa). The combined use of the DNA glycosylase and deaminases enabled co-editing of target guanines with adenines or cytosines.}, }
@article {pmid38695644, year = {2024}, author = {Liu, Y and Cheng, Z and Chen, W and Wu, C and Chen, J and Sui, Y}, title = {Establishment of genome-editing system and assembly of a near-complete genome in broomcorn millet.}, journal = {Journal of integrative plant biology}, volume = {66}, number = {8}, pages = {1688-1702}, doi = {10.1111/jipb.13664}, pmid = {38695644}, issn = {1744-7909}, support = {2022YFD1400800//the National Key Research and Development Program of China/ ; }, mesh = {*Gene Editing/methods ; *Genome, Plant ; *Panicum/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Oxidoreductases ; }, abstract = {The ancient crop broomcorn millet (Panicum miliaceum L.) is an indispensable orphan crop in semi-arid regions due to its short life cycle and excellent abiotic stress tolerance. These advantages make it an important alternative crop to increase food security and achieve the goal of zero hunger, particularly in light of the uncertainty of global climate change. However, functional genomic and biotechnological research in broomcorn millet has been hampered due to a lack of genetic tools such as transformation and genome-editing techniques. Here, we successfully performed genome editing of broomcorn millet. We identified an elite variety, Hongmi, that produces embryogenic callus and has high shoot regeneration ability in in vitro culture. We established an Agrobacterium tumefaciens-mediated genetic transformation protocol and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome-editing system for Hongmi. Using these techniques, we produced herbicide-resistant transgenic plants and edited phytoene desaturase (PmPDS), which is involved in chlorophyll biosynthesis. To facilitate the rapid adoption of Hongmi as a model line for broomcorn millet research, we assembled a near-complete genome sequence of Hongmi and comprehensively annotated its genome. Together, our results open the door to improving broomcorn millet using biotechnology.}, }
@article {pmid38695642, year = {2024}, author = {Bai, Y and Liu, S and Bai, Y and Xu, Z and Zhao, H and Zhao, H and Lai, J and Liu, Y and Song, W}, title = {Application of CRISPR/Cas12i.3 for targeted mutagenesis in broomcorn millet (Panicum miliaceum L.).}, journal = {Journal of integrative plant biology}, volume = {66}, number = {8}, pages = {1544-1547}, doi = {10.1111/jipb.13669}, pmid = {38695642}, issn = {1744-7909}, support = {32271541//National Natural Science Foundation of China/ ; 2021YFD1200701//National Key Research and Development Program of China/ ; 2022010202//Key project of maize germplasm improvement/ ; B21HJ0509//Key project of maize germplasm improvement/ ; PC2023A01004//This research is supported by Pinduoduo-China Agricultural University Research Fund/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Panicum/genetics ; *Mutagenesis/genetics ; *Gene Editing/methods ; }, abstract = {A CRISPR/Cas12i.3-based gene editing platform is established in broomcorn millet (Panicum miliaceum) and used to create new elite germplasm for this ancient crop.}, }
@article {pmid39147496, year = {2024}, author = {da Silva, MF and Flaibam, B and de Mélo, AHF and Sampaio, U and Clerici, MTPS and Goldbeck, R}, title = {Optimization of enzymatic hydrolysis of cellulose extracted from bamboo culm for bioethanol production by Saccharomyces cerevisiae modified via CRISPR/Cas9.}, journal = {Food research international (Ottawa, Ont.)}, volume = {192}, number = {}, pages = {114768}, doi = {10.1016/j.foodres.2024.114768}, pmid = {39147496}, issn = {1873-7145}, mesh = {*Saccharomyces cerevisiae/metabolism/genetics ; Hydrolysis ; *Cellulose/metabolism ; *Ethanol/metabolism ; *Fermentation ; *CRISPR-Cas Systems ; *Biofuels ; Cellulase/metabolism ; Sasa ; Glucose/metabolism ; Hydrogen-Ion Concentration ; Biomass ; }, abstract = {In the context of biorefinery, researchers have been looking for lignocellulosic biomasses and ideal treatments to produce economically viable biofuels. In this scenario, the bamboo culm appears as a plant matrix of great potential, given the high cellulose content of low crystallinity. Thus, the objective and differential of this work was to determine the best conditions for enzymatic hydrolysis of cellulose extracted from bamboo culm and to evaluate its potential application in the production of bioethanol through Separate Hydrolysis and Fermentation (SHF) and Saccharification and Simultaneous Fermentation (SSF) by Saccharomyces cerevisiae modified via CRISPR/Cas9. The average cellulose extraction yield was 41.87 % with an extraction efficiency of 86.76 %. In general, as the hydrolysis time increased, an increase in glucose production was observed in almost all assays, with higher hydrolysis efficiency values at 72 h. The results ranged from 2.09 to 19.8 g/L of glucose obtained with efficiency values of 10.47 to 99 %. The best conditions were found in test 5 (temperature of 36 °C and pH 5.0, with only 10 FPU/g of substrate Cellic Ctec2 Novozymes ® cocktail). It is observed that for all hydrolysis times the independent variables pH and temperature were significant under the hydrolysis efficiency, showing a negative effect, indicating that higher values of the same promote lower values of the response variable. For bioethanol production, a maximum concentration of 7.84 g/L was observed for the SSH process after 4 h of fermentation, while for the SSF process it was 12.6 g/L after 24 h of fermentation, indicating the large potential of the simultaneous process together with the application of bamboo culm biomass for high production of biofuel.}, }
@article {pmid39146002, year = {2024}, author = {Zheng, Y and Huang, W and Runyanga Tinashe, J and Clemence, T and Shumbayaonda Chiyedzo, V and Enklebert, T and Deng, X and Zheng, Z}, title = {Genomic analysis of "Candidatus Liberibacter africanus" strain from Zimbabwe reveals unique virulence and prophage characteristics compared to "Ca. Liberibacter asiaticus".}, journal = {Plant disease}, volume = {}, number = {}, pages = {}, doi = {10.1094/PDIS-05-24-1141-SC}, pmid = {39146002}, issn = {0191-2917}, abstract = {Citrus Huanglongbing (HLB) is caused by the phloem-limited α-proteobacterium "Candidatus Liberibacter spp.", among which "Ca. Liberibacter africanus" (CLaf) have posed a significant threat to citrus production in Africa near a century. CLaf is closely related to the globally prevalent "Ca. Liberibacter asiaticus" (CLas), whereas little is known about the virulence of CLaf, primarily due to limited genome resources. In this study, we completed the whole-genome assembly and annotation of CLaf strain Zim (from Zimbabwe). Compared to CLas, a total of 102 CLaf unique genes were identified, including 14 potential Sec-dependent effectors (SDEs) genes, 29 phage-associated genes, and 59 genes with hypothetical function. Among 14 SDEs, V9J15_03810 was able to induce a significant hypersensitive response (HR) in Nicotiana benthamiana, indicating its potential as a virulence factor for CLaf. Genome analysis showed that CLaf strain Zim genome harbored a complete prophage region (named P-Zim-1, 42,208 bp). P-Zim-1 retained two immunosuppressive peroxidase genes (V9J15_02125 and V9J15_02130) homologous to CLas prophage SC1/SC2, whereas the lysogen-associated genes encoding integrase (V9J15_01970) and repressor (V9J15_02080) were homologous to the prophage of "Ca. Liberibacter solanacearum", the causal agent of potato zebra chip disease. In addition, P-Zim-1 carried a novel CRISPR/Cas system, including a CRISPR array (located within V9J15_02040, ranging from 443,643 to 443,897) and five CRISPR-related Cas proteins (V9J15_02005, 02010, 02015, 02025 and 02035). This study first characterized the unique genomic feature of CLaf related to virulence and prophage, which will facilitate future research on CLaf biology and African HLB management.}, }
@article {pmid39145933, year = {2024}, author = {Ren, W and Fu, C and Zhang, Y and Ju, X and Jiang, X and Song, J and Gong, M and Li, Z and Fan, W and Yao, J and Ding, Q}, title = {Zika virus NS5 protein inhibits type I interferon signaling via CRL3 E3 ubiquitin ligase-mediated degradation of STAT2.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {34}, pages = {e2403235121}, doi = {10.1073/pnas.2403235121}, pmid = {39145933}, issn = {1091-6490}, support = {82272302//MOST | National Natural Science Foundation of China (NSFC)/ ; 82241077//MOST | National Natural Science Foundation of China (NSFC)/ ; 32070153//MOST | National Natural Science Foundation of China (NSFC)/ ; 82341084//MOST | National Natural Science Foundation of China (NSFC)/ ; Z220018//| Beijing Municipal Natural Science Foundation ()/ ; 2021YFC2300200-04//MOST | National Key Research and Development Program of China (NKPs)/ ; 2023YFC2305900//MOST | National Key Research and Development Program of China (NKPs)/ ; 2022Z82WKJ013//Tsinghua University Vanke Special Fund for Public Health and Health Discipline Development/ ; NA//SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine/ ; 20231080039//Tsinghua University Dushi Program/ ; }, mesh = {Humans ; *STAT2 Transcription Factor/metabolism ; *Zika Virus/immunology/physiology/metabolism ; *Viral Nonstructural Proteins/metabolism/genetics ; *Interferon Type I/metabolism ; *Ubiquitin-Protein Ligases/metabolism/genetics ; *Zika Virus Infection/metabolism/immunology/virology ; *Signal Transduction ; *Ubiquitination ; *Cullin Proteins/metabolism ; *Proteolysis ; A549 Cells ; HEK293 Cells ; CRISPR-Cas Systems ; }, abstract = {The ZIKA virus (ZIKV) evades the host immune response by degrading STAT2 through its NS5 protein, thereby inhibiting type I interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism underlying this process has remained elusive. In this study, we performed a genome-wide CRISPR/Cas9 screen, revealing that ZSWIM8 as the substrate receptor of Cullin3-RING E3 ligase is required for NS5-mediated STAT2 degradation. Genetic depletion of ZSWIM8 and CUL3 substantially impeded NS5-mediated STAT2 degradation. Biochemical analysis illuminated that NS5 enhances the interaction between STAT2 and the ZSWIM8-CUL3 E3 ligase complex, thereby facilitating STAT2 ubiquitination. Moreover, ZSWIM8 knockout endowed A549 and Huh7 cells with partial resistance to ZIKV infection and protected cells from the cytopathic effects induced by ZIKV, which was attributed to the restoration of STAT2 levels and the activation of IFN signaling. Subsequent studies in a physiologically relevant model, utilizing human neural progenitor cells, demonstrated that ZSWIM8 depletion reduced ZIKV infection, resulting from enhanced IFN signaling attributed to the sustained levels of STAT2. Our findings shed light on the role of ZIKV NS5, serving as the scaffold protein, reprograms the ZSWIM8-CUL3 E3 ligase complex to orchestrate STAT2 proteasome-dependent degradation, thereby facilitating evasion of IFN antiviral signaling. Our study provides unique insights into ZIKV-host interactions and holds promise for the development of antivirals and prophylactic vaccines.}, }
@article {pmid39145745, year = {2024}, author = {Sharma, L and Rahman, F and Sharma, RA}, title = {The emerging role of biotechnological advances and artificial intelligence in tackling gluten sensitivity.}, journal = {Critical reviews in food science and nutrition}, volume = {}, number = {}, pages = {1-17}, doi = {10.1080/10408398.2024.2392158}, pmid = {39145745}, issn = {1549-7852}, abstract = {Gluten comprises an intricate network of hundreds of related but distinct proteins, mainly "gliadins" and "glutenins," which play a vital role in determining the rheological properties of wheat dough. However, ingesting gluten can trigger severe conditions in susceptible individuals, including celiac disease, wheat allergy, or non-celiac gluten sensitivity, collectively known as gluten-related disorders. This review provides a panoramic view, delving into the various aspects of gluten-triggered disorders, including symptoms, diagnosis, mechanism, and management. Though a gluten-free diet remains the primary option to manage gluten-related disorders, the emerging microbial and plant biotechnology tools are playing a transformative role in reducing the immunotoxicity of gluten. The enzymatic hydrolysis of gluten and the development of gluten-reduced/free wheat lines using RNAi and CRISPR/Cas technology are laying the foundation for creating safer wheat products. In addition to biotechnological interventions, the emerging artificial intelligence technologies are also bringing about a paradigm shift in the diagnosis and management of gluten-related disorders. Here, we provide a comprehensive overview of the latest developments and the potential these technologies hold for tackling gluten sensitivity.}, }
@article {pmid39145508, year = {2024}, author = {Lei, W and Hao, L and Qiu, H and Bian, K and Cui, T and Zeng, W and Zhang, Y and Yang, W and Zhang, B}, title = {Quantum-Dot-Encoded Beads-Enhanced CRISPR/Cas-Based Lateral-Flow Assay for the Amplification-Free, Sensitive, and Rapid Detection of Nucleic Acids in Breast Cancer.}, journal = {ACS applied materials &amp; interfaces}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsami.4c05388}, pmid = {39145508}, issn = {1944-8252}, abstract = {Nucleic acid detection plays a pivotal role in the accurate diagnosis of diseases. The CRISPR/Cas detection system, noted for its significant utility in a variety of applications, often necessitates enhanced sensitivity or specific signal amplification strategies, particularly for detecting low-abundance biomarkers. In this study, we present a quantum-dot-encoded beads (QDB)-energized CRISPR/Cas12-based lateral-flow assay (QDB-CRISPR-LFA). This method enables amplification-free, sensitive, and rapid detection (<40 min) of BRCA-1. We validated our method using contrived reference samples and nucleic acids extracted from tumor cells. The QDB-CRISPR-LFA provides a visual, more rapid alternative to the traditional BRCA-1 real-time RT-PCR assay. Significantly, through the integration of CRISPR's specificity and the high signal output of QDB, the detection threshold for BRCA-1 has been reduced to the femtomolar level, representing an enhancement of 2-4 orders of magnitude over existing CRISPR/Cas detection methods. This advancement underscores the potential of our approach in advancing nucleic acid detection techniques, which is crucial for the early and precise diagnosis of diseases.}, }
@article {pmid39143337, year = {2024}, author = {Kalim, AS and Nagata, K and Toriigahara, Y and Shirai, T and Kirino, K and Xiu-Ying, Z and Kondo, T and Kawakubo, N and Miyata, J and Matsuura, T and Tajiri, T}, title = {A lmod1a mutation causes megacystis microcolon intestinal hypoperistalsis in a CRISPR/Cas9-modified zebrafish model.}, journal = {Pediatric surgery international}, volume = {40}, number = {1}, pages = {225}, pmid = {39143337}, issn = {1437-9813}, support = {JP24K11785//Japan Society for the Promotion of Science/ ; }, mesh = {Animals ; *Zebrafish/genetics ; *Intestinal Pseudo-Obstruction/genetics ; *Disease Models, Animal ; *CRISPR-Cas Systems ; *Colon/abnormalities ; Mutation ; Urinary Bladder/abnormalities ; Abnormalities, Multiple/genetics ; Muscle Proteins/genetics ; Zebrafish Proteins/genetics ; }, abstract = {PURPOSE: Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is defined as a congenital visceral myopathy with genetic mutations. However, the etiology and pathophysiology are not fully understood. We aimed to generate a gene leiomodin-1a (lmod1a) modification technique to establish a zebrafish model of MMIHS.<br><br>METHODS: We targeted lmod1a in zebrafish using CRISPR/Cas9. After confirming the genotype, we measured the expression levels of the target gene and protein associated with MMIHS. A gut transit assay and spatiotemporal mapping were conducted to analyze the intestinal function.<br><br>RESULTS: Genetic confirmation showed a 5-base-pair deletion in exon 1 of lmod1a, which caused a premature stop codon. We observed significant mRNA downregulation of lmod1a, myh11, myod1, and acta2 and the protein expression of Lmod1 and Acta2 in the mutant group. A functional analysis of the lmod1a mutant zebrafish showed that its intestinal peristalsis was fewer, slower, and shorter in comparison to the wild type.<br><br>CONCLUSION: This study showed that targeted deletion of lmod1a in zebrafish resulted in depletion of MMIHS-related genes and proteins, resulting in intestinal hypoperistalsis. This model may have the potential to be utilized in future therapeutic approaches, such as drug discovery screening and gene repair therapy for MMIHS.}, }
@article {pmid39116631, year = {2024}, author = {Wang, T and Bai, L and Wang, G and Han, J and Wu, L and Chen, X and Zhang, H and Feng, J and Wang, Y and Wang, R and Zhang, X}, title = {SATCAS: A CRISPR/Cas13a-based simultaneous amplification and testing platform for one-pot RNA detection and SNPs distinguish in clinical diagnosis.}, journal = {Biosensors &amp; bioelectronics}, volume = {263}, number = {}, pages = {116636}, doi = {10.1016/j.bios.2024.116636}, pmid = {39116631}, issn = {1873-4235}, mesh = {*Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; *SARS-CoV-2/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; COVID-19/diagnosis/virology ; RNA, Viral/genetics ; Molecular Diagnostic Techniques/methods ; Hepatitis B virus/genetics/isolation &amp; purification ; }, abstract = {The clinical diagnosis of pathogen infectious diseases increasingly requires sensitive and rapid RNA detection technologies. The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system has shown immense potential in molecular diagnostics due to its trans-cleavage activity. However, most Cas13a-based detection methods require an amplicon transcription step, and the multi-step open-tube operations are prone to contamination, limiting their widespread application. Here, we propose an ultrasensitive (single-copy range, ∼aM) and rapid (within 40 min) isothermal one-pot RNA detection platform, termed SATCAS (Simultaneous Amplification and Testing platform based on Cas13a). This method effectively distinguishes viable bacteria (0%-100%) under constant total bacterial conditions, demonstrating its robustness and universality. SATCAS excels in identifying single nucleotide polymorphisms (SNPs), particularly detecting 0.5% drug-resistant mutations. We validated SATCAS by detecting infections in biological samples from 68 HBV, 23 EBV, and 48 SARS-CoV-2 patients, achieving 100% sensitivity, 92.86% specificity, and 97.06% accuracy in HBV infection testing. We anticipate that SATCAS has broad application potential in the early diagnosis, subtyping, drug resistance detection, and point-of-care monitoring of pathogen infectious diseases.}, }
@article {pmid39116629, year = {2024}, author = {Chen, C and Chen, S and Fu, Y and Wei, Y and Xie, L and Chen, M}, title = {Electrochemical sensing technology based on a ligation-initiated LAMP-assisted CRISPR/Cas12a system for high-specificity detection of EGFR E746-A750 deletion mutation.}, journal = {Biosensors &amp; bioelectronics}, volume = {263}, number = {}, pages = {116635}, doi = {10.1016/j.bios.2024.116635}, pmid = {39116629}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *ErbB Receptors/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Sequence Deletion ; Bacterial Proteins ; Endodeoxyribonucleases ; Molecular Diagnostic Techniques ; CRISPR-Associated Proteins ; }, abstract = {Epidermal growth factor receptor (EGFR) mutation status is pivotal in predicting the efficacy of tyrosine kinase inhibitor treatments against tumors. Among EGFR mutations, the E746-A750 deletion is particularly common and accurately quantifying it can guide targeted therapies. This study introduces a novel visual sensing technology using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system guided by ligation-initiated loop-mediated isothermal amplification (LAMP) to detect the del E746-A750 mutation in EGFR. Conventional LAMP primers were simplified by designing a pair of target-specific stem-loop DNA probes, enabling selective amplification of the target DNA. The CRISPR/Cas12a system was employed to identify the target nucleic acid and activate Cas12a trans-cleavage activity, thereby enhancing the specificity of the assay. Furthermore, the biosensor utilized high-performance nanomaterials such as triangular gold nanoparticles and graphdiyne, known for their large specific surface area, to enhance sensitivity effectively as a sensing platform. The proposed biosensor demonstrated outstanding specificity, achieving a low detection limit of 17 fM (S/N = 3). Consequently, this innovative strategy not only expands the application scope of CRISPR/Cas12a technology but also introduces a promising approach for clinical diagnostics in modern medicine.}, }
@article {pmid39111252, year = {2024}, author = {Zheng, B and Yan, J and Li, T and Zhao, Y and Xu, Z and Rao, R and Zhu, J and Hu, R and Li, Y and Yang, Y}, title = {Hydrophilic/hydrophobic modified microchip for detecting multiple gene doping candidates using CRISPR-Cas12a and RPA.}, journal = {Biosensors &amp; bioelectronics}, volume = {263}, number = {}, pages = {116631}, doi = {10.1016/j.bios.2024.116631}, pmid = {39111252}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems/genetics ; *Doping in Sports/prevention &amp; control ; Animals ; *Biosensing Techniques/instrumentation/methods ; Mice ; Humans ; *Hydrophobic and Hydrophilic Interactions ; *Lab-On-A-Chip Devices ; Erythropoietin/genetics/analysis ; Equipment Design ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {With significant advancements in understanding gene functions and therapy, the potential misuse of gene technologies, particularly in the context of sports through gene doping (GD), has come to the forefront. This raises concerns regarding the need for point-of-care testing of various GD candidates to counter illicit practices in sports. However, current GD detection techniques, such as PCR, lack the portability required for on-site multiplexed detection. In this study, we introduce an integrated microfluidics-based chip for multiplexed gene doping detection, termed MGD-Chip. Through the strategic design of hydrophilic and hydrophobic channels, MGD-Chip enables the RPA and CRISPR-Cas12a assays to be sequentially performed on the device, ensuring minimal interference and cross-contamination. Six potential GD candidates were selected and successfully tested simultaneously on the platform within 1 h. Demonstrating exceptional specificity, the platform achieved a detection sensitivity of 0.1 nM for unamplified target plasmids and 1 aM for amplified ones. Validation using mouse models established by injecting IGFI and EPO transgenes confirmed the platform's efficacy in detecting gene doping in real samples. This technology, capable of detecting multiple targets using portable elements, holds promise for real-time GD detection at sports events, offering a rapid, highly sensitive, and user-friendly solution to uphold the integrity of sports competitions.}, }
@article {pmid39102774, year = {2024}, author = {Yu, Y and Zhang, Y and Zhao, Y and Lv, K and Ai, L and Wu, Z and Song, Z and Zhang, J}, title = {Probiotic bacterial adsorption coupled with CRISPR/Cas12a system for mercury (II) ions detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {263}, number = {}, pages = {116627}, doi = {10.1016/j.bios.2024.116627}, pmid = {39102774}, issn = {1873-4235}, mesh = {*Mercury/analysis/chemistry ; *Biosensing Techniques/methods ; *Probiotics/chemistry ; *CRISPR-Cas Systems ; *Lacticaseibacillus rhamnosus/isolation &amp; purification/genetics ; Adsorption ; Limit of Detection ; }, abstract = {The complex sample matrix poses significant challenges in accurately detecting heavy metals. In view of its superior performance for the biological adsorption of heavy metals, probiotic bacteria can be explored for functional unit to eliminate matrix interference. Herein, Lactobacillus rhamnosus (LGG) demonstrates a remarkable tolerance and can adsorb up to 300 μM of Hg[2+], following the Freundlich isotherm model with the correlation coefficient (R[2]) value of 0.9881. Subsequently, by integrating the CRISPR/Cas12a system, a sensitive and specific fluorescent biosensor, "Cas12a-MB," has been developed for Hg[2+] detection. Specifically, Hg[2+] adsorbed onto LGG can specifically bind to the nucleic acid probe, thereby inhibiting the binding of the probe to LGG and the subsequent activation of the CRISPR/Cas12a system. Under optimal experimental conditions, with the detection time of 90 min and the detection limit of 0.44 nM, the "Cas12a-MB" biosensor offers a novel, eco-friendly approach for Hg[2+] detection, showcasing the innovative application of probiotics in biosensor.}, }
@article {pmid39089531, year = {2024}, author = {Bharathi Achudhan, A and Saleena, LM}, title = {Genomic reconstruction of unclassified microorganisms: Analysis of CRISPR arrays and genes involved in defense mechanisms.}, journal = {Gene}, volume = {928}, number = {}, pages = {148808}, doi = {10.1016/j.gene.2024.148808}, pmid = {39089531}, issn = {1879-0038}, mesh = {*Bacteriophages/genetics ; *Bacteria/genetics/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genome, Bacterial ; Soil Microbiology ; Metagenome ; Phylogeny ; CRISPR-Cas Systems ; }, abstract = {The constant battle between bacteria and viruses has led to the development of sophisticated antiviral defense strategies by bacteria to defend themselves against phages. This study analyzed a marshland metagenome to identify and characterize bacterial antiviral defense systems and phage interactions. We assembled 210 metagenome-assembled genomes (MAGs) from environmental DNA extracted from Pallikaranai marshland soil and 37 unclassified MAGs were filtered. MIMAG standards were followed, 2 high-quality and 15 medium-quality unclassified MAGs were picked. MINCED was used to identify 137 CRISPR arrays in the quality MAGs, and ViroBLAST was used to identify the phages that interact with the bacteria. About 242 spacer sequences were extracted from the CRISPR arrays, of which 54 had significant matches in the ViroBLAST database. 7 unverified bacteriophage species were also detected in the MAGs. The viral group of Caudoviricetes phage elements were identified as a frequent genome terminal repeat. The PADLOC identified 11 genes involved as a defense system in the MAGs. The PD-T4-6 defense system was found to be prevalent in 15 different unclassified MAGs. This study presents valuable insights intothe adaptations of unclassified bacteria to bacteriophages, as well as the genes used by these bacteria as a defense mechanism.}, }
@article {pmid39072477, year = {2024}, author = {Wu, Q and Xie, L and Ma, L and Long, X and Liu, L and Chen, A and Cui, Y and Zhang, Y and He, Y}, title = {A CRISPR/Cas12a-based fluorescence method for the amplified detection of total antioxidant capacity.}, journal = {Analytical methods : advancing methods and applications}, volume = {16}, number = {32}, pages = {5564-5570}, doi = {10.1039/d4ay01150c}, pmid = {39072477}, issn = {1759-9679}, mesh = {*CRISPR-Cas Systems/genetics ; *Antioxidants/analysis/chemistry ; Spectrometry, Fluorescence/methods ; Ascorbic Acid/analysis/chemistry ; Limit of Detection ; Oxides/chemistry ; Manganese Compounds/chemistry ; Beverages/analysis ; Fluorescence ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {The CRISPR/Cas12a system is a powerful signal amplification tool that has been widely used in nucleic acid detection. It has also been applied to the assay of non-nucleic acid targets, mainly relying on strategies for converting target determination into nucleic acid detection. Herein, we describe a CRISPR/Cas12a-based fluorescence method for sensitive detection of the total antioxidant capacity (TAC) by utilizing a strategy of converting TAC determination into Mn[2+] detection. Specifically, the reduction of MnO2 nanosheets by antioxidants produces plenty of Mn[2+], which accelerates the trans-cleavage activity of CRISPR/Cas12a. Thus, a fluorescence enhanced detection method for TAC was established, with a detection limit as low as 0.04 mg L[-1] for a typical antioxidant, ascorbic acid. More importantly, this method has been proven to successfully analyze TAC in beverages. The excellent analytical performance of this method demonstrates the great potential of the CRISPR/Cas12a system in simple and sensitive TAC analysis.}, }
@article {pmid39067412, year = {2024}, author = {Lei, X and Ding, L and Yang, X and Xu, F and Wu, Y and Yu, S}, title = {PAIT effect: Padlock activator inhibits the trans-cleavage activity of CRISPR/Cas12a.}, journal = {Biosensors &amp; bioelectronics}, volume = {263}, number = {}, pages = {116607}, doi = {10.1016/j.bios.2024.116607}, pmid = {39067412}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Calcium/metabolism/chemistry ; CRISPR-Associated Proteins/chemistry/metabolism ; DNA, Catalytic/chemistry ; Animals ; Milk/chemistry ; Endodeoxyribonucleases/metabolism/chemistry ; Bacterial Proteins/chemistry/metabolism ; Limit of Detection ; }, abstract = {The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a padlock activator which could inhibit the trans-cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca[2+] sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca[2+] dependent. In the presence of Ca[2+], DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the trans-cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca[2+] concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg[2+], Mn[2+], Cd[2+], Cu[2+], Na[+], Al[3+], K[+], Fe[2+], and Fe[3+] indicated high selectivity. Recovery ranged from 93.32% to 103.28% with RSDs from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca[2+] in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system.}, }
@article {pmid39053455, year = {2024}, author = {Sugiyama, Y and Okada, S and Daigaku, Y and Kusumoto, E and Ito, T}, title = {Strategic targeting of Cas9 nickase induces large segmental duplications.}, journal = {Cell genomics}, volume = {4}, number = {8}, pages = {100610}, doi = {10.1016/j.xgen.2024.100610}, pmid = {39053455}, issn = {2666-979X}, mesh = {*Saccharomyces cerevisiae/genetics ; Humans ; DNA Replication ; CRISPR-Associated Protein 9/genetics/metabolism ; Gene Duplication ; Replication Origin/genetics ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems/genetics ; }, abstract = {Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.}, }
@article {pmid39049599, year = {2024}, author = {Chen, Y and Zhang, X and Hu, G and Pan, Y and Guan, Y and Yang, J and Chen, H}, title = {A LAMP-CRISPR/Cas12b rapid detection platform for canine parvovirus detection.}, journal = {Analytical methods : advancing methods and applications}, volume = {16}, number = {32}, pages = {5519-5526}, doi = {10.1039/d4ay00977k}, pmid = {39049599}, issn = {1759-9679}, mesh = {Animals ; *Parvovirus, Canine/genetics/isolation &amp; purification ; Dogs ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods/veterinary ; *Parvoviridae Infections/veterinary/diagnosis/virology ; Dog Diseases/virology/diagnosis ; Molecular Diagnostic Techniques/methods/veterinary ; Sensitivity and Specificity ; Limit of Detection ; }, abstract = {Canine parvovirus (CPV) is one of the main pathogens causing toxic diarrhea in Chinese dogs, is the cause of large-scale epidemic of dogs, and poses a great threat to the dog industry in China. Rapid, sensitive, and specific CPV testing facilitates the timely diagnosis and treatment of sick dogs. The aim of this study was to build a LAMP-CRISPR/Cas12b platform for CPV detection. The loop mediated isothermal amplification (LAMP) technique was combined with CRISPR-Cas12b analysis to establish a "two-step" and "one-tube" CRISPR/Cas12b rapid CPV method, respectively. The detection system was constructed with specific LAMP primers and single guide RNA (sgRNA) for the highly conserved short fragment of the CPV gene, which could be detected within 1 h without cross-reaction with the other viruses causing canine diarrhea. The detection limits of both "two-step" and "one-tube" CRISPR/Cas12b reactions were 10[-1] copies per μL, which was 100 times more sensitive than qPCR and LAMP. In order to achieve point-of-care testing (POCT) of CPV, a one-tube LAMP-CRISPR/Cas12b nucleic acid extraction and detection platform based on magnetic nanoparticle enrichment technology was established to achieve "sample in-result out". The results of this method for simulated samples were compared with those of quantitative real-time PCR; the results showed 100% consistency, and the time was shorter, which could be used to detect the diseased dogs earlier and provide a basis for clinical diagnosis. The LAMP-CRISPR/Cas12b method established in this study provides a sensitive and specific method for rapid detection of CPV, and provides technical support for rapid diagnosis of CPV.}, }
@article {pmid38981565, year = {2024}, author = {Gao, Y and Zhang, X and Wang, X and Sun, R and Li, Y and Li, J and Quan, W and Yao, Y and Hou, Y and Li, D and Sun, Z}, title = {The clinical value of rapidly detecting urinary exosomal lncRNA RMRP in bladder cancer with an RT-RAA-CRISPR/Cas12a method.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {562}, number = {}, pages = {119855}, doi = {10.1016/j.cca.2024.119855}, pmid = {38981565}, issn = {1873-3492}, mesh = {Humans ; *Urinary Bladder Neoplasms/diagnosis/urine/genetics ; *RNA, Long Noncoding/urine/genetics ; *Exosomes/genetics ; *CRISPR-Cas Systems/genetics ; Male ; Middle Aged ; Female ; Aged ; }, abstract = {BACKGROUND AND AIMS: Bladder cancer (BCa) is a highly aggressive malignancy of the urinary system. Timely detection is imperative for enhancing BCa patient prognosis.<br><br>MATERIALS AND METHODS: This study introduces a novel approach for detecting long non-coding RNA (lncRNA) Mitochondrial RNA Processing Endoribonuclease (RMRP) in urine exosomes from BCa patients using the reverse transcription recombinase-aided amplification (RT-RAA) and clustered regularly interspaced short palindromic repeats and associated Cas12a proteins (CRISPR/Cas12a) technique. Various statistical methods were used to evaluate its diagnostic value for BCa.<br><br>RESULTS: The specificity of urine exosomal RMRP detection for BCa diagnosis was enhanced by using RT-RAA combined with CRISPR/Cas12a. The testing process duration was reduced to 30&#xa0;min, which supports rapid detection. Moreover, this approach allows the identification of target signals in real-time using blue light, facilitating immediate detection. In clinical sample analysis, this methodology exhibited a high level of diagnostic efficacy. This was evidenced by larger area under the curve values with receiver operating characteristic curve analysis compared with using traditional RT-qPCR methods, indicating superior diagnostic accuracy and sensitivity. Furthermore, the combined analysis of RMRP expression in urine exosomes detected by RT-RAA-CRISPR/Cas12a and NMP-22 expression may further enhance diagnostic accuracy.<br><br>CONCLUSIONS: The RT-RAA-CRISPR/Cas12a technology is a swift, sensitive, and uncomplicated method for nucleic acid detection. Because of its convenient and non-invasive sampling approach, user-friendly operation, and reproducibility, this technology is very promising for automated detection and holds favorable application possibilities within clinical environments.}, }
@article {pmid38964731, year = {2024}, author = {Allevato, MM and Trinh, S and Koshizuka, K and Nachmanson, D and Nguyen, TC and Yokoyama, Y and Wu, X and Andres, A and Wang, Z and Watrous, J and Molinolo, AA and Mali, P and Harismendy, O and Jain, M and Wild, R and Gutkind, JS}, title = {A genome-wide CRISPR screen reveals that antagonism of glutamine metabolism sensitizes head and neck squamous cell carcinoma to ferroptotic cell death.}, journal = {Cancer letters}, volume = {598}, number = {}, pages = {217089}, doi = {10.1016/j.canlet.2024.217089}, pmid = {38964731}, issn = {1872-7980}, mesh = {*Glutamine/metabolism ; Humans ; *Squamous Cell Carcinoma of Head and Neck/genetics/drug therapy/metabolism/pathology ; *Ferroptosis/drug effects/genetics ; Animals ; *Head and Neck Neoplasms/genetics/drug therapy/pathology/metabolism ; *Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Mice ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; }, abstract = {Glutamine is a conditionally essential amino acid for the growth and survival of rapidly proliferating cancer cells. Many cancers are addicted to glutamine, and as a result, targeting glutamine metabolism has been explored clinically as a therapeutic approach. Glutamine-catalyzing enzymes are highly expressed in primary and metastatic head and neck squamous cell carcinoma (HNSCC). However, the nature of the glutamine-associated pathways in this aggressive cancer type has not been elucidated. Here, we explored the therapeutic potential of a broad glutamine antagonist, DRP-104 (sirpiglenastat), in HNSCC tumors and aimed at shedding light on glutamine-dependent pathways in this disease. We observed a potent antitumoral effect of sirpiglenastat in HPV- and HPV + HNSCC xenografts. We conducted a whole-genome CRISPR screen and metabolomics analyses to identify mechanisms of sensitivity and resistance to glutamine metabolism blockade. These approaches revealed that glutamine metabolism blockade results in the rapid buildup of polyunsaturated fatty acids (PUFAs) via autophagy nutrient-sensing pathways. Finally, our analysis demonstrated that GPX4 mediates the protection of HNSCC cells from accumulating toxic lipid peroxides; hence, glutamine blockade sensitizes HNSCC cells to ferroptosis cell death upon GPX4 inhibition. These findings demonstrate the therapeutic potential of sirpiglenastat in HNSCC and establish a novel link between glutamine metabolism and ferroptosis, which may be uniquely translated into targeted glutamine-ferroptosis combination therapies.}, }
@article {pmid38900911, year = {2024}, author = {Pavani, R and Tripathi, V and Vrtis, KB and Zong, D and Chari, R and Callen, E and Pankajam, AV and Zhen, G and Matos-Rodrigues, G and Yang, J and Wu, S and Reginato, G and Wu, W and Cejka, P and Walter, JC and Nussenzweig, A}, title = {Structure and repair of replication-coupled DNA breaks.}, journal = {Science (New York, N.Y.)}, volume = {385}, number = {6710}, pages = {eado3867}, doi = {10.1126/science.ado3867}, pmid = {38900911}, issn = {1095-9203}, mesh = {*DNA Replication ; *DNA Breaks, Double-Stranded ; *Rad51 Recombinase/metabolism ; *Tumor Suppressor p53-Binding Protein 1/metabolism ; Humans ; *DNA Breaks, Single-Stranded ; *BRCA1 Protein/metabolism/genetics ; *CRISPR-Cas Systems ; Recombinational DNA Repair ; Homologous Recombination ; DNA Repair ; }, abstract = {Using CRISPR-Cas9 nicking enzymes, we examined the interaction between the replication machinery and single-strand breaks, one of the most common forms of endogenous DNA damage. We show that replication fork collapse at leading-strand nicks generates resected single-ended double-strand breaks (seDSBs) that are repaired by homologous recombination (HR). If these seDSBs are not promptly repaired, arrival of adjacent forks creates double-ended DSBs (deDSBs), which could drive genomic scarring in HR-deficient cancers. deDSBs can also be generated directly when the replication fork bypasses lagging-strand nicks. Unlike deDSBs produced independently of replication, end resection at nick-induced seDSBs and deDSBs is BRCA1-independent. Nevertheless, BRCA1 antagonizes 53BP1 suppression of RAD51 filament formation. These results highlight distinctive mechanisms that maintain replication fork stability.}, }
@article {pmid38775809, year = {2024}, author = {Sui, Y and Wang, T and Mei, Y and Zhu, Y and Jiang, W and Shen, J and Yan, S and Lu, W and Zhao, K and Mo, J and Wang, C and Tang, Y}, title = {Targeting Super-Enhancer-Driven Transcriptional Dependencies Suppresses Aberrant Hedgehog Pathway Activation and Overcomes Smoothened Inhibitor Resistance.}, journal = {Cancer research}, volume = {84}, number = {16}, pages = {2690-2706}, doi = {10.1158/0008-5472.CAN-23-3306}, pmid = {38775809}, issn = {1538-7445}, support = {81972646//National Natural Science Foundation of China (NSFC)/ ; 82002978//National Natural Science Foundation of China (NSFC)/ ; 82141131//National Natural Science Foundation of China (NSFC)/ ; 22ZR1436300//Science and Technology Commission of Shanghai Municipality (STCSM)/ ; SHSMU-ZDCX20212700//Innovative Research Team of High-Level Local University in Shanghai (Innovative Research Team of High-Level Local Universities in Shanghai)/ ; 2019M651527//China Postdoctoral Science Foundation (China Postdoctoral Foundation Project)/ ; 23YF1421300//Shanghai Sailing Program/ ; 2021cs001//Shanghai Charity Foundation/ ; }, mesh = {*Hedgehog Proteins/metabolism/genetics ; *Smoothened Receptor/genetics/metabolism/antagonists &amp; inhibitors ; Animals ; Humans ; Mice ; *Drug Resistance, Neoplasm/genetics ; *Signal Transduction/drug effects ; *Cyclin-Dependent Kinase 9/metabolism/antagonists &amp; inhibitors/genetics ; Xenograft Model Antitumor Assays ; Enhancer Elements, Genetic ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic/drug effects ; Small Molecule Libraries/pharmacology ; Neoplasms/drug therapy/genetics/metabolism/pathology ; CRISPR-Cas Systems ; }, abstract = {Aberrant activation of the Hedgehog (Hh) signaling pathway plays important roles in oncogenesis and therapeutic resistance in several types of cancer. The clinical application of FDA-approved Hh-targeted smoothened inhibitors (SMOi) is hindered by the emergence of primary or acquired drug resistance. Epigenetic and transcriptional-targeted therapies represent a promising direction for developing improved anti-Hh therapies. In this study, we integrated epigenetic/transcriptional-targeted small-molecule library screening with CRISPR/Cas9 knockout library screening and identified CDK9 and CDK12, two transcription elongation regulators, as therapeutic targets for antagonizing aberrant Hh activation and overcoming SMOi resistance. Inhibition of CDK9 or CDK12 potently suppressed Hh signaling and tumor growth in various SMOi responsive or resistant Hh-driven tumor models. Systemic epigenomic profiling elucidated the Hh-driven super-enhancer (SE) landscape and identified IRS1, encoding a critical component and cytoplasmic adaptor protein of the insulin-like growth factor (IGF) pathway, as an oncogenic Hh-driven SE target gene and effective therapeutic target in Hh-driven tumor models. Collectively, this study identifies SE-driven transcriptional dependencies that represent promising therapeutic vulnerabilities for suppressing the Hh pathway and overcoming SMOi resistance. As CDK9 and IRS inhibitors have already entered human clinical trials for cancer treatment, these findings provide comprehensive preclinical support for developing trials for Hh-driven cancers. Significance: Dissecting transcriptional dependencies driven by super-enhancers uncovers therapeutic targets in Hedgehog-driven cancers and identifies strategies for overcoming resistance to smoothened inhibitors.}, }
@article {pmid38763850, year = {2024}, author = {Chen, B and Deng, Y and Ren, X and Zhao, J and Jiang, C}, title = {CRISPR/Cas9 screening: unraveling cancer immunotherapy's 'Rosetta Stone'.}, journal = {Trends in molecular medicine}, volume = {30}, number = {8}, pages = {736-749}, doi = {10.1016/j.molmed.2024.04.014}, pmid = {38763850}, issn = {1471-499X}, mesh = {Humans ; *CRISPR-Cas Systems ; *Neoplasms/therapy/genetics/immunology/diagnosis ; *Immunotherapy/methods ; Animals ; Gene Editing/methods ; Biomarkers, Tumor/genetics ; }, abstract = {Clustered regularly interspaced palindromic repeats (CRISPR)-based technology, a powerful toolset for the unbiased functional genomic screening of biological processes, has facilitated several scientific breakthroughs in the biomedical field. Cancer immunotherapy has advanced the treatment of numerous malignancies that previously had restricted treatment options or unfavorable outcomes. In the realm of cancer immunotherapy, the application of CRISPR/CRISPR-associated protein 9 (Cas9)-based genetic perturbation screening has enabled the identification of genes, biomarkers, and signaling pathways that govern various cancer immunoreactivities, as well as the development of effective immunotherapeutic targets. In this review, we summarize the advances in CRISPR/Cas9-based screening for cancer immunotherapy and outline the immunotherapeutic targets identified via CRISPR screening based on cancer-type classification.}, }
@article {pmid39142774, year = {2024}, author = {Wu, Y and Chang, Y and Sun, Y and Wang, Y and Li, K and Lu, Z and Liu, Q and Wang, F and Wei, L}, title = {A multi-AS-PCR-coupled CRISPR/Cas12a assay for the detection of ten single-base mutations.}, journal = {Analytica chimica acta}, volume = {1320}, number = {}, pages = {343027}, doi = {10.1016/j.aca.2024.343027}, pmid = {39142774}, issn = {1873-4324}, mesh = {*Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems/genetics ; Humans ; Polymerase Chain Reaction/methods ; Mutation ; Mycobacterium tuberculosis/genetics ; Rifampin/pharmacology ; Limit of Detection ; Multiplex Polymerase Chain Reaction/methods ; Tuberculosis, Multidrug-Resistant/diagnosis/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Single-nucleotide polymorphism (SNP) detection is critical for diagnosing diseases, and the development of rapid and accurate diagnostic tools is essential for treatment and prevention. Allele-specific polymerase chain reaction (AS-PCR) is widely used for detecting SNPs with multiplexing capabilities, while CRISPR-based technologies provide high sensitivity and specificity in targeting mutation sites through specific guide RNAs (gRNAs). In this study, we have integrated the high sensitivity and specificity of CRISPR technology with the multiplexing capabilities of AS-PCR, achieving the simultaneous detection of ten single-base mutations. As for Multi-AS-PCR, our research identified that competitive inhibition of primers targeting the same loci, coupled with divergent amplification efficiencies of these primers, could result in diminished amplification efficiency. Consequently, we adjusted and optimized primer combinations and ratios to enhance the amplification efficacy of Multi-AS-PCR. Finally, we successfully developed a novel nested Multi-AS-PCR-Cas12a method for multiplex SNPs detection. To evaluate the clinical utility of this method in a real-world setting, we applied it to diagnose rifampicin-resistant tuberculosis (TB). The limit of detection (LoD) for the nested Multi-AS-PCR-Cas12a was 10[2] aM, achieving sensitivity, specificity, positive predictive value, and negative predictive value of 100 %, 93.33 %, 90.00 %, and 100 %, respectively, compared to sequencing. In summary, by employing an innovative design that incorporates a universal reverse primer alongside ten distinct forward allele-specific primers, the nested Multi-AS-PCR-Cas12a technique facilitates the parallel detection of ten rpoB gene SNPs. This method also holds broad potential for the detection of drug-resistant gene mutations in infectious diseases and tumors, as well as for the screening of specific genetic disorders.}, }
@article {pmid39142013, year = {2024}, author = {Martínez-Martínez, A and Amo, J and Jiménez-Estévez, E and Lara, A and Martínez, V and Rubio, F and Nieves-Cordones, M}, title = {SlCIPK9 regulates pollen tube elongation in tomato plants via a K[+]-independent mechanism.}, journal = {Plant physiology and biochemistry : PPB}, volume = {215}, number = {}, pages = {109039}, doi = {10.1016/j.plaphy.2024.109039}, pmid = {39142013}, issn = {1873-2690}, abstract = {Potassium (K[+]) is an essential macronutrient which contributes to osmotic- and turgor-related processes in plants. Calcineurin-B like Interacting Protein Kinases (CIPKs) play crucial roles in plants under low-K[+] supply since they activate root K[+] uptake transport systems such as AKT1 and AtHAK5. In Arabidopsis, AtCIPK9 is important for low-K[+] tolerance since atcipk9 plants exhibited poor growth and leaf chlorosis when K[+] was scarce. Part of these phenotypes could be ascribed to the activation of AtHAK5 by AtCIPK9. It has been reported that important differences exist between Arabidopsis and other plant species such as tomato with respect to the regulation of K[+] uptake systems. Thus, our aim was to evaluate the contribution of SlCIPK9, the homologous protein of AtCIPK9 in tomato, to K[+] nutrition. Unexpectedly, phenotyping experiments carried out with slcipk9 loss-of-function mutants revealed that SlCIPK9 did not play a clear role in tomato K[+] homeostasis. By contrast, it was found that SlCIPK9 contributed to pollen tube elongation, but not to pollen germination, via a K[+]-independent mechanism. Therefore, our results highlight the remarkable differences that exist in Ca[2+] signaling pathways between plant species and encourage the realization of more comparative studies as the one presented here.}, }
@article {pmid39143216, year = {2024}, author = {Yang, J and Li, X and He, Q and Wang, X and Tang, J and Wang, T and Zhang, Y and Yu, F and Zhang, S and Liu, Z and Zhang, L and Liao, F and Yin, H and Zhao, H and Deng, Z and Zhang, H}, title = {Structural basis for the activity of the type VII CRISPR-Cas system.}, journal = {Nature}, volume = {}, number = {}, pages = {}, pmid = {39143216}, issn = {1476-4687}, abstract = {The newly identified type VII CRISPR-Cas candidate system uses a CRISPR RNA-guided ribonucleoprotein complex formed by Cas5 and Cas7 proteins to target RNA[1]. However, the RNA cleavage is executed by a dedicated Cas14 nuclease, which is distinct from the effector nucleases of the other CRISPR-Cas systems. Here we report seven cryo-electron microscopy structures of the Cas14-bound interference complex at different functional states. Cas14, a tetrameric protein in solution, is recruited to the Cas5-Cas7 complex in a target RNA-dependent manner. The N-terminal catalytic domain of Cas14 binds a stretch of the substrate RNA for cleavage, whereas the C-terminal domain is primarily responsible for tethering Cas14 to the Cas5-Cas7 complex. The biochemical cleavage assays corroborate the captured functional conformations, revealing that Cas14 binds to different sites on the Cas5-Cas7 complex to execute individual cleavage events. Notably, a plugged-in arginine of Cas7 sandwiched by a C-shaped clamp of C-terminal domain precisely modulates Cas14 binding. More interestingly, target RNA cleavage is altered by a complementary protospacer flanking sequence at the 5' end, but not at the 3' end. Altogether, our study elucidates critical molecular details underlying the assembly of the interference complex and substrate cleavage in the type VII CRISPR-Cas system, which may help rational engineering of the type VII CRISPR-Cas system for biotechnological applications.}, }
@article {pmid39143188, year = {2024}, author = {Li, XD and Liu, LM and Xi, YC and Sun, QW and Luo, Z and Huang, HL and Wang, XW and Jiang, HB and Chen, W}, title = {Development of a base editor for convenient and multiplex genome editing in cyanobacteria.}, journal = {Communications biology}, volume = {7}, number = {1}, pages = {994}, pmid = {39143188}, issn = {2399-3642}, support = {No. 32170108 and No. 42188102//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Synechocystis/genetics ; *Anabaena/genetics/metabolism ; Genome, Bacterial ; Cyanobacteria/genetics/metabolism ; }, abstract = {Cyanobacteria are important primary producers, contributing to 25% of the global carbon fixation through photosynthesis. They serve as model organisms to study the photosynthesis, and are important cell factories for synthetic biology. To enable efficient genetic dissection and metabolic engineering in cyanobacteria, effective and accurate genetic manipulation tools are required. However, genetic manipulation in cyanobacteria by the conventional homologous recombination-based method and the recently developed CRISPR-Cas gene editing system require complicated cloning steps, especially during multi-site editing and single base mutation. This restricts the extensive research on cyanobacteria and reduces its application potential. In this study, a highly efficient and convenient cytosine base editing system was developed which allows rapid and precise C → T point mutation and gene inactivation in the genomes of Synechocystis and Anabaena. This base editing system also enables efficient multiplex editing and can be easily cured after editing by sucrose counter-selection. This work will expand the knowledge base regarding the engineering of cyanobacteria. The findings of this study will encourage the biotechnological applications of cyanobacteria.}, }
@article {pmid39138316, year = {2024}, author = {Park, HS and Gross, AC and Oh, S and Kim, NC}, title = {Orange maker: a CRISPR/Cas9-mediated genome editing and screening project to generate orange-eyed DarkJedi GAL4 lines by undergraduate students.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {18778}, pmid = {39138316}, issn = {2045-2322}, mesh = {*CRISPR-Cas Systems ; Animals ; *Gene Editing/methods ; *Drosophila Proteins/genetics ; *Eye Color/genetics ; Animals, Genetically Modified ; Transcription Factors/genetics ; Drosophila/genetics ; Students ; Drosophila melanogaster/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Eye Proteins ; ATP-Binding Cassette Transporters ; }, abstract = {One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research.}, }
@article {pmid39087644, year = {2024}, author = {Yang, J and Qin, G and Liu, Z and Zhang, H and Du, X and Ren, J and Qu, X}, title = {A Nanozyme-Boosted MOF-CRISPR Platform for Treatment of Alzheimer's Disease.}, journal = {Nano letters}, volume = {24}, number = {32}, pages = {9906-9915}, doi = {10.1021/acs.nanolett.4c02272}, pmid = {39087644}, issn = {1530-6992}, mesh = {*Alzheimer Disease/drug therapy/metabolism/genetics ; Animals ; Mice ; *Oxidative Stress/drug effects ; Humans ; NF-E2-Related Factor 2/metabolism ; Metal-Organic Frameworks/chemistry ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; Cerium/chemistry/therapeutic use/pharmacology ; Blood-Brain Barrier/metabolism ; Oxidation-Reduction ; Antioxidants/chemistry/pharmacology/therapeutic use ; }, abstract = {Rectifying the aberrant microenvironment of a disease through maintenance of redox homeostasis has emerged as a promising perspective with significant therapeutic potential for Alzheimer's disease (AD). Herein, we design and construct a novel nanozyme-boosted MOF-CRISPR platform (CMOPKP), which can maintain redox homeostasis and rescue the impaired microenvironment of AD. By modifying the targeted peptides KLVFFAED, CMOPKP can traverse the blood-brain barrier and deliver the CRISPR activation system for precise activation of the Nrf2 signaling pathway and downstream redox proteins in regions characterized by oxidative stress, thereby reinstating neuronal antioxidant capacity and preserving redox homeostasis. Furthermore, cerium dioxide possessing catalase enzyme-like activity can synergistically alleviate oxidative stress. Further in vivo studies demonstrate that CMOPKP can effectively alleviate cognitive impairment in 3xTg-AD mouse models. Therefore, our design presents an effective way for regulating redox homeostasis in AD, which shows promise as a therapeutic strategy for mitigating oxidative stress in AD.}, }
@article {pmid39075148, year = {2024}, author = {McCutcheon, SR and Rohm, D and Iglesias, N and Gersbach, CA}, title = {Epigenome editing technologies for discovery and medicine.}, journal = {Nature biotechnology}, volume = {42}, number = {8}, pages = {1199-1217}, pmid = {39075148}, issn = {1546-1696}, support = {U01AI146356//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; UM1HG012053//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01MH125236//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; RM1HG011123//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01CA289574//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; EFMA-1830957//National Science Foundation (NSF)/ ; HR0011-19-2-0008//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; }, mesh = {Humans ; *Epigenome/genetics ; *Gene Editing/methods ; Epigenomics/methods ; Epigenesis, Genetic ; CRISPR-Cas Systems/genetics ; Animals ; }, abstract = {Epigenome editing has rapidly evolved in recent years, with diverse applications that include elucidating gene regulation mechanisms, annotating coding and noncoding genome functions and programming cell state and lineage specification. Importantly, given the ubiquitous role of epigenetics in complex phenotypes, epigenome editing has unique potential to impact a broad spectrum of diseases. By leveraging powerful DNA-targeting technologies, such as CRISPR, epigenome editing exploits the heritable and reversible mechanisms of epigenetics to alter gene expression without introducing DNA breaks, inducing DNA damage or relying on DNA repair pathways.}, }
@article {pmid38963727, year = {2024}, author = {Li, X and Zhang, Y and Wen, X and Pan, J}, title = {Utilizing codon degeneracy in the design of donor DNA for CRISPR/Cas9-mediated gene editing to streamline the screening process for single amino acid mutations.}, journal = {The Plant journal : for cell and molecular biology}, volume = {119}, number = {4}, pages = {2133-2143}, doi = {10.1111/tpj.16903}, pmid = {38963727}, issn = {1365-313X}, support = {2018YFA0902500//the National Key R&amp;D Program of China/ ; IS23092//Beijing Natural Science Foundation/ ; 31991191//The National Natural Science Foundation of China/ ; 32370813//The National Natural Science Foundation of China/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Chlamydomonas reinhardtii/genetics ; *Codon/genetics ; Point Mutation/genetics ; }, abstract = {Chlamydomonas reinhardtii, a unicellular green alga, has been widely used as a model organism for studies of algal, plant and ciliary biology. The generation of targeted amino acid mutations is often necessary, and this can be achieved using CRISPR/Cas9 induced homology-directed repair to install genomic modifications from exogenous donor DNA. Due to the low gene editing efficiency, the technical challenge lies in identifying the mutant cells. Direct sequencing is not practical, and pre-screening is required. Here, we report a strategy for generating and screening for amino acid point mutations using the CRISPR/Cas9 gene editing system. The strategy is based on designing donor DNA using codon degeneracy, which enables the design of specific primers to facilitate mutant screening by PCR. An in vitro assembled RNP complex, along with a dsDNA donor and an antibiotic resistance marker, was electroporated into wild-type cells, followed by PCR screening. To demonstrate this principle, we have generated the E102K mutation in centrin and the K40R mutation in α-tubulin. The editing efficiencies at the target sites for Centrin, TUA1, TUA2 were 4, 24 and 8% respectively, based on PCR screening. More than 80% of the mutants with the expected size of PCR products were precisely edited, as revealed by DNA sequencing. Subsequently, the precision-edited mutants were biochemically verified. The introduction of codon degeneracy did not affect the gene expression of centrin and α-tubulins. Thus, this approach can be used to facilitate the identification of point mutations, especially in genes with low editing rates.}, }
@article {pmid38953638, year = {2024}, author = {Quan, J and Fan, Q and Simons, LM and Smukowski, SN and Pegg, C and Longnecker, R and Savas, JN and Hultquist, JF and Smith, GA}, title = {Leveraging biotin-based proximity labeling to identify cellular factors governing early alphaherpesvirus infection.}, journal = {mBio}, volume = {15}, number = {8}, pages = {e0144524}, doi = {10.1128/mbio.01445-24}, pmid = {38953638}, issn = {2150-7511}, support = {T32AI007476//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI148780//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; P30AI117943//Third Coast Center for AIDS Research (TC CFAR)/ ; R01AI14878//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; }, mesh = {Humans ; *Biotin/metabolism ; Zyxin/metabolism/genetics ; Animals ; Cell Line ; Herpesvirus 1, Human/genetics/physiology ; Herpesvirus 1, Suid/genetics/physiology ; Host-Pathogen Interactions ; Alphaherpesvirinae/genetics/metabolism ; CRISPR-Cas Systems ; Epithelial Cells/virology/metabolism ; }, abstract = {Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system in vivo. To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection.}, }
@article {pmid38953375, year = {2024}, author = {Bjånes, E and Stream, A and Janssen, AB and Gibson, PS and Bravo, AM and Dahesh, S and Baker, JL and Varble, A and Nizet, V and Veening, J-W}, title = {An efficient in vivo-inducible CRISPR interference system for group A Streptococcus genetic analysis and pathogenesis studies.}, journal = {mBio}, volume = {15}, number = {8}, pages = {e0084024}, doi = {10.1128/mbio.00840-24}, pmid = {38953375}, issn = {2150-7511}, support = {IZSEZ0_213879, 310030_192517, 310030_200792, 51NF40_180541//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (SNF)/ ; TMPFP3_210202//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (SNF)/ ; }, mesh = {*Streptococcus pyogenes/genetics/pathogenicity ; Animals ; Mice ; *Streptococcal Infections/microbiology ; *CRISPR-Cas Systems ; Virulence/genetics ; Gene Expression Regulation, Bacterial ; Disease Models, Animal ; Female ; Bacterial Proteins/genetics/metabolism ; }, abstract = {UNLABELLED: While genome-wide transposon mutagenesis screens have identified numerous essential genes in the significant human pathogen Streptococcus pyogenes (group A Streptococcus or GAS), many of their functions remain elusive. This knowledge gap is attributed in part to the limited molecular toolbox for controlling GAS gene expression and the bacterium's poor genetic transformability. CRISPR interference (CRISPRi), using catalytically inactive GAS Cas9 (dCas9), is a powerful approach to specifically repress gene expression in both bacteria and eukaryotes, but ironically, it has never been harnessed for controlled gene expression in GAS. In this study, we present a highly transformable and fully virulent serotype M1T1 GAS strain and introduce a doxycycline-inducible CRISPRi system for efficient repression of bacterial gene expression. We demonstrate highly efficient, oligo-based single guide RNA cloning directly to GAS, enabling the construction of a gene knockdown strain in just 2 days, in contrast to the several weeks typically required. The system is shown to be titratable and functional both in vitro and in vivo using a murine model of GAS infection. Furthermore, we provide direct in vivo evidence that the expression of the conserved cell division gene ftsZ is essential for GAS virulence, highlighting its promise as a target for emerging FtsZ inhibitors. Finally, we introduce SpyBrowse (https://veeninglab.com/SpyBrowse), a comprehensive and user-friendly online resource for visually inspecting and exploring GAS genetic features. The tools and methodologies described in this work are poised to facilitate fundamental research in GAS, contribute to vaccine development, and aid in the discovery of antibiotic targets.<br><br>IMPORTANCE: While group A Streptococcus (GAS) remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference (CRISPRi) for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS and, together with SpyBrowse, represents a valuable resource for researchers in the GAS field.}, }
@article {pmid37872410, year = {2024}, author = {Yao, D and Binan, L and Bezney, J and Simonton, B and Freedman, J and Frangieh, CJ and Dey, K and Geiger-Schuller, K and Eraslan, B and Gusev, A and Regev, A and Cleary, B}, title = {Scalable genetic screening for regulatory circuits using compressed Perturb-seq.}, journal = {Nature biotechnology}, volume = {42}, number = {8}, pages = {1282-1295}, pmid = {37872410}, issn = {1546-1696}, support = {R00 HG012203/HG/NHGRI NIH HHS/United States ; R01 HG006399/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 HG012133/HG/NHGRI NIH HHS/United States ; 1745303//National Science Foundation (NSF)/ ; R00HG012203//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30 CA008748/CA/NCI NIH HHS/United States ; HG006399//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; RM1HG006193//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; }, mesh = {Humans ; *Algorithms ; *Gene Regulatory Networks ; Single-Cell Analysis/methods ; Sequence Analysis, RNA/methods ; Genetic Testing/methods ; Genomics/methods ; Genome-Wide Association Study/methods ; CRISPR-Cas Systems/genetics ; }, abstract = {Pooled CRISPR screens with single-cell RNA sequencing readout (Perturb-seq) have emerged as a key technique in functional genomics, but they are limited in scale by cost and combinatorial complexity. In this study, we modified the design of Perturb-seq by incorporating algorithms applied to random, low-dimensional observations. Compressed Perturb-seq measures multiple random perturbations per cell or multiple cells per droplet and computationally decompresses these measurements by leveraging the sparse structure of regulatory circuits. Applied to 598 genes in the immune response to bacterial lipopolysaccharide, compressed Perturb-seq achieves the same accuracy as conventional Perturb-seq with an order of magnitude cost reduction and greater power to learn genetic interactions. We identified known and novel regulators of immune responses and uncovered evolutionarily constrained genes with downstream targets enriched for immune disease heritability, including many missed by existing genome-wide association studies. Our framework enables new scales of interrogation for a foundational method in functional genomics.}, }
@article {pmid37749268, year = {2024}, author = {Xu, Z and Sziraki, A and Lee, J and Zhou, W and Cao, J}, title = {Dissecting key regulators of transcriptome kinetics through scalable single-cell RNA profiling of pooled CRISPR screens.}, journal = {Nature biotechnology}, volume = {42}, number = {8}, pages = {1218-1223}, pmid = {37749268}, issn = {1546-1696}, support = {DP2 HG012522/HG/NHGRI NIH HHS/United States ; R01 AG076932/AG/NIA NIH HHS/United States ; RM1 HG011014/HG/NHGRI NIH HHS/United States ; 1DP2HG012522//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; RM1HG011014//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1R01AG076932//U.S. Department of Health &amp; Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)/ ; }, mesh = {*Single-Cell Analysis/methods ; Humans ; *Transcriptome/genetics ; CRISPR-Cas Systems/genetics ; Gene Expression Profiling/methods ; Kinetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation ; RNA/genetics/metabolism ; }, abstract = {We present a combinatorial indexing method, PerturbSci-Kinetics, for capturing whole transcriptomes, nascent transcriptomes and single guide RNA (sgRNA) identities across hundreds of genetic perturbations at the single-cell level. Profiling a pooled CRISPR screen targeting various biological processes, we show the gene expression regulation during RNA synthesis, processing and degradation, miRNA biogenesis and mitochondrial mRNA processing, systematically decoding the genome-wide regulatory network that underlies RNA temporal dynamics at scale.}, }
@article {pmid37697151, year = {2024}, author = {Kim, HS and Grimes, SM and Chen, T and Sathe, A and Lau, BT and Hwang, GH and Bae, S and Ji, HP}, title = {Direct measurement of engineered cancer mutations and their transcriptional phenotypes in single cells.}, journal = {Nature biotechnology}, volume = {42}, number = {8}, pages = {1254-1262}, pmid = {37697151}, issn = {1546-1696}, support = {R33 CA247700/CA/NCI NIH HHS/United States ; R33 CA278469/CA/NCI NIH HHS/United States ; S10 OD021763/OD/NIH HHS/United States ; R01 HG006137/HG/NHGRI NIH HHS/United States ; R01 HG006137/HG/NHGRI NIH HHS/United States ; R33 CA247700/CA/NCI NIH HHS/United States ; R33 CA278469/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Single-Cell Analysis/methods ; *Mutation ; *Neoplasms/genetics ; *Phenotype ; Cell Line, Tumor ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Tumor Suppressor Protein p53/genetics ; Transcriptome/genetics ; High-Throughput Nucleotide Sequencing/methods ; }, abstract = {Genome sequencing studies have identified numerous cancer mutations across a wide spectrum of tumor types, but determining the phenotypic consequence of these mutations remains a challenge. Here, we developed a high-throughput, multiplexed single-cell technology called TISCC-seq to engineer predesignated mutations in cells using CRISPR base editors, directly delineate their genotype among individual cells and determine each mutation's transcriptional phenotype. Long-read sequencing of the target gene's transcript identifies the engineered mutations, and the transcriptome profile from the same set of cells is simultaneously analyzed by short-read sequencing. Through integration, we determine the mutations' genotype and expression phenotype at single-cell resolution. Using cell lines, we engineer and evaluate the impact of >100 TP53 mutations on gene expression. Based on the single-cell gene expression, we classify the mutations as having a functionally significant phenotype.}, }
@article {pmid39137841, year = {2024}, author = {Zhao, W and Zhu, J and Yang, S and Liu, J and Sun, Z and Sun, H}, title = {Microalgal metabolic engineering facilitates precision nutrition and dietary regulation.}, journal = {The Science of the total environment}, volume = {}, number = {}, pages = {175460}, doi = {10.1016/j.scitotenv.2024.175460}, pmid = {39137841}, issn = {1879-1026}, abstract = {Microalgae have gained considerable attention as promising candidates for precision nutrition and dietary regulation due to their versatile metabolic capabilities. This review innovatively applies system metabolic engineering to utilize microalgae for precision nutrition and sustainable diets, encompassing the construction of microalgal cell factories, cell cultivation and practical application of microalgae. Manipulating the metabolic pathways and key metabolites of microalgae through multi-omics analysis and employing advanced metabolic engineering strategies, including ZFNs, TALENs, and the CRISPR/Cas system, enhances the production of valuable bioactive compounds, such as omega-3 fatty acids, antioxidants, and essential amino acids. This work begins by providing an overview of the metabolic diversity of microalgae and their ability to thrive in diverse environmental conditions. It then delves into the principles and strategies of metabolic engineering, emphasizing the genetic modifications employed to optimize microalgal strains for enhanced nutritional content. Enhancing PSY, BKT, and CHYB benefits carotenoid synthesis, whereas boosting ACCase, fatty acid desaturases, and elongases promotes polyunsaturated fatty acid production. Here, advancements in synthetic biology, evolutionary biology and machine learning are discussed, offering insights into the precision and efficiency of metabolic pathway manipulation. Also, this review highlights the potential impact of microalgal precision nutrition on human health and aquaculture. The optimized microalgal strains could serve as sustainable and cost-effective sources of nutrition for both human consumption and aquaculture feed, addressing the growing demand for functional foods and environmentally friendly feed alternatives. The tailored microalgal strains are anticipated to play a crucial role in meeting the nutritional needs of diverse populations and contributing to sustainable food production systems.}, }
@article {pmid39137494, year = {2024}, author = {Laub, MT and Typas, A}, title = {Principles of bacterial innate immunity against viruses.}, journal = {Current opinion in immunology}, volume = {89}, number = {}, pages = {102445}, doi = {10.1016/j.coi.2024.102445}, pmid = {39137494}, issn = {1879-0372}, abstract = {All organisms must defend themselves against viral predators. This includes bacteria, which harbor immunity factors such as restriction-modification systems and CRISPR-Cas systems. More recently, a plethora of additional defense systems have been identified, revealing a richer, more sophisticated immune system than previously appreciated. Some of these newly identified defense systems have distant homologs in mammals, suggesting an ancient evolutionary origin of some facets of mammalian immunity. An even broader conservation exists at the level of how these immunity systems operate. Here, we focus at this level, reviewing key principles and high-level attributes of innate immunity in bacteria that are shared with mammalian immunity, while also noting key differences, with a particular emphasis on how cells sense viral infection.}, }
@article {pmid39134917, year = {2024}, author = {Khan, FA and Ali, A and Wu, D and Huang, C and Zulfiqar, H and Ali, M and Ahmed, B and Yousaf, MR and Putri, EM and Negara, W and Imran, M and Pandupuspitasari, NS}, title = {Editing microbes to mitigate enteric methane emissions in livestock.}, journal = {World journal of microbiology &amp; biotechnology}, volume = {40}, number = {10}, pages = {300}, pmid = {39134917}, issn = {1573-0972}, mesh = {*Methane/metabolism ; Animals ; *Livestock ; *Rumen/microbiology/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Greenhouse Gases/metabolism ; Cattle ; Goats ; Microbial Consortia ; Sheep ; Bacteria/metabolism/genetics/classification ; Gastrointestinal Microbiome ; Ruminants/microbiology ; }, abstract = {Livestock production significantly contributes to greenhouse gas (GHG) emissions particularly methane (CH4) emissions thereby influencing climate change. To address this issue further, it is crucial to establish strategies that simultaneously increase ruminant productivity while minimizing GHG emissions, particularly from cattle, sheep, and goats. Recent advancements have revealed the potential for modulating the rumen microbial ecosystem through genetic selection to reduce methane (CH4) production, and by microbial genome editing including CRISPR/Cas9, TALENs (Transcription Activator-Like Effector Nucleases), ZFNs (Zinc Finger Nucleases), RNA interference (RNAi), Pime editing, Base editing and double-stranded break-free (DSB-free). These technologies enable precise genetic modifications, offering opportunities to enhance traits that reduce environmental impact and optimize metabolic pathways. Additionally, various nutrition-related measures have shown promise in mitigating methane emissions to varying extents. This review aims to present a future-oriented viewpoint on reducing methane emissions from ruminants by leveraging CRISPR/Cas9 technology to engineer the microbial consortia within the rumen. The ultimate objective is to develop sustainable livestock production methods that effectively decrease methane emissions, while maintaining animal health and productivity.}, }
@article {pmid39133651, year = {2024}, author = {Su, C and Kent, CL and Pierpoint, M and Floyd, W and Luo, L and Williams, NT and Ma, Y and Peng, B and Lazarides, AL and Subramanian, A and Himes, JE and Perez, VM and Hernansaiz-Ballesteros, RD and Roche, KE and Modliszewski, JL and Selitsky, SR and Shinohara, ML and Wisdom, AJ and Moding, EJ and Mowery, YM and Kirsch, DG}, title = {Enhancing radiotherapy response via intratumoral injection of a TLR9 agonist in autochthonous murine sarcomas.}, journal = {JCI insight}, volume = {9}, number = {14}, pages = {}, doi = {10.1172/jci.insight.178767}, pmid = {39133651}, issn = {2379-3708}, mesh = {Animals ; *Toll-Like Receptor 9/agonists ; Mice ; *Oligodeoxyribonucleotides/pharmacology/administration &amp; dosage ; *CD8-Positive T-Lymphocytes/immunology/drug effects ; Sarcoma/radiotherapy/therapy/pathology ; Injections, Intralesional ; CRISPR-Cas Systems ; Sarcoma, Experimental/pathology/radiotherapy ; Female ; }, abstract = {Radiation therapy (RT) is frequently used to treat cancers, including soft-tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to RT in transplanted tumors, but the mechanisms of this enhancement remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft-tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and 2 doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to CpG+RT, we performed bulk RNA-Seq, single-cell RNA-Seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and IFN-γ. CpG+RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG+RT, TCR clonality analysis suggests an increase in clonal T cell dominance. Collectively, these findings demonstrate that CpG+RT significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft-tissue sarcoma.}, }
@article {pmid39093040, year = {2024}, author = {Pataer, P and Zhang, P and Li, Z}, title = {Single Methylation Sensitive Restriction Endonuclease-Based Cascade Exponential Amplification Assay for Visual Detection of DNA Methylation at Single-Molecule Level.}, journal = {Analytical chemistry}, volume = {96}, number = {32}, pages = {13335-13343}, doi = {10.1021/acs.analchem.4c03638}, pmid = {39093040}, issn = {1520-6882}, mesh = {*DNA Methylation ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *DNA Restriction Enzymes/metabolism ; CRISPR-Cas Systems/genetics ; DNA/chemistry/genetics ; }, abstract = {Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 μL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.}, }
@article {pmid39092917, year = {2024}, author = {Yang, R and Ji, J and Ding, L and Yuan, X and Qu, L and Wu, Y and Li, Y}, title = {CRISPR-Enhanced Photocurrent Polarity Switching for Dual-lncRNA Detection Combining Deep Learning for Cancer Diagnosis.}, journal = {Analytical chemistry}, volume = {96}, number = {32}, pages = {13278-13284}, doi = {10.1021/acs.analchem.4c02617}, pmid = {39092917}, issn = {1520-6882}, mesh = {*RNA, Long Noncoding/genetics ; Humans ; *Deep Learning ; *Biosensing Techniques ; Quantum Dots/chemistry ; Electrochemical Techniques ; CRISPR-Cas Systems/genetics ; Neoplasms/diagnosis/genetics ; Cadmium Compounds/chemistry ; Sulfides/chemistry ; Limit of Detection ; Photochemical Processes ; }, abstract = {Abnormal expression in long noncoding RNAs (lncRNAs) is closely associated with cancers. Herein, a novel CRISPR/Cas13a-enhanced photocurrent-polarity-switching photoelectrochemical (PEC) biosensor was engineered for the joint detection of dual lncRNAs, using deep learning (DL) to assist in cancer diagnosis. After target lncRNA-activated CRISPR/Cas13a cleaves to induce DNAzyme bidirectional walkers with the help of cofactor Mg[2+], nitrogen-doped carbon-Cu/Cu2O octahedra are introduced into the biosensor, producing a photocurrent in the opposite direction of CdS quantum dots (QDs). The developed PEC biosensor shows high specificity and sensitivity with limits of detection down to 25.5 aM for lncRNA HOTAIR and 53.1 aM for lncRNA MALAT1. More importantly, this platform for the lncRNA joint assay in whole blood can successfully differentiate cancers from healthy people. Furthermore, the DL model is applied to explore the potential pattern hidden in data of the established technology, and the accuracy of DL cancer diagnosis can acquire 93.3%. Consequently, the developed platform offers a new avenue for lncRNA joint detection and early intelligent diagnosis of cancer.}, }
@article {pmid39082193, year = {2024}, author = {Peng, X and Mei, X and Liu, X and Zhang, G and Li, Y}, title = {Exonuclease III/Cas12a Cascade Amplification Strategy and Smartphone-Based Portable Fluorescence Detector to Repurpose the Commercial AFP Strip for the POCT of Multiple RNAs.}, journal = {Analytical chemistry}, volume = {96}, number = {32}, pages = {13252-13259}, doi = {10.1021/acs.analchem.4c02366}, pmid = {39082193}, issn = {1520-6882}, mesh = {*Smartphone ; *alpha-Fetoproteins/analysis ; *Exodeoxyribonucleases/metabolism/chemistry ; *Nucleic Acid Amplification Techniques ; *Point-of-Care Testing ; Humans ; CRISPR-Cas Systems ; Fluorescence ; CRISPR-Associated Proteins/metabolism ; SARS-CoV-2/isolation &amp; purification/genetics ; Limit of Detection ; Biosensing Techniques/methods ; RNA, Viral/analysis ; Spectrometry, Fluorescence ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Point of care testing (POCT) of nucleic acid (NA) contributes to the timely disease diagnosis, like bacteria and virus screening in households or resource-constrained areas, but its development has always been stagnant. Herein, we proposed an exonuclease III cascaded with CRISPR/Cas12a (Exo-III/Cas12a) amplification strategy and constructed a smartphone-based portable fluorescence detector (SPFD) to repurpose the commercial alpha-fetoprotein (AFP) strip for the ultrasensitive and hand-held detection of NA samples. In detail, the target-initiated-Exo-III/Cas12a strategy realizes the signal amplification and liberates AFP from magnetic beads through the trans-cleavages of activated Cas12a toward the AFP aptamer. After magnetic separation and migration, the fluorescence signals of the test (FT) and control (FC) lines on the AFP strip were digitally output by the SPFD, and the FT/FC was employed for the quantitative analysis to minimize external disturbances and improve accuracy. We experimentally assessed the universe applicability of the proposed NA-POCT platform toward miRNA-155, 16S rRNA of Staphylococcus aureus, and ORF1a/b RNA of Covid-19 pseudovirus, achieving favorable detection limits of 42 aM, 18 CFU/mL, and 87 copies/μL, respectively. Moreover, its simplicity, universality, and admirable detection performance demonstrate a great potential in the aspect of rapidly transforming the existing POCT devices for multiple new applications at the time of need.}, }
@article {pmid39073893, year = {2024}, author = {Elkayam, S and Tziony, I and Orenstein, Y}, title = {DeepCRISTL: deep transfer learning to predict CRISPR/Cas9 on-target editing efficiency in specific cellular contexts.}, journal = {Bioinformatics (Oxford, England)}, volume = {40}, number = {8}, pages = {}, doi = {10.1093/bioinformatics/btae481}, pmid = {39073893}, issn = {1367-4811}, support = {//supported by the Israel Science Foundation/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Deep Learning ; Humans ; Computational Biology/methods ; Machine Learning ; }, abstract = {MOTIVATION: CRISPR/Cas9 technology has been revolutionizing the field of gene editing. Guide RNAs (gRNAs) enable Cas9 proteins to target specific genomic loci for editing. However, editing efficiency varies between gRNAs and so computational methods were developed to predict editing efficiency for any gRNA of interest. High-throughput datasets of Cas9 editing efficiencies were produced to train machine-learning models to predict editing efficiency. However, these high-throughput datasets have a low correlation with functional and endogenous datasets, which are too small to train accurate machine-learning models on.<br><br>RESULTS: We developed DeepCRISTL, a deep-learning model to predict the editing efficiency in a specific cellular context. DeepCRISTL takes advantage of high-throughput datasets to learn general patterns of gRNA editing efficiency and then fine-tunes the model on functional or endogenous data to fit a specific cellular context. We tested two state-of-the-art models trained on high-throughput datasets for editing efficiency prediction, our newly improved DeepHF and CRISPRon, combined with various transfer-learning approaches. The combination of CRISPRon and fine-tuning all model weights was the overall best performer. DeepCRISTL outperformed state-of-the-art methods in predicting editing efficiency in a specific cellular context on functional and endogenous datasets. Using saliency maps, we identified and compared the important features learned by DeepCRISTL across cellular contexts. We believe DeepCRISTL will improve prediction performance in many other CRISPR/Cas9 editing contexts by leveraging transfer learning to utilize both high-throughput datasets and smaller and more biologically relevant datasets.<br><br>DeepCRISTL is available via https://github.com/OrensteinLab/DeepCRISTL.}, }
@article {pmid39020048, year = {2024}, author = {Du Toit, A}, title = {Tearing CRISPR apart.}, journal = {Nature reviews. Microbiology}, volume = {22}, number = {9}, pages = {524}, doi = {10.1038/s41579-024-01084-4}, pmid = {39020048}, issn = {1740-1534}, mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Bacteria/genetics ; }, }
@article {pmid38874509, year = {2024}, author = {Hsiao, S and Chen, S and Jiang, Y and Wang, Q and Yang, Y and Lai, Y and Zhong, T and Liao, J and Wu, Y}, title = {Library-Assisted Evolution in Eukaryotic Cells Yield Adenine Base Editors with Enhanced Editing Specificity.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {11}, number = {30}, pages = {e2309004}, doi = {10.1002/advs.202309004}, pmid = {38874509}, issn = {2198-3844}, support = {2023YFC3403401//National Key R&amp;D Program of China/ ; 82270125//National Natural Science Foundation of China/ ; 32300667//National Natural Science Foundation of China/ ; 32371535//National Natural Science Foundation of China/ ; 23HC1400400//Project of Shanghai Municipal Science and Technology Commission/ ; //National Program for Support of Top-Notch Young Professionals/ ; }, mesh = {*Gene Editing/methods ; Humans ; *Adenine/metabolism ; *Eukaryotic Cells/metabolism ; CRISPR-Cas Systems/genetics ; Gene Library ; HEK293 Cells ; }, abstract = {The current-generation adenine base editor (ABE) ABE8e, which has evolved from the prokaryotic evolution system, exhibits high efficiency in mediating A-to-G conversion and is presumed to be promising for gene therapy. However, its much wider editing window and substantially higher off-target editing activity restricted its applications in precise base editing for therapeutic use. This study uses a library-assisted protein evolution approach using eukaryotic cells to generate ABE variants with improved specificity and reduced off-target editing while maintaining high activity in human cells. The study generated an expanded set of ABEs with efficient editing activities and chose four evolved variants that offered either similar or modestly higher efficiency within a narrower editing window of protospacer position ≈4-7 compared to that of ABE8e in human cells, which would enable minimized bystander editing. Moreover, these variants resulted in reduced off-target editing events when delivered as plasmid or mRNA into human cells. Finally, these variants can install both disease-suppressing mutations and disease-correcting mutations efficiently with minimal undesired bystander editing making them promising approaches for specific therapeutic edits. In summary, the work establishes a mutant-library-assisted protein evolution method in eukaryotic cells and generates alternative ABE variants as efficient tools for precise human genome editing.}, }
@article {pmid38725246, year = {2024}, author = {Yu, M and Kuang, Y and Wang, C and Wu, X and Li, S and Zhang, D and Sun, W and Zhou, X and Ren, B and Zhou, H}, title = {Diverse nucleotide substitutions in rice base editing mediated by novel TadA variants.}, journal = {Plant communications}, volume = {5}, number = {8}, pages = {100926}, doi = {10.1016/j.xplc.2024.100926}, pmid = {38725246}, issn = {2590-3462}, mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Plant Proteins/genetics ; }, abstract = {CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops. The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes. Compared with the well-developed adenine base editors (ABEs), cytosine base editors (CBEs) and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice, significantly limiting their application. Here, we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice. We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID∗Δ, and TadA-CDd outperformed TadA-E27R/N46L. A C-to-G base editor (CGBE) engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P. In addition, a dual base editor constructed with a single protein, TadDE, enabled simultaneous, highly efficient C-to-T and A-to-G editing in rice. Collectively, our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice, providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.}, }
@article {pmid38570712, year = {2024}, author = {Wang, S and Xiong, Y and Luo, Y and Shen, Y and Zhang, F and Lan, H and Pang, Y and Wang, X and Li, X and Zheng, X and Lu, X and Liu, X and Cheng, Y and Wu, T and Dong, Y and Lu, Y and Cui, J and Jia, X and Yang, S and Wang, L and Wang, Y}, title = {Genome-wide CRISPR screens identify PKMYT1 as a therapeutic target in pancreatic ductal adenocarcinoma.}, journal = {EMBO molecular medicine}, volume = {16}, number = {5}, pages = {1115-1142}, pmid = {38570712}, issn = {1757-4684}, support = {82072974//the National Natural Science Foundation of China/ ; 82120108020//the National Natural Science Foundation of China/ ; 20JC1419200//the Basic Research Project of Shanghai Science and Technology Commission/ ; 20Z11900300//the Innovation Program of Shanghai Science and Technology Committee/ ; 2023YFE0117900//the National Key Research and Development Program of China/ ; }, mesh = {Animals ; Humans ; Mice ; Apoptosis/genetics ; *Carcinoma, Pancreatic Ductal/genetics/drug therapy/pathology ; Cell Cycle Proteins/metabolism/genetics/antagonists &amp; inhibitors ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Membrane Proteins ; *Pancreatic Neoplasms/genetics/drug therapy/pathology ; *Protein Serine-Threonine Kinases/metabolism/genetics/antagonists &amp; inhibitors ; *Protein-Tyrosine Kinases/antagonists &amp; inhibitors/genetics/metabolism ; Proto-Oncogene Proteins/metabolism/genetics/antagonists &amp; inhibitors ; CRISPR-Cas Systems ; }, abstract = {Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with an overall 5-year survival rate of <12% due to the lack of effective treatments. Novel treatment strategies are urgently needed. Here, PKMYT1 is identified through genome-wide CRISPR screens as a non-mutant, genetic vulnerability of PDAC. Higher PKMYT1 expression levels indicate poor prognosis in PDAC patients. PKMYT1 ablation inhibits tumor growth and proliferation in vitro and in vivo by regulating cell cycle progression and inducing apoptosis. Moreover, pharmacological inhibition of PKMYT1 shows efficacy in multiple PDAC cell models and effectively induces tumor regression without overt toxicity in PDAC cell line-derived xenograft and in more clinically relevant patient-derived xenograft models. Mechanistically, in addition to its canonical function of phosphorylating CDK1, PKMYT1 functions as an oncogene to promote PDAC tumorigenesis by regulating PLK1 expression and phosphorylation. Finally, TP53 function and PRKDC activation are shown to modulate the sensitivity to PKMYT1 inhibition. These results define PKMYT1 dependency in PDAC and identify potential therapeutic strategies for clinical translation.}, }
@article {pmid37919237, year = {2024}, author = {Zhang, MQ and Gong, LL and Zhao, YQ and Ma, YF and Long, GJ and Guo, H and Liu, XZ and Hull, JJ and Dewer, Y and Yang, C and Zhang, NN and He, M and He, P}, title = {Efficient DIPA-CRISPR-mediated knockout of an eye pigment gene in the white-backed planthopper, Sogatella furcifera.}, journal = {Insect science}, volume = {31}, number = {4}, pages = {1015-1025}, doi = {10.1111/1744-7917.13286}, pmid = {37919237}, issn = {1744-7917}, support = {Qianjiaohe KY number (2020) 004//Frontiers Science Center for Asymmetric Synthesis and Medicinal Molecules, Department of Education, Guizhou Province/ ; 111 Program D20023//Program of Introducing Talents of Discipline to Universities of China/ ; QKH[2017]2956//Science and Technology Support of Guizhou province/ ; 32260671//National Natural Science Foundation of China/ ; 32370527//National Natural Science Foundation of China/ ; [2019]05//Program of talent cultivation of Guizhou University/ ; 2017-33//Scientific Research Foundation of Guizhou University of China/ ; }, mesh = {Animals ; *Hemiptera/genetics ; *CRISPR-Cas Systems ; Female ; *Gene Editing/methods ; Gene Knockout Techniques ; }, abstract = {Although CRISPR/Cas9 has been widely used in insect gene editing, the need for the microinjection of preblastoderm embryos can preclude the technique being used in insect species with eggs that are small, have hard shells, and/or are difficult to collect and maintain outside of their normal environment. Such is the case with Sogatella furcifera, the white-backed planthopper (WBPH), a significant pest of Oryza sativa (rice) that oviposits inside rice stems. Egg extraction from the stem runs the risk of mechanical damage and hatching is heavily influenced by the micro-environment of the rice stem. To bypass these issues, we targeted embryos prior to oviposition via direct parental (DIPA)-CRISPR, in which Cas9 and single-guide RNAs (sgRNAs) for the WBPH eye pigment gene tryptophan 2,3-dioxygenase were injected into the hemocoel of adult females. Females at varying numbers of days posteclosion were evaluated to determine at what stage their oocyte might be most capable of taking up the gene-editing components. An evaluation of the offspring indicated that the highest G0 gene-edited efficacy (56.7%) occurred in females injected 2 d posteclosion, and that those mutations were heritably transmitted to the G1 generation. This study demonstrates the potential utility of DIPA-CRISPR for future gene-editing studies in non-model insect species and can facilitate the development of novel pest management applications.}, }
@article {pmid39133581, year = {2024}, author = {Attaway, C and Mathison, BA and Misra, A}, title = {No longer stuck in the past: new advances in artificial intelligence and molecular assays for parasitology screening and diagnosis.}, journal = {Current opinion in infectious diseases}, volume = {}, number = {}, pages = {}, pmid = {39133581}, issn = {1473-6527}, abstract = {PURPOSE OF REVIEW: Emerging technologies are revolutionizing parasitology diagnostics and challenging traditional methods reliant on microscopic analysis or serological confirmation, which are known for their limitations in sensitivity and specificity. This article sheds light on the transformative potential of artificial intelligence and molecular assays in the field, promising more accurate and efficient detection methods.<br><br>RECENT FINDINGS: Artificial intelligence has emerged as a promising tool for blood and stool parasite review, when paired with comprehensive databases and expert oversight result in heightened specificity and sensitivity of diagnoses while also increasing efficiency. Significant strides have been made in nucleic acid testing for multiplex panels for enteric pathogen. Both multiplex and single target panels for Plasmodium, Babesia, filaria, and kinetoplastids have been developed and garnered regulatory approval, notably for blood donor screening in the United States. Additional technologies such as MALDI-TOF, metagenomics, flow cytometry, and CRISPR-Cas are under investigation for their diagnostic utility and are currently in the preliminary stages of research and feasibility assessment.<br><br>SUMMARY: Recent implementation of artificial intelligence and digital microscopy has enabled swift smear screening and diagnosis, although widespread implementation remains limited. Simultaneously, molecular assays - both targeted and multiplex panels are promising and have demonstrated promise in numerous studies with some assays securing regulatory approval recently. Additional technologies are under investigation for their diagnostic utility and are compelling avenues for future proof-of-concept diagnostics.}, }
@article {pmid39132997, year = {2024}, author = {Khorshid Sokhangouy, S and Alizadeh, F and Lotfi, M and Sharif, S and Ashouri, A and Yoosefi, Y and Bozorg Qomi, S and Abbaszadegan, MR}, title = {Recent advances in CRISPR-Cas systems for colorectal cancer research and therapeutics.}, journal = {Expert review of molecular diagnostics}, volume = {}, number = {}, pages = {1-26}, doi = {10.1080/14737159.2024.2388777}, pmid = {39132997}, issn = {1744-8352}, abstract = {INTRODUCTION: Colon cancer, ranked as the fourth leading global cause of cancer death, exhibits a complex progression marked by genetic variations. Over the past decade, the utilization of diverse CRISPR systems has propelled accelerated research into colorectal cancer (CRC) treatment.<br><br>AREAS COVERED: CRISPR/Cas9, a key player in this research, identifies new oncogenes, tumor suppressor genes (TSGs), and drug-resistance genes. Additionally, it facilitates the construction of experimental models, conducts genome-wide library screening, and develops new therapeutic targets, especially for targeted knockout in vivo or molecular targeted drug delivery, contributing to personalized treatments and significantly enhancing the care of colon cancer patients. In this review, we provide insights into the mechanism of the CRISPR/Cas9 system, offering a comprehensive exploration of its applications in CRC, spanning screening, modeling, gene functions, diagnosis, and gene therapy. While acknowledging its transformative potential, the article&#xa0; highlights the challenges and limitations of CRISPR systems.<br><br>EXPERT OPINION: The application of CRISPR/Cas9 in CRC research provides a promising avenue for personalized treatments. Its potential for identifying key genes and enabling experimental models and genome-wide screening enhances patient care. This review underscores the significance of CRISPR-Cas9 gene editing technology across basic research, diagnosis, and the treatment landscape of colon cancer.}, }
@article {pmid39131396, year = {2024}, author = {Soczek, KM and Cofsky, JC and Tuck, OT and Shi, H and Doudna, JA}, title = {CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.07.31.606079}, pmid = {39131396}, issn = {2692-8205}, abstract = {RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA engineering begins with local helix distortion by transient CRISPR-Cas protein binding.}, }
@article {pmid39130943, year = {2024}, author = {Balasubramanian, A and Veluswami, K and Rao, S and Aggarwal, S and Mani, S}, title = {Exploring Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated Protein 9 (CRISPR-Cas9) as a Therapeutic Modality for Cancer: A Scoping Review.}, journal = {Cureus}, volume = {16}, number = {7}, pages = {e64324}, pmid = {39130943}, issn = {2168-8184}, abstract = {The global burden of cancer and the limitations of conventional therapies highlight the potential of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) in reshaping cancer treatment paradigms. In this review, we have investigated the mechanism of CRISPR, an adaptive immune system in bacteria that enables highly precise gene editing at the molecular level. This versatile tool demonstrates its efficacy in human cancer therapy through gene knockout, metabolic disruption, base editing, screening, and immunotherapy enhancement without affecting normal bodily domains. Despite its superiority over other nucleases like zinc-finger nucleases and transcription activator-like effector nucleases, hurdles such as off-target effects, inefficient delivery of the system to target cells, the emergence of escapers, and the ethical debate surrounding genome editing are discussed. In this article, we have reviewed the promising approaches of CRISPR-Cas9 in cancer treatment while exploring the underlying mechanism, advantages, and associated challenges.}, }
@article {pmid39128990, year = {2024}, author = {Liang, M and Wang, Y and Liu, L and Deng, D and Yan, Z and Feng, L and Kong, C and Li, C and Li, Y and Li, G}, title = {Synergistic intravesical instillation for bladder cancer: CRISPR-Cas13a and fenbendazole combination therapy.}, journal = {Journal of experimental &amp; clinical cancer research : CR}, volume = {43}, number = {1}, pages = {223}, pmid = {39128990}, issn = {1756-9966}, mesh = {*Urinary Bladder Neoplasms/drug therapy/genetics/pathology/therapy ; Humans ; Animals ; Administration, Intravesical ; Mice ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; }, abstract = {BACKGROUND: CRISPR-Cas13a is renowned for its precise and potent RNA editing capabilities in cancer therapy. While various material systems have demonstrated efficacy in supporting CRISPR-Cas13a to execute cellular functions in vitro efficiently and specifically, the development of CRISPR-Cas13a-based therapeutic agents for intravesical instillation in bladder cancer (BCa) remains unexplored.<br><br>METHODS: In this study, we introduce a CRISPR-Cas13a nanoplatform, which effectively inhibits PDL1 expression following intravesical instillation. This system utilizes a fusion protein CAST, created through the genetic fusion of CRISPR-Cas13 and the transmembrane peptide TAT. CAST acts as a potent transmembrane RNA editor and is assembled with the transepithelial delivery carrier fluorinated chitosan (FCS). Upon intravesical administration into the bladder, the CAST-crRNAa/FCS nanoparticles (NPs) exhibit remarkable transepithelial capabilities, significantly suppressing PDL1 expression in tumor tissues.To augment immune activation within the tumor microenvironment, we integrated a fenbendazole (FBZ) intravesical system (FBZ@BSA/FCS NPs). This system is formulated through BSA encapsulation followed by FCS coating, positioning FBZ as a powerful chemo-immunological agent.<br><br>RESULTS: In an orthotropic BCa model, the FBZ@BSA/FCS NPs demonstrated pronounced tumor cell apoptosis, synergistically reduced PDL1 expression, and restructured the immune microenvironment. This culminated in an enhanced synergistic intravesical instillation approach for BCa. Consequently, our study unveils a novel RNA editor nanoagent formulation and proposes a potential synergistic therapeutic strategy. This approach significantly bolsters therapeutic efficacy, holding promise for the clinical translation of CRISPR-Cas13-based cancer perfusion treatments.}, }
@article {pmid39087761, year = {2024}, author = {Du, H and Wang, F and Zhang, R and Yan, X and Zheng, J and Zhou, T and Wang, X and Zhang, G and Zhang, Z}, title = {Rolling Circle Amplification-Based Self-Assembly to Form a "GPS-Nanoconveyor" for In Vitro Targeted Imaging and Enhanced Gene/Chemo (CRISPR/DOX) Synergistic Therapy.}, journal = {Biomacromolecules}, volume = {25}, number = {8}, pages = {4991-5007}, doi = {10.1021/acs.biomac.4c00415}, pmid = {39087761}, issn = {1526-4602}, mesh = {*Doxorubicin/pharmacology/chemistry/administration &amp; dosage ; *CRISPR-Cas Systems ; Animals ; Mice ; Humans ; Aptamers, Nucleotide/chemistry ; Nucleic Acid Amplification Techniques/methods ; Cell Line, Tumor ; Antibiotics, Antineoplastic/pharmacology/administration &amp; dosage/chemistry ; }, abstract = {The GPS-Nanoconveyor (MA-NV@DOX-Cas13a) is a targeted nanoplatform designed for the imaging and gene/chemotherapy synergistic treatment of melanoma. It utilizes rolling circle amplification (RCA) products as a scaffold to construct a DNA "Nanoconveyor" (NV), which incorporates a multivalent aptamer (MA) as a "GPS", encapsulates doxorubicin (DOX) in the transporter, and equips it with CRISPR/Cas13a ribonucleoproteins (Cas13a RNP). Carrying MA enhances the ability to recognize the overexpressed receptor nucleolin on B16 cells, enabling targeted imaging and precise delivery of MA-NV@DOX-Cas13a through receptor-mediated endocytosis. The activation of signal transducer and activator of transcription 3 (STAT3) in cancer cells triggers cis-cleavage of CRISPR/Cas13a, initiating its trans-cleavage function. Additionally, deoxyribonuclease I (DNase I) degrades MA-NV, releasing DOX for intracellular imaging and as a chemotherapeutic agent. Experiments demonstrate the superior capabilities of this versatile nanoplatform for cellular imaging and co-treatment while highlighting the advantages of these nanodrug delivery systems in mitigating DOX side effects.}, }
@article {pmid39007397, year = {2024}, author = {Emmerich, K and Hageter, J and Hoang, T and Lyu, P and Sharrock, AV and Ceisel, A and Thierer, J and Chunawala, Z and Nimmagadda, S and Palazzo, I and Matthews, F and Zhang, L and White, DT and Rodriguez, C and Graziano, G and Marcos, P and May, A and Mulligan, T and Reibman, B and Saxena, MT and Ackerley, DF and Qian, J and Blackshaw, S and Horstick, E and Mumm, JS}, title = {A large-scale CRISPR screen reveals context-specific genetic regulation of retinal ganglion cell regeneration.}, journal = {Development (Cambridge, England)}, volume = {151}, number = {15}, pages = {}, doi = {10.1242/dev.202754}, pmid = {39007397}, issn = {1477-9129}, support = {G2020315//BrightFocus Foundation/ ; P30 EY001765/EY/NEI NIH HHS/United States ; R01 OD020376/OD/NIH HHS/United States ; R01 EY033009/EY/NEI NIH HHS/United States ; F31EY032790/NH/NIH HHS/United States ; R01 EY022810/EY/NEI NIH HHS/United States ; OIA2242771//U.S. National Science Foundation/ ; F31 EY032790/EY/NEI NIH HHS/United States ; EY032790-01/NH/NIH HHS/United States ; G2020315//Brightfocus Foundation/ ; OIA-2242771//National Science Foundation/ ; //Johns Hopkins University School of Medicine/ ; }, mesh = {Animals ; *Zebrafish/genetics ; *Retinal Ganglion Cells/metabolism/cytology/physiology ; *Zebrafish Proteins/genetics/metabolism ; *Animals, Genetically Modified ; Nerve Regeneration/genetics/physiology ; Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Regeneration/genetics/physiology ; Retina/metabolism/cytology ; Stem Cells/metabolism/cytology ; Transcription Factors ; }, abstract = {Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 100 genes identified seven knockouts that inhibited and 11 that promoted RGC regeneration. Surprisingly, 35 out of 36 genes known and/or implicated as being required for regeneration after widespread retinal damage were not required for RGC regeneration. The loss of seven even enhanced regeneration kinetics, including the proneural factors neurog1, olig2 and ascl1a. Mechanistic analyses revealed that ascl1a disruption increased the propensity of progenitor cells to produce RGCs, i.e. increased 'fate bias'. These data demonstrate plasticity in the mechanism through which Müller glia convert to a stem-like state and context specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics.}, }
@article {pmid36610033, year = {2024}, author = {Yi, JY and Kim, M and Jeon, M and Min, H and Kim, BG and Son, J and Sung, C}, title = {Simple visualization method for the c.577del of erythropoietin variant: CRISPR/dCas9-based single nucleotide polymorphism diagnosis.}, journal = {Drug testing and analysis}, volume = {16}, number = {8}, pages = {786-791}, doi = {10.1002/dta.3438}, pmid = {36610033}, issn = {1942-7611}, support = {2V09270//Korea Institute of Science and Technology/ ; 2V0935A//Korea Institute of Science and Technology/ ; }, mesh = {*Erythropoietin/genetics ; Humans ; *Polymorphism, Single Nucleotide ; *Doping in Sports/prevention &amp; control ; *CRISPR-Cas Systems ; Substance Abuse Detection/methods ; Polymerase Chain Reaction/methods ; }, abstract = {One of the single nucleotide polymorphisms (SNPs) in human erythropoietin (hEPO), the c.577del variant, can produces 26 amino acids longer than the wild-type hEPO, posing a risk of misinterpretation in routine doping analysis. To prevent this, the World Anti-Doping Agency (WADA) included a procedure for reporting the sequencing results regarding the presence or absence of SNPs for suspected cases in the new version of the technical document for recombinant EPO in 2022. However, it is very expensive for anti-doping laboratories to purchase a gene sequencing analyzer, which costs hundreds of thousands of dollars, and only a few companies provide specific gene sequencing services with accredited certification. Therefore, in this study, we developed a simple visualization method for the c.577del of the EPO variant at the gene level. The gene fragment of the EPO gene, including c.577del, was amplified using a fast polymerase chain reaction (PCR), and the PCR products were incubated with the clustered regularly interspaced short palindromic repeats (CRISPR)/deadCas9 system using variant-specific single-guide RNA (sgRNA). This ribonucleoprotein complex binds specifically to the EPO variant gene fragment, causing a band shift on native-PAGE. We designed 4 sgRNAs that can bind only to the EPO variant or wild-type gene. In addition, an electrophoretic mobility shift assay on a gel demonstrated that one of the sgRNAs had a high level of specificity. Consequently, the c.577del variant was selectively detected by visualizing the target fragment of the gene on the gel within 3 h using only 3 μl of the whole blood.}, }
@article {pmid39127705, year = {2024}, author = {Park, H and Bulzu, PA and Shabarova, T and Kavagutti, VS and Ghai, R and Kasalický, V and Jezberová, J}, title = {Uncovering the genomic basis of symbiotic interactions and niche adaptations in freshwater picocyanobacteria.}, journal = {Microbiome}, volume = {12}, number = {1}, pages = {150}, pmid = {39127705}, issn = {2049-2618}, support = {20&#x2011;12496X//Grantov&#xe1; Agentura Cesk&#xe9; Republiky (GA&#x10c;R)/ ; 20&#x2011;12496X//Grantov&#xe1; Agentura Cesk&#xe9; Republiky (GA&#x10c;R)/ ; 23-05081S//Grantov&#xe1; Agentura Cesk&#xe9; Republiky (GA&#x10c;R)/ ; 19-23261S//Grantov&#xe1; Agentura Cesk&#xe9; Republiky (GA&#x10c;R)/ ; 20&#x2011;12496X//Grantov&#xe1; Agentura Cesk&#xe9; Republiky (GA&#x10c;R)/ ; 19-23261S//Grantov&#xe1; Agentura Cesk&#xe9; Republiky (GA&#x10c;R)/ ; 19-23261S//Grantov&#xe1; Agentura Cesk&#xe9; Republiky (GA&#x10c;R)/ ; }, mesh = {*Symbiosis ; *Fresh Water/microbiology ; *Genome, Bacterial ; *Phylogeny ; *Cyanobacteria/genetics/classification ; Adaptation, Physiological/genetics ; Europe ; Ecosystem ; Gene Transfer, Horizontal ; Genomics ; }, abstract = {BACKGROUND: Picocyanobacteria from the genera Prochlorococcus, Synechococcus, and Cyanobium are the most widespread photosynthetic organisms in aquatic ecosystems. However, their freshwater populations remain poorly explored, due to uneven and insufficient sampling across diverse inland waterbodies.<br><br>RESULTS: In this study, we present 170 high-quality genomes of freshwater picocyanobacteria from non-axenic cultures collected across Central Europe. In addition, we recovered 33 genomes of their potential symbiotic partners affiliated with four genera, Pseudomonas, Mesorhizobium, Acidovorax, and Hydrogenophaga. The genomic basis of symbiotic interactions involved heterotrophs benefiting from picocyanobacteria-derived nutrients while providing detoxification of ROS. The global abundance patterns of picocyanobacteria revealed ecologically significant ecotypes, associated with trophic status, temperature, and pH as key environmental factors. The adaptation of picocyanobacteria in (hyper-)eutrophic waterbodies could be attributed to their colonial lifestyles and CRISPR-Cas systems. The prevailing CRISPR-Cas subtypes in picocyanobacteria were I-G and I-E, which appear to have been acquired through horizontal gene transfer from other bacterial phyla.<br><br>CONCLUSIONS: Our findings provide novel insights into the population diversity, ecology, and evolutionary strategies of the most widespread photoautotrophs within freshwater ecosystems. Video Abstract.}, }
@article {pmid39127411, year = {2024}, author = {Bai, S and Luo, H and Tong, H and Wu, Y and Yuan, Y}, title = {Advances on transfer and maintenance of large DNA in bacteria, fungi, and mammalian cells.}, journal = {Biotechnology advances}, volume = {76}, number = {}, pages = {108421}, doi = {10.1016/j.biotechadv.2024.108421}, pmid = {39127411}, issn = {1873-1899}, abstract = {Advances in synthetic biology allow the design and manipulation of DNA from the scale of genes to genomes, enabling the engineering of complex genetic information for application in biomanufacturing, biomedicine and other areas. The transfer and subsequent maintenance of large DNA are two core steps in large scale genome rewriting. Compared to small DNA, the high molecular weight and fragility of large DNA make its transfer and maintenance a challenging process. This review outlines the methods currently available for transferring and maintaining large DNA in bacteria, fungi, and mammalian cells. It highlights their mechanisms, capabilities and applications. The transfer methods are categorized into general methods (e.g., electroporation, conjugative transfer, induced cell fusion-mediated transfer, and chemical transformation) and specialized methods (e.g., natural transformation, mating-based transfer, virus-mediated transfection) based on their applicability to recipient cells. The maintenance methods are classified into genomic integration (e.g., CRISPR/Cas-assisted insertion) and episomal maintenance (e.g., artificial chromosomes). Additionally, this review identifies the major technological advantages and disadvantages of each method and discusses the development for large DNA transfer and maintenance technologies.}, }
@article {pmid39127142, year = {2024}, author = {Ng, BW and Kaukonen, MK and McClements, ME and Shamsnajafabadi, H and MacLaren, RE and Cehajic-Kapetanovic, J}, title = {Genetic therapies and potential therapeutic applications of CRISPR activators in the eye.}, journal = {Progress in retinal and eye research}, volume = {}, number = {}, pages = {101289}, doi = {10.1016/j.preteyeres.2024.101289}, pmid = {39127142}, issn = {1873-1635}, abstract = {Conventional gene therapy involving supplementation only treats loss-of-function diseases and is limited by viral packaging sizes, precluding therapy of large genes. The discovery of CRISPR/Cas has led to a paradigm shift in the field of genetic therapy, with the promise of precise gene editing, thus broadening the range of diseases that can be treated. The initial uses of CRISPR/Cas have focused mainly on gene editing or silencing of abnormal variants via utilising Cas endonuclease to trigger the target cell endogenous non-homologous end joining. Subsequently, the technology has evolved to modify the Cas enzyme and even its guide RNA, leading to more efficient editing tools in the form of base and prime editing. Further advancements of this CRISPR/Cas technology itself have expanded its functional repertoire from targeted editing to programmable transactivation, shifting the therapeutic focus to precise endogenous gene activation or upregulation with the potential for epigenetic modifications. In vivo experiments using this platform have demonstrated the potential of CRISPR-activators (CRISPRa) to treat various loss-of-function diseases, as well as in regenerative medicine, highlighting their versatility to overcome limitations associated with conventional strategies. This review summarises the molecular mechanisms of CRISPRa platforms, the current applications of this technology in vivo, and discusses potential solutions to translational hurdles for this therapy, with a focus on ophthalmic diseases.}, }
@article {pmid39126615, year = {2024}, author = {Li, P and Dong, D and Gao, F and Xie, Y and Huang, H and Sun, S and Ma, Z and He, C and Lai, J and Du, X and Wu, S}, title = {Versatile and efficient mammalian genome editing with Type I-C CRISPR System of Desulfovibrio vulgaris.}, journal = {Science China. Life sciences}, volume = {}, number = {}, pages = {}, pmid = {39126615}, issn = {1869-1889}, abstract = {CRISPR-Cas tools for mammalian genome editing typically rely on single Cas9 or Cas12a proteins. While type I CRISPR systems in Class I may offer greater specificity and versatility, they are not well-developed for genome editing. Here, we present an alternative type I-C CRISPR system from Desulfovibrio vulgaris (Dvu) for efficient and precise genome editing in mammalian cells and animals. We optimized the Dvu type I-C editing complex to generate precise deletions at multiple loci in various cell lines and pig primary fibroblast cells using a paired PAM-in crRNA strategy. These edited pig cells can serve as donors for generating transgenic cloned piglets. The Dvu type I-C editor also enabled precise large fragment replacements with homology-directed repair. Additionally, we adapted the Dvu-Cascade effector for cytosine and adenine base editing, developing Dvu-CBE and Dvu-ABE systems. These systems efficiently induced C-to-T and A-to-G substitutions in human genes without double-strand breaks. Off-target analysis confirmed the high specificity of the Dvu type I-C editor. Our findings demonstrate the Dvu type I-C editor's potential for diverse mammalian genome editing applications, including deletions, fragment replacement, and base editing, with high efficiency and specificity for biomedicine and agriculture.}, }
@article {pmid38994565, year = {2024}, author = {Li, M and Cai, Z and Song, S and Yue, X and Lu, W and Rao, S and Zhang, C and Xue, C}, title = {EcCas6e-based antisense crRNA for gene repression and RNA editing in microorganisms.}, journal = {Nucleic acids research}, volume = {52}, number = {14}, pages = {8628-8642}, doi = {10.1093/nar/gkae612}, pmid = {38994565}, issn = {1362-4962}, support = {2023YFC3402301//National Key R&amp;D Program of China/ ; 2021-I2M-1-041//CAMS Innovation Fund for Medical Sciences (CIFMS)/ ; 32101203//National Natural Science Foundation of China/ ; 2021-RC310-015//Chinese Academy of Medical Sciences/ ; TSBICIP-CXRC-025//Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project/ ; 22HHSWSS00024//Haihe Laboratory/ ; }, mesh = {*RNA Editing ; *Saccharomyces cerevisiae/genetics ; *Escherichia coli/genetics/metabolism ; *RNA, Antisense/genetics/metabolism ; *RNA, Messenger/genetics/metabolism ; Adenosine Deaminase/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; CRISPR-Associated Proteins/metabolism/genetics ; }, abstract = {Precise gene regulation and programmable RNA editing are vital RNA-level regulatory mechanisms. Gene repression tools grounded in small non-coding RNAs, microRNAs, and CRISPR-dCas proteins, along with RNA editing tools anchored in Adenosine Deaminases acting on RNA (ADARs), have found extensive application in molecular biology and cellular engineering. Here, we introduced a novel approach wherein we developed an EcCas6e mediated crRNA-mRNA annealing system for gene repression in Escherichia coli and RNA editing in Saccharomyces cerevisiae. We found that EcCas6e possesses inherent RNA annealing ability attributed to a secondary positively charged cleft, enhancing crRNA-mRNA hybridization and stability. Based on this, we demonstrated that EcCas6e, along with its cognate crRNA repeat containing a complementary region to the ribosome binding site of a target mRNA, effectively represses gene expression up to 25-fold. Furthermore, we demonstrated that multiple crRNAs can be easily assembled and can simultaneously target up to 13 genes. Lastly, the EcCas6e-crRNA system was developed as an RNA editing tool by fusing it with the ADAR2 deaminase domain. The EcCas6e-crRNA mediated gene repression and RNA editing tools hold broad applications for research and biotechnology.}, }
@article {pmid38967023, year = {2024}, author = {Zhang, D and Du, L and Gao, H and Yuan, C and Lin, Z}, title = {Structural insight into the Csx1-Crn2 fusion self-limiting ribonuclease of type III CRISPR system.}, journal = {Nucleic acids research}, volume = {52}, number = {14}, pages = {8419-8430}, doi = {10.1093/nar/gkae569}, pmid = {38967023}, issn = {1362-4962}, support = {31971222//National Natural Science Foundation of China/ ; }, mesh = {*Bacterial Proteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; Ribonucleases/metabolism/chemistry/genetics ; CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Models, Molecular ; Protein Binding ; Adenine Nucleotides/metabolism/chemistry ; Protein Multimerization ; Protein Domains ; Oligoribonucleotides ; }, abstract = {In the type III CRISPR system, cyclic oligoadenylate (cOA) molecules act as second messengers, activating various promiscuous ancillary nucleases that indiscriminately degrade host and viral DNA/RNA. Conversely, ring nucleases, by specifically cleaving cOA molecules, function as off-switches to protect host cells from dormancy or death, and allow viruses to counteract immune responses. The fusion protein Csx1-Crn2, combining host ribonuclease with viral ring nuclease, represents a unique self-limiting ribonuclease family. Here, we describe the structures of Csx1-Crn2 from the organism of Marinitoga sp., in both its full-length and truncated forms, as well as in complex with cA4. We show that Csx1-Crn2 operates as a homo-tetramer, a configuration crucial for preserving the structural integrity of the HEPN domain and ensuring effective ssRNA cleavage. The binding of cA4 to the CARF domain triggers significant conformational changes across the CARF, HTH, and into the HEPN domains, leading the two R-X4-6-H motifs to form a composite catalytic site. Intriguingly, an acetate ion was found to bind at this composite site by mimicking the scissile phosphate. Further molecular docking analysis reveals that the HEPN domain can accommodate a single ssRNA molecule involving both R-X4-6-H motifs, underscoring the importance of HEPN domain dimerization for its activation.}, }
@article {pmid38936209, year = {2024}, author = {Zhou, W and Xiang, Y and Yang, J and Chen, T}, title = {Metal ion-complexed DNA probe coupled with CRISPR/Cas12a amplification and AuNPs for sensitive colorimetric assay of metallothionein in fish.}, journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy}, volume = {321}, number = {}, pages = {124682}, doi = {10.1016/j.saa.2024.124682}, pmid = {38936209}, issn = {1873-3557}, mesh = {*Colorimetry/methods ; Animals ; *Metallothionein/metabolism/genetics ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; *DNA Probes/chemistry/metabolism/genetics ; *Fishes/metabolism ; Mercury/analysis ; CRISPR-Cas Systems ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Biosensing Techniques/methods ; }, abstract = {The accurate and sensitive detection of metallothionein (MT) is of great significance in the fields of biomedical, toxicological and environmental sciences. In this work, based on the high affinity interaction between MT and the heavy metal ions of Hg[2+] and the significant signal amplification capability of Cas12a/crRNA enzyme as well, we report a simple and highly sensitive method for visual detection of MT, a biomarker in fish for heavy metal ion-induced water bio-pollution. The target MT molecules bind Hg[2+] in the Hg[2+]- complexed hairpin DNA probes to unfold the hairpin structure into ssDNAs, which hybridize with the partial dsDNA duplexes via strand displacement to yield specific sequence-containing dsDNAs. Cas12a/crRNA recognizes these specific sequences to activate its enzyme activity to cyclically cleave the ssDNA linkers in the blue colored gold nanoparticle aggregates to transit their color into red to realize visual detection of MT. Owing to the signal amplification by Cas12a/crRNA, as low as 25 nM of MT can be visually detected with naked eye. In addition, our colorimetric detection method has high selectivity for MT against other interference proteins and can detect MT in the livers and kidneys of crucian carps bought from a local supermarket. Moreover, the developed assay overcomes the limitations of conventional MT detection methods in terms of complexity, high cost and low sensitivity and can therefore offer new methods for monitoring water bio-pollutions.}, }
@article {pmid38874474, year = {2024}, author = {Ohtani, H and Liu, M and Liang, G and Jang, HJ and Jones, PA}, title = {Efficient activation of hundreds of LTR12C elements reveals cis-regulatory function determined by distinct epigenetic mechanisms.}, journal = {Nucleic acids research}, volume = {52}, number = {14}, pages = {8205-8217}, doi = {10.1093/nar/gkae498}, pmid = {38874474}, issn = {1362-4962}, support = {R35CA209859/CA/NCI NIH HHS/United States ; 20K22808//Japan Society for the Promotion of Science/ ; //Aichi Cancer Research Foundation/ ; //Suzuken Memorial Foundation/ ; //Foundation of Kinoshita Memorial Enterprise/ ; //Terumo Life Science Foundation/ ; //Kato Memorial Bioscience Foundation/ ; R35CA209859/CA/NCI NIH HHS/United States ; R35CA209859/CA/NCI NIH HHS/United States ; }, mesh = {*Terminal Repeat Sequences/genetics ; Humans ; *Epigenesis, Genetic ; *Promoter Regions, Genetic ; *Endogenous Retroviruses/genetics ; CRISPR-Cas Systems ; Enhancer Elements, Genetic ; Transcriptional Activation ; HEK293 Cells ; Histones/metabolism/genetics ; }, abstract = {Long terminal repeats (LTRs), which often contain promoter and enhancer sequences of intact endogenous retroviruses (ERVs), are known to be co-opted as cis-regulatory elements for fine-tuning host-coding gene expression. Since LTRs are mainly silenced by the deposition of repressive epigenetic marks, substantial activation of LTRs has been found in human cells after treatment with epigenetic inhibitors. Although the LTR12C family makes up the majority of ERVs activated by epigenetic inhibitors, how these epigenetically and transcriptionally activated LTR12C elements can regulate the host-coding gene expression remains unclear due to genome-wide alteration of transcriptional changes after epigenetic inhibitor treatments. Here, we specifically transactivated >600 LTR12C elements by using single guide RNA-based dCas9-SunTag-VP64, a site-specific targeting CRISPR activation (CRISPRa) system, with minimal off-target events. Interestingly, most of the transactivated LTR12C elements acquired the H3K27ac-marked enhancer feature, while only 20% were co-marked with promoter-associated H3K4me3 modifications. The enrichment of the H3K4me3 signal was intricately associated with downstream regions of LTR12C, such as internal regions of intact ERV9 or other types of retrotransposons. Here, we leverage an optimized CRISPRa system to identify two distinct epigenetic signatures that define LTR12C transcriptional activation, which modulate the expression of proximal protein-coding genes.}, }
@article {pmid39126469, year = {2024}, author = {Sánchez-Martín, M and Sánchez-Sáez, F and Llano, E and Pendás, AM}, title = {Generation of Meiotic Mouse Models Using CRISPR/Cas9 Technology.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2818}, number = {}, pages = {93-112}, pmid = {39126469}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems ; *Meiosis/genetics ; Mice ; *Gene Editing/methods ; Male ; Models, Animal ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {In recent years, targeted genome editing has emerged as an indispensable tool for creating animal models, facilitating a comprehensive exploration of the molecular mechanisms governing a myriad of biological processes. Within this scientific landscape, the investigation of meiosis in mice has attracted considerable attention across numerous research laboratories. The precision and versatility of the CRISPR/Cas9 genome editing system have revolutionized our ability to generate mice with tailored genetic alterations, including point mutations and null mutations. These genetic modifications have provided invaluable insights into the intricate functionality of various meiotic genes and their associated variants. In this context, we present a detailed state of the art protocol for the creation of novel mouse models, each bearing specific genetic modifications within key meiotic genes, through the application of CRISPR/Cas9 technology. Furthermore, we showcase two distinct genetic modifications, accomplished within our laboratory, that can serve as valuable reference points for researchers seeking to elucidate the molecular intricacies of meiosis in mammals.}, }
@article {pmid39125980, year = {2024}, author = {Pessoa, J and Carvalho, C}, title = {Human RNA Polymerase II Segregates from Genes and Nascent RNA and Transcribes in the Presence of DNA-Bound dCas9.}, journal = {International journal of molecular sciences}, volume = {25}, number = {15}, pages = {}, doi = {10.3390/ijms25158411}, pmid = {39125980}, issn = {1422-0067}, support = {SFRH/DPD/102323/2014, PTDC/SAU-GMG/118180/2010, PTDC/BEX-BCM/5899/2014, 2022.01199.PTDC//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e Tecnologia/ ; 734825//European Commission/ ; }, mesh = {Humans ; *RNA Polymerase II/metabolism/genetics ; *Transcription, Genetic ; *beta-Globins/genetics/metabolism ; DNA/metabolism/genetics ; Promoter Regions, Genetic ; CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Cas Systems ; RNA/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics/metabolism ; Cell Line ; }, abstract = {RNA polymerase II (Pol II) dysfunction is frequently implied in human disease. Understanding its functional mechanism is essential for designing innovative therapeutic strategies. To visualize its supra-molecular interactions with genes and nascent RNA, we generated a human cell line carrying ~335 consecutive copies of a recombinant β-globin gene. Confocal microscopy showed that Pol II was not homogeneously concentrated around these identical gene copies. Moreover, Pol II signals partially overlapped with the genes and their nascent RNA, revealing extensive compartmentalization. Using a cell line carrying a single copy of the β-globin gene, we also tested if the binding of catalytically dead CRISPR-associated system 9 (dCas9) to different gene regions affected Pol II transcriptional activity. We assessed Pol II localization and nascent RNA levels using chromatin immunoprecipitation and droplet digital reverse transcription PCR, respectively. Some enrichment of transcriptionally paused Pol II accumulated in the promoter region was detected in a strand-specific way of gRNA binding, and there was no decrease in nascent RNA levels. Pol II preserved its transcriptional activity in the presence of DNA-bound dCas9. Our findings contribute further insight into the complex mechanism of mRNA transcription in human cells.}, }
@article {pmid39125884, year = {2024}, author = {Yang, X and Zhu, P and Gui, J}, title = {Advancements of CRISPR-Mediated Base Editing in Crops and Potential Applications in Populus.}, journal = {International journal of molecular sciences}, volume = {25}, number = {15}, pages = {}, doi = {10.3390/ijms25158314}, pmid = {39125884}, issn = {1422-0067}, support = {32022055//National Natural Science Foundation of China/ ; 2023ZD04057//Science and Technology Innovation 2030--Major Project/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; *Populus/genetics ; Genome, Plant ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; }, abstract = {Base editing represents a cutting-edge genome editing technique that utilizes the CRISPR system to guide base deaminases with high precision to specific genomic sites, facilitating the targeted alteration of individual nucleotides. Unlike traditional gene editing approaches, base editing does not require DNA double-strand breaks or donor templates. It functions independently of the cellular DNA repair machinery, offering significant advantages in terms of both efficiency and accuracy. In this review, we summarize the core design principles of various DNA base editors, their distinctive editing characteristics, and tactics to refine their efficacy. We also summarize their applications in crop genetic improvement and explore their potential contributions to forest genetic engineering.}, }
@article {pmid39125741, year = {2024}, author = {Huang, Y and Fu, L and Gan, Y and Qi, G and Hao, L and Xin, T and Xu, W and Song, J}, title = {Analysis of Whole-Genome for Identification of Seven Penicillium Species with Significant Economic Value.}, journal = {International journal of molecular sciences}, volume = {25}, number = {15}, pages = {}, doi = {10.3390/ijms25158172}, pmid = {39125741}, issn = {1422-0067}, support = {2022-I2M-2-001//Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences/ ; }, mesh = {*Penicillium/genetics/classification/isolation &amp; purification ; *Genome, Fungal ; CRISPR-Cas Systems ; Whole Genome Sequencing/methods ; Computational Biology/methods ; Sequence Analysis, DNA/methods/economics ; Phylogeny ; }, abstract = {The Penicillium genus exhibits a broad global distribution and holds substantial economic value in sectors including agriculture, industry, and medicine. Particularly in agriculture, Penicillium species significantly impact plants, causing diseases and contamination that adversely affect crop yields and quality. Timely detection of Penicillium species is crucial for controlling disease and preventing mycotoxins from entering the food chain. To tackle this issue, we implement a novel species identification approach called Analysis of whole GEnome (AGE). Here, we initially applied bioinformatics analysis to construct specific target sequence libraries from the whole genomes of seven Penicillium species with significant economic impact: P. canescens, P. citrinum, P. oxalicum, P. polonicum, P. paneum, P. rubens, and P. roqueforti. We successfully identified seven Penicillium species using the target we screened combined with Sanger sequencing and CRISPR-Cas12a technologies. Notably, based on CRISPR-Cas12a technology, AGE can achieve rapid and accurate identification of genomic DNA samples at a concentration as low as 0.01 ng/µL within 30 min. This method features high sensitivity and portability, making it suitable for on-site detection. This robust molecular approach provides precise fungal species identification with broad implications for agricultural control, industrial production, clinical diagnostics, and food safety.}, }
@article {pmid39123212, year = {2024}, author = {Wang, X and Pan, W and Sun, C and Yang, H and Cheng, Z and Yan, F and Ma, G and Shang, Y and Zhang, R and Gao, C and Liu, L and Zhang, H}, title = {Creating large-scale genetic diversity in Arabidopsis via base editing-mediated deep artificial evolution.}, journal = {Genome biology}, volume = {25}, number = {1}, pages = {215}, pmid = {39123212}, issn = {1474-760X}, support = {TSQN202103160//Taishan Scholar Foundation of Shandong Province/ ; ZR202103010168//Excellent Youth Foundation of Shandong Scientific Committee/ ; 2022YFD1201700//National Key R&amp;D Program of China/ ; }, mesh = {*Arabidopsis/genetics ; *Gene Editing/methods ; *Genetic Variation ; CRISPR-Cas Systems ; Directed Molecular Evolution ; Alleles ; Mutation ; Plant Breeding/methods ; Herbicide Resistance/genetics ; }, abstract = {BACKGROUND: Base editing is a powerful tool for artificial evolution to create allelic diversity and improve agronomic traits. However, the great evolutionary potential for every sgRNA target has been overlooked. And there is currently no high-throughput method for generating and characterizing as many changes in a single target as possible based on large mutant pools to permit rapid gene directed evolution in plants.<br><br>RESULTS: In this study, we establish an efficient germline-specific evolution system to screen beneficial alleles in Arabidopsis which could be applied for crop improvement. This system is based on a strong egg cell-specific cytosine base editor and the large seed production of Arabidopsis, which enables each T1 plant with unedited wild type alleles to produce thousands of independent T2 mutant lines. It has the ability of creating a wide range of mutant lines, including those containing atypical base substitutions, and as well&#xa0;providing a space- and labor-saving way to store and screen the resulting mutant libraries. Using this system, we efficiently generate herbicide-resistant EPSPS, ALS, and HPPD variants that could be used in crop breeding.<br><br>CONCLUSIONS: Here, we demonstrate the significant potential of base editing-mediated artificial evolution for each sgRNA target and devised an efficient system for conducting deep evolution to harness this potential.}, }
@article {pmid39123110, year = {2024}, author = {Li, R and Cui, L and Martina, M and Bracuto, V and Meijer-Dekens, F and Wolters, AA and Moglia, A and Bai, Y and Acquadro, A}, title = {Less is more: CRISPR/Cas9-based mutations in DND1 gene enhance tomato resistance to powdery mildew with low fitness costs.}, journal = {BMC plant biology}, volume = {24}, number = {1}, pages = {763}, pmid = {39123110}, issn = {1471-2229}, support = {PROSPEcT research project, www.crispr-plants.unito.it//Cassa di Risparmio di Cuneo (CRC) Foundation/ ; PROSPEcT research project, www.crispr-plants.unito.it//Cassa di Risparmio di Cuneo (CRC) Foundation/ ; PROSPEcT research project, www.crispr-plants.unito.it//Cassa di Risparmio di Cuneo (CRC) Foundation/ ; PROSPEcT research project, www.crispr-plants.unito.it//Cassa di Risparmio di Cuneo (CRC) Foundation/ ; PROSPEcT research project, www.crispr-plants.unito.it//Cassa di Risparmio di Cuneo (CRC) Foundation/ ; }, mesh = {*Solanum lycopersicum/genetics/microbiology ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology/genetics ; *Disease Resistance/genetics ; *Ascomycota/physiology ; *Mutation ; Plant Proteins/genetics/metabolism ; Gene Editing ; Genes, Plant ; }, abstract = {Powdery mildew (PM), triggered by Oidium neolycopersici, represents a significant threat and a major concern for the productivity of tomato plants (Solanum lycopersicum L.). The presence of susceptibility (S) genes in plants facilitates pathogen proliferation and their dysfunction can lead to a recessively inherited broad-spectrum and durable type of resistance. Past studies have demonstrated that disrupting the function of DND1 (Defense No Death 1) increases plant resilience against various pathogens, such as powdery mildew (PM), but this comes at the cost of negatively affecting the overall health and vigor of the plant. To investigate the possibility of minimizing the adverse effects of the dnd1 mutation while boosting disease resistance, a CRISPR-Cas9 construct with four single guide RNAs targeting three exons of SlDND1 (Solyc02g088560.4.1) was designed and introduced into the tomato variety Moneymaker (MM) through Agrobacterium tumefaciens-mediated transformation. Three T1 lines (named E1, E3 and E4) were crossed with MM and then selfed to produce TF2 families. All the TF2 plants in homozygous state dnd1/dnd1, showed reduced PM symptoms compared to the heterozygous (DND1/dnd1) and wild type (DND1/DND1) ones. Two full knock-out (KO) mutant events (E1 and E4) encoding truncated DND1 proteins, exhibited clear dwarfness and auto-necrosis phenotypes, while mutant event E3 harbouring deletions of 3 amino acids, showed normal growth in height with less auto-necrotic spots. Analysis of the 3D structures of both the reference and the mutant proteins revealed significant conformational alterations in the protein derived from E3, potentially impacting its function. A dnd1/dnd1 TF2 line (TV181848-9, E3) underwent whole-genome sequencing using Illumina technology, which confirmed the absence of off-target mutations in selected genomic areas. Additionally, no traces of the Cas9 gene were detected, indicating its elimination through segregation. Our findings confirm the role of DND1 as an S-gene in tomato because impairment of this gene leads to a notable reduction in susceptibility to O. neolycopersici. Moreover, we provide, for the first time, a dnd1 mutant allele (E3) that exhibits fitness advantages in comparison with previously reported dnd1 mutant alleles, indicating a possible way to breed with dnd1 mutants.}, }
@article {pmid39122956, year = {2024}, author = {Tang, L}, title = {Epigenetic editing with CHARM.}, journal = {Nature methods}, volume = {21}, number = {8}, pages = {1410}, doi = {10.1038/s41592-024-02387-3}, pmid = {39122956}, issn = {1548-7105}, mesh = {Humans ; *Epigenesis, Genetic ; *Gene Editing/methods ; CRISPR-Cas Systems ; Epigenomics/methods ; Animals ; }, }
@article {pmid39122734, year = {2024}, author = {Pescod, P and Bevivino, G and Anthousi, A and Shepherd, J and Shelton, R and Lombardo, F and Nolan, T}, title = {Homing gene drives can transfer rapidly between Anopheles gambiae strains with minimal carryover of flanking sequences.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6846}, pmid = {39122734}, issn = {2041-1723}, support = {SBF006\1183/AMS_/Academy of Medical Sciences/United Kingdom ; SBF006\1183/AMS_/Academy of Medical Sciences/United Kingdom ; MR/W002159/1//RCUK | Medical Research Council (MRC)/ ; Bando Torno Subito 2019 (ID 18848)//Regione Lazio (Region of Lazio)/ ; MR/W002159/1//RCUK | MRC | Medical Research Foundation/ ; }, mesh = {*Anopheles/genetics ; Animals ; *Gene Drive Technology/methods ; *CRISPR-Cas Systems ; Female ; Alleles ; Gene Conversion ; Meiosis/genetics ; Male ; DNA Breaks, Double-Stranded ; Chromosomes, Insect/genetics ; }, abstract = {CRISPR-Cas9 homing gene drives are designed to induce a targeted double-stranded DNA break at a wild type allele ('recipient'), which, when repaired by the host cell, is converted to the drive allele from the homologous ('donor') chromosome. Germline localisation of this process leads to super-Mendelian inheritance of the drive and the rapid spread of linked traits, offering a novel strategy for population control through the deliberate release of drive individuals. During the homology-based DNA repair, additional segments of the recipient chromosome may convert to match the donor, potentially impacting carrier fitness and strategy success. Using Anopheles gambiae strains with variations around the drive target site, here we assess the extent and nature of chromosomal conversion. We show both homing and meiotic drive contribute as mechanisms of inheritance bias. Additionally, over 80% of homing events resolve within 50 bp of the chromosomal break, enabling rapid gene drive transfer into locally-adapted genetic backgrounds.}, }
@article {pmid39122729, year = {2024}, author = {Shu, Z and Dwivedi, B and Switchenko, JM and Yu, DS and Deng, X}, title = {PD-L1 deglycosylation promotes its nuclear translocation and accelerates DNA double-strand-break repair in cancer.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6830}, pmid = {39122729}, issn = {2041-1723}, support = {R01CA255257//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01CA200905//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; }, mesh = {Humans ; *DNA Breaks, Double-Stranded/radiation effects ; *DNA End-Joining Repair ; *B7-H1 Antigen/metabolism/genetics ; *Ku Autoantigen/metabolism/genetics ; Cell Line, Tumor ; *Cell Nucleus/metabolism ; Animals ; Lung Neoplasms/genetics/metabolism/radiotherapy/pathology ; Mice ; Glycosylation ; Radiation, Ionizing ; CRISPR-Cas Systems ; }, abstract = {Resistance to radiotherapy is a major barrier during cancer treatment. Here using genome-scale CRISPR/Cas9 screening, we identify CD274 gene, which encodes PD-L1, to confer lung cancer cell resistance to ionizing radiation (IR). Depletion of endogenous PD-L1 delays the repair of IR-induced DNA double-strand breaks (DSBs) and PD-L1 loss downregulates non-homologous end joining (NHEJ) while overexpression of PD-L1 upregulates NHEJ. IR induces translocation of PD-L1 from the membrane into nucleus dependent on deglycosylation of PD-L1 at N219 and CMTM6 and leads to PD-L1 recruitment to DSBs foci. PD-L1 interacts with Ku in the nucleus and enhances Ku binding to DSB DNA. The interaction between the IgC domain of PD-L1 and the core domain of Ku is required for PD-L1 to accelerate NHEJ-mediated DSB repair and produce radioresistance. Thus, PD-L1, in addition to its immune inhibitory activity, acts as mechanistic driver for NHEJ-mediated DSB repair in cancer.}, }
@article {pmid39122707, year = {2024}, author = {Gao, Y and Shonai, D and Trn, M and Zhao, J and Soderblom, EJ and Garcia-Moreno, SA and Gersbach, CA and Wetsel, WC and Dawson, G and Velmeshev, D and Jiang, YH and Sloofman, LG and Buxbaum, JD and Soderling, SH}, title = {Proximity analysis of native proteomes reveals phenotypic modifiers in a mouse model of autism and related neurodevelopmental conditions.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6801}, pmid = {39122707}, issn = {2041-1723}, support = {MH111684//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; }, mesh = {Animals ; *Proteome/metabolism ; *Disease Models, Animal ; Mice ; Humans ; *Brain/metabolism ; *Proteomics/methods ; *Autistic Disorder/genetics/metabolism ; *Autism Spectrum Disorder/metabolism/genetics ; Phenotype ; Gene Editing ; Male ; Genetic Predisposition to Disease ; Mice, Inbred C57BL ; Female ; CRISPR-Cas Systems ; }, abstract = {One of the main drivers of autism spectrum disorder is risk alleles within hundreds of genes, which may interact within shared but unknown protein complexes. Here we develop a scalable genome-editing-mediated approach to target 14 high-confidence autism risk genes within the mouse brain for proximity-based endogenous proteomics, achieving the identification of high-specificity spatial proteomes. The resulting native proximity proteomes are enriched for human genes dysregulated in the brain of autistic individuals, and reveal proximity interactions between proteins from high-confidence risk genes with those of lower-confidence that may provide new avenues to prioritize genetic risk. Importantly, the datasets are enriched for shared cellular functions and genetic interactions that may underlie the condition. We test this notion by spatial proteomics and CRISPR-based regulation of expression in two autism models, demonstrating functional interactions that modulate mechanisms of their dysregulation. Together, these results reveal native proteome networks in vivo relevant to autism, providing new inroads for understanding and manipulating the cellular drivers underpinning its etiology.}, }
@article {pmid39122671, year = {2024}, author = {Jin, YY and Zhang, P and Liu, LL and Zhao, X and Hu, XQ and Liu, SZ and Li, ZK and Liu, Q and Wang, JQ and Hao, DL and Zhang, ZQ and Chen, HZ and Liu, DP}, title = {Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6843}, pmid = {39122671}, issn = {2041-1723}, mesh = {*DNA, Single-Stranded/metabolism/genetics ; Humans ; *Gene Editing/methods ; *Recombinational DNA Repair ; *Rad51 Recombinase/metabolism/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; CRISPR-Associated Proteins/metabolism/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; DNA End-Joining Repair ; }, abstract = {Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.}, }
@article {pmid39121843, year = {2024}, author = {Versluis, P and Graham, TGW and Eng, V and Ebenezer, J and Darzacq, X and Zipfel, WR and Lis, JT}, title = {Live-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation.}, journal = {Molecular cell}, volume = {84}, number = {15}, pages = {2856-2869.e9}, doi = {10.1016/j.molcel.2024.07.009}, pmid = {39121843}, issn = {1097-4164}, mesh = {*RNA Polymerase II/metabolism/genetics ; Animals ; *Drosophila melanogaster/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Transcription, Genetic ; *Transcriptional Elongation Factors/metabolism/genetics ; HSP70 Heat-Shock Proteins/metabolism/genetics ; Positive Transcriptional Elongation Factor B/metabolism/genetics ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; Polytene Chromosomes/genetics/metabolism ; Gene Expression Regulation ; Phosphorylation ; Protein Binding ; Heat-Shock Response/genetics ; Nuclear Proteins/metabolism/genetics ; Nucleosomes/metabolism/genetics ; }, abstract = {RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.}, }
@article {pmid39120640, year = {2024}, author = {Adibzadeh, S and Amiri, S and Barkhordari, F and Mowla, SJ and Bayat, H and Ghanbari, S and Faghihi, F and Davami, F}, title = {CHO cell engineering via targeted integration of circular miR-21 decoy using CRISPR/RMCE hybrid system.}, journal = {Applied microbiology and biotechnology}, volume = {108}, number = {1}, pages = {434}, pmid = {39120640}, issn = {1432-0614}, support = {IR.PII.REC.1400.075//Pasteur Institute of Iran/ ; }, mesh = {CHO Cells ; *Cricetulus ; Animals ; *MicroRNAs/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Engineering/methods ; Gene Editing/methods ; Recombinant Proteins/genetics/metabolism ; Recombinases/genetics/metabolism ; Cricetinae ; }, abstract = {Chinese hamster ovary (CHO) cells, widely acknowledged as the preferred host system for industrial recombinant protein manufacturing, play a crucial role in developing pharmaceuticals, including anticancer therapeutics. Nevertheless, mammalian cell-based biopharmaceutical production methods are still beset by cellular constraints such as limited growth and poor productivity. MicroRNA-21 (miR-21) has a major impact on a variety of malignancies, including glioblastoma multiforme (GBM). However, reduced productivity and growth rate have been linked to miR-21 overexpression in CHO cells. The current study aimed to engineer a recombinant CHO (rCHO) cell using the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system coupled with the Bxb1 recombinase-mediated cassette exchange (RMCE) to express a circular miR-21 decoy (CM21D) with five bulged binding sites for miR-21 sponging. Implementing the ribonucleoprotein (RNP) delivery method, a landing pad was inserted into the genome utilizing the CRIS-PITCh technique. Subsequently, the CM21D cassette flanked by Bxb1 attB was then retargeted into the integrated landing pad using the RMCE/Bxb1 system. This strategy raised the targeting efficiency by 1.7-fold, and off-target effects were decreased. The miR-21 target genes (Pdcd4 and Atp11b) noticed a significant increase in expression upon the miR-21 sponging through CM21D. Following the expression of CM21D, rCHO cells showed a substantial decrease in doubling time and a 1.3-fold increase in growth rate. Further analysis showed an increased yield of hrsACE2, a secretory recombinant protein, by 2.06-fold. Hence, we can conclude that sponging-induced inhibition of miR-21 may lead to a growth rate increase that could be linked to increased CHO cell productivity. For industrial cell lines, including CHO cells, an increase in productivity is crucial. The results of our research indicate that CM21D is an auspicious CHO engineering approach. KEY POINTS: • CHO is an ideal host cell line for producing industrial therapeutics manufacturing, and miR-21 is downregulated in CHO cells, which produce recombinant proteins. • The miR-21 target genes noticed a significant increase in expression upon the miR-21 sponging through CM21D. Additionally, sponging of miR-21 by CM21D enhanced the growth rate of CHO cells. • Productivity and growth rate were increased in CHO cells expressing recombinant hrs-ACE2 protein after CM21D knocking in.}, }
@article {pmid39084224, year = {2024}, author = {Nicolas-Martinez, EC and Robinson, O and Pflueger, C and Gardner, A and Corbett, MA and Ritchie, T and Kroes, T and van Eyk, CL and Scheffer, IE and Hildebrand, MS and Barnier, JV and Rousseau, V and Genevieve, D and Haushalter, V and Piton, A and Denommé-Pichon, AS and Bruel, AL and Nambot, S and Isidor, B and Grigg, J and Gonzalez, T and Ghedia, S and Marchant, RG and Bournazos, A and Wong, WK and Webster, RI and Evesson, FJ and Jones, KJ and ,  and Cooper, ST and Lister, R and Gecz, J and Jolly, LA}, title = {RNA variant assessment using transactivation and transdifferentiation.}, journal = {American journal of human genetics}, volume = {111}, number = {8}, pages = {1673-1699}, doi = {10.1016/j.ajhg.2024.06.018}, pmid = {39084224}, issn = {1537-6605}, mesh = {Humans ; *Cell Transdifferentiation/genetics ; *Fibroblasts/metabolism/cytology ; *Transcriptional Activation ; *Neurons/metabolism/cytology ; RNA/genetics/metabolism ; CRISPR-Cas Systems ; }, abstract = {Understanding the impact of splicing and nonsense variants on RNA is crucial for the resolution of variant classification as well as their suitability for precision medicine interventions. This is primarily enabled through RNA studies involving transcriptomics followed by targeted assays using RNA isolated from clinically accessible tissues (CATs) such as blood or skin of affected individuals. Insufficient disease gene expression in CATs does however pose a major barrier to RNA based investigations, which we show is relevant to 1,436 Mendelian disease genes. We term these "silent" Mendelian genes (SMGs), the largest portion (36%) of which are associated with neurological disorders. We developed two approaches to induce SMG expression in human dermal fibroblasts (HDFs) to overcome this limitation, including CRISPR-activation-based gene transactivation and fibroblast-to-neuron transdifferentiation. Initial transactivation screens involving 40 SMGs stimulated our development of a highly multiplexed transactivation system culminating in the 6- to 90,000-fold induction of expression of 20/20 (100%) SMGs tested in HDFs. Transdifferentiation of HDFs directly to neurons led to expression of 193/516 (37.4%) of SMGs implicated in neurological disease. The magnitude and isoform diversity of SMG expression following either transactivation or transdifferentiation was comparable to clinically relevant tissues. We apply transdifferentiation and/or gene transactivation combined with short- and long-read RNA sequencing to investigate the impact that variants in USH2A, SCN1A, DMD, and PAK3 have on RNA using HDFs derived from affected individuals. Transactivation and transdifferentiation represent rapid, scalable functional genomic solutions to investigate variants impacting SMGs in the patient cell and genomic context.}, }
@article {pmid39025072, year = {2024}, author = {Wang, PY and Bartel, DP}, title = {The guide-RNA sequence dictates the slicing kinetics and conformational dynamics of the Argonaute silencing complex.}, journal = {Molecular cell}, volume = {84}, number = {15}, pages = {2918-2934.e11}, doi = {10.1016/j.molcel.2024.06.026}, pmid = {39025072}, issn = {1097-4164}, mesh = {*Argonaute Proteins/metabolism/genetics/chemistry ; Humans ; *RNA-Induced Silencing Complex/metabolism/genetics/chemistry ; Kinetics ; *Nucleic Acid Conformation ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; RNA Interference ; Base Sequence ; HEK293 Cells ; }, abstract = {The RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that, for different guide-RNA sequences, slicing rates of perfectly complementary bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6-7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3'-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3' complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action.}, }
@article {pmid39019044, year = {2024}, author = {Kim, S and Tan, S and Ku, J and Widowati, TA and Ku, D and Lee, K and You, K and Kim, Y}, title = {RNA 5-methylcytosine marks mitochondrial double-stranded RNAs for degradation and cytosolic release.}, journal = {Molecular cell}, volume = {84}, number = {15}, pages = {2935-2948.e7}, doi = {10.1016/j.molcel.2024.06.023}, pmid = {39019044}, issn = {1097-4164}, support = {R01 DE032707/DE/NIDCR NIH HHS/United States ; }, mesh = {Humans ; *Cytosol/metabolism ; *5-Methylcytosine/metabolism ; *Mitochondria/metabolism/genetics ; *RNA Stability ; *RNA, Double-Stranded/metabolism/genetics ; *RNA, Mitochondrial/genetics/metabolism ; HEK293 Cells ; HeLa Cells ; Methyltransferases/metabolism/genetics ; Immunity, Innate ; RNA, Long Noncoding/genetics/metabolism ; Animals ; RNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; }, abstract = {Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m[5]C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m[5]C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release.}, }
@article {pmid39119666, year = {2024}, author = {Cardiff, RAL and Carothers, JM and Zalatan, JG and Sauro, HM}, title = {Systems-Level Modeling for CRISPR-Based Metabolic Engineering.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.4c00053}, pmid = {39119666}, issn = {2161-5063}, abstract = {The CRISPR-Cas system has enabled the development of sophisticated, multigene metabolic engineering programs through the use of guide RNA-directed activation or repression of target genes. To optimize biosynthetic pathways in microbial systems, we need improved models to inform design and implementation of transcriptional programs. Recent progress has resulted in new modeling approaches for identifying gene targets and predicting the efficacy of guide RNA targeting. Genome-scale and flux balance models have successfully been applied to identify targets for improving biosynthetic production yields using combinatorial CRISPR-interference (CRISPRi) programs. The advent of new approaches for tunable and dynamic CRISPR activation (CRISPRa) promises to further advance these engineering capabilities. Once appropriate targets are identified, guide RNA prediction models can lead to increased efficacy in gene targeting. Developing improved models and incorporating approaches from machine learning may be able to overcome current limitations and greatly expand the capabilities of CRISPR-Cas9 tools for metabolic engineering.}, }
@article {pmid39118151, year = {2024}, author = {Li, T and Li, S and Kang, Y and Zhou, J and Yi, M}, title = {Harnessing the evolving CRISPR/Cas9 for precision oncology.}, journal = {Journal of translational medicine}, volume = {22}, number = {1}, pages = {749}, pmid = {39118151}, issn = {1479-5876}, support = {LQ24H160007//Natural Science Foundation of Zhejiang Province/ ; 2022M722766//Postdoctoral Research Foundation of China/ ; 2023M743016//Postdoctoral Research Foundation of China/ ; GZB20230642//National Postdoctoral Program for Innovative Talents/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Precision Medicine/methods ; *Neoplasms/genetics/therapy ; Gene Editing/methods ; Animals ; Medical Oncology/methods/trends ; }, abstract = {The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system, a groundbreaking innovation in genetic engineering, has revolutionized our approach to surmounting complex diseases, culminating in CASGEVY™ approved for sickle cell anemia. Derived from a microbial immune defense mechanism, CRISPR/Cas9, characterized as precision, maneuverability and universality in gene editing, has been harnessed as a versatile tool for precisely manipulating DNA in mammals. In the process of applying it to practice, the consecutive exploitation of novel orthologs and variants never ceases. It's conducive to understanding the essentialities of diseases, particularly cancer, which is crucial for diagnosis, prevention, and treatment. CRISPR/Cas9 is used not only to investigate tumorous genes functioning but also to model disparate cancers, providing valuable insights into tumor biology, resistance, and immune evasion. Upon cancer therapy, CRISPR/Cas9 is instrumental in developing individual and precise cancer therapies that can selectively activate or deactivate genes within tumor cells, aiming to cripple tumor growth and invasion and sensitize cancer cells to treatments. Furthermore, it facilitates the development of innovative treatments, enhancing the targeting efficiency of reprogrammed immune cells, exemplified by advancements in CAR-T regimen. Beyond therapy, it is a potent tool for screening susceptible genes, offering the possibility of intervening before the tumor initiative or progresses. However, despite its vast potential, the application of CRISPR/Cas9 in cancer research and therapy is accompanied by significant efficacy, efficiency, technical, and safety considerations. Escalating technology innovations are warranted to address these issues. The CRISPR/Cas9 system is revolutionizing cancer research and treatment, opening up new avenues for advancements in our understanding and management of cancers. The integration of this evolving technology into clinical practice promises a new era of precision oncology, with targeted, personalized, and potentially curative therapies for cancer patients.}, }
@article {pmid39116547, year = {2024}, author = {Ali, M and Xu, C and Wang, J and Kulyar, MF and Li, K}, title = {Emerging therapeutic avenues against Cryptosporidium: A comprehensive review.}, journal = {Veterinary parasitology}, volume = {331}, number = {}, pages = {110279}, doi = {10.1016/j.vetpar.2024.110279}, pmid = {39116547}, issn = {1873-2550}, abstract = {Cryptosporidium is among the top causes of life-threatening diarrheal infection in public health and livestock sectors. Despite its high prevalence and economic importance, currently, there is no vaccine. Control of this protozoan is difficult due to the excretion of many resistant oocysts in the feces of the infected host, which contaminate the environment. Paromomycin shows inconsistent results and isn't considered a reliable therapy for cryptosporidiosis. Nitazoxanide (NTZ), the only FDA-approved drug against this parasite, is less productive in impoverished children and PLWHA (people living with HIV/AIDS). The absence of mitochondria and apicoplast, its unique location inside enterocytes separated by parasitophorous vacuole, and, most importantly, challenges in its genetic manipulations are some hurdles to the drug-discovery process. A library of compounds has been tested against Cryptosporidium during in vitro and in vivo trials. However, there has still not been sufficient success in finding the drug of choice against this parasite. Recent genome editing technologies based on CRISPR/Cas-9 have explored the functions of the vital genes by producing transgenic parasites that help to screen a collection of compounds to find target-specific drugs, provided the sufficient availability of in vitro culturing platforms, efficient transfection methods, and analytic techniques. The use of herbal remedies against Cryptosporidium is also an emerging area of interest with sufficient clinical success due to enhanced concern regarding anthelmintic resistance. Here, we highlighted present treatment options with their associated limitations, the use of genetic tools and natural products against it to find safe, effective, and inexpensive drugs to control the ever-increasing global burden of this disease.}, }
@article {pmid38676538, year = {2024}, author = {Wang, Y and Xu, HQ and Han, HL and Chen, D and Jiang, H and Smagghe, G and Wang, JJ and Wei, D}, title = {CRISPR/Cas9-mediated knockout of a male accessory glands-specific gene takeout1 decreases the fecundity of Zeugodacus cucurbitae female.}, journal = {Pest management science}, volume = {80}, number = {9}, pages = {4399-4409}, doi = {10.1002/ps.8145}, pmid = {38676538}, issn = {1526-4998}, support = {SWU-XJLJ202304//Special Fund for Youth Team of the Southwest University/ ; CARS-26//The earmarked fund for China Agriculture Research System/ ; 2021YFC2600100//National Key Research and Development Program of China/ ; QJJ[2023]024//Program for Natural Science Research in Guizhou Education Department/ ; B18044//111 Project/ ; }, mesh = {Animals ; Male ; Female ; *CRISPR-Cas Systems ; *Fertility ; *Insect Proteins/genetics/metabolism ; *Tephritidae/genetics/physiology ; Gene Knockout Techniques ; Phylogeny ; }, abstract = {BACKGROUND: The melon fly, Zeugodacus cucurbitae (Coquillett), is an invasive Tephritidae pest with robust fertility. The male accessory glands (MAGs) form a vital organ that ensures insect reproductive efficiency. Most of the secreted proteins by MAGs exhibit a male bias expression. Takeout, one of these proteins, is abundantly present in the MAGs of many insects.<br><br>RESULTS: In this study, we identified 32 takeout genes in Z. cucurbitae. The phylogenetic analysis and multiple sequence alignment results showed that Zctakeout1 is the most related homolog to the MAGs-specific takeout in Tephritidae. The real-time quantitative PCR results showed that Zctakeout1 was exclusively expressed in the male adult stage, and its expression level gradually increased with the increase in age and then remained stable at the sexually matured stage. The distribution among tissues demonstrated the specific expression of Zctakeout1 in the MAGs, and fluorescence immunohistochemical results confirmed the presence of Zctakeout1 in close proximity to binuclear cells of the mesoderm epidermal MAGs. In continuation, CRISPR/Cas9-mediated genome editing was employed, resulting in successfully generating a homozygous strain with an +8&#x2009;bp insertion. The mating experiments with the Zctakeout1[-/-] males resulted in significant reductions in both the mating rate and egg production of females.<br><br>CONCLUSION: These findings prove that the MAGs-specific Zctakeout1 is essential in regulating fecundity in female Z. cucurbitae fruit flies. Our data suggests its utilization in future essential insect-specific gene-directed sterility insect technique (SIT) by the genetic manipulation to keep these important Tephritidae populations under control. &#xa9; 2024 Society of Chemical Industry.}, }
@article {pmid39116129, year = {2024}, author = {Liu, J and Zheng, Q and Yao, R and Wang, M}, title = {Lung-specific supramolecular nanoparticles for efficient delivery of therapeutic proteins and genome editing nucleases.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {33}, pages = {e2406654121}, doi = {10.1073/pnas.2406654121}, pmid = {39116129}, issn = {1091-6490}, support = {22077125//MOST | National Natural Science Foundation of China (NSFC)/ ; Z220023//| Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; }, mesh = {Animals ; *Gene Editing/methods ; *Lung/metabolism ; Mice ; *Nanoparticles/chemistry ; Dogs ; Rabbits ; Humans ; CRISPR-Cas Systems ; Drug Delivery Systems/methods ; }, abstract = {Protein therapeutics play a critical role in treating a large variety of diseases, ranging from infections to genetic disorders. However, their delivery to target tissues beyond the liver, such as the lungs, remains a great challenge. Here, we report a universally applicable strategy for lung-targeted protein delivery by engineering Lung-Specific Supramolecular Nanoparticles (LSNPs). These nanoparticles are designed through the hierarchical self-assembly of metal-organic polyhedra (MOP), featuring a customized surface chemistry that enables protein encapsulation and specific lung affinity after intravenous administration. Our design of LSNPs not only addresses the hurdles of cell membrane impermeability of protein and nonspecific tissue distribution of protein delivery, but also shows exceptional versatility in delivering various proteins, including those vital for anti-inflammatory and CRISPR-based genome editing to the lung, and across multiple animal species, including mice, rabbits, and dogs. Notably, the delivery of antimicrobial proteins using LSNPs effectively alleviates acute bacterial pneumonia, demonstrating a significant therapeutic potential. Our strategy not only surmounts the obstacles of tissue-specific protein delivery but also paves the way for targeted treatments in genetic disorders and combating antibiotic resistance, offering a versatile solution for precision protein therapy.}, }
@article {pmid39115550, year = {2024}, author = {Kaur, R and Gupta, S and Chauhan, A and Mishra, V and Sharma, MK and Singh, J}, title = {Harnessing the power of clustered regularly interspaced short palindromic repeats (CRISPR) based microfluidics for next-generation molecular diagnostics.}, journal = {Molecular biology reports}, volume = {51}, number = {1}, pages = {896}, pmid = {39115550}, issn = {1573-4978}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Microfluidics/methods ; Pathology, Molecular/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Molecular Diagnostic Techniques/methods ; Gene Editing/methods ; Lab-On-A-Chip Devices ; }, abstract = {CRISPR-based (Clustered regularly interspaced short palindromic repeats-based) technologies have revolutionized molecular biology and diagnostics, offering unprecedented precision and versatility. However, challenges remain, such as high costs, demanding technical expertise, and limited quantification capabilities. To overcome these limitations, innovative microfluidic platforms are emerging as powerful tools for enhancing CRISPR diagnostics. This review explores the exciting intersection of CRISPR and microfluidics, highlighting their potential to revolutionize healthcare diagnostics. By integrating CRISPR's specificity with microfluidics' miniaturization and automation, researchers are developing more sensitive and portable diagnostic tools for a range of diseases. These microfluidic devices streamline sample processing, improve diagnostic performance, and enable point-of-care applications, allowing for rapid and accurate detection of pathogens, genetic disorders, and other health conditions. The review discusses various CRISPR/Cas systems, including Cas9, Cas12, and Cas13, and their integration with microfluidic platforms. It also examines the advantages and limitations of these systems, highlighting their potential for detecting DNA and RNA biomarkers. The review also explores the key challenges in developing and implementing CRISPR-driven microfluidic diagnostics, such as ensuring robustness, minimizing cross-contamination, and achieving robust quantification. Finally, it highlights potential future directions for this rapidly evolving field, emphasizing the transformative potential of these technologies for personalized medicine and global health.}, }
@article {pmid39111310, year = {2024}, author = {Hirano, S and Altae-Tran, H and Kannan, S and Macrae, RK and Zhang, F}, title = {Structural determinants of DNA cleavage by a CRISPR HNH-Cascade system.}, journal = {Molecular cell}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.molcel.2024.07.026}, pmid = {39111310}, issn = {1097-4164}, abstract = {Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. Recently, a highly precise subtype I-F1 CRISPR-Cas system (HNH-Cascade) was found that lacks Cas3, the absence of which is compensated for by the insertion of an HNH endonuclease domain in the Cas8 Cascade component. Here, we describe the cryo-EM structure of Selenomonas sp. HNH-Cascade (SsCascade) in complex with target DNA and characterize its mechanism of action. The Cascade scaffold is complemented by the HNH domain, creating a ring-like structure in which the unwound target DNA is precisely cleaved. This structure visualizes a unique hybrid of two extensible biological systems-Cascade, an evolutionary platform for programmable DNA effectors, and an HNH nuclease, an adaptive domain with a spectrum of enzymatic activity.}, }
@article {pmid39084223, year = {2024}, author = {Collin, RWJ and Leroy, BP}, title = {In vivo genome editing for inherited retinal disease: Opportunities and challenges.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {32}, number = {8}, pages = {2433-2434}, doi = {10.1016/j.ymthe.2024.07.001}, pmid = {39084223}, issn = {1525-0024}, mesh = {*Gene Editing/methods ; Humans ; *Retinal Diseases/therapy/genetics ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; Animals ; }, }
@article {pmid38996675, year = {2024}, author = {Zhu, QY and Ren, ML and Jiang, YJ and He, C and Ding, ZJ and Zheng, SJ and Wang, ZG and Jin, CW}, title = {Co-mutation of OsLPR1/3/4/5 provides a promising strategy to minimize Cd contamination in rice grains.}, journal = {Journal of hazardous materials}, volume = {476}, number = {}, pages = {135165}, doi = {10.1016/j.jhazmat.2024.135165}, pmid = {38996675}, issn = {1873-3336}, mesh = {*Oryza/genetics/metabolism ; *Cadmium ; *Mutation ; *Plant Proteins/genetics/metabolism ; Soil Pollutants/metabolism ; Edible Grain ; Plants, Genetically Modified/genetics/metabolism ; CRISPR-Cas Systems ; Oxidoreductases/genetics/metabolism ; Food Contamination/analysis ; }, abstract = {Minimizing cadmium (Cd) contamination in rice grains is crucial for ensuring food security and promoting sustainable agriculture. Utilizing genetic modification to generate rice varieties with low Cd accumulation is a promising strategy due to its cost-effectiveness and operational simplicity. Our study demonstrated that the CRISPR-Cas9-mediated quadruple mutation of the multicopper oxidase genes OsLPR1/3/4/5 in the japonica rice cultivar Tongjing 981 had little effect on yields. However, a notable increase was observed in the cell wall functional groups that bind with Cd. As a result, the quadruple mutation of OsLPR1/3/4/5 enhanced Cd sequestration within the cell wall while reducing Cd concentrations in both xylem and phloem sap, thereby inhibiting Cd transport from roots to shoots. Consequently, Cd concentrations in brown rice and husk in oslpr1/3/4/5 quadruple mutants (qm) decreased by 52% and 55%, respectively, compared to the wild-type. These findings illustrate that the quadruple mutation of OsLPR1/3/4/5 is an effective method for minimizing Cd contamination in rice grains without compromising yields. Therefore, the quadruple mutation of OsLPR1/3/4/5 via biotechnological pathways may represent a valuable strategy for the generation of new rice varieties with low Cd accumulation.}, }
@article {pmid38987000, year = {2024}, author = {Zhang, H and Wang, Y and Chen, C and Xing, W and Xia, W and Fu, W and Liu, A and Zhang, C and Guan, Q and Zhao, Y and Sun, G and Lu, D and Dong, Z and Li, Z and Zhou, Y and Zhang, S and Du, Y and Zheng, C and Xu, D}, title = {A novel rapid visual nucleic acid detection technique for tick-borne encephalitis virus by combining RT-recombinase-aided amplification and CRISPR/Cas13a coupled with a lateral flow dipstick.}, journal = {International journal of biological macromolecules}, volume = {275}, number = {Pt 2}, pages = {133720}, doi = {10.1016/j.ijbiomac.2024.133720}, pmid = {38987000}, issn = {1879-0003}, mesh = {*Encephalitis Viruses, Tick-Borne/genetics ; *CRISPR-Cas Systems ; Humans ; *Recombinases/metabolism ; Nucleic Acid Amplification Techniques/methods ; Encephalitis, Tick-Borne/diagnosis/virology/blood ; Sensitivity and Specificity ; RNA, Viral/genetics ; Molecular Diagnostic Techniques/methods ; }, abstract = {Tick-borne encephalitis virus (TBEV), a zoonotic pathogen, can cause severe neurological complications and fatal outcomes in humans. Early diagnosis of TBEV infection is crucial for clinical practice. Although serological assays are frequently employed for detection, the lack of antibodies in the early stages of infection and the cross-reactivity of antibodies limit their efficacy. Conventional molecular diagnostic methods such as RT-qPCR can achieve early and accurate identification but require specialized instrumentation and professionals, hindering their application in resource-limited areas. Our study developed a rapid and visual TBEV molecular detection method by combining RT-recombinase-aided amplification, the CRISPR/Cas13a system, and lateral flow dipsticks. The diagnostic sensitivity of this method is 50 CFU/ml, with no cross-reactivity with a variety of viruses. The detection can be carried out within 1 h at a temperature between 37 and 42 °C, and the results can be visually determined without the need for complex instruments and professionals. Subsequently, this assay was used to analyze clinical samples from 15 patients suspected of TBEV infection and 10 healthy volunteers, and its sensitivity and specificity reached 100 %, which was consistent with the results of RT-qPCR. These results indicate that this new method can be a promising point-of-care test for the diagnosis of tick-borne encephalitis.}, }
@article {pmid38949306, year = {2024}, author = {Higgins, SA and Kara Murdoch, F and Clifton, JM and Brooks, JH and Fillinger, KL and Middleton, JK and Heater, BS}, title = {CRISPR-Cas9-mediated barcode insertion into Bacillus thuringiensis for surrogate tracking.}, journal = {Microbiology spectrum}, volume = {12}, number = {8}, pages = {e0000324}, pmid = {38949306}, issn = {2165-0497}, support = {FA8075-14-D-0003-FA807518F1414//DOD | OSD | Defense Technical Information Center (DTIC)/ ; 70RSAT19KPM0000470001//U.S. Department of Homeland Security (DHS)/ ; }, mesh = {*Bacillus thuringiensis/genetics ; *CRISPR-Cas Systems ; *DNA Barcoding, Taxonomic/methods ; Genome, Bacterial/genetics ; Bacillus anthracis/genetics ; Whole Genome Sequencing/methods ; Plasmids/genetics ; Gene Editing/methods ; }, abstract = {UNLABELLED: The use of surrogate organisms can enable researchers to safely conduct research on pathogens and in a broader set of conditions. Being able to differentiate between the surrogates used in the experiments and background contamination as well as between different experiments will further improve research efforts. One effective approach is to introduce unique genetic barcodes into the surrogate genome and track their presence using the quantitative polymerase chain reaction (qPCR). In this report, we utilized the CRISPR-Cas9 methodology, which employs a single plasmid and a transformation step to insert five distinct barcodes into Bacillus thuringiensis, a well-established surrogate for Bacillus anthracis when Risk Group 1 organisms are needed. We subsequently developed qPCR assays for barcode detection and successfully demonstrated the stability of the barcodes within the genome through five cycles of sporulation and germination. Additionally, we conducted whole-genome sequencing on these modified strains and analyzed 187 potential Cas9 off-target sites. We found no correlation between the mutations observed in the engineered strains and the predicted off-target sites, suggesting this genome engineering strategy did not directly result in off-target mutations in the genome. This simple approach has the potential to streamline the creation of barcoded B. thuringiensis strains for use in future studies on surrogate genomes.<br><br>IMPORTANCE: The use of Bacillus anthracis as a biothreat agent poses significant challenges for public health and national security. Bacillus anthracis surrogates, like Bacillus thuringiensis, are invaluable tools for safely understanding Bacillus anthracis properties without the safety concerns that would arise from using a virulent strain of Bacillus anthracis. We report a simple method for barcode insertion into Bacillus thuringiensis using the CRISPR-Cas9 methodology and subsequent tracking by quantitative polymerase chain reaction (qPCR). Moreover, whole-genome sequencing data and CRISPR-Cas9 off-target analyses in Bacillus thuringiensis suggest that this gene-editing method did not directly cause unwanted mutations in the genome. This study should assist in the facile development of barcoded Bacillus thuringiensis surrogate strains, among other biotechnological applications in Bacillus species.}, }
@article {pmid38943249, year = {2024}, author = {Gong, Y and Germeraad, WTV and Zhang, X and Wu, N and Li, B and Janssen, L and He, Z and Gijbels, MJJ and Wu, B and Gijsbers, BLMG and Olieslagers, TI and Bos, GMJ and Zheng, L and Klein Wolterink, RGJ}, title = {NKG2A genetic deletion promotes human primary NK cell anti-tumor responses better than an anti-NKG2A monoclonal antibody.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {32}, number = {8}, pages = {2711-2727}, doi = {10.1016/j.ymthe.2024.06.034}, pmid = {38943249}, issn = {1525-0024}, mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *NK Cell Lectin-Like Receptor Subfamily C/genetics/metabolism ; Animals ; Mice ; *Xenograft Model Antitumor Assays ; Cell Line, Tumor ; HLA-E Antigens ; Neoplasms/immunology/therapy/genetics ; Antibodies, Monoclonal/pharmacology ; CRISPR-Cas Systems ; Gene Deletion ; Histocompatibility Antigens Class I/immunology/genetics/metabolism ; Cytotoxicity, Immunologic ; }, abstract = {Natural killer (NK) cells eliminate infected or cancer cells via their cytotoxic capacity. NKG2A is an inhibitory receptor on NK cells and cancer cells often overexpress its ligand HLA-E to evade NK cell surveillance. Given the successes of immune checkpoint blockade in cancer therapy, NKG2A is an interesting novel target. However, anti-NKG2A antibodies have shown limited clinical response. In the pursuit of enhancing NK cell-mediated anti-tumor responses, we devised a Cas9-based strategy to delete KLRC1, encoding NKG2A, in human primary NK cells. Our approach involved electroporation of KLRC1-targeting Cas9 ribonucleoprotein resulting in effective ablation of NKG2A expression. Compared with anti-NKG2A antibody blockade, NKG2A[KO] NK cells exhibited enhanced activation, reduced suppressive signaling, and elevated expression of key transcription factors. NKG2A[KO] NK cells overcame inhibition from HLA-E, significantly boosting NK cell activity against solid and hematologic cancer cells. We validated this efficacy across multiple cell lines, a xenograft mouse model, and primary human leukemic cells. Combining NKG2A knockout with antibody coating of tumor cells further enhanced cytotoxicity through ADCC. Thus, we provide a comprehensive comparison of inhibition of the NKG2A pathway using genetic ablation and antibodies and provide novel insight in the observed differences in molecular mechanisms, which can be translated to enhance adoptive NK cell immunotherapy.}, }
@article {pmid38937969, year = {2024}, author = {Porreca, I and Blassberg, R and Harbottle, J and Joubert, B and Mielczarek, O and Stombaugh, J and Hemphill, K and Sumner, J and Pazeraitis, D and Touza, JL and Francescatto, M and Firth, M and Selmi, T and Collantes, JC and Strezoska, Z and Taylor, B and Jin, S and Wiggins, CM and van Brabant Smith, A and Lambourne, JJ}, title = {An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {32}, number = {8}, pages = {2692-2710}, doi = {10.1016/j.ymthe.2024.06.033}, pmid = {38937969}, issn = {1525-0024}, mesh = {Humans ; *Gene Editing/methods ; *Gene Knockout Techniques ; *CRISPR-Cas Systems ; *Aptamers, Nucleotide/genetics ; *T-Lymphocytes/metabolism/immunology ; Immunotherapy, Adoptive/methods ; Receptors, Chimeric Antigen/genetics/metabolism ; Gene Knock-In Techniques/methods ; Transgenes ; }, abstract = {Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.}, }
@article {pmid38901249, year = {2024}, author = {Yang, J and Zhao, Y and Qian, L and Yu, Y and Zhang, Y and Zhang, J}, title = {Modularization of dual recognized CRISPR/Cas12a system for the detection of Staphylococcus aureus assisted by hydrazone chemistry.}, journal = {Journal of hazardous materials}, volume = {476}, number = {}, pages = {134877}, doi = {10.1016/j.jhazmat.2024.134877}, pmid = {38901249}, issn = {1873-3336}, mesh = {*Staphylococcus aureus/drug effects/genetics ; *Hydrazones/chemistry/pharmacology ; *CRISPR-Cas Systems ; }, abstract = {In this work, a dual recognized CRISPR/Cas12a system has been proposed, in which the activation chain is cleverly divided into two parts that can serve for precise dual target recognition, and hydrazone chemistry is introduced for the formation of a whole activation chain. It has been further explored to construct a new method for the specific and sensitive detection of Staphylococcus aureus (SA) as one of the most common pathogens in infectious diseases. In virtue of proximity effect contributed by complementary base pairing, hydrazone chemistry accelerates the formation of the whole activation strand and improves the specificity of the CRISPR/Cas12a system, serving for the accurate analysis of SA. Moreover, the temporary aggregation of CRISPR/Cas12a around SA enhances its catalytical efficiency so as to further amplify signal. With high sensitivity, stability, reproducibility and specificity, the established method has been successfully applied to detect SA in complex substrates. Meanwhile, our established method can well evaluate the inhibition effect of chlorogenic acid and congo red in comparison with flow cytometry. ENVIRONMENTAL IMPLICATION: Bacterial pathogens exist widely in the environment and seriously threaten the safety of human health. Staphylococcus aureus (SA) is the most common pathogen of human suppurative infection, which can cause local suppurative infection, pneumonia, and even systemic infections such as sepsis. In this work, a dual recognized CRISPR/Cas12a system mediated by hydrazone chemistry has been proposed. With high sensitivity and low detection limit, the established method can specifically detect SA and effectively evaluate the antibacterial effect of inhibitors. This method is expected to be further developed into a detection method in different scenarios such as environmental monitoring and clinical diagnosis.}, }
@article {pmid38879753, year = {2024}, author = {Zhao, L and Lai, Y and Jiao, H and Li, J and Lu, K and Huang, J}, title = {CRISPR-mediated Sox9 activation and RelA inhibition enhance cell therapy for osteoarthritis.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {32}, number = {8}, pages = {2549-2562}, doi = {10.1016/j.ymthe.2024.06.016}, pmid = {38879753}, issn = {1525-0024}, support = {R01 AR070222/AR/NIAMS NIH HHS/United States ; }, mesh = {*SOX9 Transcription Factor/genetics/metabolism ; *Osteoarthritis/therapy/genetics/metabolism ; *Mesenchymal Stem Cells/metabolism ; Animals ; *Transcription Factor RelA/metabolism/genetics ; Mice ; *CRISPR-Cas Systems ; Humans ; Disease Models, Animal ; Cartilage, Articular/metabolism/pathology ; Mesenchymal Stem Cell Transplantation/methods ; Chondrogenesis/genetics ; Gene Editing ; Cell- and Tissue-Based Therapy/methods ; Chondrocytes/metabolism ; }, abstract = {Osteoarthritis (OA) is a painful and debilitating disease affecting over 500 million people worldwide. Intraarticular injection of mesenchymal stromal cells (MSCs) shows promise for the clinical treatment of OA, but the lack of consistency in MSC preparation and application makes it difficult to further optimize MSC therapy and to properly evaluate the clinical outcomes. In this study, we used Sox9 activation and RelA inhibition, both mediated by the CRISPR-dCas9 technology simultaneously, to engineer MSCs with enhanced chondrogenic potential and downregulated inflammatory responses. We found that both Sox9 and RelA could be fine-tuned to the desired levels, which enhances the chondrogenic and immunomodulatory potentials of the cells. Intraarticular injection of modified cells significantly attenuated cartilage degradation and palliated OA pain compared with the injection of cell culture medium or unmodified cells. Mechanistically, the modified cells promoted the expression of factors beneficial to cartilage integrity, inhibited the production of catabolic enzymes in osteoarthritic joints, and suppressed immune cells. Interestingly, a substantial number of modified cells could survive in the cartilaginous tissues including articular cartilage and meniscus. Together, our results suggest that CRISPR-dCas9-based gene regulation is useful for optimizing MSC therapy for OA.}, }
@article {pmid38876330, year = {2024}, author = {Wei, Y and Shi, D and Chen, T and Zhou, S and Yang, Z and Li, H and Yang, D and Li, J and Jin, M}, title = {CRISPR/Cas9-based engineered Escherichia coli biosensor for sensitive and specific detection of Cd(II) in drinking water.}, journal = {Chemosphere}, volume = {362}, number = {}, pages = {142607}, doi = {10.1016/j.chemosphere.2024.142607}, pmid = {38876330}, issn = {1879-1298}, mesh = {*Drinking Water/microbiology ; *Biosensing Techniques/methods ; *Escherichia coli/genetics ; *Cadmium/analysis ; *CRISPR-Cas Systems ; *Water Pollutants, Chemical/analysis ; Gene Editing ; Limit of Detection ; Environmental Monitoring/methods ; }, abstract = {Cadmium (Cd) is a ubiquitous pollutant that poses a potential threat to human health. Monitoring Cd(II) in drinking water has significant implications for preventing potential threats of Cd(II) to human. However, the weak signal output and response to nontarget interference limit the detection of Cd(II) using bacterial biosensors. In this study, to enable sensitive and specific detection of Cd(II) in water, a stable whole-cell biosensor, K12-PMP-luxCDABE-△cysI, was constructed in a dual-promoter mode by fusing the mercury promoter Pmer, regulatory gene merR(m), and luciferase gene luxCDABE into the E.coli chromosome based on CRISPR/Cas9 gene editing technology. By knocking out the cadmium-resistance-gene cysI, the sensitivity of the biosensor to Cd(II) was further enhanced. The constructed E. coli biosensor K12-PMP-luxCDABE-△cysI exhibited good nonlinear responses to 0.005-2 mg/L Cd(II). Notably, among the three constructed E. coli biosensor, it exhibited the strongest fluorescence intensity, with the limit of detection meeting the allowable limit for Cd(II) in drinking water. Simultaneously, it could specifically detect Cd(II). Nontarget metal ions, such as Zn(II), Hg(II), and Pb(II), did not affect its performance. Furthermore, it exhibited superior performance in detecting Cd(II) in real drinking water samples by avoiding background interference, and showed excellent stability with the relative standard deviation under 5%. Thus, K12-PMP-luxCDABE-△cysI holds promise as a potential tool for the detection of Cd(II) in drinking water.}, }
@article {pmid38867450, year = {2024}, author = {Sartori-Maldonado, R and Montaser, H and Soppa, I and Eurola, S and Juutila, J and Balaz, M and Puttonen, H and Otonkoski, T and Saarimäki-Vire, J and Wartiovaara, K}, title = {Thymidylate synthase disruption to limit cell proliferation in cell therapies.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {32}, number = {8}, pages = {2535-2548}, doi = {10.1016/j.ymthe.2024.06.014}, pmid = {38867450}, issn = {1525-0024}, mesh = {Humans ; Animals ; *Cell Proliferation ; Mice ; *Thymidine/metabolism ; *Cell Differentiation ; *Thymidylate Synthase/genetics/metabolism ; Insulin-Secreting Cells/metabolism/cytology ; Cell- and Tissue-Based Therapy/methods ; Cell Line ; Pluripotent Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; }, abstract = {Stem and progenitor cells hold great promise for regenerative medicine and gene therapy approaches. However, transplantation of living cells entails a fundamental risk of unwanted growth, potentially exacerbated by CRISPR-Cas9 or other genetic manipulations. Here, we describe a safety system to control cell proliferation while allowing robust and efficient cell manufacture, without any added genetic elements. Inactivating TYMS, a key nucleotide metabolism enzyme, in several cell lines resulted in cells that proliferate only when supplemented with exogenous thymidine. Under supplementation, TYMS[-/-]-pluripotent stem cells proliferate, produce teratomas, and successfully differentiate into potentially therapeutic cell types such as pancreatic β cells. Our results suggest that supplementation with exogenous thymidine affects stem cell proliferation, but not the function of stem cell-derived cells. After differentiation, postmitotic cells do not require thymidine in vitro or in vivo, as shown by the production of functional human insulin in mice up to 5 months after implantation of stem cell-derived pancreatic tissue.}, }
@article {pmid38822173, year = {2024}, author = {}, title = {Paired CRISPR screening libraries for studying the function of the non-coding genome at scale.}, journal = {Nature biomedical engineering}, volume = {8}, number = {7}, pages = {806-807}, pmid = {38822173}, issn = {2157-846X}, support = {11707//KWF Kankerbestrijding (Dutch Cancer Society)/ ; 950655-Silencer//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Library ; Gene Editing/methods ; Genome/genetics ; }, }
@article {pmid38778183, year = {2024}, author = {Li, Y and Tan, M and Akkari-Henić, A and Zhang, L and Kip, M and Sun, S and Sepers, JJ and Xu, N and Ariyurek, Y and Kloet, SL and Davis, RP and Mikkers, H and Gruber, JJ and Snyder, MP and Li, X and Pang, B}, title = {Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs.}, journal = {Nature biomedical engineering}, volume = {8}, number = {7}, pages = {890-908}, pmid = {38778183}, issn = {2157-846X}, support = {11707//KWF Kankerbestrijding (Dutch Cancer Society)/ ; 950655-Silencer//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; }, mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; K562 Cells ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; Genome, Human/genetics ; Cell Differentiation/genetics ; Myocytes, Cardiac/metabolism ; RNA, Untranslated/genetics ; Human Embryonic Stem Cells/metabolism ; PAX6 Transcription Factor/genetics/metabolism ; Gene Library ; }, abstract = {The functions of non-coding regulatory elements (NCREs), which constitute a major fraction of the human genome, have not been systematically studied. Here we report a method involving libraries of paired single-guide RNAs targeting both ends of an NCRE as a screening system for the Cas9-mediated deletion of thousands of NCREs genome-wide to study their functions in distinct biological contexts. By using K562 and 293T cell lines and human embryonic stem cells, we show that NCREs can have redundant functions, and that many ultra-conserved elements have silencer activity and play essential roles in cell growth and in cellular responses to drugs (notably, the ultra-conserved element PAX6_Tarzan may be critical for heart development, as removing it from human embryonic stem cells led to defects in cardiomyocyte differentiation). The high-throughput screen, which is compatible with single-cell sequencing, may allow for the identification of druggable NCREs.}, }
@article {pmid38685159, year = {2024}, author = {Wirdnam, CD and Warmus, D and Faso, C}, title = {Nourseothricin as a novel drug for selection of transgenic Giardia lamblia.}, journal = {International journal for parasitology. Drugs and drug resistance}, volume = {25}, number = {}, pages = {100543}, pmid = {38685159}, issn = {2211-3207}, mesh = {*Giardia lamblia/genetics/drug effects ; *Streptothricins/pharmacology ; Acetyltransferases/genetics ; Drug Resistance/genetics ; Streptomyces/genetics/drug effects ; Antiprotozoal Agents/pharmacology ; Organisms, Genetically Modified ; CRISPR-Cas Systems ; }, abstract = {Functional gene and protein characterizations in parasitic protists are often limited by their genetic tractability. Despite the development of CRISPR-Cas9-derived or inspired approaches for a handful of protist parasites, the overall genetic tractability of these organisms remains limited. The intestinal parasite Giardia lamblia is one such species, with the added challenge of a paucity of reliable selection markers. To address this limitation, we tested the feasibility of using Nourseothricin as an effective selection agent in Giardia. Here, we report that axenically-grown WB Giardia cells are sensitive to Nourseothricin and that engineering expression of the streptothricin acetyltransferase (SAT-1) gene from Streptomyces rochei in transgenic parasites confers resistance to this antibiotic. Furthermore, we determine that SAT-1-expressing parasites are cross-resistant neither to Neomycin nor Puromycin, which are widely used to select for transgenic parasites. Consequently, we show that Nourseothricin can be used in sequential combination with both Neomycin and Puromycin to select for dual transfection events. This work increases the number of reliable selection agents and markers for Giardia genetic manipulation, expanding the limited molecular toolbox for this species of global medical importance.}, }
@article {pmid38493051, year = {2024}, author = {Clark, T and Waller, MA and Loo, L and Moreno, CL and Denes, CE and Neely, GG}, title = {CRISPR activation screens: navigating technologies and applications.}, journal = {Trends in biotechnology}, volume = {42}, number = {8}, pages = {1017-1034}, doi = {10.1016/j.tibtech.2024.02.007}, pmid = {38493051}, issn = {1879-3096}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Testing/methods ; Animals ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) has become an integral part of the molecular biology toolkit. CRISPRa genetic screens are an exciting high-throughput means of identifying genes the upregulation of which is sufficient to elicit a given phenotype. Activation machinery is continually under development to achieve greater, more robust, and more consistent activation. In this review, we offer a succinct technological overview of available CRISPRa architectures and a comprehensive summary of pooled CRISPRa screens. Furthermore, we discuss contemporary applications of CRISPRa across broad fields of research, with the aim of presenting a view of exciting emerging applications for CRISPRa screening.}, }
@article {pmid39110781, year = {2024}, author = {Han, J and Sul, JH and Lee, J and Kim, E and Kim, HK and Chae, M and Lim, J and Kim, J and Kim, C and Kim, JS and Cho, Y and Park, JH and Cho, YW and Jo, DG}, title = {Engineered exosomes with a photoinducible protein delivery system enable CRISPR-Cas-based epigenome editing in Alzheimer's disease.}, journal = {Science translational medicine}, volume = {16}, number = {759}, pages = {eadi4830}, doi = {10.1126/scitranslmed.adi4830}, pmid = {39110781}, issn = {1946-6242}, mesh = {*Exosomes/metabolism ; Animals ; *Alzheimer Disease/genetics/therapy/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Mice ; Epigenesis, Genetic ; Drug Delivery Systems ; Amyloid Precursor Protein Secretases/metabolism ; Amyloid beta-Peptides/metabolism ; Disease Models, Animal ; Integrases/metabolism ; }, abstract = {Effective intracellular delivery of therapeutic proteins can potentially treat a wide array of diseases. However, efficient delivery of functional proteins across the cell membrane remains challenging. Exosomes are nanosized vesicles naturally secreted by various types of cells and may serve as promising nanocarriers for therapeutic biomolecules. Here, we engineered exosomes equipped with a photoinducible cargo protein release system, termed mMaple3-mediated protein loading into and release from exosome (MAPLEX), in which cargo proteins can be loaded into the exosomes by fusing them with photocleavable protein (mMaple3)-conjugated exosomal membrane markers and subsequently released from the exosomal membrane by inducing photocleavage with blue light illumination. Using this system, we first induced transcriptional regulation by delivering octamer-binding transcription factor 4 and SRY-box transcription factor 2 to fibroblasts in vitro. Second, we induced in vivo gene recombination in Cre reporter mice by delivering Cre recombinase. Last, we achieved targeted epigenome editing in the brains of 5xFAD and 3xTg-AD mice, two models of Alzheimer's disease. Administration of MAPLEXs loaded with β-site amyloid precursor protein cleaving enzyme 1 (Bace1)-targeting single guide RNA-incorporated dCas9 ribonucleoprotein complexes, coupled with the catalytic domain of DNA methyltransferase 3A, resulted in successful methylation of the targeted CpG sites within the Bace1 promoter. This approach led to a significant reduction in Bace1 expression, improved recognition memory impairment, and reduced amyloid pathology in 5xFAD and 3xTg-AD mice. These results suggest that MAPLEX is an efficient intracellular protein delivery system that can deliver diverse therapeutic proteins for multiple diseases.}, }
@article {pmid39109857, year = {2024}, author = {Zhang, M and Lang, Y and Li, W}, title = {Multiplexed CRISPR-Cas system targeting ASFV genes in vivo: solution lies within.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0071424}, doi = {10.1128/spectrum.00714-24}, pmid = {39109857}, issn = {2165-0497}, abstract = {The emergence and spread of the African swine fever virus (ASFV) posed a significant threat to the global swine breeding industry, calling for innovative approaches benefiting viral containment and control. A recent study (Z. Zheng, L. Xu, H. Dou, Y. Zhou, X., et al., Microbiol Spectr 12: e02164-23, 2024, https://doi.org/10.1128/spectrum.02164-23) established a multiplexed CRISPR-Cas system targeting the genome of ASFV and tested the consequent antiviral activity both in vitro and in vivo. Application of this system showed a significant reduction of viral replication in vitro, while the germline-edited pigs expressing this system exhibited normal growth with continuous guide RNA expression. Although no survival advantage was observed upon ASFV challenge compared with nonengineered pigs, this marks the first attempt of germline editing to pursue ASFV resistance and paves the way for future disease-resistant animal breeding approaches utilizing CRISPR-Cas technology.}, }
@article {pmid39108851, year = {2024}, author = {Wang, J and Liu, F and Long, J and Jin, Y and Chen, S and Duan, G and Yang, H}, title = {The application of CRISPR-Cas system in Staphylococcus aureus infection.}, journal = {Heliyon}, volume = {10}, number = {14}, pages = {e34383}, pmid = {39108851}, issn = {2405-8440}, abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated nuclease (Cas) system has been proven to play an irreplaceable role in bacteria immunity activity against exogenous genetic elements. In recent years, this system has emerged as a valid gene engineering method and could be used to detect and treat various microorganisms such as bacteria and viruses, etc. Staphylococcus aureus, as a Gram-positive, opportunistic human and animal pathogen, can cause a variety of diseases greatly threatening human health. Here, we mainly reviewed the applications of the CRISPR-Cas system in Staphylococcus aureus infections in detail. Furthermore, the prospects and drawbacks of the CRISPR-Cas system were also discussed.}, }
@article {pmid39107894, year = {2024}, author = {Müller, T and Bronkhorst, J and Müller, J and Safari, N and Hahn, M and Sprakel, J and Scheuring, D}, title = {Plant infection by the necrotrophic fungus Botrytis requires actin-dependent generation of high invasive turgor pressure.}, journal = {The New phytologist}, volume = {}, number = {}, pages = {}, doi = {10.1111/nph.20025}, pmid = {39107894}, issn = {1469-8137}, support = {101000981/ERC_/European Research Council/International ; SCHE 1836/5-1//Deutsche Forschungsgemeinschaft/ ; }, abstract = {The devastating pathogen Botrytis cinerea infects a broad spectrum of host plants, causing great socio-economic losses. The necrotrophic fungus rapidly kills plant cells, nourishing their wall and cellular contents. To this end, necrotrophs secrete a cocktail of cell wall degrading enzymes, phytotoxic proteins and metabolites. Additionally, many fungi produce specialized invasion organs that generate high invasive pressures to force their way into the plant cell. However, for most necrotrophs, including Botrytis, the biomechanics of penetration and its contribution to virulence are poorly understood. Here, we use a combination of quantitative micromechanical imaging and CRISPR-Cas-guided mutagenesis to show that Botrytis uses substantial invasive pressure, in combination with strong surface adherence, for penetration. We found that the fungus establishes a unique mechanical geometry of penetration that develops over time during penetration events, and which is actin cytoskeleton dependent. Furthermore, interference of force generation by blocking actin polymerization was found to decrease Botrytis virulence, indicating that also for necrotrophs, mechanical pressure is important in host colonization. Our results demonstrate for the first time mechanistically how a necrotrophic fungus such as Botrytis employs this 'brute force' approach, in addition to the secretion of lytic proteins and phytotoxic metabolites, to overcome plant host resistance.}, }
@article {pmid39107797, year = {2024}, author = {Tsiami, F and Lago, C and Pozza, N and Piccioni, F and Zhao, X and Lülsberg, F and Root, DE and Tiberi, L and Kool, M and Schittenhelm, J and Bandopadhayay, P and Segal, RA and Tabatabai, G and Merk, DJ}, title = {Genome-wide CRISPR-Cas9 knockout screens identify DNMT1 as a druggable dependency in sonic hedgehog medulloblastoma.}, journal = {Acta neuropathologica communications}, volume = {12}, number = {1}, pages = {125}, pmid = {39107797}, issn = {2051-5960}, support = {2022.094.1//Wilhelm Sander-Stiftung/ ; EXC 2180-390900677//German Research Foundation/ ; }, mesh = {*Medulloblastoma/genetics/metabolism/pathology ; Animals ; *DNA (Cytosine-5-)-Methyltransferase 1/genetics/metabolism ; *Hedgehog Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; *Cerebellar Neoplasms/genetics/metabolism/pathology ; Humans ; Mice ; Cell Line, Tumor ; Smoothened Receptor/genetics/metabolism ; Gene Knockout Techniques/methods ; }, abstract = {Sonic hedgehog subgroup of medulloblastoma (SHH-MB) is characterized by aberrant activation of the SHH signaling pathway. An inhibition of the positive SHH regulator Smoothened (SMO) has demonstrated promising clinical efficacy. Yet, primary and acquired resistance to SMO inhibitors limit their efficacy. An understanding of underlying molecular mechanisms of resistance to therapy is warranted to bridge this unmet need. Here, we make use of genome-wide CRISPR-Cas9 knockout screens in murine SMB21 and human DAOY cells, in order to unravel genetic dependencies and drug-related genetic interactors that could serve as alternative therapeutic targets for SHH-MB. Our screens reinforce SMB21 cells as a faithful model system for SHH-MB, as opposed to DAOY cells, and identify members of the epigenetic machinery including DNA methyltransferase 1 (DNMT1) as druggable targets in SHH-dependent tumors. We show that Dnmt1 plays a crucial role in normal murine cerebellar development and is required for SHH-MB growth in vivo. Additionally, DNMT1 pharmacological inhibition alone and in combination with SMO inhibition effectively inhibits tumor growth in murine and human SHH-MB cell models and prolongs survival of SHH-MB mouse models by inhibiting SHH signaling output downstream of SMO. In conclusion, our data highlight the potential of inhibiting epigenetic regulators as a novel therapeutic avenue in SMO-inhibitor sensitive as well as resistant SHH-MBs.}, }
@article {pmid39103341, year = {2024}, author = {Hu, K and Chou, CW and Wilke, CO and Finkelstein, IJ}, title = {Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6653}, pmid = {39103341}, issn = {2041-1723}, support = {R01GM124141//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; F-1808//Welch Foundation/ ; R01GM088344//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, mesh = {*DNA Transposable Elements/genetics ; *Escherichia coli/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Transfer, Horizontal ; *CRISPR-Associated Proteins/metabolism/genetics ; Escherichia coli Proteins/genetics/metabolism ; }, abstract = {CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π - π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.}, }
@article {pmid39048831, year = {2024}, author = {Borrelli, C and Roberts, M and Eletto, D and Hussherr, MD and Fazilaty, H and Valenta, T and Lafzi, A and Kretz, JA and Guido Vinzoni, E and Karakatsani, A and Adivarahan, S and Mannhart, A and Kimura, S and Meijs, A and Baccouche Mhamedi, F and Acar, IE and Handler, K and Ficht, X and Platt, RJ and Piscuoglio, S and Moor, AE}, title = {In vivo interaction screening reveals liver-derived constraints to metastasis.}, journal = {Nature}, volume = {632}, number = {8024}, pages = {411-418}, pmid = {39048831}, issn = {1476-4687}, mesh = {Animals ; Mice ; *Liver Neoplasms/secondary/metabolism/pathology ; Humans ; *Colorectal Neoplasms/pathology/metabolism ; Male ; Female ; *Liver/pathology/metabolism ; *Kruppel-Like Factor 4 ; *Semaphorins/metabolism ; *Hepatocytes/metabolism/cytology/pathology ; Nerve Tissue Proteins/metabolism/genetics ; Cell Line, Tumor ; Disease Models, Animal ; Kruppel-Like Transcription Factors/metabolism/genetics ; Neoplasm Metastasis/pathology ; Pancreatic Neoplasms/pathology/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Melanoma/pathology/metabolism/genetics ; }, abstract = {It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases[1]. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology[2] and fluorescence niche labelling[3], we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.}, }
@article {pmid39048823, year = {2024}, author = {Dai, Y and Idorn, M and Serrero, MC and Pan, X and Thomsen, EA and Narita, R and Maimaitili, M and Qian, X and Iversen, MB and Reinert, LS and Flygaard, RK and Chen, M and Ding, X and Zhang, BC and Carter-Timofte, ME and Lu, Q and Jiang, Z and Zhong, Y and Zhang, S and Da, L and Zhu, J and Denham, M and Nissen, P and Mogensen, TH and Mikkelsen, JG and Zhang, SY and Casanova, JL and Cai, Y and Paludan, SR}, title = {TMEFF1 is a neuron-specific restriction factor for herpes simplex virus.}, journal = {Nature}, volume = {632}, number = {8024}, pages = {383-389}, pmid = {39048823}, issn = {1476-4687}, mesh = {Animals ; *Neurons/virology/metabolism ; Mice ; Humans ; *Herpesvirus 1, Human/physiology ; *Virus Replication ; *Herpes Simplex/virology/metabolism ; Female ; Brain/virology/metabolism ; Male ; Virus Internalization ; Antiviral Restriction Factors ; Membrane Proteins/metabolism/deficiency/genetics ; Viral Load ; Host Specificity ; CRISPR-Cas Systems/genetics ; Cell Death ; }, abstract = {The brain is highly sensitive to damage caused by infection and inflammation[1,2]. Herpes simplex virus 1 (HSV-1) is a neurotropic virus and the cause of herpes simplex encephalitis[3]. It is unknown whether neuron-specific antiviral factors control virus replication to prevent infection and excessive inflammatory responses, hence protecting the brain. Here we identify TMEFF1 as an HSV-1 restriction factor using genome-wide CRISPR screening. TMEFF1 is expressed specifically in neurons of the central nervous system and is not regulated by type I interferon, the best-known innate antiviral system controlling virus infections. Depletion of TMEFF1 in stem-cell-derived human neurons led to elevated viral replication and neuronal death following HSV-1 infection. TMEFF1 blocked the HSV-1 replication cycle at the level of viral entry through interactions with nectin-1 and non-muscle myosin heavy chains IIA and IIB, which are core proteins in virus-cell binding and virus-cell fusion, respectively[4-6]. Notably, Tmeff1[-/-] mice exhibited increased susceptibility to HSV-1 infection in the brain but not in the periphery. Within the brain, elevated viral load was observed specifically in neurons. Our study identifies TMEFF1 as a neuron-specific restriction factor essential for prevention of HSV-1 replication in the central nervous system.}, }
@article {pmid39020166, year = {2024}, author = {Renz, PF and Ghoshdastider, U and Baghai Sain, S and Valdivia-Francia, F and Khandekar, A and Ormiston, M and Bernasconi, M and Duré, C and Kretz, JA and Lee, M and Hyams, K and Forny, M and Pohly, M and Ficht, X and Ellis, SJ and Moor, AE and Sendoel, A}, title = {In vivo single-cell CRISPR uncovers distinct TNF programmes in tumour evolution.}, journal = {Nature}, volume = {632}, number = {8024}, pages = {419-428}, pmid = {39020166}, issn = {1476-4687}, mesh = {*Single-Cell Analysis ; Animals ; Mice ; Humans ; *Epithelial-Mesenchymal Transition/genetics ; Female ; *Carcinoma, Squamous Cell/genetics/pathology ; Signal Transduction/genetics ; Macrophages/metabolism ; Tumor Necrosis Factor-alpha/metabolism/genetics ; Receptors, Tumor Necrosis Factor, Type I/genetics/metabolism ; Male ; CRISPR-Cas Systems/genetics ; Carcinogenesis/genetics/pathology ; Mutation ; Gene Expression Regulation, Neoplastic ; Clonal Evolution/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Transformation, Neoplastic/genetics ; Transcriptome/genetics ; Clone Cells/metabolism ; Tumor Necrosis Factors/genetics/metabolism ; Neoplasm Invasiveness/genetics ; }, abstract = {The tumour evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions can remodel entire tissues[1-3], the mechanisms that result in only a small number of clones transforming into malignant tumours remain unknown. Here we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing and guide capture to longitudinally monitor clonal expansions and document their underlying gene programmes at single-cell transcriptomic resolution. We uncover a tumour necrosis factor (TNF) signalling module, which is dependent on TNF receptor 1 and involving macrophages, that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF signalling module is downregulated. Instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF gene programme associated with epithelial-mesenchymal transition. Finally, we provide in vivo evidence that the autocrine TNF gene programme is sufficient to mediate invasive properties and show that the TNF signature correlates with shorter overall survival of patients with squamous cell carcinoma. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues, unveils distinct TNF programmes in tumour evolution and highlights the importance of understanding the relationship between clonal expansions in epithelia and tumorigenesis.}, }
@article {pmid38925443, year = {2024}, author = {Pardi, N and Ivics, Z}, title = {An LNP-CRISPR gene editing drug demonstrates efficacy and safety in patients with hereditary angioedema following in vivo administration.}, journal = {The Journal of allergy and clinical immunology}, volume = {154}, number = {2}, pages = {272-274}, doi = {10.1016/j.jaci.2024.06.011}, pmid = {38925443}, issn = {1097-6825}, mesh = {Humans ; *Angioedemas, Hereditary/genetics/drug therapy ; *Gene Editing ; Female ; Male ; Adult ; Middle Aged ; CRISPR-Cas Systems ; }, }
@article {pmid38878270, year = {2024}, author = {Hooper, AJ and Tang, XL and Burnett, JR}, title = {VERVE-101, a CRISPR base-editing therapy designed to permanently inactivate hepatic PCSK9 and reduce LDL-cholesterol.}, journal = {Expert opinion on investigational drugs}, volume = {33}, number = {8}, pages = {753-756}, doi = {10.1080/13543784.2024.2369747}, pmid = {38878270}, issn = {1744-7658}, mesh = {*Proprotein Convertase 9/genetics ; Humans ; *Cholesterol, LDL/blood ; Animals ; *Gene Editing/methods ; Genetic Therapy/methods ; CRISPR-Cas Systems ; Liver ; }, }
@article {pmid38839984, year = {2024}, author = {Basu, M and Zurla, C and Auroni, TT and Vanover, D and Chaves, LCS and Sadhwani, H and Pathak, H and Basu, R and Beyersdorf, JP and Amuda, OO and Elsharkawy, A and Mosur, V and Arthur, RA and Claussen, H and Sasser, LE and Wroe, JA and Peck, HE and Kumar, M and Brinton, MA and Santangelo, PJ}, title = {mRNA-encoded Cas13 can be used to treat dengue infections in mice.}, journal = {Nature microbiology}, volume = {9}, number = {8}, pages = {2160-2172}, pmid = {38839984}, issn = {2058-5276}, support = {HR0011-19-2-0008//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; }, mesh = {Animals ; *Dengue/drug therapy ; Mice ; *Dengue Virus/genetics/drug effects ; *RNA, Messenger/genetics/metabolism ; *Nanoparticles/chemistry ; *Antiviral Agents/pharmacology/administration &amp; dosage ; *Disease Models, Animal ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; CRISPR-Associated Proteins/metabolism/genetics ; Lipids/chemistry ; Viral Load/drug effects ; Female ; Liposomes ; }, abstract = {Dengue is a major global health threat, and there are no approved antiviral agents. Prior research using Cas13 only demonstrated dengue mitigation in vitro. Here we demonstrate that systemic delivery of mRNA-encoded Cas13a and guide RNAs formulated in lipid nanoparticles can be used to treat dengue virus (DENV) 2 and 3 in mice. First, we identified guides against DENV 2 and 3 that demonstrated in vitro efficacy. Next, we confirmed that Cas13 enzymatic activity is necessary for DENV 2 or DENV 3 mitigation in vitro. Last, we show that a single dose of lipid-nanoparticle-formulated mRNA-encoded Cas13a and guide RNA, administered 1 day post-infection, promotes survival of all infected animals and serum viral titre decreases on days 2 and 3 post-infection after lethal challenge in mice. Off-target analysis in mice using RNA sequencing showed no collateral cleavage. Overall, these data demonstrate the potential of mRNA-encoded Cas13 as a pan-DENV drug.}, }
@article {pmid38839055, year = {2024}, author = {Patel, R and Bryant, AS and Castelletto, ML and Walsh, B and Akimori, D and Hallem, EA}, title = {The generation of stable transgenic lines in the human-infective nematode Strongyloides stercoralis.}, journal = {G3 (Bethesda, Md.)}, volume = {14}, number = {8}, pages = {}, doi = {10.1093/g3journal/jkae122}, pmid = {38839055}, issn = {2160-1836}, support = {F32AI174816/GF/NIH HHS/United States ; F30AI179222//University of Washington/ ; T32GM145388//UCLA/ ; }, mesh = {*Strongyloides stercoralis/genetics ; Animals ; *Animals, Genetically Modified ; Humans ; CRISPR-Cas Systems ; Strongyloidiasis/parasitology/genetics ; Transgenes ; Rats ; Larva ; }, abstract = {The skin-penetrating gastrointestinal parasitic nematode Strongyloides stercoralis causes strongyloidiasis, which is a neglected tropical disease that is associated with severe chronic illness and fatalities. Unlike other human-infective nematodes, S. stercoralis cycles through a single free-living generation and thus serves as a genetically tractable model organism for understanding the mechanisms that enable parasitism. Techniques such as CRISPR/Cas9-mediated mutagenesis and transgenesis are now routinely performed in S. stercoralis by introducing exogenous DNA into free-living adults and then screening their F1 progeny for transgenic or mutant larvae. However, transgenesis in S. stercoralis has been severely hindered by the inability to establish stable transgenic lines that can be propagated for multiple generations through a host; to date, studies of transgenic S. stercoralis have been limited to heterogeneous populations of transgenic F1 larvae. Here, we develop an efficient pipeline for the generation of stable transgenic lines in S. stercoralis. We also show that this approach can be used to efficiently generate stable transgenic lines in the rat-infective nematode Strongyloides ratti. The ability to generate stable transgenic lines circumvents the limitations of working with heterogeneous F1 populations, such as variable transgene expression and the inability to generate transgenics of all life stages. Our transgenesis approach will enable novel lines of inquiry into parasite biology, such as transgene-based comparisons between free-living and parasitic generations.}, }
@article {pmid38701221, year = {2024}, author = {Nyberg, KG and Navales, FG and Keles, E and Nguyen, JQ and Hertz, LM and Carthew, RW}, title = {Robust and heritable knockdown of gene expression using a self-cleaving ribozyme in Drosophila.}, journal = {Genetics}, volume = {227}, number = {4}, pages = {}, doi = {10.1093/genetics/iyae067}, pmid = {38701221}, issn = {1943-2631}, support = {R35GM118144/NH/NIH HHS/United States ; 1764421//National Science Foundation/ ; 597491//Simons Foundation/ ; RRID:SCR_017767//Northwestern's Biological Imaging Facility/ ; //Chemistry for Life Processes Institute/ ; //NU Office for Research/ ; //Department of Molecular Biosciences, and the Rice Foundation/ ; CA060553//NCI Cancer Center Support Grant/ ; }, mesh = {Animals ; *RNA, Catalytic/genetics/metabolism ; *Drosophila melanogaster/genetics ; *Gene Knockdown Techniques ; CRISPR-Cas Systems ; Male ; }, abstract = {The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111-bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into 4 independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3' fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila, with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes.}, }
@article {pmid38679667, year = {2024}, author = {Tian, J and Tang, Z and Niu, R and Zhou, Y and Yang, D and Chen, D and Luo, M and Mou, R and Yuan, M and Xu, G}, title = {Engineering disease-resistant plants with alternative translation efficiency by switching uORF types through CRISPR.}, journal = {Science China. Life sciences}, volume = {67}, number = {8}, pages = {1715-1726}, pmid = {38679667}, issn = {1869-1889}, mesh = {*Oryza/genetics ; *Disease Resistance/genetics ; *Open Reading Frames/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Plant Diseases/genetics ; Arabidopsis/genetics ; Plants, Genetically Modified/genetics ; Protein Biosynthesis/genetics ; Gene Expression Regulation, Plant ; Crops, Agricultural/genetics ; Translations ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Engineering disease-resistant plants can be a powerful solution to the issue of food security. However, it requires addressing two fundamental questions: what genes to express and how to control their expressions. To find a solution, we screen CRISPR-edited upstream open reading frame (uORF) variants in rice, aiming to optimize translational control of disease-related genes. By switching uORF types of the 5'-leader from Arabidopsis TBF1, we modulate the ribosome accessibility to the downstream firefly luciferase. We assume that by switching uORF types using CRISPR, we could generate uORF variants with alternative translation efficiency (CRISPR-aTrE-uORF). These variants, capable of boosting translation for resistance-associated genes and dampening it for susceptible ones, can help pinpoint previously unidentified genes with optimal expression levels. To test the assumption, we screened edited uORF variants and found that enhanced translational suppression of the plastic glutamine synthetase 2 can provide broad-spectrum disease resistance in rice with minimal fitness costs. This strategy, which involves modifying uORFs from none to some, or from some to none or different ones, demonstrates how translational agriculture can speed up the development of disease-resistant crops. This is vital for tackling the food security challenges we face due to growing populations and changing climates.}, }
@article {pmid39052979, year = {2024}, author = {Liu, R and Li, C and Zhu, L and Wang, S and Liu, D and Xie, L and Ge, S and Yu, J}, title = {Cu Single-Atom Nanozyme-Mediated Electrochemiluminescence Biosensor for Highly Sensitive Detection of MicroRNA-622.}, journal = {Analytical chemistry}, volume = {96}, number = {31}, pages = {12838-12845}, doi = {10.1021/acs.analchem.4c02514}, pmid = {39052979}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; Humans ; *MicroRNAs/analysis/blood ; *Copper/chemistry ; *Electrochemical Techniques ; *Luminescent Measurements ; *Graphite/chemistry ; *Luminol/chemistry ; Limit of Detection ; Hydrogen Peroxide/chemistry ; Nitrogen Compounds/chemistry ; Catalysis ; CRISPR-Associated Proteins ; CRISPR-Cas Systems ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {MicroRNA (miRNA) detection is a critical aspect of disease diagnosis, and recent studies indicate that miRNA-622 could be a potential target for lung cancer. Herein, Cu single atoms were anchored on graphitic carbon nitride (Cu SAs@CN) as a coreaction accelerator applied in luminol-H2O2 system, thereby establishing an efficient and sensitive electrochemiluminescence (ECL) biosensor for miRNA-622 detection. Cu SAs@CN was explored to possess excellent enzyme-like activities that promote the generation of abundant reactive oxygen species, which amplified ECL emission. Meanwhile, in order to improve the accuracy and sensitivity for miRNA-622 detection, the highly specific trans-cleavage ability of CRISPR/Cas12a was combined with a catalytic hairpin assembly strategy. Therefore, an ECL biosensor for miRNA-622 detection was systematically constructed as a proof of concept, achieving an ultralow limit of detection of 1.09 fM, and the feasibility was demonstrated in human serum samples. The findings of this research provide a promising strategy to enhance the ECL response using versatile single-atom catalysts, thus advancing the development of ECL biosensing applications.}, }
@article {pmid39047235, year = {2024}, author = {Wang, Y and Xu, X and Que, J and Wang, X and Ni, W and Wu, Y and Yang, L and Li, Y}, title = {Ratiometric Readout of Bacterial Infections via a Lyophilized CRISPR-Cas12a Sensor with Color-Changeable Bioluminescence.}, journal = {Analytical chemistry}, volume = {96}, number = {31}, pages = {12776-12783}, doi = {10.1021/acs.analchem.4c02114}, pmid = {39047235}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems/genetics ; *Luminescent Measurements ; *Pseudomonas aeruginosa/isolation &amp; purification/genetics ; Humans ; Freeze Drying ; Color ; Bacterial Infections/diagnosis/microbiology ; Biosensing Techniques ; CRISPR-Associated Proteins/metabolism ; Luminescence ; }, abstract = {The healthcare burden imposed by bacterial infections demands robust and accessible diagnostic methods that can be performed outside hospitals and centralized laboratories. Here, we report Pathogen Assay with Ratiometric Luminescence (PEARL), a sensitive and easy-to-operate platform for detecting pathogenic bacteria. The PEARL leveraged a color-changeable CRISPR-Cas12a sensor and recombinase polymerase amplification to elicit ratiometric bioluminescence responses to target inputs. This platform enabled robust and visualized identification of attomolar bacteria genome deoxyribonucleic acid according to the color changes of the reactions. In addition, the components of the color-changeable Cas12a sensor could be lyophilized for 3 month storage at ambient temperature and then be fully activated with the amplicons derived from crude bacterial lysates, reducing the requirements for cold-chain storage and tedious handling steps. We demonstrated that the PEARL assay is applicable for identifying the infections caused by Pseudomonas aeruginosa in different clinical specimens, including sputa, urines, and swabs derived from wounds. These results revealed the potential of PEARL to be used by untrained personnel, which will facilitate decentralized pathogen diagnosis in community- and resource-limited regions.}, }
@article {pmid39037484, year = {2024}, author = {Park, A and Lee, JY}, title = {Adenoviral Vector System: A Comprehensive Overview of Constructions, Therapeutic Applications and Host Responses.}, journal = {Journal of microbiology (Seoul, Korea)}, volume = {62}, number = {7}, pages = {491-509}, pmid = {39037484}, issn = {1976-3794}, support = {2021R1F1A1063240//Ministry of Science and ICT, South Korea/ ; }, mesh = {*Genetic Vectors/genetics ; Humans ; *Adenoviridae/genetics ; *Genetic Therapy/methods ; *Gene Editing/methods ; Animals ; CRISPR-Cas Systems ; Gene Transfer Techniques ; Vaccine Development ; }, abstract = {Adenoviral vectors are crucial for gene therapy and vaccine development, offering a platform for gene delivery into host cells. Since the discovery of adenoviruses, first-generation vectors with limited capacity have evolved to third-generation vectors flacking viral coding sequences, balancing safety and gene-carrying capacity. The applications of adenoviral vectors for gene therapy and anti-viral treatments have expanded through the use of in vitro ligation and homologous recombination, along with gene editing advancements such as CRISPR-Cas9. Current research aims to maintain the efficacy and safety of adenoviral vectors by addressing challenges such as pre-existing immunity against adenoviral vectors and developing new adenoviral vectors from rare adenovirus types and non-human species. In summary, adenoviral vectors have great potential in gene therapy and vaccine development. Through continuous research and technological advancements, these vectors are expected to lead to the development of safer and more effective treatments.}, }
@article {pmid39037392, year = {2024}, author = {Jiang, H and Chang, W and Zhu, X and Liu, G and Liu, K and Chen, W and Wang, H and Qin, P}, title = {Development of a Colorimetric and SERS Dual-Signal Platform via dCas9-Mediated Chain Assembly of Bifunctional Au@Pt Nanozymes for Ultrasensitive and Robust Salmonella Assay.}, journal = {Analytical chemistry}, volume = {96}, number = {31}, pages = {12684-12691}, doi = {10.1021/acs.analchem.4c01474}, pmid = {39037392}, issn = {1520-6882}, mesh = {*Colorimetry ; *Gold/chemistry ; *Spectrum Analysis, Raman ; *Platinum/chemistry ; *Nucleic Acid Amplification Techniques ; Salmonella/isolation &amp; purification/genetics ; Metal Nanoparticles/chemistry ; Limit of Detection ; Benzidines/chemistry ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Molecular Diagnostic Techniques ; }, abstract = {Timely screening for harmful pathogens is a great challenge in emergencies where traditional culture methods suffer from long assay time and alternative methods are limited by poor accuracy and low robustness. Herein, we present a dCas9-mediated colorimetric and surface-enhanced Raman scattering (SERS) dual-signal platform (dCas9-CSD) to address this challenge. Strategically, the platform used dCas9 to accurately recognize the repetitive sequences in amplicons produced by loop-mediated isothermal amplification (LAMP), forming nucleic acid frameworks that assemble numerous bifunctional gold-platinum (Au@Pt) nanozymes into chains on the surface of streptavidin-magnetic beads (SA-MB). The collected Au@Pt converted colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB (oxTMB) via its Pt shell and then enhanced the Raman signal of oxTMB by its Au core. Therefore, the presence of Salmonella could be dexterously converted into cross-validated colorimetric and SERS signals, providing more reliable conclusions. Notably, dCas9-mediated secondary recognition of amplicons reduced background signal caused by nontarget amplification, and two-round signal amplification consisting of LAMP reaction and Au@Pt catalysis greatly improved the sensitivity. With this design, Salmonella as low as 1 CFU/mL could be detected within 50 min by colorimetric and SERS modes. The robustness of dCas9-CSD was further confirmed by various real samples such as lake water, cabbage, milk, orange juice, beer, and eggs. This work provides a promising point-of-need tool for pathogen detection.}, }
@article {pmid39037068, year = {2024}, author = {Palanki, R and Han, EL and Murray, AM and Maganti, R and Tang, S and Swingle, KL and Kim, D and Yamagata, H and Safford, HC and Mrksich, K and Peranteau, WH and Mitchell, MJ}, title = {Optimized microfluidic formulation and organic excipients for improved lipid nanoparticle mediated genome editing.}, journal = {Lab on a chip}, volume = {24}, number = {16}, pages = {3790-3801}, doi = {10.1039/d4lc00283k}, pmid = {39037068}, issn = {1473-0189}, mesh = {*Gene Editing ; *Nanoparticles/chemistry ; *Lipids/chemistry ; Humans ; Animals ; Excipients/chemistry ; CRISPR-Cas Systems ; Mice ; Lab-On-A-Chip Devices ; Liposomes ; }, abstract = {mRNA-based gene editing platforms have tremendous promise in the treatment of genetic diseases. However, for this potential to be realized in vivo, these nucleic acid cargos must be delivered safely and effectively to cells of interest. Ionizable lipid nanoparticles (LNPs), the most clinically advanced non-viral RNA delivery system, have been well-studied for the delivery of mRNA but have not been systematically optimized for the delivery of mRNA-based CRISPR-Cas9 platforms. In this study, we investigated the effect of microfluidic and lipid excipient parameters on LNP gene editing efficacy. Through in vitro screening in liver cells, we discovered distinct trends in delivery based on phospholipid, cholesterol, and lipid-PEG structure in LNP formulations. Combination of top-performing lipid excipients produced an LNP formulation that resulted in 3-fold greater gene editing in vitro and facilitated 3-fold greater reduction of a therapeutically-relevant protein in vivo relative to the unoptimized LNP formulation. Thus, systematic optimization of LNP formulation parameters revealed a novel LNP formulation that has strong potential for delivery of gene editors to the liver to treat metabolic disease.}, }
@article {pmid38971118, year = {2024}, author = {Xia, X and Wu, X and Jiang, D and Hu, Y and Cong, X and Li, J and Dai, M and Du, Y and Qi, J}, title = {The inhibitory effect of swine TAB1 on the replication of pseudorabies virus.}, journal = {Veterinary microbiology}, volume = {296}, number = {}, pages = {110172}, doi = {10.1016/j.vetmic.2024.110172}, pmid = {38971118}, issn = {1873-2542}, mesh = {Animals ; *Herpesvirus 1, Suid/genetics/physiology ; *Virus Replication ; Swine ; *Pseudorabies/virology ; Swine Diseases/virology ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; Cell Line ; CRISPR-Cas Systems ; Interferon-beta/genetics/metabolism ; }, abstract = {TAK1-binding protein 1 (TAB1) assembles with TAK1 through its C-terminal domain, leading to the self-phosphorylation and activation of TAK1, which plays an important role in the activation of NF-κB and MAPK signaling pathway. Pseudorabies virus (PRV) is the pathogen of Pseudorabies (PR), which belongs to the Alphaherpesvirus subfamily and causes serious economic losses to the global pig industry. However, the impact of swine TAB1 (sTAB1) on PRV infection has not been reported. In this study, evidence from virus DNA copies, virus titer and western blotting confirmed that sTAB1 could inhibit PRV replication and knockout of sTAB1 by CRISPR-Cas9 gene editing system could promote PRV replication. Further mechanistic studies by real-time PCR and luciferase reporter gene assay demonstrated that sTAB1 could enhance the production of inflammatory factors and chemokines, IFN-β transcription level and IFN-β promoter activity after PRV infection. In summary, we clarify the underlying mechanism of sTAB1 in inhibiting PRV replication for the first time, which provides a new idea for preventing PRV infection and lays a foundation for PRV vaccine development.}, }
@article {pmid38924982, year = {2024}, author = {Li, X and Liu, M and Men, D and Duan, Y and Deng, L and Zhou, S and Hou, J and Hou, C and Huo, D}, title = {Rapid, portable, and sensitive detection of CaMV35S by RPA-CRISPR/Cas12a-G4 colorimetric assays with high accuracy deep learning object recognition and classification.}, journal = {Talanta}, volume = {278}, number = {}, pages = {126441}, doi = {10.1016/j.talanta.2024.126441}, pmid = {38924982}, issn = {1873-3573}, mesh = {*Colorimetry/methods ; *CRISPR-Cas Systems/genetics ; *Deep Learning ; *G-Quadruplexes ; Promoter Regions, Genetic ; CRISPR-Associated Proteins/genetics/metabolism ; Limit of Detection ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; }, abstract = {Fast, sensitive, and portable detection of genetic modification contributes to agricultural security and food safety. Here, we developed RPA-CRISPR/Cas12a-G-quadruplex colorimetric assays that can combine with intelligent recognition by deep learning algorithms to achieve sensitive, rapid, and portable detection of the CaMV35S promoter. When the crRNA-Cas12a complex recognizes the RPA amplification product, Cas12 cleaves the G-quadruplex, causing the G4-Hemin complex to lose its peroxide mimetic enzyme function and be unable to catalyze the conversion of ABTS[2-] to ABTS, allowing CaMV35S concentration to be determined based on ABTS absorbance. By utilizing the RPA-CRISPR/Cas12a-G4 assay, we achieved a CaMV35S limit of detection down to 10 aM and a 0.01 % genetic modification sample in 45 min. Deep learning algorithms are designed for highly accurate classification of color results. Yolov5 objective finding and Resnet classification algorithms have been trained to identify trace (0.01 %) CaMV35S more accurately than naked eye colorimetry. We also coupled deep learning algorithms with a smartphone app to achieve portable and rapid photo identification. Overall, our findings enable low cost ($0.43), high accuracy, and intelligent detection of the CaMV35S promoter.}, }
@article {pmid38908140, year = {2024}, author = {Hu, J and Yu, Y and Pan, X and Yue Han,  and She, X and Liu, X and Zhang, Q and Gai, H and Zong, C}, title = {Highly sensitive and specific detection of human papillomavirus type 16 using CRISPR/Cas12a assay coupled with an enhanced single nanoparticle dark-field microscopy imaging technique.}, journal = {Talanta}, volume = {278}, number = {}, pages = {126449}, doi = {10.1016/j.talanta.2024.126449}, pmid = {38908140}, issn = {1873-3573}, mesh = {*Human papillomavirus 16/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; Silver/chemistry ; Microscopy/methods ; Limit of Detection ; Papillomavirus Infections/diagnosis/virology ; DNA, Viral/analysis/genetics ; CRISPR-Associated Proteins/metabolism/chemistry ; }, abstract = {Human papillomavirus (HPV) is a prevalent sexually transmitted pathogen associated with cervical cancer. Detecting high-risk HPV (hr-HPV) infections is crucial for cervical cancer prevention, particularly in resource-limited settings. Here, we present a highly sensitive and specific sensor for HPV-16 detection based on CRISPR/Cas12a coupled with enhanced single nanoparticle dark-field microscopy (DFM) imaging techniques. Ag-Au satellites were assembled through the hybridization of AgNPs-based spherical nucleic acid (Ag-SNA) and AuNPs-based spherical nucleic acid (Au-SNA), and their disassembly upon target-mediated cleavage by the Cas12a protein was monitored using DFM for HPV-16 quantification. To enhance the cleavage efficiency and detection sensitivity, the composition of the ssDNA sequences on Ag-SNA and Au-SNA was optimized. Additionally, we explored using the SynSed technique (synergistic sedimentation of Brownian motion suppression and dehydration transfer) as an alternative particle transfer method in DFM imaging to traditional electrostatic deposition. This addresses the issue of inconsistent deposition efficiency of Ag-Au satellites and their disassembly due to their size and charge differences. The sensor achieved a remarkable limit of detection (LOD) of 10 fM, lowered by 9-fold compared to traditional electrostatic deposition methods. Clinical testing in DNA extractions from 10 human cervical swabs demonstrated significant response differences between the positive and negative samples. Our sensor offers a promising solution for sensitive and specific HPV-16 detection, with implications for cancer screening and management.}, }
@article {pmid38771392, year = {2024}, author = {Lazovic, B and Nguyen, HT and Ansarizadeh, M and Wigge, L and Kohl, F and Li, S and Carracedo, M and Kettunen, J and Krimpenfort, L and Elgendy, R and Richter, K and De Silva, L and Bilican, B and Singh, P and Saxena, P and Jakobsson, L and Hong, X and Eklund, L and Hicks, R}, title = {Human iPSC and CRISPR targeted gene knock-in strategy for studying the somatic TIE2[L914F] mutation in endothelial cells.}, journal = {Angiogenesis}, volume = {27}, number = {3}, pages = {523-542}, pmid = {38771392}, issn = {1573-7209}, support = {814316//European Union's Horizon 2020 research and innovation program under the Marie Sk&#x142;odowska-Curie grant agreement/ ; }, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Receptor, TIE-2/genetics/metabolism ; *Endothelial Cells/metabolism ; *Gene Knock-In Techniques ; Mutation/genetics ; CRISPR-Cas Systems/genetics ; Vascular Malformations/genetics/pathology/metabolism ; }, abstract = {Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases, including those with genetic origins. Currently, primary ECs are the main source for disease modelling in this field. However, they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level, we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor, known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2[L914F] iECs showed a significantly lower expression of TIE2 on mRNA and protein level, which has not been observed before due to a lack of models with endogenous edited TIE2[L914F] and sparse patient data. Interestingly, the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2[L914F] iECs exhibited dysregulated angiogenesis markers and upregulated migration capability, while proliferation was not affected. Under shear stress TIE2[L914F] iECs showed reduced alignment in the flow direction and a larger cell area than TIE2[WT] iECs. In summary, we developed a novel TIE2[L914F] iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs.}, }
@article {pmid39103226, year = {2024}, author = {Toddes, CA}, title = {An Accessible Intersectional Transgenic Single-Vector CRISPR/Cas9 Platform for Precise Gene Editing and Functional Analysis.}, journal = {eNeuro}, volume = {11}, number = {8}, pages = {}, doi = {10.1523/ENEURO.0264-24.2024}, pmid = {39103226}, issn = {2373-2822}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Genetic Vectors ; }, }
@article {pmid39101073, year = {2024}, author = {Mishu, MA and Nath, SK and Sohidullah, M and Hossain, MT}, title = {Advancement of animal and poultry nutrition: Harnessing the power of CRISPR-Cas genome editing technology.}, journal = {Journal of advanced veterinary and animal research}, volume = {11}, number = {2}, pages = {483-493}, pmid = {39101073}, issn = {2311-7710}, abstract = {CRISPR-associated proteins and clustered regularly interspaced short palindromic repeats (CRISPR-Cas) technology has emerged as a groundbreaking advancement in animal and poultry nutrition to improve feed conversion efficiency, enhance disease resistance, and improve the nutritional quality of animal products. Despite significant advancements, there is a research gap in the systematic understanding and comprehensive use of the CRISPR-Cas method in animal and poultry nutrition. The purpose of this study is to elucidate the latest advancements in animal and poultry nutrition through CRISPR-Cas genome editing technology, focusing on gene manipulation in metabolism, immunity, and growth. Following preferred reporting items in meta-analysis and systematic reviews guidelines, we conducted a systematic search using several databases, including Scopus, PubMed, and Web of Science, until May 2024, and finally, we included a total of 108 articles in this study. This article explores the use of the CRISPR-Cas system in the advancement of feed additives like probiotics and enzymes, which could reduce the use of antibiotics in animal production. Furthermore, the article discusses ethical and regulatory issues related to gene editing in animal and poultry nutrition, including concerns about animal welfare, food safety, and environmental impacts. Overall, the CRISPR-Cas system holds substantial promise to overcome the challenges in modern animal agriculture. By enriching the nutritional quality of animal products, increasing disease resistance, and improving feed efficiency, it offers sustainable and cost-effective solutions that can revolutionize animal and poultry nutrition.}, }
@article {pmid39100665, year = {2024}, author = {Geng, Y and Jiang, C and Yang, H and Xia, Q and Xu, X and Yang, K and Yuan, X and Chen, J and Chen, Y and Chen, X and Zhang, L and Hu, C and Guo, A}, title = {Construction of an IFNAR1 knockout MDBK cell line using CRISPR/Cas9 and its effect on bovine virus replication.}, journal = {Frontiers in immunology}, volume = {15}, number = {}, pages = {1404649}, pmid = {39100665}, issn = {1664-3224}, mesh = {Animals ; Cattle ; *Receptor, Interferon alpha-beta/genetics ; *Virus Replication ; *CRISPR-Cas Systems ; Cell Line ; *Gene Knockout Techniques ; Diarrhea Viruses, Bovine Viral/physiology/genetics ; }, abstract = {The type I interferon (IFN) pathway is important for eukaryotic cells to resist viral infection, as well as an impediment to efficient virus replication. Therefore, this study aims to create an IFNAR1 knockout (KO) Madin-Darby bovine kidney (MDBK) cell line using CRISPR/Cas9 and investigate its application and potential mechanism in increasing viral replication of bovines. The IFNAR1 KO cells showed increased titers of bovine viral diarrhea virus (BVDV) (1.5 log10), with bovine enterovirus and bovine parainfluenza virus type 3 (0.5-0.8 log10). RNA-seq revealed reduced expression of the genes related IFN-I pathways including IFNAR1, STAT3, IRF9, and SOCS3 in IFNAR1 KO cells compared with WT cells. In WT cells, 306 differentially expressed genes (DEGs) were identified between BVDV-infected and -uninfected cells. Of these, 128 up- and 178 down-regulated genes were mainly associated with growth cycle and biosynthesis, respectively. In IFNAR1 KO cells, 286 DEGs were identified, with 82 up-regulated genes were associated with signaling pathways, and 204 down-regulated genes. Further, 92 DEGs were overlapped between WT and IFNAR1 KO cells including ESM1, IL13RA2, and SLC25A34. Unique DEGs in WT cells were related to inflammation and immune regulation, whereas those unique in IFNAR1 KO cells involved in cell cycle regulation through pathways such as MAPK. Knocking down SLC25A34 and IL13RA2 in IFNAR1 KO cells increased BVDV replication by 0.3 log10 and 0.4 log10, respectively. Additionally, we constructed an IFNAR1/IFNAR2 double-knockout MDBK cell line, which further increased BVDV viral titers compared with IFNAR1 KO cells (0.6 log10). Overall, the IFNAR1 KO MDBK cell line can support better replication of bovine viruses and therefore provides a valuable tool for bovine virus research on viral pathogenesis and host innate immune response.}, }
@article {pmid39100447, year = {2024}, author = {Kakkar, A and Kandwal, G and Nayak, T and Jaiswal, LK and Srivastava, A and Gupta, A}, title = {Engineered bacteriophages: A panacea against pathogenic and drug resistant bacteria.}, journal = {Heliyon}, volume = {10}, number = {14}, pages = {e34333}, pmid = {39100447}, issn = {2405-8440}, abstract = {Antimicrobial resistance (AMR) is a major global concern; antibiotics and other regular treatment methods have failed to overcome the increasing number of infectious diseases. Bacteriophages (phages) are viruses that specifically target/kill bacterial hosts without affecting other human microbiome. Phage therapy provides optimism in the current global healthcare scenario with a long history of its applications in humans that has now reached various clinical trials. Phages in clinical trials have specific requirements of being exclusively lytic, free from toxic genes with an enhanced host range that adds an advantage to this requisite. This review explains in detail the various phage engineering methods and their potential applications in therapy. To make phages more efficient, engineering has been attempted using techniques like conventional homologous recombination, Bacteriophage Recombineering of Electroporated DNA (BRED), clustered regularly interspaced short palindromic repeats (CRISPR)-Cas, CRISPY-BRED/Bacteriophage Recombineering with Infectious Particles (BRIP), chemically accelerated viral evolution (CAVE), and phage genome rebooting. Phages are administered in cocktail form in combination with antibiotics, vaccines, and purified proteins, such as endolysins. Thus, phage therapy is proving to be a better alternative for treating life-threatening infections, with more specificity and fewer detrimental consequences.}, }
@article {pmid39097877, year = {2024}, author = {Zhu, Y and Wang, G and Xu, H and Guo, Y}, title = {CRISPR-Cas9 Mediated AGO2 Knockout inhibits tumorigenesis in human colorectal cancer cells.}, journal = {Cellular and molecular biology (Noisy-le-Grand, France)}, volume = {70}, number = {7}, pages = {174-179}, doi = {10.14715/cmb/2024.70.7.25}, pmid = {39097877}, issn = {1165-158X}, mesh = {Humans ; *Argonaute Proteins/metabolism/genetics ; *Colorectal Neoplasms/genetics/pathology/metabolism ; *Cell Proliferation/genetics ; *CRISPR-Cas Systems/genetics ; *Cell Movement/genetics ; *Carcinogenesis/genetics/pathology ; HCT116 Cells ; *MicroRNAs/genetics/metabolism ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; }, abstract = {AGO2 plays a vital role in small RNA-guided gene silencing, which has been implied in the tumorigenesis of different types of tumors. Fundamentally, increased expression of AGO2 protein is associated with cancer progression and metastasis. This study aims to investigate the molecular mechanism by which AGO2 promotes tumorigenesis in colorectal cancer (CRC). Databases were used to analyze the expression levels of AGO2 in CRC and confirmed by a quantitative reverse transcriptase-PCR (qRT-PCR) assay in CRC tissues and normal adjacent tissues collected from 25 CRC patients. CRISPR/Cas9-mediated genome editing was used to knockout the AGO2 in HCT116 cells as a model system for colorectal cancers. The cell proliferation, migration and invasion ability of HCT116 cells were detected by CCK-8 assay, Wound scratch assay and Transwell assay. Moreover, the quantities of miRNA binding with AGO2 were detected by RNA-Binding Protein Immunoprecipitation (RIP-Assay). We demonstrated that AGO2 was aberrantly high-expressed in 25 matched-tissue pairs of colorectal cancer and para-carcinoma tissue. The following functional experiments verified that knockout of AGO2 suppressed cell proliferation, migration and tumorigenesis to hamper the aggressiveness of CRC. Our study also suggests a possible link between AGO2 and miRNA in RISC. AGO2 was elevated in CRC and knockout of AGO2 suppressed proliferation and tumorigenicity of CRC cells. Moreover, RISC formation and the function of miRNAs are also subject to AGO2. AGO2 may be a meaningful target for CRC therapy.}, }
@article {pmid38878426, year = {2024}, author = {Xu, X and Meng, Y and Su, B and Lin, J}, title = {Development of whole cell biocatalytic system for asymmetric synthesis of esomeprazole with enhancing coenzyme biosynthesis pathway.}, journal = {Enzyme and microbial technology}, volume = {179}, number = {}, pages = {110469}, doi = {10.1016/j.enzmictec.2024.110469}, pmid = {38878426}, issn = {1879-0909}, mesh = {*Esomeprazole/metabolism ; *Biocatalysis ; *Escherichia coli/genetics/metabolism ; *Burkholderia/genetics/enzymology/metabolism ; Coenzymes/metabolism ; Biosynthetic Pathways ; Metabolic Engineering ; Formate Dehydrogenases/metabolism/genetics ; CRISPR-Cas Systems ; Mixed Function Oxygenases/metabolism/genetics ; }, abstract = {Esomeprazole is the most popular proton pump inhibitor for treating gastroesophageal reflux disease. Previously, a phenylacetone monooxygenase mutant LnPAMOmu15 (LM15) was obtained by protein engineering for asymmetric synthesis of esomeprazole using pyrmetazole as substrate. To scale up the whole cell asymmetric synthesis of esomeprazole and reduce the cost, in this work, an Escherichia coli whole-cell catalyst harboring LM15 and formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) were constructed through optimized gene assembly patterns. CRISPR/Cas9 mediated insertion of Ptrc promoter in genome was done to enhance the expression of key genes to increase the cellular NADP supply in the whole cell catalyst, by which the amount of externally added NADP[+] for the asymmetric synthesis of esomeprazole decreased to 0.05 mM from 0.3 mM for reducing the cost. After the optimization of reaction conditions in the reactor, the scalable synthesis of esomeprazole was performed using the efficient LM15-BstFDH whole-cell as catalyst, which showed the highest reported space-time yield of 3.28 g/L/h with 50 mM of pyrmetazole loading. Isolation procedure was conducted to obtain esomeprazole sodium of 99.55 % purity and > 99.9 % ee with 90.1 % isolation yield. This work provides the basis for production of enantio-pure esomeprazole via cost-effective whole cell biocatalysis.}, }
@article {pmid37915126, year = {2024}, author = {Mamrutha, HM and Zeenat, W and Kapil, D and Budhagatapalli, N and Tikaniya, D and Rakesh, K and Krishnappa, G and Singh, G and Singh, GP}, title = {Evidence and opportunities for developing non-transgenic genome edited crops using site-directed nuclease 1 approach.}, journal = {Critical reviews in biotechnology}, volume = {44}, number = {6}, pages = {1140-1150}, doi = {10.1080/07388551.2023.2270581}, pmid = {37915126}, issn = {1549-7801}, mesh = {*Crops, Agricultural/genetics ; *Gene Editing/methods ; *Plants, Genetically Modified/genetics ; Genome, Plant ; CRISPR-Cas Systems/genetics ; }, abstract = {The innovations and progress in genome editing/new breeding technologies have revolutionized research in the field of functional genomics and crop improvement. This revolution has expanded the horizons of agricultural research, presenting fresh possibilities for creating novel plant varieties equipped with desired traits that can effectively combat the challenges posed by climate change. However, the regulation and social acceptance of genome-edited crops still remain as major barriers. Only a few countries considered the site-directed nuclease 1 (SDN1) approach-based genome-edited plants under less or no regulation. Hence, the present review aims to comprise information on the research work conducted using SDN1 in crops by various genome editing tools. It also elucidates the promising candidate genes that can be used for editing and has listed the studies on non-transgenic crops developed through SDN1 either by Agrobacterium-mediated transformation or by ribo nucleoprotein (RNP) complex. The review also hoards the existing regulatory landscape of genome editing and provides an overview of globally commercialized genome-edited crops. These compilations will enable confidence in researchers and policymakers, across the globe, to recognize the full potential of this technology and reconsider the regulatory aspects associated with genome-edited crops. Furthermore, this compilation serves as a valuable resource for researchers embarking on the development of customized non-transgenic crops through the utilization of SDN1.}, }
@article {pmid39097869, year = {2024}, author = {Ahmadifard, A and Maroofi, N and Maleki Tehrani, M and Dabestani, T and Sadat Mousavi Maleki, M and Bayrami, S and Banan, M}, title = {Genome editing in K562 cells suggests a functional role for the XmnI Gg polymorphism: a widely used genetic marker in β-thalassemia and sickle cell disease patients.}, journal = {Cellular and molecular biology (Noisy-le-Grand, France)}, volume = {70}, number = {7}, pages = {230-236}, doi = {10.14715/cmb/2024.70.7.33}, pmid = {39097869}, issn = {1165-158X}, mesh = {Humans ; *beta-Thalassemia/genetics ; K562 Cells ; *Gene Editing/methods ; *Anemia, Sickle Cell/genetics ; *CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide/genetics ; *Alleles ; Genetic Markers/genetics ; Deoxyribonucleases, Type II Site-Specific/metabolism/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Fetal Hemoglobin/genetics/metabolism ; Base Sequence ; }, abstract = {The XmnI Gg -158 C/T polymorphism has been widely associated with fetal hemoglobin (HbF) levels, the severity of disease, and the response to the drug hydroxyurea (HU) in both β-thalassemia (β-thal) and sickle cell disease (SCD) patients. However, the functional significance of this single nucleotide polymorphism (SNP) remains unclear. To gain insight, green fluorescence protein (GFP) cassettes harboring the XmnI C or T alleles in their left homology arms (i.e. Gg promoters) were knocked into the Gg gene(s) of K562 cells via CRISPR/Cas9. Subsequently, the GFP fluorescence levels were compared in the ensuing cell populations and isolated clones. In both instances, median fluorescence intensities (MFI) of the knockin cells having the inserted XmnI T allele were higher than those having the XmnI C allele. Our results suggest that the XmnI T allele can increase Gg expression in K562 cells. The possible functional significance of the XmnI Gg -158 C/T polymorphism provides a rationale for the aforementioned associations. Furthermore, the XmnI polymorphism as a functional SNP substantiates its importance as a prognostic marker.}, }
@article {pmid39095793, year = {2024}, author = {Oh, Y and Kim, S and Kim, Y and Kim, H and Jang, D and Shin, S and Lee, SJ and Kim, J and Lee, SE and Oh, J and Yang, Y and Kim, D and Jung, HR and Kim, S and Kim, J and Min, K and Cho, B and Seo, H and Han, D and Park, H and Cho, SY}, title = {Genome-wide CRISPR screening identifies tyrosylprotein sulfotransferase-2 as a target for augmenting anti-PD1 efficacy.}, journal = {Molecular cancer}, volume = {23}, number = {1}, pages = {155}, pmid = {39095793}, issn = {1476-4598}, support = {HN21C0196000021//Korea Drug Development Fund/ ; HN21C0196000021//Korea Drug Development Fund/ ; 2021R1C1C2009923//National Research Foundation of Korea/ ; 2021R1A2C3008021//National Research Foundation of Korea/ ; 800-20230479//Seoul National University/ ; }, mesh = {Animals ; Humans ; Mice ; *Sulfotransferases/genetics/metabolism ; *Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/metabolism ; Cell Line, Tumor ; Interferon-gamma/metabolism ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; CRISPR-Cas Systems ; Xenograft Model Antitumor Assays ; Neoplasms/genetics/drug therapy/immunology/pathology/metabolism ; Disease Models, Animal ; }, abstract = {BACKGROUND: Immune checkpoint therapy (ICT) provides durable responses in select cancer patients, yet resistance remains a significant challenge, prompting the exploration of underlying molecular mechanisms. Tyrosylprotein sulfotransferase-2 (TPST2), known for its role in protein tyrosine O-sulfation, has been suggested to modulate the extracellular protein-protein interactions, but its specific role in cancer immunity remains largely unexplored.<br><br>METHODS: To explore tumor cell-intrinsic factors influencing anti-PD1 responsiveness, we conducted a pooled loss-of-function genetic screen in humanized mice engrafted with human immune cells. The responsiveness of cancer cells to interferon-&#x3b3; (IFN&#x3b3;) was estimated by evaluating IFN&#x3b3;-mediated induction of target genes, STAT1 phosphorylation, HLA expression, and cell growth suppression. The sulfotyrosine-modified target gene of TPST2 was identified by co-immunoprecipitation and mass spectrometry. The in vivo effects of TPST2 inhibition were evaluated using mouse syngeneic tumor models and corroborated by bulk and single-cell RNA sequencing analyses.<br><br>RESULTS: Through in vivo genome-wide CRISPR screening, TPST2 loss-of-function emerged as a potential enhancer of anti-PD1 treatment efficacy. TPST2 suppressed IFN&#x3b3; signaling by sulfating IFN&#x3b3; receptor 1 at Y397 residue, while its downregulation boosted IFN&#x3b3;-mediated signaling and antigen presentation. Depletion of TPST2 in cancer cells augmented anti-PD1 antibody efficacy in syngeneic mouse tumor models by enhancing tumor-infiltrating lymphocytes. RNA sequencing data revealed TPST2's inverse correlation with antigen presentation, and increased TPST2 expression is associated with poor prognosis and altered cancer immunity across cancer types.<br><br>CONCLUSIONS: We propose TPST2's novel role as a suppressor of cancer immunity and advocate for its consideration as a therapeutic target in ICT-based treatments.}, }
@article {pmid38908546, year = {2024}, author = {Aquino-Jarquin, G}, title = {CircRNA knockdown based on antisense strategies.}, journal = {Drug discovery today}, volume = {29}, number = {8}, pages = {104066}, doi = {10.1016/j.drudis.2024.104066}, pmid = {38908546}, issn = {1878-5832}, mesh = {*RNA, Circular/genetics ; Animals ; Humans ; *Oligonucleotides, Antisense/genetics ; Gene Knockdown Techniques/methods ; CRISPR-Cas Systems/genetics ; RNA Interference ; }, abstract = {Circular RNAs (circRNAs) are a type of noncoding RNA that are formed by back-splicing from eukaryotic protein-coding genes. The most frequently reported and well-characterized function of circRNAs is their ability to act as molecular decoys, most often as miRNA and protein sponges. However, the functions of most circRNAs still need to be better understood. To more fully understand the biological relevance of validated circRNAs, knockdown functional analyses can be performed using antisense oligonucleotides, RNA interference (RNAi) experiments (e.g., targeting back-splicing junction sites), the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)-9 system (e.g., generating circRNA-specific knockouts), and CRISPR-Cas13 technology to effectively target circRNAs without affecting host genes. In this review, I summarize the feasibility and effectiveness of circRNA knockdown through antisense strategies for investigating the biological roles of circRNAs in cultured cells and animal models.}, }
@article {pmid38105607, year = {2024}, author = {Kendal, E}, title = {Whose (germ) line is it anyway? Reproductive technologies and kinship.}, journal = {Bioethics}, volume = {38}, number = {7}, pages = {632-642}, doi = {10.1111/bioe.13254}, pmid = {38105607}, issn = {1467-8519}, mesh = {Humans ; Female ; Male ; *Parents ; Reproductive Techniques, Assisted/ethics ; Germ Cells ; Sexual and Gender Minorities ; Family ; Pregnancy ; Gene Editing/ethics ; Surrogate Mothers ; Fertilization in Vitro/ethics ; Gametogenesis ; Reproductive Techniques/ethics ; CRISPR-Cas Systems ; }, abstract = {Reproductive biotechnologies can separate concepts of parenthood into genetic, gestational and social dimensions, often leading to a fragmentation of heteronormative kinship models and posing a challenge to historical methods of establishing legal and/or moral parenthood. Using fictional cases, this article will demonstrate that the issues surrounding the intersection of current and emerging reproductive biotechnologies with definitions of parenthood are already leading to confusion regarding social and legal family ties for offspring, which is only expected to increase as new technologies develop. Rather than opposing these new technologies to reassert traditional concepts of the family, however, this article will explore the opportunities that these technologies represent for re-imagining various culturally cherished values of family-making in a way that is inclusive of diverse genders, sexualities and cultures. It will consider IVF, gametogenesis, mitochondrial donation, surrogacy, artificial gestation, CRISPR-Cas9 gene editing, foster care and adoption as some of many possible pathways to parenthood, including for members of the LGBTIAUQ+ community.}, }
@article {pmid39095367, year = {2024}, author = {Collins, M and Lau, MB and Ma, W and Shen, A and Wang, B and Cai, S and La Russa, M and Jewett, MC and Qi, LS}, title = {A frugal CRISPR kit for equitable and accessible education in gene editing and synthetic biology.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6563}, pmid = {39095367}, issn = {2041-1723}, support = {2046650//National Science Foundation (NSF)/ ; }, mesh = {*Gene Editing/methods ; *Synthetic Biology/methods ; Humans ; *CRISPR-Cas Systems ; Students ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Schools ; }, abstract = {Equitable and accessible education in life sciences, bioengineering, and synthetic biology is crucial for training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology can benefit a wide-ranging population. As a groundbreaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology in educational settings remains limited due to the extensive resources required for CRISPR experiments. Here, we develop CRISPRkit, an affordable kit designed for gene editing and regulation in high school education. CRISPRkit eliminates the need for specialized equipment, prioritizes biosafety, and utilizes cost-effective reagents. By integrating CRISPRi gene regulation, colorful chromoproteins, cell-free transcription-translation systems, smartphone-based quantification, and an in-house automated algorithm (CRISPectra), our kit offers an inexpensive (~$2) and user-friendly approach to performing and analyzing CRISPR experiments, without the need for a traditional laboratory setup. Experiments conducted by high school students in classroom settings highlight the kit's utility for reliable CRISPRkit experiments. Furthermore, CRISPRkit provides a modular and expandable platform for genome engineering, and we demonstrate its applications for controlling fluorescent proteins and metabolic pathways such as melanin production. We envision CRISPRkit will facilitate biotechnology education for communities of diverse socioeconomic and geographic backgrounds.}, }
@article {pmid39095257, year = {2024}, author = {Chen, L and Hu, M and Zhou, X}, title = {Trends in developing one-pot CRISPR diagnostics strategies.}, journal = {Trends in biotechnology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tibtech.2024.07.007}, pmid = {39095257}, issn = {1879-3096}, abstract = {The integration of nucleic acid amplification (NAA) with the CRISPR detection system has led to significant advancements and opportunities for development in molecular diagnostics. Nevertheless, the incompatibility between CRISPR cleavage and NAA has significantly impeded the commercialization of this technology. Currently, several one-pot detection strategies based on CRISPR systems have been devised to address concerns regarding aerosol contamination risk and operational complexity associated with step-by-step detection as well as the sensitivity limitation of conventional one-pot methods. In this review, we provide a comprehensive introduction and outlook of the various solutions of the one-pot CRISPR assay for practitioners who are committed to developing better CRISPR nucleic acid detection technologies to promote the progress of molecular diagnostics.}, }
@article {pmid39094583, year = {2024}, author = {Maestri, A and Pons, BJ and Pursey, E and Chong, CE and Gandon, S and Custodio, R and Olina, A and Agapov, A and Chisnall, MAW and Grasso, A and Paterson, S and Szczelkun, MD and Baker, KS and van Houte, S and Chevallereau, A and Westra, ER}, title = {The bacterial defense system MADS interacts with CRISPR-Cas to limit phage infection and escape.}, journal = {Cell host &amp; microbe}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.chom.2024.07.005}, pmid = {39094583}, issn = {1934-6069}, abstract = {The constant arms race between bacteria and their parasites has resulted in a large diversity of bacterial defenses, with many bacteria carrying multiple systems. Here, we report the discovery of a phylogenetically widespread defense system, coined methylation-associated defense system (MADS), which is distributed across gram-positive and gram-negative bacteria. MADS interacts with a CRISPR-Cas system in its native host to provide robust and durable resistance against phages. While phages can acquire epigenetic-mediated resistance against MADS, co-existence of MADS and a CRISPR-Cas system limits escape emergence. MADS comprises eight genes with predicted nuclease, ATPase, kinase, and methyltransferase domains, most of which are essential for either self/non-self discrimination, DNA restriction, or both. The complex genetic architecture of MADS and MADS-like systems, relative to other prokaryotic defenses, points toward highly elaborate mechanisms of sensing infections, defense activation, and/or interference.}, }
@article {pmid39094478, year = {2024}, author = {Pandey, S and Divakar, S and Singh, A}, title = {Genome editing prospects for heat stress tolerance in cereal crops.}, journal = {Plant physiology and biochemistry : PPB}, volume = {215}, number = {}, pages = {108989}, doi = {10.1016/j.plaphy.2024.108989}, pmid = {39094478}, issn = {1873-2690}, abstract = {The world population is steadily growing, exerting increasing pressure to feed in the future, which would need additional production of major crops. Challenges associated with changing and unpredicted climate (such as heat waves) are causing global food security threats. Cereal crops are a staple food for a large portion of the world's population. They are mostly affected by these environmentally generated abiotic stresses. Therefore, it is imperative to develop climate-resilient cultivars to support the sustainable production of main cereal crops (Rice, wheat, and maize). Among these stresses, heat stress causes significant losses to major cereals. These issues can be solved by comprehending the molecular mechanisms of heat stress and creating heat-tolerant varieties. Different breeding and biotechnology techniques in the last decade have been employed to develop heat-stress-tolerant varieties. However, these time-consuming techniques often lack the pace required for varietal improvement in climate change scenarios. Genome editing technologies offer precise alteration in the crop genome for developing stress-resistant cultivars. CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/Cas9), one such genome editing platform, recently got scientists' attention due to its easy procedures. It is a powerful tool for functional genomics as well as crop breeding. This review will focus on the molecular mechanism of heat stress and different targets that can be altered using CRISPR/Cas genome editing tools to generate climate-smart cereal crops. Further, heat stress signaling and essential players have been highlighted to provide a comprehensive overview of the topic.}, }
@article {pmid39093358, year = {2024}, author = {Zhou, Y and Che, S and Wang, Z and Zhang, X and Yuan, X}, title = {Primer exchange reaction assisted CRISPR/Cas9 cleavage for detection of dual microRNAs with electrochemistry method.}, journal = {Mikrochimica acta}, volume = {191}, number = {8}, pages = {502}, pmid = {39093358}, issn = {1436-5073}, support = {22276103//National Natural Science Foundation of China/ ; ZR2023QH081//Natural Science Foundation of Shandong Province/ ; }, mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; Humans ; *Limit of Detection ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {An electrochemical sensor assisted by primer exchange reaction (PER) and CRISPR/Cas9 system (PER-CRISPR/Cas9-E) was established for the sensitive detection of dual microRNAs (miRNAs). Two PER hairpin (HP) were designed to produce a lot of extended PER products, which could hybridize with two kinds of hairpin probes modified on the electrode and initiate the cleavage of two CRISPR/Cas9 systems guided by single guide RNAs (sgRNAs) with different recognition sequences. The decrease of the two electrochemical redox signals indicated the presence of dual-target miRNAs. With the robustness and high specificity of PER amplification and CRISPR/Cas9 cleavage system, simultaneous detection of two targets was achieved and the detection limits for miRNA-21 and miRNA-155 were 0.43 fM and 0.12 fM, respectively. The developed biosensor has the advantages of low cost, easy operation, and in-situ detection, providing a promising platform for point-of-care detection of multiple miRNAs.}, }
@article {pmid39032428, year = {2024}, author = {Takagi, D and Tsukamoto, S and Nakade, K and Shimizu, T and Arai, Y and Matsuo-Takasaki, M and Noguchi, M and Nakamura, Y and Yumoto, N and Kawada, J and Hayata, T and Hayashi, Y}, title = {Generation of MBP-tdTomato reporter human induced pluripotent stem cell line for live myelin visualization.}, journal = {Stem cell research}, volume = {79}, number = {}, pages = {103493}, doi = {10.1016/j.scr.2024.103493}, pmid = {39032428}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Myelin Basic Protein/metabolism/genetics ; Myelin Sheath/metabolism ; Cell Line ; Cell Differentiation ; CRISPR-Cas Systems ; Genes, Reporter ; Luminescent Proteins/metabolism/genetics ; Gene Editing ; Red Fluorescent Protein ; }, abstract = {Myelin basic protein (MBP) is a major component of the myelin sheaths of oligodendrocytes in the central nervous system and Schwann cells of the peripheral nervous system. Here we generated heterozygous fluorescent reporter of MBP gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock in fused tdTomato fluorescent protein and EF1 alpha promoter-driven Bleomycin (Zeocin) resistance gene to the translational MBP C-terminal region. The resulting line, MBP-TEZ, showed tdTomato fluorescence upon oligodendrocyte differentiation. This reporter hiPSC line provides a precedential opportunity for monitoring human myelin formation and degeneration and purifying MBP-expressing cell lineages.}, }
@article {pmid39018827, year = {2024}, author = {Wang, X and Gao, J and Liu, C and Sun, J}, title = {Establishment of human embryonic stem cell lines carrying LQT1 mutations by CRISPR base editing.}, journal = {Stem cell research}, volume = {79}, number = {}, pages = {103496}, doi = {10.1016/j.scr.2024.103496}, pmid = {39018827}, issn = {1876-7753}, mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *KCNQ1 Potassium Channel/genetics/metabolism ; *Mutation ; Cell Line ; CRISPR-Cas Systems ; Long QT Syndrome/genetics/metabolism ; Myocytes, Cardiac/metabolism/cytology ; Cell Differentiation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {The KCNQ1 gene encodes a voltage-gated potassium channel required for cardiac action potentials. Mutations in this gene have been associated with hereditary long QT syndrome 1, Jervell and Lange-Nielsen syndromes, and familial atrial fibrillation. The NM_000218.3(KCNQ1): c.604 + 2T > C mutation has been categorized as the causative variant leading to LQT1. In this study, we generated a KCNQ1 (c.644 + 2T > C) mutation human embryonic stem cell line WAe009-A-1L based on CRISPR base editing system. WAe009-A-1L cell has the potential to differentiate cardiomyocytes and would be used as an in vitro disease model for mechanism exploration and drug screening.}, }
@article {pmid39003885, year = {2024}, author = {Pachernegg, S and Robevska, G and Ferreira, LGA and van den Bergen, JA and Vlahos, K and Howden, SE and Sinclair, AH and Ayers, KL}, title = {Generation of a homozygous (MCRIi031-A-3) WT1 knockout human iPSC line.}, journal = {Stem cell research}, volume = {79}, number = {}, pages = {103494}, doi = {10.1016/j.scr.2024.103494}, pmid = {39003885}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *WT1 Proteins/genetics/metabolism ; *Homozygote ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; Gene Knockout Techniques ; Gene Editing ; }, abstract = {The transcription factor WT1 plays a critical role in several embryonic developmental processes such as gonadogenesis, nephrogenesis, and cardiac development. We generated a homozygous (MCRIi031-A-3) WT1 knockout induced pluripotent stem cell (iPSC) line from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. The cells exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of WT1 in critical developmental processes.}, }
@article {pmid38924973, year = {2024}, author = {Pachernegg, S and Robevska, G and G A Ferreira, L and van den Bergen, JA and Vlahos, K and Howden, SE and Sinclair, AH and Ayers, KL}, title = {Generation of heterozygous (MCRIi031-A-1) and homozygous (MCRIi031-A-2) SOX9 knockout human iPSC lines.}, journal = {Stem cell research}, volume = {79}, number = {}, pages = {103484}, doi = {10.1016/j.scr.2024.103484}, pmid = {38924973}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *SOX9 Transcription Factor/metabolism/genetics ; *Homozygote ; *Heterozygote ; Cell Line ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Editing ; Cell Differentiation ; }, abstract = {The transcription factor SOX9 plays a critical role in several embryonic developmental processes such as gonadogenesis, chrondrogenesis, and cardiac development. We generated heterozygous (MCRIi031-A-1) and homozygous (MCRIi031-A-2) SOX9 knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of SOX9 in critical developmental processes.}, }
@article {pmid38917806, year = {2024}, author = {Saurat, N and Minotti, AP and Rahman, MT and Sikder, T and Zhang, C and Cornacchia, D and Jungverdorben, J and Ciceri, G and Betel, D and Studer, L}, title = {Genome-wide CRISPR screen identifies neddylation as a regulator of neuronal aging and AD neurodegeneration.}, journal = {Cell stem cell}, volume = {31}, number = {8}, pages = {1162-1174.e8}, doi = {10.1016/j.stem.2024.06.001}, pmid = {38917806}, issn = {1875-9777}, support = {R01 AG054720/AG/NIA NIH HHS/United States ; }, mesh = {Humans ; *Alzheimer Disease/pathology/genetics/metabolism ; Cellular Senescence/genetics ; Neurons/metabolism/pathology ; NEDD8 Protein/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; tau Proteins/metabolism/genetics ; Parkinson Disease/genetics/pathology/metabolism ; Aging/genetics/pathology/metabolism ; Dopaminergic Neurons/metabolism/pathology ; CRISPR-Cas Systems/genetics ; }, abstract = {Aging is the biggest risk factor for the development of Alzheimer's disease (AD). Here, we performed a whole-genome CRISPR screen to identify regulators of neuronal age and show that the neddylation pathway regulates both cellular age and AD neurodegeneration in a human stem cell model. Specifically, we demonstrate that blocking neddylation increased cellular hallmarks of aging and led to an increase in Tau aggregation and phosphorylation in neurons carrying the APP[swe/swe] mutation. Aged APP[swe/swe] but not isogenic control neurons also showed a progressive decrease in viability. Selective neuronal loss upon neddylation inhibition was similarly observed in other isogenic AD and in Parkinson's disease (PD) models, including PSEN[M146V/M146V] cortical and LRRK2[G2019S][/G2019S] midbrain dopamine neurons, respectively. This study indicates that cellular aging can reveal late-onset disease phenotypes, identifies new potential targets to modulate AD progression, and describes a strategy to program age-associated phenotypes into stem cell models of disease.}, }
@article {pmid38905814, year = {2024}, author = {Juchem, M and Lehmann, N and Behrens, YL and Bär, C and Thum, T and Hoepfner, J}, title = {CRISPR/Cas9-based GLA knockout to generate the female Fabry disease human induced pluripotent stem cell line MHHi001-A-15.}, journal = {Stem cell research}, volume = {79}, number = {}, pages = {103478}, doi = {10.1016/j.scr.2024.103478}, pmid = {38905814}, issn = {1876-7753}, mesh = {*Fabry Disease/genetics/pathology/metabolism ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *CRISPR-Cas Systems ; *alpha-Galactosidase/genetics/metabolism ; Female ; Cell Line ; Gene Knockout Techniques ; Cell Differentiation ; }, abstract = {The X-linked lysosomal storage disorder Fabry disease originates from GLA gene mutations causing α-galactosidase A enzyme deficiency. Here we generated the GLA knockout hiPSC line MHHi001-A-15 (GLA-KOhiPSC) as an in vitro Fabry disease model by targeting exon 2 of the GLA gene by CRISPR/Cas9 in the established control hiPSC line MHHi001-A. GLA-KOhiPSCs retained the expression of pluripotency markers, trilineage differentiation potential, as well as normal karyotype and stem cell morphology but lacked α-galactosidase A enzyme activity. The GLA-KOhiPSCs represent a potent resource to not only study the Fabry disease manifestation but also screen for novel treatment options.}, }
@article {pmid38772378, year = {2024}, author = {Zhang, K and Wan, P and Wang, L and Wang, Z and Tan, F and Li, J and Ma, X and Cen, J and Yuan, X and Liu, Y and Sun, Z and Cheng, X and Liu, Y and Liu, X and Hu, J and Zhong, G and Li, D and Xia, Q and Hui, L}, title = {Efficient expansion and CRISPR-Cas9-mediated gene correction of patient-derived hepatocytes for treatment of inherited liver diseases.}, journal = {Cell stem cell}, volume = {31}, number = {8}, pages = {1187-1202.e8}, doi = {10.1016/j.stem.2024.04.022}, pmid = {38772378}, issn = {1875-9777}, mesh = {*Hepatocytes/metabolism/transplantation ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Liver Diseases/therapy/genetics/pathology ; *Gene Editing ; Mice ; *Genetic Therapy/methods ; Tyrosinemias/therapy/genetics ; Cell Proliferation ; Hydrolases ; }, abstract = {Cell-based ex vivo gene therapy in solid organs, especially the liver, has proven technically challenging. Here, we report a feasible strategy for the clinical application of hepatocyte therapy. We first generated high-quality autologous hepatocytes through the large-scale expansion of patient-derived hepatocytes. Moreover, the proliferating patient-derived hepatocytes, together with the AAV2.7m8 variant identified through screening, enabled CRISPR-Cas9-mediated targeted integration efficiently, achieving functional correction of pathogenic mutations in FAH or OTC. Importantly, these edited hepatocytes repopulated the injured mouse liver at high repopulation levels and underwent maturation, successfully treating mice with tyrosinemia following transplantation. Our study combines ex vivo large-scale cell expansion and gene editing in patient-derived transplantable hepatocytes, which holds potential for treating human liver diseases.}, }
@article {pmid38599514, year = {2024}, author = {Hou, S and Chen, J and Feng, R and Xu, X and Liang, N and Champer, J}, title = {A homing rescue gene drive with multiplexed gRNAs reaches high frequency in cage populations but generates functional resistance.}, journal = {Journal of genetics and genomics = Yi chuan xue bao}, volume = {51}, number = {8}, pages = {836-843}, doi = {10.1016/j.jgg.2024.04.001}, pmid = {38599514}, issn = {1673-8527}, mesh = {Animals ; *Gene Drive Technology/methods ; *Drosophila melanogaster/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Alleles ; Drosophila Proteins/genetics ; Gene Editing ; }, abstract = {CRISPR homing gene drives have considerable potential for managing populations of medically and agriculturally significant insects. They operate by Cas9 cleavage followed by homology-directed repair, copying the drive allele to the wild-type chromosome and thus increasing in frequency and spreading throughout a population. However, resistance alleles formed by end-joining repair pose a significant obstacle. To address this, we create a homing drive targeting the essential hairy gene in Drosophila melanogaster. Nonfunctional resistance alleles are recessive lethal, while drive carriers have a recoded "rescue" version of hairy. The drive inheritance rate is moderate, and multigenerational cage studies show drive spread to 96%-97% of the population. However, the drive does not reach 100% due to the formation of functional resistance alleles despite using four gRNAs. These alleles have a large deletion but likely utilize an alternate start codon. Thus, revised designs targeting more essential regions of a gene may be necessary to avoid such functional resistance. Replacement of the rescue element's native 3' UTR with a homolog from another species increases drive inheritance by 13%-24%. This was possibly because of reduced homology between the rescue element and surrounding genomic DNA, which could also be an important design consideration for rescue gene drives.}, }
@article {pmid39091309, year = {2024}, author = {Yang, N and Zhang, H and Han, X and Liu, Z and Lu, Y}, title = {Advancements and applications of loop-mediated isothermal amplification technology: a comprehensive overview.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1406632}, doi = {10.3389/fmicb.2024.1406632}, pmid = {39091309}, issn = {1664-302X}, abstract = {Loop-mediated isothermal amplification (LAMP) is a novel method for nucleic acid detection known for its isothermal properties, high efficiency, sensitivity, and specificity. LAMP employs 4 to 6 primers targeting 6 to 8 regions of the desired sequence, allowing for amplification at temperatures between 60 and 65°C and the production of up to 10[9] copies within a single hour. The product can be monitored by various methods such as turbidimetry, fluorometry, and colorimetry. However, it faces limitations such as the risk of non-specific amplification, challenges in primer design, unsuitability for short gene sequences, and difficulty in multiplexing. Recent advancements in polymerase and primer design have enhanced the speed and convenience of the LAMP reaction. Additionally, integrating LAMP with technologies like rolling circle amplification (RCA), recombinase polymerase amplification (RPA), and CRISPR-Cas systems has enhanced its efficiency. The combination of LAMP with various biosensors has enabled real-time analysis, broadening its application in point-of-care testing (POCT). Microfluidic technology has further facilitated the automation and miniaturization of LAMP assays, allowing for the simultaneous detection of multiple targets and preventing contamination. This review highlights advancements in LAMP, focusing on primer design, polymerase engineering, and its integration with other technologies. Continuous improvements and integration of LAMP with complementary technologies have significantly enhanced its diagnostic capabilities, making it a robust tool for rapid, sensitive, and specific nucleic acid detection with promising implications for healthcare, agriculture, and environmental monitoring.}, }
@article {pmid39089060, year = {2024}, author = {Su, CW and Hsu, YC and Tsai, LC and Lee, JC and Linacre, A and Hsieh, HM}, title = {Rapid detection of blood using a novel application of RT-RPA integrated with CRISPR-Cas: ALAS2 detection as a model.}, journal = {Forensic science international. Genetics}, volume = {73}, number = {}, pages = {103098}, doi = {10.1016/j.fsigen.2024.103098}, pmid = {39089060}, issn = {1878-0326}, abstract = {A rapid, sensitive and specific test for blood is reported based on a novel application of recombinase polymerase amplification integrated with CRISPR-Cas and lateral flow assay (LFA). The blood specific marker ALAS2 was used as the target to record the presence of blood. The assay used either RNA extracted from a body fluid as a template, or omitting this extraction step and using a direct approach where the questioned body fluid was added directly to the assay. The assay only detected blood (all peripheral blood and some menstrual blood samples) and no other body fluid (semen, saliva, or vaginal fluid). The limit of detection varied from an initial template of 0.195 ng extracted RNA (2[7] dilution) or 0.0218 μL (2[6] dilution) liquid peripheral blood. The assay gave the expected result when peripheral blood was mixed with saliva: ratios of peripheral blood/saliva at 19:1, 3:1, 1:1, 1:3 and 1:19 all gave a positive result using extracted RNA. By contrast, only three ratios of peripheral blood and saliva gave a positive result for blood (19:1, 3:1 and 1:1) when adding these two body fluids directly. When peripheral blood was mixed with semen there was a strong inhibition of the assay and ALAS2 could only be detected at ratio of 19:1 using RNA. Using reconstituted peripheral bloodstains gave comparable results to liquid peripheral blood. This is the first application of RT-RPA integrated CRISPR and combined with a LFA assay to detect body fluid-specific RNA. The proposed method opens up the potential to perform this method remote from laboratories such as at crime scenes.}, }
@article {pmid39052141, year = {2024}, author = {Richman, J and Phelps, M}, title = {Activin Signaling Pathway Specialization During Embryonic and Skeletal Muscle Development in Rainbow Trout (Oncorhynchus mykiss).}, journal = {Marine biotechnology (New York, N.Y.)}, volume = {26}, number = {4}, pages = {766-775}, pmid = {39052141}, issn = {1436-2236}, support = {2021-67015-33400//U.S. Department of Agriculture/ ; }, mesh = {Animals ; *Oncorhynchus mykiss/genetics/growth &amp; development/metabolism/embryology ; *Signal Transduction ; *Muscle, Skeletal/metabolism/growth &amp; development ; Female ; Gene Expression Regulation, Developmental ; Activins/metabolism/genetics ; Embryonic Development/genetics ; Muscle Development/genetics ; Gene Editing ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Activin Receptors/metabolism/genetics ; Fish Proteins/genetics/metabolism ; }, abstract = {Activin signaling is essential for proper embryonic, skeletal muscle, and reproductive development. Duplication of the pathway in teleost fish has enabled diversification of gene function across the pathway but how gene duplication influences the function of activin signaling in non-mammalian species is poorly understood. Full characterization of activin receptor signaling pathway expression was performed across embryonic development and during early skeletal muscle growth in rainbow trout (RBT, Oncorhynchus mykiss). Rainbow trout are a model salmonid species that have undergone two additional rounds of whole genome duplication. A small number of genes were expressed early in development and most genes increased expression throughout development. There was limited expression of activin Ab in RBT embryos despite these genes exhibiting significantly elevated expression in post-hatch skeletal muscle. CRISPR editing of the activin Aa1 ohnolog and subsequent production of meiotic gynogenetic offspring revealed that biallelic disruption of activin Aa1 did not result in developmental defects, as occurs with knockout of activin A in mammals. The majority of gynogenetic offspring exhibited homozygous activin Aa1 genotypes (wild type, in-frame, or frameshift) derived from the mosaic founder female. The research identifies mechanisms of specialization among the duplicated activin ohnologs across embryonic development and during periods of high muscle growth in larval and juvenile fish. The knowledge gained provides insights into potential viable gene-targeting approaches for engineering the activin receptor signaling pathway and establishes the feasibility of employing meiotic gynogenesis as a tool for producing homozygous F1 genome-edited fish for species with long-generation times, such as salmonids.}, }
@article {pmid38833208, year = {2024}, author = {Liu, B and Wittayarat, M and Takebayashi, K and Lin, Q and Torigoe, N and Namula, Z and Hirata, M and Nagahara, M and Tanihara, F and Otoi, T}, title = {Effects of centrifugation treatment before electroporation on gene editing in pig embryos.}, journal = {In vitro cellular &amp; developmental biology. Animal}, volume = {60}, number = {7}, pages = {732-739}, pmid = {38833208}, issn = {1543-706X}, support = {JP22H02499//JSPS KAKENHI/ ; JP22K19896//JSPS KAKENHI/ ; Uzushio Program//University of Tokushima/ ; }, mesh = {Animals ; *Gene Editing/methods ; *Centrifugation ; Swine ; *Electroporation/methods ; *CRISPR-Cas Systems/genetics ; Embryo, Mammalian/metabolism ; Zona Pellucida/metabolism ; Zygote/metabolism ; Blastocyst/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation/genetics ; Galactosyltransferases ; }, abstract = {Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used.}, }
@article {pmid38833200, year = {2024}, author = {Zhang, Y and Xia, H and Peng, W and Liu, L and Liu, L and Yang, P}, title = {Application of Repetitive Sequences in Fish Cell Depletion as a Target for the CRISPR/Cas9 System.}, journal = {Marine biotechnology (New York, N.Y.)}, volume = {26}, number = {4}, pages = {639-648}, pmid = {38833200}, issn = {1436-2236}, support = {32172965//National&#xa0;Natural&#xa0;Science&#xa0;Foundation&#xa0;of&#xa0;China/ ; 32360922//National&#xa0;Natural&#xa0;Science&#xa0;Foundation&#xa0;of&#xa0;China/ ; 23A0499//Hunan&#xa0;Provincial&#xa0;Education&#xa0;Department&#xa0;key&#xa0;project/ ; 22A0488//Hunan&#xa0;Provincial&#xa0;Education&#xa0;Department&#xa0;key&#xa0;project/ ; 32360922//National Natural Science Foundation of China/ ; 2022JJA130338//Guangxi&#xa0;Natural&#xa0;Science&#xa0;Foundation/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Apoptosis ; Germ Cells/metabolism ; Gene Knockout Techniques ; Repetitive Sequences, Nucleic Acid/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Caspase 3/metabolism/genetics ; Tumor Suppressor Protein p53/genetics/metabolism ; Caspase 9/genetics/metabolism ; Zygote/metabolism ; Embryo, Nonmammalian/metabolism ; RNA, Messenger/metabolism/genetics ; }, abstract = {Specific cell depletion is a common means to study the physiological function of cell lineages and tissue regeneration. However, 100% depletion is difficult to achieve with existing cell depletion strategies. With the increasing maturity of CRISPR/Cas9 technology, it is increasingly used for the depletion of various cells. However, even with this technology, it is difficult to complete the depletion of specific gene knockout cells. For this reason, cell depletion with the use of repetitive sequences as the target of CRISPR/Cas9 was explored using zebrafish. All cells were used as the target cells for the first set of experiments. The results showed that injection of a mixture of DANA-gRNA and Cas9 mRNA into zygotes resulted in substantial cell apoptosis. Cells are almost invisible in the embryonic animal pole during the dome stage. The activities of the caspase-3 and caspase-9 proteins and the mRNA level of the P53 gene were significantly increased. Then, primordial germ cells (PGCs) in embryos were used as the target cells in subsequent experiments. To specifically knock out PGCs, we injected the mix of DANA-gRNA, pkop: Cas9 plasmid (the kop promotor allows Cas9 expression only in PGCs), and eGFP-nos3'UTR mRNA into zebrafish fertilized eggs. The results revealed that the activity of the caspase-3 protein was significantly increased, and the mRNA levels of P53, ku70, and ku80 were significantly upregulated, while the number of PGCs decreased gradually. Few PGCs labeled with GFP could be seen 20 h post-fertilization (hpf), and no PGCs could be seen at the germinal ridge 24 hpf. Therefore, the combination of CRISPR/Cas9 technology and repetitive sequences can achieve efficient cell depletion regardless of whether there is generalized expression or expression in specific cells. These results indicate that it is feasible to eliminate cells by using repeat sequences as CRISPR/Cas9 system target sites.}, }
@article {pmid38813985, year = {2024}, author = {Gadhia, A and Barker, E and Morgan, A and Barclay, JW}, title = {Functional analysis of epilepsy-associated GABAA receptor mutations using Caenorhabditis elegans.}, journal = {Epilepsia open}, volume = {9}, number = {4}, pages = {1458-1466}, doi = {10.1002/epi4.12982}, pmid = {38813985}, issn = {2470-9239}, support = {WT reference 102172/B/13/Z/WT_/Wellcome Trust/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; }, mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Receptors, GABA-A/genetics ; *Epilepsy/genetics ; *Caenorhabditis elegans Proteins/genetics ; *Disease Models, Animal ; *Mutation ; Phenotype ; CRISPR-Cas Systems ; Gene Editing ; Locomotion ; }, abstract = {OBJECTIVE: GABAA receptor subunit mutations pose a significant risk for genetic generalized epilepsy; however, there are over 150 identified variants, many with unknown or unvalidated pathogenicity. We aimed to develop in vivo models for testing GABAA receptor variants using the model organism, Caenorhabditis elegans.<br><br>METHODS: CRISPR-Cas9 gene editing was used to create a complete deletion of unc-49, a C. elegans GABAA receptor, and to create homozygous epilepsy-associated mutations in the endogenous unc-49 gene. The unc-49 deletion strain was rescued with transgenes for either the C. elegans unc-49B subunit or the &#x3b1;1, &#x3b2;3, and &#x3b3;2 subunits for the human GABAA receptor. All newly created strains were analyzed for phenotype and compared against existing unc-49 mutations.<br><br>RESULTS: Nematodes with a full genetic deletion of the entire unc-49 locus were compared with existing unc-49 mutations in three separate phenotypic assays-coordinated locomotion, shrinker frequency and seizure-like convulsions. The full unc-49 deletion exhibited reduced locomotion and increased shrinker frequency and PTZ-induced convulsions, but were not found to be phenotypically stronger than existing unc-49 mutations. Rescue with the unc-49B subunit or creation of humanized worms for the GABAA receptor both showed partial phenotypic rescue for all three phenotypes investigated. Finally, two epilepsy-associated variants were analyzed and deemed to be loss of function, thus validating their pathogenicity.<br><br>SIGNIFICANCE: These findings establish C. elegans as a genetic model to investigate GABAA receptor mutations and delineate a platform for validating associated variants in any epilepsy-associated gene.<br><br>PLAIN LANGUAGE SUMMARY: Epilepsy is a complex human disease that can be caused by mutations in specific genes. Many possible mutations have been identified, but it is unknown for most of them whether they cause the disease. We tested the role of mutations in one specific gene using a small microscopic worm as an animal model. Our results establish this worm as a model for epilepsy and confirm that the two unknown mutations are likely to cause the disease.}, }
@article {pmid38485817, year = {2024}, author = {Nguyen, TV and Do, LTK and Lin, Q and Nagahara, M and Namula, Z and Wittayarat, M and Hirata, M and Otoi, T and Tanihara, F}, title = {Programmed cell death-1-modified pig developed using electroporation-mediated gene editing for in vitro fertilized zygotes.}, journal = {In vitro cellular &amp; developmental biology. Animal}, volume = {60}, number = {7}, pages = {716-724}, pmid = {38485817}, issn = {1543-706X}, mesh = {Animals ; *Gene Editing/methods ; *Electroporation/methods ; *Zygote/metabolism ; *Fertilization in Vitro/methods ; Swine ; *Programmed Cell Death 1 Receptor/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Animals, Genetically Modified ; RNA, Guide, CRISPR-Cas Systems/genetics ; Female ; Base Sequence ; Mutation/genetics ; Phenotype ; }, abstract = {Programmed cell death-1 (PD-1) is an immunoinhibitory receptor required to suppress inappropriate immune responses such as autoimmunity. Immune checkpoint antibodies that augment the PD-1 pathway lead to immune-related adverse events (irAEs), organ non-specific side effects due to autoimmune activation in humans. In this study, we generated a PD-1 mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes to evaluate the PD-1 gene deficiency phenotype. We optimized the efficient guide RNAs (gRNAs) targeting PD-1 in zygotes and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. One recipient gilt became pregnant and gave birth to two piglets. Sequencing analysis revealed that both piglets were biallelic mutants. At 18 mo of age, one pig showed non-purulent arthritis of the left elbow/knee joint and oligozoospermia, presumably related to PD-1 modification. Although this study has a limitation because of the small number of cases, our phenotypic analysis of PD-1 modification in pigs will provide significant insight into human medicine and PD-1-deficient pigs can be beneficial models for studying human irAEs.}, }
@article {pmid38334880, year = {2024}, author = {Matsuzaki, S and Sakuma, T and Yamamoto, T}, title = {REMOVER-PITCh: microhomology-assisted long-range gene replacement with highly multiplexed CRISPR-Cas9.}, journal = {In vitro cellular &amp; developmental biology. Animal}, volume = {60}, number = {7}, pages = {697-707}, pmid = {38334880}, issn = {1543-706X}, support = {21K06137//Japan Society for the Promotion of Science/ ; 17H01409//Japan Society for the Promotion of Science/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; Base Sequence ; HEK293 Cells ; }, abstract = {A variety of CRISPR-Cas9-based gene editing technologies have been developed, including gene insertion and gene replacement, and applied to the study and treatment of diseases. While numerous studies have been conducted to improve the efficiency of gene insertion and to expand the system in various ways, there have been relatively few reports on gene replacement technology; therefore, further improvements are still needed in this context. Here, we developed the REMOVER-PITCh system to establish an efficient long-range gene replacement method and demonstrated its utility at two genomic loci in human cultured cells. REMOVER-PITCh depends on microhomology-assisted gene insertion technology called PITCh with highly multiplexed CRISPR-Cas9. First, we achieved gene replacement of about 20-kb GUSB locus using this system. Second, by applying the previously established knock-in-enhancing platform, the LoAD system, along with REMOVER-PITCh, we achieved the replacement of a longer gene region of about 200 kb at the ARSB locus. Our REMOVER-PITCh system will make it possible to remove and incorporate a variety of sequences from and into the genome, respectively, which will facilitate the generation of various disease and humanized models.}, }
@article {pmid39086277, year = {2024}, author = {Parums, DV}, title = {Editorial: Genome Editing Goes Beyond CRISPR with the Emergence of 'Bridge' RNA Editing.}, journal = {Medical science monitor : international medical journal of experimental and clinical research}, volume = {30}, number = {}, pages = {e945933}, doi = {10.12659/MSM.945933}, pmid = {39086277}, issn = {1643-3750}, mesh = {*Gene Editing/methods ; *RNA Editing/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome/genetics ; DNA Transposable Elements/genetics ; }, abstract = {Therapeutic human gene editing technologies continue to advance, with the endonuclease, clustered regularly interspaced short palindromic repeats (CRISPR) being one of the most rapidly developing technologies. Recently, in 2024, a method of RNA editing called 'bridge editing' has been described in bacteria, which is more powerful and has broader applications than CRISPR to reshape the genome. The term 'bridge editing' is used because the method physically links, or bridges, two sections of DNA and can alter large sections of a genome. 'Bridge editing' relies on insertion sequence (IS) elements, the simplest autonomous transposable elements in prokaryotic genomes. This method provides a unified mechanism for the three fundamental types of DNA rearrangement required for genome design: inversion, insertion, and excision. The 'bridge' recombination system could expand the range and diversity of nucleic acid-guided therapeutic systems beyond RNA interference and CRISPR. This editorial aims to introduce new developments in 'bridge' RNA editing that have the increased potential to reshape the genome.}, }
@article {pmid39085844, year = {2024}, author = {Dalvie, NC and Lorgeree, TR and Yang, Y and Rodriguez-Aponte, SA and Whittaker, CA and Hinckley, JA and Clark, JJ and Del Rosario, AM and Love, KR and Love, JC}, title = {CRISPR-Cas9 knockout screen informs efficient reduction of the Komagataella phaffii secretome.}, journal = {Microbial cell factories}, volume = {23}, number = {1}, pages = {217}, pmid = {39085844}, issn = {1475-2859}, support = {P30-CA14051/CA/NCI NIH HHS/United States ; P30-CA14051/CA/NCI NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; *Saccharomycetales/genetics/metabolism ; *Recombinant Proteins/genetics/biosynthesis/metabolism ; Humans ; Gene Knockout Techniques/methods ; Fungal Proteins/genetics/metabolism ; Proteome/metabolism ; Antibodies, Monoclonal/biosynthesis ; Serum Albumin, Human/genetics/metabolism ; }, abstract = {BACKGROUND: The yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K. phaffii, however, genetic engineering is limited by lack gene annotation and knowledge of gene essentiality.<br><br>RESULTS: We identified a set of endogenous secreted proteins in K. phaffii by mass spectrometry and signal peptide prediction. Our efforts to disrupt these genes were hindered by limited annotation of essential genes. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a&#x2009;~20&#xd7;&#x2009;increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody.<br><br>CONCLUSIONS: We demonstrated that disruption of as few as six genes can increase production of recombinant proteins. Further reduction of the endogenous proteome of K. phaffii may further improve strain performance. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes.}, }
@article {pmid39085666, year = {2024}, author = {Xiang, Z and Ye, Q and Zhao, Z and Wang, N and Li, J and Zou, M and Lau, CH and Zhu, H and Wang, S and Ding, Y}, title = {Development of a baculoviral CRISPR/Cas9 vector system for beta-2-microglobulin knockout in human pluripotent stem cells.}, journal = {Molecular genetics and genomics : MGG}, volume = {299}, number = {1}, pages = {74}, pmid = {39085666}, issn = {1617-4623}, support = {NTF20030//Shantou University Research Initiation Fund Project/ ; 2023A1515011906//Guangdong Provincial Natural Science Foundation General Project/ ; QN2023021001L//Xiamen Municipal Bureau of Science and Technology-National Foreign Expert Program/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *beta 2-Microglobulin/genetics ; *Pluripotent Stem Cells/cytology/metabolism ; *Baculoviridae/genetics ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *Cell Differentiation/genetics ; Gene Knockout Techniques/methods ; Animals ; Fibroblasts/metabolism/cytology ; Human Embryonic Stem Cells/metabolism/cytology ; Mice ; }, abstract = {Derivation of hypoimmunogenic human cells from genetically manipulated pluripotent stem cells holds great promise for future transplantation medicine and adoptive immunotherapy. Disruption of beta-2-microglobulin (B2M) in pluripotent stem cells followed by differentiation into specialized cell types is a promising approach to derive hypoimmunogenic cells. Given the attractive features of CRISPR/Cas9-based gene editing tool and baculoviral delivery system, baculovirus can deliver CRISPR/Cas9 components for site-specific gene editing of B2M. Herein, we report the development of a baculoviral CRISPR/Cas9 vector system for the B2M locus disruption in human cells. When tested in human embryonic stem cells (hESCs), the B2M gene knockdown/out was successfully achieved, leading to the stable down-regulation of human leukocyte antigen class I expression on the cell surface. Fibroblasts derived from the B2M gene-disrupted hESCs were then used as stimulator cells in the co-cultures with human peripheral blood mononuclear cells. These fibroblasts triggered significantly reduced alloimmune responses as assessed by sensitive Elispot assays. The B2M-negative hESCs maintained the pluripotency and the ability to differentiate into three germ lineages in vitro and in vivo. These findings demonstrated the feasibility of using the baculoviral-CRISPR/Cas9 system to establish B2M-disrupted pluripotent stem cells. B2M knockdown/out sufficiently leads to hypoimmunogenic conditions, thereby supporting the potential use of B2M-negative cells as universal donor cells for allogeneic cell therapy.}, }
@article {pmid38987628, year = {2024}, author = {Villanueva, MT}, title = {A rush of CRISPR to the lungs.}, journal = {Nature reviews. Drug discovery}, volume = {23}, number = {8}, pages = {580}, pmid = {38987628}, issn = {1474-1784}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Lung/drug effects ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Lung Diseases/genetics ; }, }
@article {pmid39085660, year = {2024}, author = {Han, Y and Jia, Z and Xu, K and Li, Y and Lu, S and Guan, L}, title = {CRISPR-Cpf1 system and its applications in animal genome editing.}, journal = {Molecular genetics and genomics : MGG}, volume = {299}, number = {1}, pages = {75}, pmid = {39085660}, issn = {1617-4623}, support = {81801127//National Natural Science Foundation of China/ ; 242102310134//Key Technologies R&amp;D Program of Henan Province/ ; XYBSKYZZ201523//Doctoral Scientific Research Program Foundation of Xinxiang Medical University/ ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein (Cas) system is a gene editing technology guided by RNA endonuclease. The CRISPR-Cas12a (also known as CRISPR-Cpf1) system is extensively utilized in genome editing research due to its accuracy and high efficiency. In this paper, we primarily focus on the application of CRISPR-Cpf1 technology in the construction of disease models and gene therapy. Firstly, the structure and mechanism of the CRISPR-Cas system are introduced. Secondly, the similarities and differences between CRISPR-Cpf1 and CRISPR-Cas9 technologies are compared. Thirdly, the main focus is on the application of the CRISPR-Cpf1 system in cell and animal genome editing. Finally, the challenges faced by CRISPR-Cpf1 technology and corresponding strategies are analyzed. Although CRISPR-Cpf1 technology has certain off-target effects, it can effectively and accurately edit cell and animal genomes, and has significant advantages in the preclinical research.}, }
@article {pmid39083228, year = {2024}, author = {Mao, C and Zheng, H and Chen, Y and Yuan, P and Sun, D}, title = {Development of a Type I-E CRISPR-Based Programmable Repression System for Fine-Tuning Metabolic Flux toward D-Pantothenic Acid in Bacillus subtilis.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.4c00256}, pmid = {39083228}, issn = {2161-5063}, abstract = {The CRISPR-based regulation tools enable fine-tuning of gene transcription, showing potential in areas of biomanufacturing and live therapeutics. However, the cell toxicity and PAM specificity of existing CRISPR-based regulation systems limit their broad application. The development of new and less-toxic CRISPR-controlled expression systems remains highly desirable for expanding the application scope of CRISPR-based tools. Here, we reconstituted the type I CRISPR-Cas system from Escherichia coli to finely tune gene expression in Bacillus subtilis. Through engineering the 5' untranslated region (UTR) of mRNAs of cas genes, we remarkably improved the efficacy of the type I CRISPRi system. The improved type I CRISPRi system was applied in engineering the D-pantothenic acid (DPA)-producing B. subtilis, which was generated by strengthening the metabolic flux toward β-alanine and (R)-pantoate via enhancing expression of key enzymes at both transcriptional and translational levels. Through controlling the expression of pdhA with the CRISPRi system for fine-tuning the metabolic flux toward DPA and the TCA cycle, we elevated the DPA titer to 0.88 g/L in shake flasks and 12.81 g/L in fed-batch fermentations without the addition of the precursor β-alanine. The type I CRISPRi system and the strategy for fine-tuning metabolic flux reported here not only enrich the CRISPR toolbox in B. subtilis and facilitate DPA production through microbial fermentation but also provide a paradigm for programming important organisms to produce value-added chemicals with cheap raw materials.}, }
@article {pmid39083026, year = {2024}, author = {Kwon, T and Artiaga, BL and McDowell, CD and Whitworth, KM and Wells, KD and Prather, RS and Delhon, G and Cigan, M and White, SN and Retallick, J and Gaudreault, NN and Morozov, I and Richt, JA}, title = {Gene editing of pigs to control influenza A virus infections.}, journal = {Emerging microbes &amp; infections}, volume = {}, number = {}, pages = {2387449}, doi = {10.1080/22221751.2024.2387449}, pmid = {39083026}, issn = {2222-1751}, abstract = {Proteolytic activation of the hemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro. IAV infection in vivo resulted in significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to wild-type pigs. Our findings support the commercial use of GE pigs to mitigate influenza A virus infection in pigs, as an alternative approach to prevent zoonotic influenza A transmissions from pigs to human.}, }
@article {pmid39080645, year = {2024}, author = {Li, M and Li, S and Guo, Y and Hu, P and Shi, J}, title = {Magnetothermal-activated gene editing strategy for enhanced tumor cell apoptosis.}, journal = {Journal of nanobiotechnology}, volume = {22}, number = {1}, pages = {450}, pmid = {39080645}, issn = {1477-3155}, support = {2022YFB3804500//National Key Research and Development Program of China/ ; 23XD1424200//the Program of Shanghai Academic/Technology Research Leader/ ; 23DX1900202//the Biomedical Orientation Project of Shanghai Science and Technology Commission/ ; 22322509, 52072394, 22335006//the National Natural Science Foundation of China/ ; ZDBS-LY-SLH029//Key Research Program of Frontier Sciences, Chinese Academy of Sciences/ ; }, mesh = {*Gene Editing/methods ; *Apoptosis ; *CRISPR-Cas Systems ; Humans ; Cell Line, Tumor ; Animals ; Polyethyleneimine/chemistry ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Hyaluronic Acid/chemistry ; HSP70 Heat-Shock Proteins/genetics/metabolism ; Mice ; Neoplasms/therapy/genetics ; Plasmids/genetics ; Magnetite Nanoparticles/chemistry ; }, abstract = {Precise and effective initiation of the apoptotic mechanism in tumor cells is one of the most promising approaches for the treatment of solid tumors. However, current techniques such as high-temperature ablation or gene editing suffer from the risk of damage to adjacent normal tissues. This study proposes a magnetothermal-induced CRISPR-Cas9 gene editing system for the targeted knockout of HSP70 and BCL2 genes, thereby enhancing tumor cell apoptosis. The magnetothermal nanoparticulate platform is composed of superparamagnetic ZnCoFe2O4@ZnMnFe2O4 nanoparticles and the modified polyethyleneimine (PEI) and hyaluronic acid (HA) on the surface, on which plasmid DNA can be effectively loaded. Under the induction of a controllable alternating magnetic field, the mild magnetothermal effect (42℃) not only triggers dual-genome editing to disrupt the apoptosis resistance mechanism of tumor cells but also sensitizes tumor cells to apoptosis through the heat effect itself, achieving a synergistic therapeutic effect. This strategy can precisely regulate the activation of the CRISPR-Cas9 system for tumor cell apoptosis without inducing significant damage to healthy tissues, thus providing a new avenue for cancer treatment.}, }
@article {pmid39080539, year = {2024}, author = {van der Els, S and Boekhorst, J and Bron, PA and Kleerebezem, M}, title = {The lactococcal ICE-ome encodes a repertoire of exchangeable traits with potential industrial relevance.}, journal = {BMC genomics}, volume = {25}, number = {1}, pages = {734}, pmid = {39080539}, issn = {1471-2164}, mesh = {*Genome, Bacterial ; Lactococcus/genetics ; Interspersed Repetitive Sequences/genetics ; CRISPR-Cas Systems ; Conjugation, Genetic ; }, abstract = {Dairy industries apply selected lactococcal strains and mixed cultures to produce diverse fermented products with distinctive flavor and texture properties. Innovation of the starter culture functionality in cheese applications embraces natural biodiversity of the Lactococcus species to identify novel strains with alternative flavor or texture forming capacities and/or increased processing robustness and phage resistance. Mobile genetic elements (MGE), like integrative conjugative elements (ICEs) play an important role in shaping the biodiversity of bacteria. Besides the genes involved in the conjugation of ICEs from donor to recipient strains, these elements also harbor cargo genes that encode a wide range of functions. The definition of such cargo genes can only be achieved by accurate identification of the ICE boundaries (delimiting). Here, we delimited 25 ICEs in lactococcal genome sequences with low contig numbers using insertion-sites flanking single-copy core-genome genes as markers for each of the distinct ICE-integrases we identified previously within the conserved ICE-core genes. For ICEs in strains for which genome information with large numbers of contigs is available, we exemplify that CRISPR-Cas9 driven ICE-curing, followed by resequencing, allows accurate delimitation and cargo definition of ICEs. Finally, we compare and contrast the cargo gene repertoire of the 26 delimited lactococcal ICEs, identifying high plasticity among the cargo of lactococccal ICEs and a range of encoded functions that is of apparent industrial interest, including restriction modification, abortive infection, and stress adaptation genes.}, }
@article {pmid39080280, year = {2024}, author = {Machour, FE and R Abu-Zhayia, E and Kamar, J and Barisaac, AS and Simon, I and Ayoub, N}, title = {Harnessing DNA replication stress to target RBM10 deficiency in lung adenocarcinoma.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6417}, pmid = {39080280}, issn = {2041-1723}, support = {2511/19//Israel Science Foundation (ISF)/ ; 1283/21//Israel Science Foundation (ISF)/ ; 22-110-PG//Israel Cancer Research Fund (Israel Cancer Research Fund, Inc.)/ ; 2019688//United States - Israel Binational Science Foundation (BSF)/ ; 2021085//United States - Israel Binational Science Foundation (BSF)/ ; }, mesh = {Humans ; *DNA Replication/genetics ; *Adenocarcinoma of Lung/genetics/metabolism/pathology ; *Lung Neoplasms/genetics/metabolism/pathology ; *RNA-Binding Proteins/metabolism/genetics ; Animals ; Cell Line, Tumor ; Mice ; CRISPR-Cas Systems ; Histone Deacetylase 1/metabolism/genetics ; Cell Cycle Proteins/metabolism/genetics ; Protein-Tyrosine Kinases/metabolism/genetics ; R-Loop Structures ; }, abstract = {The splicing factor RNA-binding motif protein 10 (RBM10) is frequently mutated in lung adenocarcinoma (LUAD) (9-25%). Most RBM10 cancer mutations are loss-of-function, correlating with increased tumorigenesis and limiting the efficacy of current LUAD targeted therapies. Remarkably, therapeutic strategies leveraging RBM10 deficiency remain unexplored. Here, we conduct a CRISPR-Cas9 synthetic lethality (SL) screen and identify ~60 RBM10 SL genes, including WEE1 kinase. WEE1 inhibition sensitizes RBM10-deficient LUAD cells in-vitro and in-vivo. Mechanistically, we identify a splicing-independent role of RBM10 in regulating DNA replication fork progression and replication stress response, which underpins RBM10-WEE1 SL. Additionally, RBM10 interacts with active DNA replication forks, relying on DNA Primase Subunit 1 (PRIM1) that synthesizes Okazaki RNA primers. Functionally, we demonstrate that RBM10 serves as an anchor for recruiting Histone Deacetylase 1 (HDAC1) to facilitate H4K16 deacetylation and R-loop homeostasis to maintain replication fork stability. Collectively, our data reveal a role of RBM10 in fine-tuning DNA replication and provide therapeutic arsenal for targeting RBM10-deficient tumors.}, }
@article {pmid39080265, year = {2024}, author = {Yi, Z and Zhang, X and Wei, X and Li, J and Ren, J and Zhang, X and Zhang, Y and Tang, H and Chang, X and Yu, Y and Wei, W}, title = {Programmable DNA pyrimidine base editing via engineered uracil-DNA glycosylase.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6397}, pmid = {39080265}, issn = {2041-1723}, support = {NSFC31930016//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Uracil-DNA Glycosidase/metabolism/genetics ; *Gene Editing/methods ; Humans ; Protein Engineering/methods ; DNA/metabolism/genetics ; Thymine/metabolism ; Deinococcus/genetics/enzymology/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; Mutation ; HEK293 Cells ; CRISPR-Cas Systems ; }, abstract = {DNA base editing technologies predominantly utilize engineered deaminases, limiting their ability to edit thymine and guanine directly. In this study, we successfully achieve base editing of both cytidine and thymine by leveraging the translesion DNA synthesis pathway through the engineering of uracil-DNA glycosylase (UNG). Employing structure-based rational design, exploration of homologous proteins, and mutation screening, we identify a Deinococcus radiodurans UNG mutant capable of effectively editing thymine. When fused with the nickase Cas9, the engineered DrUNG protein facilitates efficient thymine base editing at endogenous sites, achieving editing efficiencies up to 55% without enrichment and exhibiting minimal cellular toxicity. This thymine base editor (TBE) exhibits high editing specificity and significantly restores IDUA enzyme activity in cells derived from patients with Hurler syndrome. TBEs represent efficient, specific, and low-toxicity approaches to base editing with potential applications in treating relevant diseases.}, }
@article {pmid39075256, year = {2024}, author = {Song, Y}, title = {RNA-guided recombination.}, journal = {Nature chemical biology}, volume = {20}, number = {8}, pages = {941}, doi = {10.1038/s41589-024-01697-z}, pmid = {39075256}, issn = {1552-4469}, mesh = {*Recombination, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; }, }
@article {pmid38739546, year = {2024}, author = {Zhang, Y and Sharma, D and Liang, Y and Downs, N and Dolman, F and Thorne, K and Black, IM and Pereira, JH and Adams, P and Scheller, HV and O'Neill, M and Urbanowicz, B and Mortimer, JC}, title = {Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing which bypasses embryo lethality.}, journal = {Plant physiology}, volume = {195}, number = {4}, pages = {2551-2565}, pmid = {38739546}, issn = {1532-2548}, support = {//DOE Joint BioEnergy Institute/ ; //U. S. Department of Energy, Office of Science/ ; DE-AC02-05CH11231//Office of Biological and Environmental Research/ ; //Lawrence Berkeley National Laboratory/ ; //University of Adelaide/ ; //Division of Chemical Sciences, Geosciences, and Biosciences/ ; DESC0008472//Office of Basic Energy Sciences of the United States Department of Energy/ ; }, mesh = {*Arabidopsis/genetics/metabolism ; *Pectins/metabolism ; *Gene Editing/methods ; *Arabidopsis Proteins/genetics/metabolism ; *Glycosyltransferases/genetics/metabolism ; Seeds/genetics/metabolism/growth &amp; development ; Cell Wall/metabolism/genetics ; CRISPR-Cas Systems ; Mutation/genetics ; }, abstract = {Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate cross-linking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect cross-linking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here, we developed a method to generate viable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in callus tissue via CRISPR/Cas9-mediated gene editing. We combined this with a candidate gene approach to characterize the male gametophyte defective 2 (MGP2) gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We showed that in the callus mutant cell walls, RG-II does not cross-link normally because it lacks 3-deoxy-D-manno-octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-D-kdop-(1→sidechain). We suggest that MGP2 encodes an inverting RG-II CMP-β-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization. Our method also provides a viable strategy for further identifying proteins involved in the biosynthesis of RG-II.}, }
@article {pmid38701041, year = {2024}, author = {Elliott, K and Veley, KM and Jensen, G and Gilbert, KB and Norton, J and Kambic, L and Yoder, M and Weil, A and Motomura-Wages, S and Bart, RS}, title = {CRISPR/Cas9-generated mutations in a sugar transporter gene reduce cassava susceptibility to bacterial blight.}, journal = {Plant physiology}, volume = {195}, number = {4}, pages = {2566-2578}, pmid = {38701041}, issn = {1532-2548}, support = {OPP1125410//Bill and Melinda Gates Foundation/ ; DGE-2139839//National Science Foundation/ ; //Donald Danforth Plant Science Center William H. Danforth Fellowship/ ; GM103757//Maximize Student Development Program/ ; }, mesh = {*Manihot/genetics/microbiology ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology/genetics ; *Xanthomonas/pathogenicity/physiology ; *Mutation/genetics ; Plant Proteins/genetics/metabolism ; }, abstract = {Bacteria from the genus Xanthomonas are prolific phytopathogens that elicit disease in over 400 plant species. Xanthomonads carry a repertoire of specialized proteins called transcription activator-like (TAL) effectors that promote disease and pathogen virulence by inducing the expression of host susceptibility (S) genes. Xanthomonas phaseoli pv. manihotis (Xpm) causes bacterial blight on the staple food crop cassava (Manihot esculenta Crantz). The Xpm effector TAL20 induces ectopic expression of the S gene Manihot esculenta Sugars Will Eventually be Exported Transporter 10a (MeSWEET10a), which encodes a sugar transporter that contributes to cassava bacterial blight (CBB) susceptibility. We used CRISPR/Cas9 to generate multiple cassava lines with edits to the MeSWEET10a TAL20 effector binding site and/or coding sequence. In several of the regenerated lines, MeSWEET10a expression was no longer induced by Xpm, and in these cases, we observed reduced CBB disease symptoms post Xpm infection. Because MeSWEET10a is expressed in cassava flowers, we further characterized the reproductive capability of the MeSWEET10a promoter and coding sequence mutants. Lines were crossed to themselves and to wild-type plants. The results indicated that expression of MeSWEET10a in female, but not male, flowers is critical to produce viable F1 seed. In the case of promoter mutations that left the coding sequence intact, viable F1 progeny were recovered. Taken together, these results demonstrate that blocking MeSWEET10a induction is a viable strategy for decreasing cassava susceptibility to CBB and that ideal lines will contain promoter mutations that block TAL effector binding while leaving endogenous expression of MeSWEET10a unaltered.}, }
@article {pmid39080269, year = {2024}, author = {Yao, M and Zeng, Z and Li, S and Zou, Z and Chen, Z and Chen, X and Gao, Q and Zhao, G and Chen, A and Li, Z and Wang, Y and Ning, R and McAlinden, C and Zhou, X and Huang, J}, title = {CRISPR-CasRx-mediated disruption of Aqp1/Adrb2/Rock1/Rock2 genes reduces intraocular pressure and retinal ganglion cell damage in mice.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6395}, pmid = {39080269}, issn = {2041-1723}, mesh = {Animals ; *Retinal Ganglion Cells/metabolism/pathology ; *rho-Associated Kinases/metabolism/genetics ; *Intraocular Pressure ; Female ; *Aquaporin 1/metabolism/genetics ; Mice ; *Glaucoma/genetics/metabolism ; *CRISPR-Cas Systems ; Aqueous Humor/metabolism ; Mice, Inbred C57BL ; Disease Models, Animal ; RNA Interference ; Humans ; }, abstract = {Glaucoma affects approximately 80 million individuals worldwide, a condition for which current treatment options are inadequate. The primary risk factor for glaucoma is elevated intraocular pressure. Intraocular pressure is determined by the balance between the secretion and outflow of aqueous humor. Here we show that using the RNA interference tool CasRx based on shH10 adenovirus-associated virus can reduce the expression of the aqueous humor circulation related genes Rock1 and Rock2, as well as aquaporin 1 and β2 adrenergic receptor in female mice. This significantly reduced intraocular pressure in female mice and provided protection to the retina ganglion cells, ultimately delaying disease progression. In addition, we elucidated the mechanisms by which the knockdown of Rock1 and Rock2, or aquaporin 1 and β2 adrenergic receptor in female mice, reduces the intraocular pressure and secures the retina ganglion cells by single-cell sequencing.}, }
@article {pmid39080117, year = {2024}, author = {Jain, D and Kalia, A and Sharma, S and Manchanda, P}, title = {Genome editing tools based improved applications in macrofungi.}, journal = {Molecular biology reports}, volume = {51}, number = {1}, pages = {873}, pmid = {39080117}, issn = {1573-4978}, support = {Prime Minister's Research Fellowship (Award no.: 0040/2021-22)//SERB-FICCI Primeminister Fellowship provided by Department of Science and Technology, New Delhi, India/ ; RKVY-18//Rashtriya Krishi Vikas yojna (RKVY), GoI, New Delhi, India/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Fungal ; *Agaricales/genetics ; }, abstract = {Macrofungi commonly referred to as Mushrooms are distributed worldwide and well known for their nutritional, medicinal, and organoleptic properties. Strain improvement in mushrooms is lagging due to paucity of efficient genome modification techniques. Thus, for advanced developments in research and commercial or economical viability and benefit, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) emerged as an efficient genome editing tool. The higher efficiency and precision of the desired genetic modification(s) are the most valuable attributes of this recent technology. The present review comprehensively summarizes various conventional methods utilized for strain improvement in mushrooms including hybridization, protoplast fusion, and di-mon mating. Furthermore, the problems associated with these techniques have been discussed besides providing the potential recluses. The significance of CRISPR/Cas9 strategies employed for improvement in various mushroom genera has been deliberated, as these strategies will paves the way forward for obtaining improved strain and effective cultivation methods for enhancing the yield and quality of the fruit bodies.}, }
@article {pmid39078677, year = {2024}, author = {Palanki, R and Riley, JS and Bose, SK and Luks, V and Dave, A and Kus, N and White, BM and Ricciardi, AS and Swingle, KL and Xue, L and Sung, D and Thatte, AS and Safford, HC and Chaluvadi, VS and Carpenter, M and Han, EL and Maganti, R and Hamilton, AG and Mrksich, K and Billingsley, MB and Zoltick, PW and Alameh, MG and Weissman, D and Mitchell, MJ and Peranteau, WH}, title = {In utero delivery of targeted ionizable lipid nanoparticles facilitates in vivo gene editing of hematopoietic stem cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {32}, pages = {e2400783121}, doi = {10.1073/pnas.2400783121}, pmid = {39078677}, issn = {1091-6490}, support = {F30HL162465-01A1//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; R01DK123049//HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/ ; DP2TR002776//HHS | NIH | National Center for Advancing Translational Sciences (NCATS)/ ; DP2HL152427//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; }, mesh = {Animals ; *Hematopoietic Stem Cells/metabolism ; *Gene Editing/methods ; *Nanoparticles/chemistry ; Mice ; Female ; Pregnancy ; Lipids/chemistry ; Leukocyte Common Antigens/metabolism/genetics ; Humans ; Genetic Therapy/methods ; CRISPR-Cas Systems ; Liposomes ; }, abstract = {Monogenic blood diseases are among the most common genetic disorders worldwide. These diseases result in significant pediatric and adult morbidity, and some can result in death prior to birth. Novel ex vivo hematopoietic stem cell (HSC) gene editing therapies hold tremendous promise to alter the therapeutic landscape but are not without potential limitations. In vivo gene editing therapies offer a potentially safer and more accessible treatment for these diseases but are hindered by a lack of delivery vectors targeting HSCs, which reside in the difficult-to-access bone marrow niche. Here, we propose that this biological barrier can be overcome by taking advantage of HSC residence in the easily accessible liver during fetal development. To facilitate the delivery of gene editing cargo to fetal HSCs, we developed an ionizable lipid nanoparticle (LNP) platform targeting the CD45 receptor on the surface of HSCs. After validating that targeted LNPs improved messenger ribonucleic acid (mRNA) delivery to hematopoietic lineage cells via a CD45-specific mechanism in vitro, we demonstrated that this platform mediated safe, potent, and long-term gene modulation of HSCs in vivo in multiple mouse models. We further optimized this LNP platform in vitro to encapsulate and deliver CRISPR-based nucleic acid cargos. Finally, we showed that optimized and targeted LNPs enhanced gene editing at a proof-of-concept locus in fetal HSCs after a single in utero intravenous injection. By targeting HSCs in vivo during fetal development, our Systematically optimized Targeted Editing Machinery (STEM) LNPs may provide a translatable strategy to treat monogenic blood diseases before birth.}, }
@article {pmid39078500, year = {2024}, author = {Choudry, MW and Riaz, R and Nawaz, P and Ashraf, M and Ijaz, B and Bakhsh, A}, title = {CRISPR-Cas9 mediated understanding of plants' abiotic stress-responsive genes to combat changing climatic patterns.}, journal = {Functional &amp; integrative genomics}, volume = {24}, number = {4}, pages = {132}, pmid = {39078500}, issn = {1438-7948}, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Stress, Physiological/genetics ; Crops, Agricultural/genetics ; Droughts ; Climate Change ; Gene Expression Regulation, Plant ; }, abstract = {Multiple abiotic stresses like extreme temperatures, water shortage, flooding, salinity, and exposure to heavy metals are confronted by crop plants with changing climatic patterns. Prolonged exposure to these adverse environmental conditions leads to stunted plant growth and development with significant yield loss in crops. CRISPR-Cas9 genome editing tool is being frequently employed to understand abiotic stress-responsive genes. Noteworthy improvements in CRISPR-Cas technology have been made over the years, including upgradation of Cas proteins fidelity and efficiency, optimization of transformation protocols for different crop species, base and prime editing, multiplex gene-targeting, transgene-free editing, and graft-based heritable CRISPR-Cas9 approaches. These developments helped to improve the knowledge of abiotic stress tolerance in crops that could potentially be utilized to develop knock-out varieties and over-expressed lines to tackle the adverse effects of altered climatic patterns. This review summarizes the mechanistic understanding of heat, drought, salinity, and metal stress-responsive genes characterized so far using CRISPR-Cas9 and provides data on potential candidate genes that can be exploited by modern-day biotechnological tools to develop transgene-free genome-edited crops with better climate adaptability. Furthermore, the importance of early-maturing crop varieties to withstand abiotic stresses is also discussed in this review.}, }
@article {pmid39077930, year = {2024}, author = {Assa, G and Kalter, N and Rosenberg, M and Beck, A and Markovich, O and Gontmakher, T and Hendel, A and Yakhini, Z}, title = {Quantifying allele-specific CRISPR editing activity with CRISPECTOR2.0.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae651}, pmid = {39077930}, issn = {1362-4962}, support = {//Israel Innovation Authority/ ; //CRISPR-IL/ ; //Yakhini research group/ ; }, abstract = {Off-target effects present a significant impediment to the safe and efficient use of CRISPR-Cas genome editing. Since off-target activity is influenced by the genomic sequence, the presence of sequence variants leads to varying on- and off-target profiles among different alleles or individuals. However, a reliable tool that quantifies genome editing activity in an allelic context is not available. Here, we introduce CRISPECTOR2.0, an extended version of our previously published software tool CRISPECTOR, with an allele-specific editing activity quantification option. CRISPECTOR2.0 enables reference-free, allele-aware, precise quantification of on- and off-target activity, by using de novo sample-specific single nucleotide variant (SNV) detection and statistical-based allele-calling algorithms. We demonstrate CRISPECTOR2.0 efficacy in analyzing samples containing multiple alleles and quantifying allele-specific editing activity, using data from diverse cell types, including primary human cells, plants, and an original extensive human cell line database. We identified instances where an SNV induced changes in the protospacer adjacent motif sequence, resulting in allele-specific editing. Intriguingly, differential allelic editing was also observed in regions carrying distal SNVs, hinting at the involvement of additional epigenetic factors. Our findings highlight the importance of allele-specific editing measurement as a milestone in the adaptation of efficient, accurate, and safe personalized genome editing.}, }
@article {pmid39076074, year = {2024}, author = {Zhang, Y and Wei, J and Wang, H and Wang, Y}, title = {Characterization of NiCas12b for In Vivo Genome Editing.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {}, number = {}, pages = {e2400469}, doi = {10.1002/advs.202400469}, pmid = {39076074}, issn = {2198-3844}, support = {2021YFC2701103//National Key Research and Development Program of China/ ; 2021YFA0910602//National Key Research and Development Program of China/ ; 82070258//National Natural Science Foundation of China/ ; 81870199//National Natural Science Foundation of China/ ; 19DZ2282100//Science and Technology Research Program of Shanghai/ ; }, abstract = {The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system represents the third family of CRISPR-Cas systems that are harnessed for genome editing. However, only a few nucleases have demonstrated activity in human cells, and their in vivo therapeutic potential remains uncertain. In this study, a green fluorescent protein (GFP)-activation assay is conducted to screen a panel of 15 Cas12b orthologs, and four of them exhibited editing activity in mammalian cells. Particularly noteworthy is the NiCas12b derived from Nitrospira sp., which recognizes a "TTN" protospacer adjacent motif (PAM) and facilitates efficient genome editing in various cell lines. Importantly, NiCas12b also exhibits a high degree of specificity, rendering it suitable for therapeutic applications. As proof of concept, the adeno-associated virus (AAV) is employed to introduce NiCas12b to target the cholesterol regulatory gene proprotein convertase subtilisin/ kexin type 9 (Pcsk9) in the mouse liver. After 4 weeks of injections, an impressive is observed over 16.0% insertion/deletion (indel) efficiency, resulting in a significant reduction in serum cholesterol levels. NiCas12b provides a novel option for both basic research and clinical applications.}, }
@article {pmid39076066, year = {2024}, author = {Hosseini, K and Fallahi, J and Aligholi, H and Heidari, Z and Nadimi, E and Safari, F and Sisakht, M and Atapour, A and Khajeh, S and Tabei, SMB and Razban, V}, title = {Creation of an in vitro model of GM1 gangliosidosis by CRISPR/Cas9 knocking-out the GLB1 gene in SH-SY5Y human neuronal cell line.}, journal = {Cell biochemistry and function}, volume = {42}, number = {6}, pages = {e4102}, doi = {10.1002/cbf.4102}, pmid = {39076066}, issn = {1099-0844}, support = {21891//Shiraz University of Medical Sciences/ ; }, mesh = {Humans ; *Gangliosidosis, GM1/genetics/metabolism ; *CRISPR-Cas Systems ; beta-Galactosidase/metabolism/genetics ; Neurons/metabolism ; Gene Knockout Techniques ; Models, Biological ; }, abstract = {GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of β-galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH-SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock-out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP-CHOP websites, and high-efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH-SY5Y cell line) by electroporation. After 48 h, GFP[+] cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X-gal and Periodic acid-Schiff (PAS) staining, and qPCR were used to confirm the knock-out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock-out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH-SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non-transfected cell, X-gal staining resulted in no blue color in GLB1 gene knock-out cells indicating the absence of β-galactosidase enzyme activity in these cells. Real-time PCR (qPCR) results confirmed the RNA-Seq analysis outcomes on the GEO data set and following the GLB1 gene knock-out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1-NEU1-CTSA complex gene was involved in suppressed proliferation and invasion ability of knock-out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH-SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease.}, }
@article {pmid39024394, year = {2024}, author = {Shen, D and Zhang, G and Weng, X and Liu, R and Liu, Z and Sheng, X and Zhang, Y and Liu, Y and Mu, Y and Zhu, Y and Sun, E and Zhang, J and Li, F and Xia, C and Ge, J and Liu, Z and Bu, Z and Zhao, D}, title = {A genome-wide CRISPR/Cas9 knockout screen identifies TMEM239 as an important host factor in facilitating African swine fever virus entry into early endosomes.}, journal = {PLoS pathogens}, volume = {20}, number = {7}, pages = {e1012256}, doi = {10.1371/journal.ppat.1012256}, pmid = {39024394}, issn = {1553-7374}, mesh = {Animals ; Swine ; *African Swine Fever Virus/genetics/physiology ; *Virus Internalization ; *CRISPR-Cas Systems ; *African Swine Fever/virology/metabolism/genetics ; *Endosomes/metabolism/virology ; *Virus Replication ; *Membrane Proteins/genetics/metabolism ; Gene Knockout Techniques ; }, abstract = {African swine fever (ASF) is a highly contagious, fatal disease of pigs caused by African swine fever virus (ASFV). The complexity of ASFV and our limited understanding of its interactions with the host have constrained the development of ASFV vaccines and antiviral strategies. To identify host factors required for ASFV replication, we developed a genome-wide CRISPR knockout (GeCKO) screen that contains 186,510 specific single guide RNAs (sgRNAs) targeting 20,580 pig genes and used genotype II ASFV to perform the GeCKO screen in wild boar lung (WSL) cells. We found that knockout of transmembrane protein 239 (TMEM239) significantly reduced ASFV replication. Further studies showed that TMEM239 interacted with the early endosomal marker Rab5A, and that TMEM239 deletion affected the co-localization of viral capsid p72 and Rab5A shortly after viral infection. An ex vivo study showed that ASFV replication was significantly reduced in TMEM239-/- peripheral blood mononuclear cells from TMEM239 knockout piglets. Our study identifies a novel host factor required for ASFV replication by facilitating ASFV entry into early endosomes and provides insights for the development of ASF-resistant breeding.}, }
@article {pmid38940507, year = {2024}, author = {Ramírez-Zavala, B and Hoffmann, A and Krüger, I and Schwanfelder, S and Barker, KS and Rogers, PD and Morschhäuser, J}, title = {Probing gene function in Candida albicans wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.}, journal = {mSphere}, volume = {9}, number = {7}, pages = {e0038824}, doi = {10.1128/msphere.00388-24}, pmid = {38940507}, issn = {2379-5042}, support = {210879364//Deutsche Forschungsgemeinschaft (DFG)/ ; R01 AI058145/AI/NIAID NIH HHS/United States ; }, mesh = {*Candida albicans/genetics/drug effects ; *CRISPR-Cas Systems ; *Loss of Heterozygosity ; *Recombination, Genetic ; Gene Deletion ; Drug Resistance, Fungal/genetics ; Antifungal Agents/pharmacology ; Fluconazole/pharmacology ; Hygromycin B/pharmacology ; CRISPR-Associated Protein 9/genetics ; Gene Editing/methods ; Streptothricins/pharmacology ; Genetic Markers ; }, abstract = {UNLABELLED: The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast Candida albicans, because homozygous mutants of this diploid fungus can now be generated in a single step, allowing the rapid screening of candidate genes for their involvement in a phenotype of interest. However, the Cas9-mediated double-strand breaks at the target site may result in an undesired loss of heterozygosity (LOH) on the affected chromosome and cause phenotypic alterations that are not related to the function of the investigated gene. In our present study, we harnessed Cas9-facilitated gene deletion to probe a set of genes that are constitutively overexpressed in strains containing hyperactive forms of the transcription factor Mrr1 for a possible contribution to the fluconazole resistance of such strains. To this aim, we used gene deletion cassettes containing two different dominant selection markers, caSAT1 and HygB, which confer resistance to nourseothricin and hygromycin, respectively, for simultaneous genomic integration in a single step, hypothesizing that this would minimize undesired LOH events at the target locus. We found that selection for resistance to both nourseothricin and hygromycin strongly increased the proportion of homozygous deletion mutants among the transformants compared with selection on media containing only one of the antibiotics, but it did not avoid undesired LOH events. Our results demonstrate that LOH on the target chromosome is a significant problem when using Cas9 for the generation of C. albicans gene deletion mutants, which demands a thorough examination of recombination events at the target site.<br><br>IMPORTANCE: Candida albicans is one of the medically most important fungi and a model organism to study fungal pathogenicity. Investigating gene function in this diploid yeast has been facilitated by the adaptation of gene deletion methods based on the bacterial CRISPR-Cas9 system, because they enable the generation of homozygous mutants in a single step. We found that, in addition to increasing the efficiency of gene replacement by selection markers, the Cas9-mediated double-strand breaks also result in frequent loss of heterozygosity on the same chromosome, even when two different selection markers were independently integrated into the two alleles of the target gene. Since loss of heterozygosity for other genes can result in phenotypic alterations that are not caused by the absence of the target gene, these findings show that it is important to thoroughly analyze recombination events at the target locus when using Cas9 to generate gene deletion mutants in C. albicans.}, }
@article {pmid38936584, year = {2024}, author = {Li, L and Li, M and Qiu, Y and Dong, Y}, title = {A sensitive CRISPR/Cas12a-assisted fluorescent aptasensor for rapid detection of food allergens.}, journal = {International journal of biological macromolecules}, volume = {274}, number = {Pt 2}, pages = {133444}, doi = {10.1016/j.ijbiomac.2024.133444}, pmid = {38936584}, issn = {1879-0003}, mesh = {*Allergens/analysis ; *Aptamers, Nucleotide/chemistry ; Bacterial Proteins/chemistry ; *Biosensing Techniques/methods ; CRISPR-Associated Proteins/chemistry ; *CRISPR-Cas Systems ; Endodeoxyribonucleases/chemistry ; Fluorescent Dyes/chemistry ; Limit of Detection ; Muramidase/chemistry ; }, abstract = {Food allergens elicit abnormal immune system responses among allergic individuals and sensitive detection for allergenic ingredient is greatly significant. To address this need, a novel fluorescent aptasensor, assisted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), have been developed for food allergens. In this study, aptamer offers distinctive recognition capabilities in binding specific targets, while CRISPR-associated-12a protein (Cas12a) holds precise cis-cleavage for cutting fluorescent signal probes. Notably, the utilization of Cas12a cis-cleavage activity, rather than trans-cleavage, eliminates the necessity for additional fluorescent probes, thus reducing interference between substances and enhancing sensitivity. Throughout the process, complementary DNA (cDNA) plays a crucial dual role in target recognition conversion and signal presentation, representing a key challenge and innovative aspect of this study. To evaluate the performance of the aptasensor, lysozyme (LYS) is employed as a representative model target of food allergens. Under optimal conditions, the developed aptasensor could achieve an exceptional low limit of detection (LOD) of 6.10 pM with a dynamic detection range of 10 pM-320 pM. The aptasensor demonstrates high selectivity and great recovery rates. This strategy yields promising outcomes, holding the potential to serve as a valuable reference for various food allergens detection.}, }
@article {pmid38925172, year = {2024}, author = {Jiang, W and Guo, K and Dong, H and Zhang, X and Guo, Z and Duan, J and Jing, X and Xia, Q and Zhao, P}, title = {Mutation in the Bombyx mori BmGMC2 gene impacts silk production and silk protein synthesis.}, journal = {International journal of biological macromolecules}, volume = {274}, number = {Pt 2}, pages = {133400}, doi = {10.1016/j.ijbiomac.2024.133400}, pmid = {38925172}, issn = {1879-0003}, mesh = {*Bombyx/genetics/metabolism ; Animals ; *Silk/genetics/biosynthesis/metabolism ; *Insect Proteins/genetics/metabolism ; *Mutation ; Larva/genetics/metabolism ; Fibroins/genetics/metabolism ; Proteomics/methods ; Protein Biosynthesis ; CRISPR-Cas Systems ; 3-Hydroxysteroid Dehydrogenases ; }, abstract = {Silk is a natural protein fiber that is predominantly comprised of fibroin and sericin. In addition, it contains seroins, protease inhibitors, enzymes, and other proteins. We found an ecdysone oxidase BmGMC2, notably, which is specifically and highly expressed only in the silk glands of silkworms (Bombyx mori L.). It is also one of the main components of non-cocoon silk, however, its precise function remains unclear. In this study, we examined the spatiotemporal expression pattern of this protein and obtained a homozygous mutant strain (K-GMC2) using the CRISPR-Cas9 system. Compared to the wild-type strain (WT), the silk production and main silk proteins significantly decreased in the larval stage, and the adhesive strength of native silk proteins decreased in the final instar. Proteomic data indicated the abundance of ribosomal proteins decreased significantly in K-GMC2, differentially expressed proteins (DEPs) were enriched in pathways related to neurodegenerative diseases and genetic information processing, indicating that knockout may lead to a certain degree of cell stress, affecting the synthesis of silk proteins. This study investigated the expression pattern and gene function of ecdysone oxidase BmGMC2 in silk and silk glands, laying the groundwork for understanding the role of enzymes in the production of silk fibers.}, }
@article {pmid38804043, year = {2024}, author = {Owens, LA and Thurber, MI and Goldberg, TL}, title = {CRISPR-Cas9-mediated host signal reduction for 18S metabarcoding of host-associated eukaryotes.}, journal = {Molecular ecology resources}, volume = {24}, number = {6}, pages = {e13980}, doi = {10.1111/1755-0998.13980}, pmid = {38804043}, issn = {1755-0998}, support = {1R01AG049395-01/HI/NHLBI NIH HHS/United States ; 1R21AI163592-01/HI/NHLBI NIH HHS/United States ; T32AI007414/HI/NHLBI NIH HHS/United States ; 1R01AG049395-01/HI/NHLBI NIH HHS/United States ; 1R21AI163592-01/HI/NHLBI NIH HHS/United States ; T32AI007414/HI/NHLBI NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; *Eukaryota/genetics ; *DNA Barcoding, Taxonomic/methods ; *RNA, Ribosomal, 18S/genetics ; Metagenomics/methods ; Humans ; Animals ; }, abstract = {Metabarcoding-based methods for identification of host-associated eukaryotes have the potential to revolutionize parasitology and microbial ecology, yet significant technical challenges remain. In particular, highly abundant host reads can mask the presence of less-abundant target organisms, especially for sample types rich in host DNA (e.g., blood and tissues). Here, we present a new CRISPR-Cas9-mediated approach designed to reduce host signal by selective amplicon digestion, thus enriching clinical samples for eukaryotic endosymbiont sequences during metabarcoding. Our method achieves a nearly 76% increased efficiency in host signal reduction compared with no treatment and a nearly 60% increased efficiency in host signal reduction compared with the most commonly used published method. Furthermore, the application of our method to clinical samples allows for the detection of parasite infections that would otherwise have been missed.}, }
@article {pmid29472270, year = {2018}, author = {Kim, S and Koo, T and Jee, HG and Cho, HY and Lee, G and Lim, DG and Shin, HS and Kim, JS}, title = {CRISPR RNAs trigger innate immune responses in human cells.}, journal = {Genome research}, volume = {28}, number = {3}, pages = {367-373}, pmid = {29472270}, issn = {1549-5469}, mesh = {Humans ; *Immunity, Innate ; *RNA, Guide, CRISPR-Cas Systems ; Animals ; *CRISPR-Cas Systems ; Mice ; Interferon Type I/metabolism/immunology ; DEAD Box Protein 58/metabolism/genetics/immunology ; CD4-Positive T-Lymphocytes/immunology/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; HEK293 Cells ; Gene Editing/methods ; }, abstract = {Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5'-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4[+] T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5'-hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.}, }
@article {pmid39075609, year = {2024}, author = {Molaei, Z and Jabbarpour, Z and Omidkhoda, A and Ahmadbeigi, N}, title = {Exploring non-viral methods for the delivery of CRISPR-Cas ribonucleoprotein to hematopoietic stem cells.}, journal = {Stem cell research &amp; therapy}, volume = {15}, number = {1}, pages = {233}, pmid = {39075609}, issn = {1757-6512}, abstract = {Gene manipulation of hematopoietic stem cells (HSCs) using the CRISPR/Cas system as a potent genome editing tool holds immense promise for addressing hematologic disorders. An essential hurdle in advancing this treatment lies in effectively delivering CRISPR/Cas to HSCs. While various delivery formats exist, Ribonucleoprotein complex (RNP) emerges as a particularly efficient option. RNP complexes offer enhanced gene editing capabilities, devoid of viral vectors, with rapid activity and minimized off-target effects. Nevertheless, novel delivery methods such as microfluidic-based techniques, filtroporation, nanoparticles, and cell-penetrating peptides are continually evolving. This study aims to provide a comprehensive review of these methods and the recent research on delivery approaches of RNP complexes to HSCs.}, }
@article {pmid39075259, year = {2024}, author = {Bayat, M and Nahand, JS}, title = {Let's make it personal: CRISPR tools in manipulating cell death pathways for cancer treatment.}, journal = {Cell biology and toxicology}, volume = {40}, number = {1}, pages = {61}, pmid = {39075259}, issn = {1573-6822}, mesh = {Humans ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Precision Medicine/methods ; *Cell Death/genetics ; Signal Transduction/genetics ; Gene Editing/methods ; Apoptosis/genetics ; Mutation/genetics ; Animals ; }, abstract = {Advancements in the CRISPR technology, a game-changer in experimental research, have revolutionized various fields of life sciences and more profoundly, cancer research. Cell death pathways are among the most deregulated in cancer cells and are considered as critical aspects in cancer development. Through decades, our knowledge of the mechanisms orchestrating programmed cellular death has increased substantially, attributed to the revolution of cutting-edge technologies. The heroic appearance of CRISPR systems have expanded the available screening platform and genome engineering toolbox to detect mutations and create precise genome edits. In that context, the precise ability of this system for identification and targeting of mutations in cell death signaling pathways that result in cancer development and therapy resistance is an auspicious choice to transform and accelerate the individualized cancer therapy. The concept of personalized cancer therapy stands on the identification of molecular characterization of the individual tumor and its microenvironment in order to provide a precise treatment with the highest possible outcome and minimum toxicity. This study explored the potential of CRISPR technology in precision cancer treatment by identifying and targeting specific cell death pathways. It showed the promise of CRISPR in finding key components and mutations involved in programmed cell death, making it a potential tool for targeted cancer therapy. However, this study also highlighted the challenges and limitations that need to be addressed in future research to fully realize the potential of CRISPR in cancer treatment.}, }
@article {pmid39075253, year = {2024}, author = {Shu, X and Wang, R and Li, Z and Xue, Q and Wang, J and Liu, J and Cheng, F and Liu, C and Zhao, H and Hu, C and Li, J and Ouyang, S and Li, M}, title = {CRISPR-repressed toxin-antitoxin provides herd immunity against anti-CRISPR elements.}, journal = {Nature chemical biology}, volume = {}, number = {}, pages = {}, pmid = {39075253}, issn = {1552-4469}, abstract = {Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are highly vulnerable to phage-encoded anti-CRISPR (Acr) factors. How CRISPR-Cas systems protect themselves remains unclear. Here we uncovered a broad-spectrum anti-anti-CRISPR strategy involving a phage-derived toxic protein. Transcription of this toxin is normally repressed by the CRISPR-Cas effector but is activated to halt cell division when the effector is inhibited by any anti-CRISPR proteins or RNAs. We showed that this abortive infection-like effect efficiently expels Acr elements from bacterial population. Furthermore, we exploited this anti-anti-CRISPR mechanism to develop a screening method for specific Acr candidates for a CRISPR-Cas system and successfully identified two distinct Acr proteins that enhance the binding of CRISPR effector to nontarget DNA. Our data highlight the broad-spectrum role of CRISPR-repressed toxins in counteracting various types of Acr factors. We propose that the regulatory function of CRISPR-Cas confers host cells herd immunity against Acr-encoding genetic invaders whether they are CRISPR targeted or not.}, }
@article {pmid39073063, year = {2024}, author = {Tung, J and Huang, L and George, G and Harding, HP and Ron, D and Ordonez, A}, title = {A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, doi = {10.7554/eLife.96979}, pmid = {39073063}, issn = {2050-084X}, support = {10.35802/224407/WT_/Wellcome Trust/United Kingdom ; }, mesh = {*Activating Transcription Factor 6/metabolism/genetics ; *Calreticulin/metabolism/genetics ; Animals ; *CRISPR-Cas Systems ; *Cricetulus ; CHO Cells ; Humans ; Unfolded Protein Response ; Endoplasmic Reticulum Stress/genetics ; Protein Serine-Threonine Kinases/metabolism/genetics ; Signal Transduction ; }, abstract = {Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6α activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6α signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6α-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6α. Purified CRT interacted with the luminal domain of ATF6α in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6α in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6α to selectively regulate one arm of the UPR.}, }
@article {pmid39073026, year = {2024}, author = {Chen, Q and Pang, M and Chen, P and Zhou, Z and Lei, J and He, B and Sun, Z and Paek, C and Jing, B and Wu, Y and Liu, S and Chen, Y and Yin, L}, title = {High-fidelity CRISPR/Cas12a dual-crRNA screening reveals novel synergistic interactions in hepatocellular carcinoma.}, journal = {Clinical and translational medicine}, volume = {14}, number = {7}, pages = {e1758}, doi = {10.1002/ctm2.1758}, pmid = {39073026}, issn = {2001-1326}, support = {2022YFA1303500//National Key R&amp;D Program of China/ ; 32171210//National Natural Science Foundation of China/ ; 32101196//National Natural Science Foundation of China/ ; 32371271//National Natural Science Foundation of China/ ; 2021TQ0253//China Postdoctoral Science Foundation/ ; 2022M712468//China Postdoctoral Science Foundation/ ; 2042022kf1189//Fundamental Research Funds for the Central Universities/ ; 2022YFA1303500//National Key Research and Development Program of China/ ; }, mesh = {*Carcinoma, Hepatocellular/genetics ; *Liver Neoplasms/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Genetic Testing/methods ; }, abstract = {: CRISPR/Cas12a-based combinational screening has shown remarkable potential for identifying genetic interactions. Here, we describe an innovative method for combinational genetic screening with rapid construction of a dual-CRISPR RNA (crRNA) library using gene splicing through overlap extension PCR (SOE PCR) and the adoption of CeCas12a, which we previously identified with strict PAM recognition and low off-targeting to guarantee fidelity and efficiency. The custom-pooled SOE crRNA array (SOCA) library for double-knockout screening could be conveniently constructed in the laboratory for widespread use, and the CeCas12a-mediated high-fidelity screen displayed good performance even under a negative selection screen. By designing a SOCA dual-crRNA library that covered most of the kinase and metabolism-associated gene targets of FDA-approved drugs implicated in hepatocellular carcinoma (HCC) tumourigenesis, novel cross-talk between the two gene sets was negatively selected to inhibit HCC cell growth in vitro and in vivo and was validated using virtual double-knockdown screening based on TCGA databases. Thus, this rapid, efficient and high-fidelity double-knockout screening system is promising for systemically identifying potential genetic interactions between multiple gene sets or combinations of FDA- approved drugs for clinical translational medicine in the future.}, }
@article {pmid39071309, year = {2024}, author = {Yousefian-Jazi, A and Kim, S and Choi, SH and Chu, J and Nguyen, PT and Park, U and Lim, K and Hwang, H and Lee, K and Kim, Y and Hyeon, SJ and Rhim, H and Ryu, HL and Lim, G and Stein, TD and Ryu, H and Lee, J}, title = {Loss of MEF2C function by enhancer mutation leads to neuronal mitochondria dysfunction and motor deficits in mice.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.07.15.603186}, pmid = {39071309}, issn = {2692-8205}, abstract = {Genetic changes and epigenetic modifications are associated with neuronal dysfunction in the pathogenesis of neurodegenerative disorders. However, the mechanism behind genetic mutations in the non-coding region of genes that affect epigenetic modifications remains unclear. Here, we identified an ALS-associated SNP located in the intronic region of MEF2C (rs304152), residing in a putative enhancer element, using convolutional neural network. The enhancer mutation of MEF2C reduces own gene expression and consequently impairs mitochondrial function in motor neurons. MEF2C localizes and binds to the mitochondria DNA, and directly modulates mitochondria-encoded gene expression. CRISPR/Cas-9-induced mutation of the MEF2C enhancer decreases expression of mitochondria-encoded genes. Moreover, MEF2C mutant cells show reduction of mitochondrial membrane potential, ATP level but elevation of oxidative stress. MEF2C deficiency in the upper and lower motor neurons of mice impairs mitochondria-encoded genes, and leads to mitochondrial metabolic disruption and progressive motor behavioral deficits. Together, MEF2C dysregulation by the enhancer mutation leads to mitochondrial dysfunction and oxidative stress, which are prevalent features in motor neuronal damage and ALS pathogenesis. This genetic and epigenetic crosstalk mechanism provides insights for advancing our understanding of motor neuron disease and developing effective treatments.}, }
@article {pmid39003740, year = {2024}, author = {Busley, AV and Gutiérrez-Gutiérrez, &#xd3; and Hammer, E and Koitka, F and Mirzaiebadizi, A and Steinegger, M and Pape, C and Böhmer, L and Schroeder, H and Kleinsorge, M and Engler, M and Cirstea, IC and Gremer, L and Willbold, D and Altmüller, J and Marbach, F and Hasenfuss, G and Zimmermann, WH and Ahmadian, MR and Wollnik, B and Cyganek, L}, title = {Mutation-induced LZTR1 polymerization provokes cardiac pathology in recessive Noonan syndrome.}, journal = {Cell reports}, volume = {43}, number = {7}, pages = {114448}, doi = {10.1016/j.celrep.2024.114448}, pmid = {39003740}, issn = {2211-1247}, mesh = {Humans ; *Noonan Syndrome/genetics/pathology/metabolism ; *Myocytes, Cardiac/metabolism/pathology ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *ras Proteins/metabolism/genetics ; Transcription Factors/metabolism/genetics ; Mutation/genetics ; Cardiomyopathy, Hypertrophic/genetics/pathology/metabolism ; Polymerization ; CRISPR-Cas Systems/genetics ; Proteolysis ; Mutation, Missense ; Protein Multimerization ; Genes, Recessive ; Phenotype ; }, abstract = {Noonan syndrome patients harboring causative variants in LZTR1 are particularly at risk to develop severe and early-onset hypertrophic cardiomyopathy. In this study, we investigate the mechanistic consequences of a homozygous variant LZTR1[L580P] by using patient-specific and CRISPR-Cas9-corrected induced pluripotent stem cell (iPSC) cardiomyocytes. Molecular, cellular, and functional phenotyping in combination with in silico prediction identify an LZTR1[L580P]-specific disease mechanism provoking cardiac hypertrophy. The variant is predicted to alter the binding affinity of the dimerization domains facilitating the formation of linear LZTR1 polymers. LZTR1 complex dysfunction results in the accumulation of RAS GTPases, thereby provoking global pathological changes of the proteomic landscape ultimately leading to cellular hypertrophy. Furthermore, our data show that cardiomyocyte-specific MRAS degradation is mediated by LZTR1 via non-proteasomal pathways, whereas RIT1 degradation is mediated by both LZTR1-dependent and LZTR1-independent pathways. Uni- or biallelic genetic correction of the LZTR1[L580P] missense variant rescues the molecular and cellular disease phenotype, providing proof of concept for CRISPR-based therapies.}, }
@article {pmid38954773, year = {2024}, author = {Goto, Y and Koshizuka, K and Ando, T and Izumi, H and Wu, X and Sato, K and Ishikawa, T and Ford, K and Feng, X and Wang, Z and Arang, N and Allevato, MM and Kishore, A and Mali, P and Gutkind, JS}, title = {A Kinome-Wide Synthetic Lethal CRISPR/Cas9 Screen Reveals That mTOR Inhibition Prevents Adaptive Resistance to CDK4/CDK6 Blockade in HNSCC.}, journal = {Cancer research communications}, volume = {4}, number = {7}, pages = {1850-1862}, doi = {10.1158/2767-9764.CRC-24-0247}, pmid = {38954773}, issn = {2767-9764}, mesh = {Humans ; *Cyclin-Dependent Kinase 4/antagonists &amp; inhibitors/genetics/metabolism ; *Cyclin-Dependent Kinase 6/antagonists &amp; inhibitors/genetics/metabolism ; *CRISPR-Cas Systems ; *Squamous Cell Carcinoma of Head and Neck/genetics/drug therapy/metabolism/pathology ; *Piperazines/pharmacology/therapeutic use ; *Pyridines/pharmacology ; Mice ; Animals ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Head and Neck Neoplasms/genetics/drug therapy/pathology/metabolism ; Cell Line, Tumor ; MTOR Inhibitors/pharmacology/therapeutic use ; Protein Kinase Inhibitors/pharmacology/therapeutic use ; TOR Serine-Threonine Kinases/metabolism ; Cyclin E/genetics/metabolism ; Xenograft Model Antitumor Assays ; Synthetic Lethal Mutations ; Oncogene Proteins ; }, abstract = {UNLABELLED: The comprehensive genomic analysis of the head and neck squamous cell carcinoma (HNSCC) oncogenome revealed the frequent loss of p16INK4A (CDKN2A) and amplification of cyclin D1 genes in most human papillomavirus-negative HNSCC lesions. However, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have shown modest effects in the clinic. The aberrant activation of the PI3K/mTOR pathway is highly prevalent in HNSCC, and recent clinical trials have shown promising clinical efficacy of mTOR inhibitors (mTORi) in the neoadjuvant and adjuvant settings but not in patients with advanced HNSCC. By implementing a kinome-wide CRISPR/Cas9 screen, we identified cell-cycle inhibition as a synthetic lethal target for mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergism in HNSCC-derived cells in vitro and in vivo. Remarkably, we found that an adaptive increase in cyclin E1 (CCNE1) expression upon palbociclib treatment underlies the rapid acquired resistance to this CDK4/6 inhibitor. Mechanistically, mTORi inhibits the formation of eIF4G-CCNE1 mRNA complexes, with the consequent reduction in mRNA translation and CCNE1 protein expression. Our findings suggest that mTORi reverts the adaptive resistance to palbociclib. This provides a multimodal therapeutic option for HNSCC by cotargeting mTOR and CDK4/6, which in turn may halt the emergence of palbociclib resistance.<br><br>SIGNIFICANCE: A kinome-wide CRISPR/Cas9 screen identified cell-cycle inhibition as a synthetic lethal target of mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergistic effects in HNSCC. Mechanistically, mTORis inhibited palbociclib-induced increase in CCNE1.}, }
@article {pmid38819758, year = {2024}, author = {Mouri, T and Usa, S and Tokumoto, T}, title = {Pax7 is involved in leucophore formation in goldfish and gene knockout improves the transparency of transparent goldfish.}, journal = {Fish physiology and biochemistry}, volume = {50}, number = {4}, pages = {1701-1710}, pmid = {38819758}, issn = {1573-5168}, support = {23K05830//Japan Society for the Promotion of Science/ ; }, mesh = {Animals ; *Goldfish/genetics ; *Gene Knockout Techniques/methods ; *PAX7 Transcription Factor/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Fish Proteins/genetics/metabolism ; }, abstract = {Lines with few or no pigment cells have been established in fishes, and these lines are useful for bioimaging. The transparent goldfish (tra) line previously established by N-ethyl-N-nitrosourea (ENU) mutagenesis is also suitable for such experiments. However, in the case of tra, leucophores form in the adult fish, making it difficult to observe the organs inside body from outside the body. In this study, we attempted to create a knockout line of the pax7a and pax7b genes, which are thought to be involved in the formation of leucophores, to further improve the transparency of tra strain.Mutations were introduced by microinjection of the CRISPR/Cas9 mixture into single-cell embryos, mutant individuals were found in F0, and the next generation was generated to confirm the mutation patterns. As a result, multiple mutation patterns, including knockout, were obtained. The same pattern of knockout F1 with pax7a and pax7b mutations was crossed to generate a homozygous knockout in F2.In the resulting pax7b[-/-] (tra) fish but not in pax7a[-/-] (tra) fish, the number of leucophores was reduced compared to that in tra, and the transparency of the body was improved. It was suggested that pax7b plays an important role in leucophore formation in goldfish. The established transparent pax7b[-/-] (tra) goldfish line will be a useful model for bioimaging of the body interior.}, }
@article {pmid39068732, year = {2024}, author = {Bustamante-Brito, R and Vera-Ponce de León, A and Rosenblueth, M and Martínez-Romero, E}, title = {Comparative genomics of the carmine cochineal symbiont Candidatus Dactylopiibacterium carminicum reveals possible protection to the host against viruses via CRISPR/Cas.}, journal = {Systematic and applied microbiology}, volume = {47}, number = {5}, pages = {126540}, doi = {10.1016/j.syapm.2024.126540}, pmid = {39068732}, issn = {1618-0984}, abstract = {We present new genomes from the bacterial symbiont Candidatus Dactylopiibacterium carminicum obtained from non-domesticated carmine cochineals belonging to the scale insect Dactylopius (Hemiptera: Coccoidea: Dactylopiidae). As Dactylopiibacterium has not yet been cultured in the laboratory, metagenomes and metatranscriptomics have been key in revealing putative symbiont functions. Dactylopiibacterium is a nitrogen-fixing beta-proteobacterium that may be vertically transmitted and shows differential gene expression inside the cochineal depending on the tissue colonized. Here we found that all cochineal species tested had Dactylopiibacterium carminicum which has a highly conserved genome. All Dactylopiibacterium genomes analyzed had genes involved in nitrogen fixation and plant polymer degradation. Dactylopiibacterium genomes resemble those from free-living plant bacteria, some found as endophytes. Notably, we found here a new putative novel function where the bacteria may protect the insect from viruses, since all Dactylopiibacterium genomes contain CRISPRs with a spacer matching nucleopolyhedrovirus that affects insects.}, }
@article {pmid39068338, year = {2024}, author = {Yousefi Taemeh, S and Dehdilani, N and Goshayeshi, L and Dehghani, H}, title = {Exploring the Function of Gene Promoter Regulatory Elements Using CRISPR Tools.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2844}, number = {}, pages = {145-156}, pmid = {39068338}, issn = {1940-6029}, mesh = {*Promoter Regions, Genetic ; *CRISPR-Cas Systems ; Animals ; Humans ; Gene Expression Regulation ; Enhancer Elements, Genetic ; Ovalbumin/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Gene promoters serve as pivotal regulators of transcription, orchestrating the initiation, rate, and specificity of gene expression, resulting in cellular diversity found among distinct cell types within multicellular organisms. Identification of the sequence and function of promoters' regulatory elements and their complex interaction with transcription factors, enhancers, silencers, and insulators is fundamental to coordinated transcriptional processes within cells. Identifying these regulatory elements and scrutinizing their functions and interactions through the use of synthetic promoters can pave the way for researchers in various fields ranging from uncovering the origins of diseases associated with promoter mutations to harnessing these regulatory components in biotechnological applications.In this chapter, we describe the manipulation of regulatory elements within promoters, with a specific focus on the use of CRISPR technology on enhancers and silencer elements of the Ovalbumin gene promoter. We explain and discuss processes for the deletion of/interference with regulatory elements within the promoter, employing CRISPR-based approaches. Furthermore, we demonstrate that a CRISPR/Cas-manipulated promoter can activate gene transcription in cell types where it is normally inactive. This confirms that CRISPR technology can be effectively used to engineer synthetic promoters with desired characteristics, such as inducibility, tissue-specificity, or enhanced transcriptional strength. Such an approach provides valuable insights into the mechanisms and dynamics of gene expression, thereby offering new opportunities in the fields of biotechnology and medicine.}, }
@article {pmid39068337, year = {2024}, author = {Liu, S and Tang, X and Qi, Y and Zhang, Y}, title = {Optimizing Rice Genomics: Employing the Hypercompact Cas12j2 System for Targeted Transcriptional Regulation and Epigenome Modification.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2844}, number = {}, pages = {133-143}, pmid = {39068337}, issn = {1940-6029}, mesh = {*Oryza/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genomics/methods ; *Plants, Genetically Modified/genetics ; Genome, Plant ; Gene Expression Regulation, Plant ; Epigenome ; Epigenesis, Genetic ; DNA Methylation ; }, abstract = {In the burgeoning field of genome engineering, the CRISPR-Cas systems have emerged as pivotal tools for precise genetic modifications in various organisms, including humans, animals, and plants. One significant obstacle in this arena is the substantial size of Cas proteins, such as SpCas9, which is approximately 190 kDa, complicating their delivery, particularly via viral vectors. To overcome this challenge, our research introduces the hypercompact Cas12j2 system, a groundbreaking development with a size of merely ~80 kDa, originally identified in Biggiephage. We demonstrate its application in plant genome editing, with a particular focus on rice. In this context, we have successfully adapted Cas12j2 for gene activation, achieving significant increases in gene expression, specifically up to a tenfold activation for OsER1 and a fourfold activation for OsNRT1.1A in stable transgenic rice plants. Moreover, we have ventured beyond mere gene editing to develop a Cas12j2-based approach for targeted epigenome editing, particularly in the context of DNA methylation. This was demonstrated through the targeted methylation of the OsGBSS1 promoter, as verified by Next-Generation Sequencing of bisulfite sequencing PCR products. This chapter presents a detailed protocol about utilizing the hypercompact Cas12j2 system in conjunction with specific effectors, such as transcriptional activation or repression domains, or methylation domains, to achieve targeted gene transcriptional regulation and epigenome modification in rice.}, }
@article {pmid39068336, year = {2024}, author = {Zhang, Y and Qi, H and Wang, M}, title = {CRISPR/Cas9-Mediated Promoter Engineering in Saccharopolyspora erythraea.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2844}, number = {}, pages = {123-132}, pmid = {39068336}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; *Saccharopolyspora/genetics ; *Gene Editing/methods ; *Promoter Regions, Genetic ; Genetic Engineering/methods ; Erythromycin ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {In situ promoter engineering is an effective way to alter target gene expression without introducing excess DNA sequences. Recently, the CRISPR/Cas9 technologies have been proved to be efficient tools for genome editing in actinomycetes, making it easier and more efficient to perform gene insertion and substitution in actinomycetes in a scarless manner. In this chapter, we describe a routine protocol for CRISPR/Cas9-mediated promoter engineering in Saccharopolyspora erythraea NRRL 23338, which is the wild-type producer of erythromycin. This protocol can be adapted to CRISPR/Cas9-mediated gene editing, not limited to promoter engineering, in other actinomycetes, with modifications.}, }
@article {pmid39068154, year = {2024}, author = {Fontana, J and Sparkman-Yager, D and Faulkner, I and Cardiff, R and Kiattisewee, C and Walls, A and Primo, TG and Kinnunen, PC and Garcia Martin, H and Zalatan, JG and Carothers, JM}, title = {Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6341}, pmid = {39068154}, issn = {2041-1723}, support = {MCB 1817623, MCB 2032794, CBET 1844152//National Science Foundation (NSF)/ ; DE-EE0008927, DE-SC0023091//U.S. Department of Energy (DOE)/ ; }, mesh = {*Escherichia coli/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems ; Metabolic Engineering/methods ; Biosynthetic Pathways/genetics ; Promoter Regions, Genetic ; Humans ; Gene Expression Regulation, Bacterial ; RNA Folding ; }, abstract = {Engineering metabolism to efficiently produce chemicals from multi-step pathways requires optimizing multi-gene expression programs to achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important tools for programming multi-gene expression, but poor predictability of guide RNA folding can disrupt expression control. Here, we correlate efficacy of modified guide RNAs (scRNAs) for CRISPR activation (CRISPRa) in E. coli with a computational kinetic parameter describing scRNA folding rate into the active structure (rS = 0.8). This parameter also enables forward design of scRNAs, allowing us to design a system of three synthetic CRISPRa promoters that can orthogonally activate (>35-fold) expression of chosen outputs. Through combinatorial activation tuning, we profile a three-dimensional design space expressing two different biosynthetic pathways, demonstrating variable production of pteridine and human milk oligosaccharide products. This RNA design approach aids combinatorial optimization of metabolic pathways and may accelerate routine design of effective multi-gene regulation programs in bacterial hosts.}, }
@article {pmid39067912, year = {2024}, author = {You, D and Xu, T and Huang, BZ and Wu, F and Deng, LS and Liu, ZY and Ge, LP and Liu, ZH and Sun, J and Zeng, X and Zhou, YC and Lai, SY and Ai, YR and Huang, JB and Zhu, L and Xu, ZW}, title = {Rapid, sensitive, and visual detection of pseudorabies virus with an RPA-CRISPR/EsCas13d-based dual-readout portable platform.}, journal = {Analytica chimica acta}, volume = {1318}, number = {}, pages = {342918}, doi = {10.1016/j.aca.2024.342918}, pmid = {39067912}, issn = {1873-4324}, mesh = {*Herpesvirus 1, Suid/genetics/isolation &amp; purification ; Animals ; Swine ; *CRISPR-Cas Systems/genetics ; Pseudorabies/diagnosis/virology ; Swine Diseases/virology/diagnosis ; }, abstract = {Pseudorabies viruses (PRV) pose a major threat to the global pig industry and public health. Rapid, intuitive, affordable, and accurate diagnostic testing is critical for controlling and eradicating infectious diseases. In this study, a portable detection platform based on RPA-CRISPR/EsCas13d was developed. The platform exhibits high sensitivity (1 copy/μL), good specificity, and no cross-reactivity with common pathogens. The platform uses rapid preamplification technology to provide visualization results (lateral flow assays or visual fluorescence) within 1 h. Fifty pig samples (including tissues, oral fluids, and serum) were tested using this platform and real-time quantitative polymerase chain reaction (qPCR), showing 34.0 % (17 of 50) PRV positivity with the portable CRISPR/EsCas13d dual-readout platform, consistent with the qPCR results. These results highlight the stability, sensitivity, efficiency, and low equipment requirements of the portable platform. Additionally, a novel point-of-care test is being developed for clinical use in remote rural and resource-limited areas, which could be a prospective measure for monitoring the progression of pseudorabies and other infectious diseases worldwide.}, }
@article {pmid39067906, year = {2024}, author = {Ren, N and Sui, B and Liu, C and Zhang, S and Liu, Z and Zhou, W and Liu, H}, title = {Specific detection of gut pathogens for one-pot chip based on RPA-CRISPR/Cas12a.}, journal = {Analytica chimica acta}, volume = {1318}, number = {}, pages = {342886}, doi = {10.1016/j.aca.2024.342886}, pmid = {39067906}, issn = {1873-4324}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; DNA, Bacterial/analysis ; Bacteria/isolation &amp; purification/genetics ; Limit of Detection ; Gastrointestinal Microbiome ; Nucleic Acid Amplification Techniques ; }, abstract = {BACKGROUND: There are billions of bacteria in the intestine, most of which are harmless and play important roles in humans. Although only a very small number of bacteria can cause diseases, once the pathogenic bacteria are ingested into the body and multiply in large quantities, it can lead to inflammatory diseases in the intestines and even other organs. Although polymerase chain reaction can specifically detect bacterial nucleic acid. However, the demand for temperature cycling limits its portability. Therefore, it is hoped to establish a high-throughput, highly specific and portable detection platform for directly detecting nucleic acid of intestinal pathogens.<br><br>RESULTS: Herein, a one-pot chip based on RPA-CRCISPR/Cas12a platform was developed. The chip is the same size as a glass slide and allows detection at the same temperature. Multiple samples could be detected simultaneously on the one chip, achieved high-throughput detection and improved the integration of detection. The specific recognition of CRISPR/Cas12a avoided the influence of non-specific amplification of RPA and enhanced the specificity of the analysis. At the same time, the one-pot chip avoided secondary contamination when the lid was opened during the analysis process. And the bacterial concentration showed good linearity at 10[2]-10[8]&#xa0;cfu&#xa0;mL[-1]. The limit of detection could be as low as 0.43&#xa0;cfu&#xa0;mL[-1]. This method has been successfully used to detect pollution samples. It can provide a reliable platform for early screening of gastrointestinal and other inflammatory diseases.<br><br>SIGNIFICANCE: The one-pot chip based on the RPA-CRISPR/Cas12a platform established can directly detect the nucleic acid of intestinal pathogens, with portability and specificity. It is worth noting that the platform has good programmability, can be used for other target detection by changing crRNA and RPA primers, it can achieve multi sample detection on the one chip.}, }
@article {pmid39066958, year = {2024}, author = {Mo, Z and Lin, S and Li, T and Yu, G and Sun, Y and Zhou, J and Xu, Z}, title = {Native CRISPR-Cas-based programmable multiplex gene repression in Klebsiella variicola.}, journal = {Biotechnology letters}, volume = {}, number = {}, pages = {}, pmid = {39066958}, issn = {1573-6776}, support = {2023A1515012775//Guangdong Basic and Applied Basic Research Foundation/ ; pdjh2023b0086//Special Funds for the Cultivation of Guangdong College Students' Scientific and Technological Innovation ("Climbing Program" Special Funds)/ ; 2021ZDJS002//Special Project for Research and Development in Key areas of Guangdong Province/ ; }, abstract = {Klebsiella variicola is a Gram-negative bacterium that is frequently isolated from a wide variety of natural niches. It is a ubiquitous opportunistic pathogen that can cause diverse infections in plants, animals, and humans. It also has significant biotechnological potential. However, due to the lack of efficient genetic tools, the molecular basis contributing to the pathogenesis and beneficial activities of K. variicola remains poorly understood. In this study, we found and characterized a native type I-E CRISPR-Cas system in a recently isolated K. variicola strain KV-1. The system cannot cleave target DNA sequences due to the inactivation of the Cas3 nuclease by a transposable element but retains the activity of the crRNA-guided Cascade binding to the target DNA sequence. A targeting plasmid carrying a mini-CRISPR to encode a crRNA was designed and introduced into the KV-1 strain, which successfully repurposed the native type I-E CRISPR-Cas system to inhibit the expression of the target gene efficiently and specifically. Moreover, by creating a mini-CRISPR to encode multiple crRNAs, multiplex gene repression was achieved by providing a single targeting plasmid. This work provides the first native CRISPR-Cas-based tool for programmable multiplex gene repression in K. variicola, which will facilitate studying the pathogenic mechanism of K. variicola and enable metabolic engineering to produce valuable bioproducts.}, }
@article {pmid39066891, year = {2024}, author = {Wang, C and Xie, Q and Xia, X and Zhang, C and Jiang, S and Wang, S and Zhang, X and Hua, R and Xue, J and Zheng, H}, title = {ZMYND12 serves as an IDAd subunit that is essential for sperm motility in mice.}, journal = {Cellular and molecular life sciences : CMLS}, volume = {81}, number = {1}, pages = {317}, pmid = {39066891}, issn = {1420-9071}, support = {32000584//National Natural Science Foundation of China/ ; 82371622//National Natural Science Foundation of China/ ; 2023AH050843//the Key Project of Natural Science Foundation for Universities of Anhui Province Education Department/ ; HAB202305//Huai'an Municipal Science and Technology Bureau/ ; 2208085Y31//Anhui Provincial Department of Science and Technology/ ; 2023rcyb022//the exceptional support plan of talent introduction of Anhui University of Chinese Medicine/ ; NMUB20220214//the Science and Technology development Fundation of Nanjing Medical Univertisy/ ; 2023Z178//Natural Science Foundation of Ningbo Municipality/ ; }, mesh = {Animals ; Male ; *Sperm Motility/genetics ; *Mice, Knockout ; Mice ; *Infertility, Male/genetics/metabolism/pathology ; Dyneins/metabolism/genetics ; Spermatozoa/metabolism ; Humans ; Asthenozoospermia/genetics/metabolism/pathology ; Sperm Capacitation/genetics ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; }, abstract = {Inner dynein arms (IDAs) are formed from a protein complex that is essential for appropriate flagellar bending and beating. IDA defects have previously been linked to the incidence of asthenozoospermia (AZS) and male infertility. The testes-enriched ZMYND12 protein is homologous with an IDA component identified in Chlamydomonas. ZMYND12 deficiency has previously been tied to infertility in males, yet the underlying mechanism remains uncertain. Here, a CRISPR/Cas9 approach was employed to generate Zmynd12 knockout (Zmynd12[-/-]) mice. These Zmynd12[-/-] mice exhibited significant male subfertility, reduced sperm motile velocity, and impaired capacitation. Through a combination of co-immunoprecipitation and mass spectrometry, ZMYND12 was found to interact with TTC29 and PRKACA. Decreases in the levels of PRKACA were evident in the sperm of these Zmynd12[-/-] mice, suggesting that this change may account for the observed drop in male fertility. Moreover, in a cohort of patients with AZS, one patient carrying a ZMYND12 variant was identified, expanding the known AZS-related variant spectrum. Together, these findings demonstrate that ZMYND12 is essential for flagellar beating, capacitation, and male fertility.}, }
@article {pmid39066330, year = {2024}, author = {Xu, Q and Zhang, Y and Sadigh, Y and Tang, N and Chai, J and Cheng, Z and Gao, Y and Qin, A and Shen, Z and Yao, Y and Nair, V}, title = {Specific and Sensitive Visual Proviral DNA Detection of Major Pathogenic Avian Leukosis Virus Subgroups Using CRISPR-Associated Nuclease Cas13a.}, journal = {Viruses}, volume = {16}, number = {7}, pages = {}, pmid = {39066330}, issn = {1999-4915}, support = {ZR2020KC006//Natural Science Foundation of Shandong Province/ ; BB/P016472/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L014262/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007032/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R012865/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/OS/NW/000007/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 2023YFE0106100//National Key Research and Development Program of China/ ; 31761133002//National Natural Science Foundation of China/ ; }, mesh = {*Avian Leukosis Virus/genetics/isolation &amp; purification/classification ; Animals ; *Proviruses/genetics/isolation &amp; purification ; *Avian Leukosis/virology/diagnosis ; *CRISPR-Cas Systems ; *DNA, Viral/genetics ; Poultry Diseases/virology/diagnosis ; Chickens/virology ; Sensitivity and Specificity ; CRISPR-Associated Proteins/genetics/metabolism ; }, abstract = {Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs' eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs.}, }
@article {pmid39063076, year = {2024}, author = {Kou, YJ and Gao, J and Li, R and Ma, ZY and Elsheikha, HM and Wu, XJ and Zheng, XN and Wang, M and Zhu, XQ}, title = {Functional Characterization of Six Eukaryotic Translation Initiation Factors of Toxoplasma gondii Using the CRISPR-Cas9 System.}, journal = {International journal of molecular sciences}, volume = {25}, number = {14}, pages = {}, pmid = {39063076}, issn = {1422-0067}, support = {32172887//National Natural Science Foundation of China/ ; U2202201//the NSFC-Yunnan Joint Fund/ ; 23JRRA553//the Natural Science Foundation of Gansu Province, China/ ; 2021XG001//the Special Research Fund of Shanxi Agricultural University for High-level Talents/ ; }, mesh = {*Toxoplasma/genetics/pathogenicity/metabolism/growth &amp; development ; *CRISPR-Cas Systems ; Animals ; Mice ; *Protozoan Proteins/genetics/metabolism ; *Eukaryotic Initiation Factors/genetics/metabolism ; Virulence/genetics ; Toxoplasmosis/parasitology/genetics ; Humans ; }, abstract = {Eukaryotic translation initiation factors (eIFs) are crucial for initiating protein translation and ensuring the correct assembly of mRNA-ribosomal subunit complexes. In this study, we investigated the effects of deleting six eIFs in the apicomplexan parasite Toxoplasma gondii using the CRISPR-Cas9 system. We determined the subcellular localization of these eIFs using C-terminal endogenous tagging and immunofluorescence analysis. Four eIFs (RH::315150-6HA, RH::286090-6HA, RH::249370-6HA, and RH::211410-6HA) were localized in the cytoplasm, while RH::224235-6HA was localized in the apicoplast. Additionally, RH::272640-6HA was found in both the basal complex and the cytoplasm of T. gondii. Functional characterization of the six RHΔeIFs strains was conducted using plaque assay, cell invasion assay, intracellular growth assay and egress assay in vitro, and virulence assay in mice. Disruption of five eIF genes (RHΔ315150, RHΔ272640, RHΔ249370, RHΔ211410, and RHΔ224235) did not affect the ability of the T. gondii RH strain to invade, replicate, form plaques and egress in vitro, or virulence in Kunming mice (p > 0.05). However, the RHΔ286090 strain showed slightly reduced invasion efficiency and virulence (p < 0.01) compared to the other five RHΔeIFs strains and the wild-type strain. The disruption of the TGGT1_286090 gene significantly impaired the ability of tachyzoites to differentiate into bradyzoites in both type I RH and type II Pru strains. These findings reveal that the eukaryotic translation initiation factor TGGT1_286090 is crucial for T. gondii bradyzoite differentiation and may serve as a potential target for drug development and an attenuated vaccine against T. gondii.}, }
@article {pmid39062879, year = {2024}, author = {Sun, Y and Wang, HY and Liu, B and Yue, B and Liu, Q and Liu, Y and Rosa, IF and Doretto, LB and Han, S and Lin, L and Gong, X and Shao, C}, title = {CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole (Cynoglossus semilaevis) Testis Cells.}, journal = {International journal of molecular sciences}, volume = {25}, number = {14}, pages = {}, pmid = {39062879}, issn = {1422-0067}, support = {2022YFD2400100//the National Key Research and Development Program of China/ ; 32002371//the National Nature Science Foundation of China/ ; LSKJ202203802//Laoshan Laboratory Project/ ; NO.tstp20221149//the Taishan Scholars Program/ ; 2024XT0103, 2023TD19//the Central Public-interest Scientific Institution Basal Research Fund CAFS/ ; 2017ASTCP-ES06//the AoShan Talents Cultivation Program Supported by Qingdao National Laboratory for Marine Scienceand Technology/ ; }, mesh = {Animals ; *DNA Methylation ; Male ; *Homeodomain Proteins/genetics/metabolism ; *Testis/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems ; Transcription Factors/genetics/metabolism ; Flatfishes/genetics ; Promoter Regions, Genetic/genetics ; Fish Proteins/genetics/metabolism ; DNA Methyltransferase 3A ; }, abstract = {DNA methylation is a key epigenetic mechanism orchestrating gene expression networks in many biological processes. Nonetheless, studying the role of specific gene methylation events in fish faces challenges. In this study, we validate the regulation of DNA methylation on empty spiracles homeobox 2 (emx2) expression with decitabine treatment in Chinese tongue sole testis cells. We used the emx2 gene as the target gene and developed a new DNA methylation editing system by fusing dnmt3a with catalytic dead Cas9 (dCas9) and demonstrated its ability for sequence-specific DNA methylation editing. Results revealed that utilizing dCas9-dnmt3a to target emx2 promoter region led to increased DNA methylation levels and decreased emx2 expression in Chinese tongue sole testis cells. More importantly, the DNA methylation editing significantly suppressed the expression of MYC proto-oncogene, bHLH transcription factor (myc), one target gene of emx2. Furthermore, we assessed the off-target effects of dCas9-dnmt3a and confirmed no significant impact on the predicted off-target gene expression. Taken together, we developed the first DNA methylation editing system in marine species and demonstrated its effective editing ability in Chinese tongue sole cells. This provides a new strategy for both epigenetic research and molecular breeding of marine species.}, }
@article {pmid39062741, year = {2024}, author = {Wu, G and Liang, Y and Chen, C and Chen, G and Zuo, Q and Niu, Y and Song, J and Han, W and Jin, K and Li, B}, title = {Identification of Two Potential Gene Insertion Sites for Gene Editing on the Chicken Z/W Chromosomes.}, journal = {Genes}, volume = {15}, number = {7}, pages = {}, pmid = {39062741}, issn = {2073-4425}, support = {31972547, 32172718 and 32202655//The National Natural Science Foundation of China/ ; 2022M722697//China Postdoctoral Science Foundation/ ; }, mesh = {Animals ; *Chickens/genetics ; *Gene Editing/methods ; *Sex Chromosomes/genetics ; Mutagenesis, Insertional ; CRISPR-Cas Systems ; Cell Line ; Gene Knockout Techniques/methods ; Female ; Male ; }, abstract = {The identification of accurate gene insertion sites on chicken sex chromosomes is crucial for advancing sex control breeding materials. In this study, the intergenic region NC_006127.4 on the chicken Z chromosome and the non-repetitive sequence EE0.6 on the W chromosome were selected as potential gene insertion sites. Gene knockout vectors targeting these sites were constructed and transfected into DF-1 cells. T7E1 enzyme cleavage and luciferase reporter enzyme analyses revealed knockout efficiencies of 80.00% (16/20), 75.00% (15/20), and 75.00% (15/20) for the three sgRNAs targeting the EE0.6 site. For the three sgRNAs targeting the NC_006127.4 site, knockout efficiencies were 70.00% (14/20), 60.00% (12/20), and 45.00% (9/20). Gel electrophoresis and high-throughput sequencing were performed to detect potential off-target effects, showing no significant off-target effects for the knockout vectors at the two sites. EdU and CCK-8 proliferation assays revealed no significant difference in cell proliferation activity between the knockout and control groups. These results demonstrate that the EE0.6 and NC_006127.4 sites can serve as gene insertion sites on chicken sex chromosomes for gene editing without affecting normal cell proliferation.}, }
@article {pmid39060399, year = {2024}, author = {Mangin, A and Dion, V and Menzies, G}, title = {Developing small Cas9 hybrids using molecular modeling.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {17233}, pmid = {39060399}, issn = {2045-2322}, support = {DRI-TAP2021-01//UK Dementia Research Institute/ ; }, mesh = {Humans ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Streptococcus pyogenes/genetics/enzymology ; Molecular Dynamics Simulation ; CRISPR-Cas Systems ; Gene Editing/methods ; Models, Molecular ; }, abstract = {The contraction of CAG/CTG repeats is an attractive approach to correct the mutation that causes at least 15 neuromuscular and neurodegenerative diseases, including Huntington's disease and Myotonic Dystrophy type 1. Contractions can be achieved in vivo using the Cas9 D10A nickase from Streptococcus pyogenes (SpCas9) using a single guide RNA (sgRNA) against the repeat tract. One hurdle on the path to the clinic is that SpCas9 is too large to be packaged together with its sgRNA into a single adeno-associated virus. Here we aimed to circumvent this problem using the smaller Cas9 orthologue, SlugCas9, and the Cas9 ancestor OgeuIscB. We found them to be ineffective in inducing contractions, despite their advertised PAM sequences being compatible with CAG/CTG repeats. Thus, we further developed smaller Cas9 hybrids, made of the PAM interacting domain of S. pyogenes and the catalytic domains of the smaller Cas9 orthologues. We also designed the cognate sgRNA hybrids using molecular dynamic simulations and binding energy calculations. We found that the four Cas9/sgRNA hybrid pairs tested in human cells failed to edit their target sequences. We conclude that in silico approaches can identify functional changes caused by point mutations but are not sufficient for designing larger scale complexes of Cas9/sgRNA hybrids.}, }
@article {pmid39060288, year = {2024}, author = {Weiss, T and Kumar, J and Chen, C and Guo, S and Schlegel, O and Lutterman, J and Ling, K and Zhang, F}, title = {Dual activities of an X-family DNA polymerase regulate CRISPR-induced insertional mutagenesis across species.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6293}, pmid = {39060288}, issn = {2041-1723}, support = {IOS-2206920//NSF | BIO | Division of Integrative Organismal Systems (IOS)/ ; }, mesh = {*CRISPR-Cas Systems ; Humans ; *Mutagenesis, Insertional ; *DNA End-Joining Repair ; Gene Editing/methods ; Arabidopsis/genetics ; DNA Polymerase beta/metabolism/genetics ; Species Specificity ; HEK293 Cells ; }, abstract = {The canonical non-homologous end joining (c-NHEJ) repair pathway, generally viewed as stochastic, has recently been shown to produce predictable outcomes in CRISPR-Cas9 mutagenesis. This predictability, mainly in 1-bp insertions and small deletions, has led to the development of in-silico prediction programs for various animal species. However, the predictability of CRISPR-induced mutation profiles across species remained elusive. Comparing CRISPR-Cas9 repair outcomes between human and plant species reveals significant differences in 1-bp insertion profiles. The high predictability observed in human cells links to the template-dependent activity of human Polλ. Yet plant Polλ exhibits dual activities, generating 1-bp insertions through both templated and non-templated manners. Polλ knockout in plants leads to deletion-only mutations, while its overexpression enhances 1-bp insertion rates. Two conserved motifs are identified to modulate plant Polλ's dual activities. These findings unveil the mechanism behind species-specific CRISPR-Cas9-induced insertion profiles and offer strategies for predictable, precise genome editing through c-NHEJ.}, }
@article {pmid39041030, year = {2024}, author = {Berchowitz, L and Wood, T and Henriques, W and Cullen, H and Romero, M and Blengini, C and Sarathy, S and Sorkin, J and Bekele, H and Jin, C and Kim, S and Chemiakine, A and Khondker, R and Isola, J and Stout, MB and Gennarino, V and Mogessie, B and Jain, D and Schindler, K and Suh, Y and Wiedenheft, B}, title = {The Retrotransposon-Derived Capsid Genes PNMA1 and PNMA4 Maintain Reproductive Capacity.}, journal = {Research square}, volume = {}, number = {}, pages = {}, pmid = {39041030}, issn = {2693-5015}, support = {R35 GM146725/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; R01 AG069750/AG/NIA NIH HHS/United States ; R01 NS109858/NS/NINDS NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; R01 AG069742/AG/NIA NIH HHS/United States ; R35 GM136340/GM/NIGMS NIH HHS/United States ; R35 GM124633/GM/NIGMS NIH HHS/United States ; R35 GM147130/GM/NIGMS NIH HHS/United States ; }, abstract = {The human genome contains 24 gag-like capsid genes derived from deactivated retrotransposons conserved among eutherians. Although some of their encoded proteins retain the ability to form capsids and even transfer cargo, their fitness benefit has remained elusive. Here we show that the gag-like genes PNMA1 and PNMA4 support reproductive capacity during aging. Analysis of donated human ovaries shows that expression of both genes declines normally with age, while several PNMA1 and PNMA4 variants identified in genome-wide association studies are causally associated with low testosterone, altered puberty onset, or obesity. Six-week-old mice lacking either Pnma1 or Pnma4 are indistinguishable from wild-type littermates, but by six months the mutant mice become prematurely subfertile, with precipitous drops in sex hormone levels, gonadal atrophy, and abdominal obesity; overall they produce markedly fewer offspring than controls. These findings expand our understanding of factors that maintain human reproductive health and lend insight into the domestication of retrotransposon-derived genes.}, }
@article {pmid38996955, year = {2024}, author = {Rasl, J and Caslavsky, J and Grusanovic, J and Chvalova, V and Kosla, J and Adamec, J and Grousl, T and Klimova, Z and Vomastek, T}, title = {Depletion of calpain2 accelerates epithelial barrier establishment and reduces growth factor-induced cell scattering.}, journal = {Cellular signalling}, volume = {121}, number = {}, pages = {111295}, doi = {10.1016/j.cellsig.2024.111295}, pmid = {38996955}, issn = {1873-3913}, mesh = {*Calpain/metabolism ; Animals ; Dogs ; Madin Darby Canine Kidney Cells ; *Epithelial Cells/metabolism/cytology ; *Cell Adhesion ; Cell Movement ; Cadherins/metabolism ; Proteolysis ; Hepatocyte Growth Factor/metabolism ; beta Catenin/metabolism ; Calcium/metabolism ; Mitogen-Activated Protein Kinase 1/metabolism ; CRISPR-Cas Systems ; }, abstract = {Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, β-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes.}, }
@article {pmid38985676, year = {2024}, author = {Feng, YY and Li, YC and Liu, HM and Xu, R and Liu, YT and Zhang, W and Yang, HY and Chen, G}, title = {Synthetic lethal CRISPR screen identifies a cancer cell-intrinsic role of PD-L1 in regulation of vulnerability to ferroptosis.}, journal = {Cell reports}, volume = {43}, number = {7}, pages = {114477}, doi = {10.1016/j.celrep.2024.114477}, pmid = {38985676}, issn = {2211-1247}, mesh = {Humans ; *Ferroptosis/genetics ; *B7-H1 Antigen/metabolism/genetics ; Cell Line, Tumor ; *Reactive Oxygen Species/metabolism ; Superoxide Dismutase/metabolism/genetics ; Squamous Cell Carcinoma of Head and Neck/genetics/pathology/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; Synthetic Lethal Mutations ; Mice ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Neoplastic ; Head and Neck Neoplasms/genetics/pathology/metabolism ; }, abstract = {Despite the success of programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibition in tumor therapy, many patients do not benefit. This failure may be attributed to the intrinsic functions of PD-L1. We perform a genome-wide CRISPR synthetic lethality screen to systematically explore the intrinsic functions of PD-L1 in head and neck squamous cell carcinoma (HNSCC) cells, identifying ferroptosis-related genes as essential for the viability of PD-L1-deficient cells. Genetic and pharmacological induction of ferroptosis accelerates cell death in PD-L1 knockout cells, which are also more susceptible to immunogenic ferroptosis. Mechanistically, nuclear PD-L1 transcriptionally activates SOD2 to maintain redox homeostasis. Lower reactive oxygen species (ROS) and ferroptosis are observed in patients with HNSCC who have higher PD-L1 expression. Our study illustrates that PD-L1 confers ferroptosis resistance in HNSCC cells by activating the SOD2-mediated antioxidant pathway, suggesting that targeting the intrinsic functions of PD-L1 could enhance therapeutic efficacy.}, }
@article {pmid38964321, year = {2024}, author = {Karasu, ME and Jahnke, L and Joseph, BJ and Amerzhanova, Y and Mironov, A and Shu, X and Schröder, MS and Gvozdenovic, A and Sala, I and Zavolan, M and Jonas, S and Corn, JE}, title = {CCAR1 promotes DNA repair via alternative splicing.}, journal = {Molecular cell}, volume = {84}, number = {14}, pages = {2634-2647.e9}, doi = {10.1016/j.molcel.2024.06.011}, pmid = {38964321}, issn = {1097-4164}, mesh = {Humans ; *Alternative Splicing ; Animals ; Mice ; *Fanconi Anemia Complementation Group A Protein/genetics/metabolism ; Recombinational DNA Repair ; Fanconi Anemia/genetics/metabolism ; HEK293 Cells ; Exons ; CRISPR-Cas Systems ; DNA Repair ; HeLa Cells ; DNA Damage ; }, abstract = {DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.}, }
@article {pmid38936359, year = {2024}, author = {Zhang, R and He, Z and Shi, Y and Sun, X and Chen, X and Wang, G and Zhang, Y and Gao, P and Wu, Y and Lu, S and Duan, J and Sun, S and Yang, N and Fan, W and Zhao, K and Yang, B and Xia, Y and Zhang, Y and Zhang, Y and Yin, H}, title = {Amplification editing enables efficient and precise duplication of DNA from short sequence to megabase and chromosomal scale.}, journal = {Cell}, volume = {187}, number = {15}, pages = {3936-3952.e19}, doi = {10.1016/j.cell.2024.05.056}, pmid = {38936359}, issn = {1097-4172}, mesh = {Humans ; *Gene Editing/methods ; *Gene Duplication ; *Genome, Human ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Animals ; Embryonic Stem Cells/metabolism ; Chromosomes, Human/genetics ; }, abstract = {Duplication is a foundation of molecular evolution and a driver of genomic and complex diseases. Here, we develop a genome editing tool named Amplification Editing (AE) that enables programmable DNA duplication with precision at chromosomal scale. AE can duplicate human genomes ranging from 20 bp to 100 Mb, a size comparable to human chromosomes. AE exhibits activity across various cell types, encompassing diploid, haploid, and primary cells. AE exhibited up to 73.0% efficiency for 1 Mb and 3.4% for 100 Mb duplications, respectively. Whole-genome sequencing and deep sequencing of the junctions of edited sequences confirm the precision of duplication. AE can create chromosomal microduplications within disease-relevant regions in embryonic stem cells, indicating its potential for generating cellular and animal models. AE is a precise and efficient tool for chromosomal engineering and DNA duplication, broadening the landscape of precision genome editing from an individual genetic locus to the chromosomal scale.}, }
@article {pmid38908633, year = {2024}, author = {Zhang, Q and Yu, G and Ding, X and Zhang, K and Sun, W and Li, Q and Yi, Y and Wang, J and Pang, X and Chen, L}, title = {A rapid simultaneous detection of duck hepatitis A virus 3 and novel duck reovirus based on RPA CRISPR Cas12a/Cas13a.}, journal = {International journal of biological macromolecules}, volume = {274}, number = {Pt 1}, pages = {133246}, doi = {10.1016/j.ijbiomac.2024.133246}, pmid = {38908633}, issn = {1879-0003}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Ducks/virology ; Hepatitis Virus, Duck/genetics/isolation &amp; purification ; Orthoreovirus, Avian/genetics/isolation &amp; purification ; }, abstract = {The mixed infection of duck hepatitis A virus 3 (DHAV-3) and novel duck reovirus (NDRV) has caused significant losses to the global duck farming industry. On-site point-of-care testing of viruses plays a crucial role in the early diagnosis, prevention, and disease control. Here, we proposed an RPA-CRISPR Cas12a/Cas13a one-pot strategy (DRCFS) for rapid and simultaneous detection of DHAV-3 and NDRV. This method integrated the reaction of RPA and CRISPR Cas12a/Cas13a in a single tube, eliminating the need to open the lid during the intermediate processes and thereby avoiding aerosol contamination. On this basis, we proposed a dual RPA-CRISPR strategy coupled with a lateral flow analysis platform (DRC-LFA). This circumvented the necessity for complex instruments, enabling direct visual interpretation of results, making the test more accessible and user-friendly. Our findings demonstrated that the DRCFS method could detect DHAV-3 and NDRV at concentrations as low as 10[0] copy/μL, while DRC-LFA achieved limit of 10[1] copies/μL within 35 min. Furthermore, when DRCFS, DRC-LFA, and qPCR were employed collectively for clinical samples analysis, all three methods yielded consistent results. The specificity, sensitivity, and user-friendly of these methods rendered them invaluable for on-site virus detection.}, }
@article {pmid38908207, year = {2024}, author = {Ji, P and Li, Y and Wang, Z and Jia, S and Jiang, X and Chen, H and Wang, Q}, title = {Advances in precision gene editing for liver fibrosis: From technology to therapeutic applications.}, journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie}, volume = {177}, number = {}, pages = {117003}, doi = {10.1016/j.biopha.2024.117003}, pmid = {38908207}, issn = {1950-6007}, mesh = {Humans ; *Liver Cirrhosis/genetics/therapy ; *Gene Editing/methods ; Animals ; *Genetic Therapy/methods ; Precision Medicine/methods ; CRISPR-Cas Systems/genetics ; }, abstract = {This review presents a comprehensive exploration of gene editing technologies and their potential applications in the treatment of liver fibrosis, a condition often leading to serious complications such as liver cancer. Through an in-depth review of current literature and critical analysis, the study delves into the intricate signaling pathways underlying liver fibrosis development and examines the promising role of gene editing in alleviating this disease burden. Gene editing technologies offer precise, efficient, and reproducible tools for manipulating genetic material, holding significant promise for basic research and clinical practice. The manuscript highlights the challenges and potential risks associated with gene editing technology. By synthesizing existing knowledge and exploring future perspectives, this study aims to provide valuable insights into the potential of precision gene editing to combat liver fibrosis and its associated complications, ultimately contributing to advances in liver fibrosis research and therapy.}, }
@article {pmid38897195, year = {2024}, author = {Jiang, X and Dimitriou, E and Grabe, V and Sun, R and Chang, H and Zhang, Y and Gershenzon, J and Rybak, J and Hansson, BS and Sachse, S}, title = {Ring-shaped odor coding in the antennal lobe of migratory locusts.}, journal = {Cell}, volume = {187}, number = {15}, pages = {3973-3991.e24}, doi = {10.1016/j.cell.2024.05.036}, pmid = {38897195}, issn = {1097-4172}, mesh = {Animals ; *Odorants ; *Olfactory Receptor Neurons/metabolism ; *Arthropod Antennae/physiology ; Smell/physiology ; Grasshoppers/physiology ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Olfactory Pathways/physiology ; Receptors, Odorant/metabolism/genetics ; Locusta migratoria/physiology ; Calcium/metabolism ; }, abstract = {The representation of odors in the locust antennal lobe with its >2,000 glomeruli has long remained a perplexing puzzle. We employed the CRISPR-Cas9 system to generate transgenic locusts expressing the genetically encoded calcium indicator GCaMP in olfactory sensory neurons. Using two-photon functional imaging, we mapped the spatial activation patterns representing a wide range of ecologically relevant odors across all six developmental stages. Our findings reveal a functionally ring-shaped organization of the antennal lobe composed of specific glomerular clusters. This configuration establishes an odor-specific chemotopic representation by encoding different chemical classes and ecologically distinct odors in the form of glomerular rings. The ring-shaped glomerular arrangement, which we confirm by selective targeting of OR70a-expressing sensory neurons, occurs throughout development, and the odor-coding pattern within the glomerular population is consistent across developmental stages. Mechanistically, this unconventional spatial olfactory code reflects the locust-specific and multiplexed glomerular innervation pattern of the antennal lobe.}, }
@article {pmid38853211, year = {2024}, author = {Oya, S and Ozawa, H and Nakamura, T and Mori, A and Ochi, S and Maehiro, Y and Umeda, M and Takaki, Y and Fukuyama, T and Yamasaki, Y and Yamaguchi, M and Aoyama, K and Mouri, F and Nagafuji, K}, title = {CRISPR/Cas9 gene editing clarifies the role of CD33 SNP rs12459419 in gemtuzumab ozogamicin-mediated cytotoxicity.}, journal = {International journal of hematology}, volume = {120}, number = {2}, pages = {194-202}, pmid = {38853211}, issn = {1865-3774}, mesh = {Humans ; *Gemtuzumab ; *Sialic Acid Binding Ig-like Lectin 3/genetics ; *Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Cell Line, Tumor ; Leukemia, Myeloid, Acute/genetics/drug therapy ; }, abstract = {The single-nucleotide polymorphism (SNP) rs12459419 is located at the intron/exon junction of CD33 exon2. When exon2 is skipped by this CD33 SNP, the full-length CD33 (CD33FL) is converted to a short CD33 isoform (CD33D2). Since gemtuzumab ozogamicin (GO) only recognizes CD33FL, the CD33 SNP may affect the clinical efficacy of GO. To elucidate the significance of CD33 SNP on GO reactivity, we leveraged the CRISPR/Cas9 genome-editing system to create OCI-AML3 cell lines with specifically modified CD33 SNPs. Levels of CD33 D2 mRNA were significantly higher in the T/T clone (p < 0.001), but CD33D2 protein was not detectable in any clones. There was no significant difference in CD33FL mRNA expression across edited clones, and CD33FL protein expression was lowest in T/T clones, followed by T/C and C/C. Cytotoxicity assays revealed that the IC50 of GO was significantly lower in T/C and C/C clones than in the T/T clone (p < 0.001). Our study demonstrated a difference in GO-induced cytotoxicity in CD33 SNP-edited clones, clearly indicating that at least one CD33 SNP allele, rs12459419 C, is important for sensitivity to GO.}, }
@article {pmid38714872, year = {2024}, author = {Tieu, V}, title = {A MEGA RNA-editing tool.}, journal = {Nature reviews. Cancer}, volume = {24}, number = {8}, pages = {519}, pmid = {38714872}, issn = {1474-1768}, mesh = {Humans ; *RNA Editing ; CRISPR-Cas Systems ; Neoplasms/genetics ; }, }
@article {pmid37867187, year = {2024}, author = {Youssry, I and Ayad, N}, title = {Sickle cell disease: combination new therapies vs. CRISPR-Cas9 potential and challenges - review article.}, journal = {Annals of hematology}, volume = {103}, number = {8}, pages = {2613-2619}, pmid = {37867187}, issn = {1432-0584}, mesh = {*Anemia, Sickle Cell/therapy/genetics ; Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Combined Modality Therapy ; }, abstract = {In 2022, sickle cell disease (SCD) continues to affect the lives of millions of people, being one of the most frequently inherited blood disorders worldwide. Recently, several new therapies have been FDA approved for the treatment of SCD. The complexity of the pathophysiology of sickling has given opportunity to the evolution of several modalities of therapies. Nonetheless, the potential for complementary targeting of HbS polymerization, vasocclusion, and other inflammatory pathways remains controversial. None of these drugs can be considered a single curative line of treatment. With the advancement of CRISPR/Cas9 technology, autologous transplant of gene-edited hematopoietic stem cells could possibly provide a cure for most patients with SCD. The advantage of this approach over the conventional stem cell transplantation is that it decreases the need for immuno-suppressive drugs and the risk of graft-versus-host disease. In addition, recent technological advances can reduce the off-target effects, but long-term monitoring is needed to ensure the reliability of these methods in the clinical setting. This review explores the efficacy and safety of combination therapies and contrasting this alternative with the challenges that exist with sickle cell gene therapy using CRISPR.}, }
@article {pmid37573566, year = {2024}, author = {Rahman, SU and Khan, MO and Ullah, R and Ahmad, F and Raza, G}, title = {Agrobacterium-Mediated Transformation for the Development of Transgenic Crops; Present and Future Prospects.}, journal = {Molecular biotechnology}, volume = {66}, number = {8}, pages = {1836-1852}, pmid = {37573566}, issn = {1559-0305}, mesh = {*Plants, Genetically Modified/genetics/growth &amp; development ; *Crops, Agricultural/genetics/growth &amp; development ; *Transformation, Genetic ; *Agrobacterium/genetics ; Transgenes ; CRISPR-Cas Systems ; Gene Editing/methods ; Genetic Engineering/methods ; }, abstract = {Plant transformation based on Agrobacterium-mediated transformation is a technique that mimics the natural agrobacterium system for gene(s) introduction into crops. Through this technique, various crop species have been improved/modified for different trait/s, showing a successful genetic transformation so far. This technique has many advantages over other transformation methods such as stable integration of transgene, cost effective. However, there are many limitations of this technology such as mostly the crops are recalcitrant to agrobacterium, low transformation efficiency, transgene integration as well as off targets. So, it's very important to explore the major limitations and possible solutions for Agrobacterium-mediated transformation in order to increase its genetic transformation efficiency. Therefore, the present review article gives a comprehensive study how the transgenic crops are developed using Agrobacterium-mediated transformation, crops that have already been modified through this method, and risks associated with transgenic plants based on Agrobacterium-mediated transformation. Moreover, the challenges and problems associated with Agrobacterium-mediated transformation and how those problems can be solved in future for a successful genetic transformation of crops using modern biotechnology techniques such as CRISPR/Cas9 systems. The present review article will be really helpful for the audience those working on Genome editing of crops using Agrobacterium-mediated transformation and will opens many ways for future plant genetic transformation.}, }
@article {pmid37572221, year = {2024}, author = {Tyagi, A and Karapurkar, JK and Colaco, JC and Sarodaya, N and Antao, AM and Kaushal, K and Haq, S and Chandrasekaran, AP and Das, S and Singh, V and Hong, SH and Suresh, B and Kim, KS and Ramakrishna, S}, title = {USP19 Negatively Regulates p53 and Promotes Cervical Cancer Progression.}, journal = {Molecular biotechnology}, volume = {66}, number = {8}, pages = {2032-2045}, pmid = {37572221}, issn = {1559-0305}, support = {2021R1I1A1A01052637//National Research Foundation of Korea/ ; 2021M3A9H3015389//National Research Foundation of Korea/ ; 22A0304L1-01//Korean Fund for Regenerative Medicine/ ; 2017R1A5A2015395//Medical Research Center/ ; }, mesh = {Humans ; *Uterine Cervical Neoplasms/genetics/metabolism/pathology ; Female ; *Tumor Suppressor Protein p53/metabolism/genetics ; *Ubiquitination ; *Cell Movement ; *Disease Progression ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Endopeptidases/metabolism/genetics ; CRISPR-Cas Systems ; HeLa Cells ; Cell Proliferation ; }, abstract = {p53 is a tumor suppressor gene activated in response to cellular stressors that inhibits cell cycle progression and induces pro-apoptotic signaling. The protein level of p53 is well balanced by the action of several E3 ligases and deubiquitinating enzymes (DUBs). Several DUBs have been reported to negatively regulate and promote p53 degradation in tumors. In this study, we identified USP19 as a negative regulator of p53 protein level. We demonstrate a direct interaction between USP19 and p53 by pull down assay. The overexpression of USP19 promoted ubiquitination of p53 and reduced its protein half-life. We also demonstrate that CRISPR/Cas9-mediated knockout of USP19 in cervical cancer cells elevates p53 protein levels, resulting in reduced colony formation, cell migration, and cell invasion. Overall, our results indicate that USP19 negatively regulates p53 protein levels in cervical cancer progression.}, }
@article {pmid39066241, year = {2024}, author = {Zhai, Y and Gnanasekaran, P and Pappu, HR}, title = {Development of a CRISPR/SHERLOCK-Based Method for Rapid and Sensitive Detection of Selected Pospiviroids.}, journal = {Viruses}, volume = {16}, number = {7}, pages = {}, doi = {10.3390/v16071079}, pmid = {39066241}, issn = {1999-4915}, support = {7721//Animal and Plant Health Inspection Service/ ; }, abstract = {Pospiviroids infect a wide range of plant species, and many pospiviroids can be transmitted to potato and tomato. Pospiviroids continue to be a major production constraint as well as of quarantine concern for the movement of germplasm, and are regulated in several countries/regions. The USDA APHIS issued a federal order requiring all imported tomato and pepper seeds be certified free of six pospiviroids of quarantine significance. The six pospiviroids of quarantine interest include CLVd, PCFVd, PSTVd, TASVd, TCDVd, TPMVd. Currently, those six viroids are detected by real-time RT-PCR. CRISPR/Cas-based genome editing has been increasingly used for virus detection in the past five years. We used a rapid Cas13-based Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) platform for pospiviroid detection, determined the limits of detection and specificity of CRISPR-Cas13a assays. This platform combines recombinase polymerase amplification (RPA) with CRISPR and CRISPR-associated (CRISPR-Cas) RNA-guided endoribonuclease that is rapid and does not require expensive equipment, and can be adapted for on-site detection.}, }
@article {pmid39065411, year = {2024}, author = {Mishra, S and Nayak, S and Tuteja, N and Poosapati, S and Swain, DM and Sahoo, RK}, title = {CRISPR/Cas-Mediated Genome Engineering in Plants: Application and Prospectives.}, journal = {Plants (Basel, Switzerland)}, volume = {13}, number = {14}, pages = {}, doi = {10.3390/plants13141884}, pmid = {39065411}, issn = {2223-7747}, abstract = {Genetic engineering has become an essential element in developing climate-resilient crops and environmentally sustainable solutions to respond to the increasing need for global food security. Genome editing using CRISPR/Cas [Clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein (Cas)] technology is being applied to a variety of organisms, including plants. This technique has become popular because of its high specificity, effectiveness, and low production cost. Therefore, this technology has the potential to revolutionize agriculture and contribute to global food security. Over the past few years, increasing efforts have been seen in its application in developing higher-yielding, nutrition-rich, disease-resistant, and stress-tolerant "crops", fruits, and vegetables. Cas proteins such as Cas9, Cas12, Cas13, and Cas14, among others, have distinct architectures and have been used to create new genetic tools that improve features that are important for agriculture. The versatility of Cas has accelerated genomic analysis and facilitated the use of CRISPR/Cas to manipulate and alter nucleic acid sequences in cells of different organisms. This review provides the evolution of CRISPR technology exploring its mechanisms and contrasting it with traditional breeding and transgenic approaches to improve different aspects of stress tolerance. We have also discussed the CRISPR/Cas system and explored three Cas proteins that are currently known to exist: Cas12, Cas13, and Cas14 and their potential to generate foreign-DNA-free or non-transgenic crops that could be easily regulated for commercialization in most countries.}, }
@article {pmid39062641, year = {2024}, author = {Kansal, R}, title = {The CRISPR-Cas System and Clinical Applications of CRISPR-Based Gene Editing in Hematology with a Focus on Inherited Germline Predisposition to Hematologic Malignancies.}, journal = {Genes}, volume = {15}, number = {7}, pages = {}, doi = {10.3390/genes15070863}, pmid = {39062641}, issn = {2073-4425}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing has begun to transform the treatment landscape of genetic diseases. The history of the discovery of CRISPR/CRISPR-associated (Cas) proteins/single-guide RNA (sgRNA)-based gene editing since the first report of repetitive sequences of unknown significance in 1987 is fascinating, highly instructive, and inspiring for future advances in medicine. The recent approval of CRISPR-Cas9-based gene therapy to treat patients with severe sickle cell anemia and transfusion-dependent β-thalassemia has renewed hope for treating other hematologic diseases, including patients with a germline predisposition to hematologic malignancies, who would benefit greatly from the development of CRISPR-inspired gene therapies. The purpose of this paper is three-fold: first, a chronological description of the history of CRISPR-Cas9-sgRNA-based gene editing; second, a brief description of the current state of clinical research in hematologic diseases, including selected applications in treating hematologic diseases with CRISPR-based gene therapy, preceded by a brief description of the current tools being used in clinical genome editing; and third, a presentation of the current progress in gene therapies in inherited hematologic diseases and bone marrow failure syndromes, to hopefully stimulate efforts towards developing these therapies for patients with inherited bone marrow failure syndromes and other inherited conditions with a germline predisposition to hematologic malignancies.}, }
@article {pmid39062568, year = {2024}, author = {Chernyi, N and Gavrilova, D and Saruhanyan, M and Oloruntimehin, ES and Karabelsky, A and Bezsonov, E and Malogolovkin, A}, title = {Recent Advances in Gene Therapy for Hemophilia: Projecting the Perspectives.}, journal = {Biomolecules}, volume = {14}, number = {7}, pages = {}, doi = {10.3390/biom14070854}, pmid = {39062568}, issn = {2218-273X}, support = {none//Academic Leadership Program Priority 2030 by the Federal State Autonomous Educational Institution of Higher Education I.M. Sechenov, First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University)/ ; none//The grant of the state program of the &#xab;Sirius&#xbb; Federal Territory &#xab;Scientific and technological development of the &#xab;Sirius&#xbb; Federal Territory&#xbb;/ ; }, abstract = {One of the well-known X-linked genetic disorders is hemophilia, which could be hemophilia A as a result of a mutation in the F8 (factor VIII) gene or hemophilia B as a result of a mutation in the F9 (factor IX) gene, leading to insufficient levels of the proteins essential for blood coagulation cascade. In patients with severe hemophilia, factor VIII or factor IX activities in the blood plasma are considerably low, estimated to be less than 1%. This is responsible for spontaneous or post-traumatic bleeding episodes, or both, leading to disease complications and death. Current treatment of hemophilia relies on the prevention of bleeding, which consists of expensive lifelong replacement infusion therapy of blood plasma clotting factors, their recombinant versions, or therapy with recombinant monoclonal antibodies. Recently emerged gene therapy approaches may be a potential game changer that could reshape the therapeutic outcomes of hemophilia A or B using a one-off vector in vivo delivery and aim to achieve long-term endogenous expression of factor VIII or IX. This review examines both traditional approaches to the treatment of hemophilia and modern methods, primarily focusing on gene therapy, to update knowledge in this area. Recent technological advances and gene therapeutics in the pipeline are critically reviewed and summarized. We consider gene therapy to be the most promising method as it may overcome the problems associated with more traditional treatments, such as the need for constant and expensive infusions and the presence of an immune response to the antibody drugs used to treat hemophilia.}, }
@article {pmid39062198, year = {2024}, author = {Costache, C and Colosi, I and Toc, DA and Daian, K and Damacus, D and Botan, A and Toc, A and Pana, AG and Panaitescu, P and Neculicioiu, V and Schiopu, P and Iordache, D and Butiuc-Keul, A}, title = {CRISPR-Cas System, Antimicrobial Resistance, and Enterococcus Genus-A Complicated Relationship.}, journal = {Biomedicines}, volume = {12}, number = {7}, pages = {}, doi = {10.3390/biomedicines12071625}, pmid = {39062198}, issn = {2227-9059}, abstract = {(1) Background: The rise in antibiotic resistant bacteria poses a significant threat to public health worldwide, necessitating innovative solutions. This study explores the role of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in the context of antibiotic resistance among different species from the Enterococcus genus. (2) Methods: The genomes of Enterococcus included in the study were analyzed using CRISPRCasFinder to distinguish between CRISPR-positive (level 4 CRISPR) and CRISPR-negative genomes. Antibiotic resistance genes were identified, and a comparative analysis explored potential associations between CRISPR presence and antibiotic resistance profiles in Enterococcus species. (3) Results: Out of ten antibiotic resistance genes found in Enterococcus species, only one, the efmA gene, showed a strong association with CRISPR-negative isolates, while the others did not significantly differ between CRISPR-positive and CRISPR-negative Enterococcus genomes. (4) Conclusion: These findings indicate that the efmA gene may be more prevalent in CRISPR-negative Enterococcus genomes, and they may contribute to a better understanding of the molecular mechanisms underlying the acquisition of antibiotic resistance genes in Enterococcus species.}, }
@article {pmid39061110, year = {2024}, author = {McFarlane, GR and Polanco, JVC and Bogema, D}, title = {CRISPR-Cas guide RNA indel analysis using CRISPResso2 with Nanopore sequencing data.}, journal = {BMC research notes}, volume = {17}, number = {1}, pages = {205}, pmid = {39061110}, issn = {1756-0500}, abstract = {OBJECTIVE: Insertion and deletion (indel) analysis of CRISPR-Cas guide RNAs (gRNAs) is crucial in gene editing to assess gRNA efficiency and indel frequency. This study evaluates the utility of CRISPResso2 with Oxford Nanopore sequencing data (nCRISPResso2) for gRNA indel screening, compared to two common Sanger sequencing-based methods, TIDE and ICE. To achieve this, sheep and horse fibroblasts were transfected with Cas9 and a gRNA targeting the myostatin (MSTN) gene. DNA was subsequently extracted, and PCR products exceeding 600 bp were sequenced using both Sanger and Nanopore sequencing. Indel profiling was then conducted using TIDE, ICE, and nCRISPResso2.<br><br>RESULTS: Comparison revealed close correspondence in indel formation among methods. For the sheep MSTN gRNA, indel percentages were 52%, 58%, and 64% for TIDE, ICE, and nCRISPResso2, respectively. Horse MSTN gRNA showed 81%, 87%, and 86% edited amplicons for TIDE, ICE, and nCRISPResso2. The frequency of each type of indel was also comparable among the three methods, with nCRISPResso2 and ICE aligning the closest. nCRISPResso2 offers a viable alternative for CRISPR-Cas gRNA indel screening, especially with large amplicons unsuitable for Illumina sequencing. CRISPResso2's compatibility with Nanopore data enables cost-effective and efficient indel profiling, yielding results comparable to common Sanger sequencing-based methods.}, }
@article {pmid39060676, year = {2024}, author = {Gill, J}, title = {Dissecting the mechanism of CRISPR-Cas technologies to design efficient biotechnologies.}, journal = {Nature structural &amp; molecular biology}, volume = {}, number = {}, pages = {}, pmid = {39060676}, issn = {1545-9985}, }
@article {pmid39059527, year = {2024}, author = {Badwal, AK and Singh, S}, title = {A comprehensive review on the current status of CRISPR based clinical trials for rare diseases.}, journal = {International journal of biological macromolecules}, volume = {}, number = {}, pages = {134097}, doi = {10.1016/j.ijbiomac.2024.134097}, pmid = {39059527}, issn = {1879-0003}, abstract = {A considerable fraction of population in the world suffers from rare diseases. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its related Cas proteins offer a modern form of curative gene therapy for treating the rare diseases. Hereditary transthyretin amyloidosis, hereditary angioedema, duchenne muscular dystrophy and Rett syndrome are a few examples of such rare diseases. CRISPR/Cas9, for example, has been used in the treatment of β-thalassemia and sickle cell disease (Frangoul et al., 2021; Pavani et al., 2021) [1,2]. Neurological diseases such as Huntington's have also been focused in some studies involving CRISPR/Cas (Yang et al., 2017; Yan et al., 2023) [3,4]. Delivery of these biologicals via vector and non vector mediated methods depends on the type of target cells, characteristics of expression, time duration of expression, size of foreign genetic material etc. For instance, retroviruses find their applicability in case of ex vivo delivery in somatic cells due to their ability to integrate in the host genome. These have been successfully used in gene therapy involving X-SCID patients although, incidence of inappropriate activation has been reported. On the other hand, ex vivo gene therapy for β-thalassemia involved use of BB305 lentiviral vector for high level expression of CRISPR biological in HSCs. The efficacy and safety of these biologicals will decide their future application as efficient genome editing tools as they go forward in further stages of human clinical trials. This review focuses on CRISPR/Cas based therapies which are at various stages of clinical trials for treatment of rare diseases and the constraints and ethical issues associated with them.}, }
@article {pmid39056796, year = {2024}, author = {Choi, W and Cha, S and Kim, K}, title = {Navigating the CRISPR/Cas Landscape for Enhanced Diagnosis and Treatment of Wilson's Disease.}, journal = {Cells}, volume = {13}, number = {14}, pages = {}, doi = {10.3390/cells13141214}, pmid = {39056796}, issn = {2073-4409}, support = {NRF- 2023R1A2C2004222, RS-2023-00220894, and RS-2023-00261905//The Bio &amp; Medical Technology Development Program of the National Research Foundation (NRF) of Korea/ ; }, mesh = {*Hepatolenticular Degeneration/genetics/therapy/diagnosis ; Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Gene Editing/methods ; Copper-Transporting ATPases/genetics/metabolism ; Animals ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system continues to evolve, thereby enabling more precise detection and repair of mutagenesis. The development of CRISPR/Cas-based diagnosis holds promise for high-throughput, cost-effective, and portable nucleic acid screening and genetic disease diagnosis. In addition, advancements in transportation strategies such as adeno-associated virus (AAV), lentiviral vectors, nanoparticles, and virus-like vectors (VLPs) offer synergistic insights for gene therapeutics in vivo. Wilson's disease (WD), a copper metabolism disorder, is primarily caused by mutations in the ATPase copper transporting beta (ATP7B) gene. The condition is associated with the accumulation of copper in the body, leading to irreversible damage to various organs, including the liver, nervous system, kidneys, and eyes. However, the heterogeneous nature and individualized presentation of physical and neurological symptoms in WD patients pose significant challenges to accurate diagnosis. Furthermore, patients must consume copper-chelating medication throughout their lifetime. Herein, we provide a detailed description of WD and review the application of novel CRISPR-based strategies for its diagnosis and treatment, along with the challenges that need to be overcome.}, }
@article {pmid39056772, year = {2024}, author = {Bailey, JK and Ma, D and Clegg, DO}, title = {Initial Characterization of WDR5B Reveals a Role in the Proliferation of Retinal Pigment Epithelial Cells.}, journal = {Cells}, volume = {13}, number = {14}, pages = {}, doi = {10.3390/cells13141189}, pmid = {39056772}, issn = {2073-4409}, support = {N/A//Garland Initiative for Vision/ ; DR1-01444//California Institute for Regenerative Medicine/ ; FA1-00616//California Institute for Regenerative Medicine/ ; TG2-01151//California Institute for Regenerative Medicine/ ; CL1-00521//California Institute for Regenerative Medicine/ ; N/A//Breaux Foundation/ ; Wynn-Gund Translational Research Acceleration Program/FFB/Foundation Fighting Blindness/United States ; }, mesh = {Humans ; *Retinal Pigment Epithelium/metabolism/cytology ; *Cell Proliferation ; CRISPR-Cas Systems/genetics ; Epithelial Cells/metabolism ; Cell Differentiation ; Intracellular Signaling Peptides and Proteins/metabolism/genetics ; Cell Line ; Animals ; Gene Editing ; }, abstract = {The chromatin-associated protein WDR5 has been widely studied due to its role in histone modification and its potential as a pharmacological target for the treatment of cancer. In humans, the protein with highest sequence homology to WDR5 is encoded by the retrogene WDR5B, which remains unexplored. Here, we used CRISPR-Cas9 genome editing to generate WDR5B knockout and WDR5B-FLAG knock-in cell lines for further characterization. In contrast to WDR5, WDR5B exhibits low expression in pluripotent cells and is upregulated upon neural differentiation. Loss or shRNA depletion of WDR5B impairs cell growth and increases the fraction of non-viable cells in proliferating retinal pigment epithelial (RPE) cultures. CUT&amp;RUN chromatin profiling in RPE and neural progenitors indicates minimal WDR5B enrichment at established WDR5 binding sites. These results suggest that WDR5 and WDR5B exhibit several divergent biological properties despite sharing a high degree of sequence homology.}, }
@article {pmid39056771, year = {2024}, author = {Na, DH and Cui, S and Fang, X and Lee, H and Eum, SH and Shin, YJ and Lim, SW and Yang, CW and Chung, BH}, title = {Advancements in Research on Genetic Kidney Diseases Using Human-Induced Pluripotent Stem Cell-Derived Kidney Organoids.}, journal = {Cells}, volume = {13}, number = {14}, pages = {}, doi = {10.3390/cells13141190}, pmid = {39056771}, issn = {2073-4409}, mesh = {Humans ; *Organoids/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; *Kidney Diseases/genetics/pathology ; *Kidney/pathology ; Animals ; CRISPR-Cas Systems/genetics ; }, abstract = {Genetic or hereditary kidney disease stands as a pivotal cause of chronic kidney disease (CKD). The proliferation and widespread utilization of DNA testing in clinical settings have notably eased the diagnosis of genetic kidney diseases, which were once elusive but are now increasingly identified in cases previously deemed CKD of unknown etiology. However, despite these diagnostic strides, research into disease pathogenesis and novel drug development faces significant hurdles, chiefly due to the dearth of appropriate animal models and the challenges posed by limited patient cohorts in clinical studies. Conversely, the advent and utilization of human-induced pluripotent stem cells (hiPSCs) offer a promising avenue for genetic kidney disease research. Particularly, the development of hiPSC-derived kidney organoid systems presents a novel platform for investigating various forms of genetic kidney diseases. Moreover, the integration of the CRISPR/Cas9 technique into this system holds immense potential for efficient research on genetic kidney diseases. This review aims to explore the applications of in vitro kidney organoids generated from hiPSCs in the study of diverse genetic kidney diseases. Additionally, it will delve into the limitations of this research platform and outline future perspectives for advancing research in this crucial area.}, }
@article {pmid39056004, year = {2024}, author = {Ghaffarian, S and Panahi, B}, title = {Occurrence and diversity pattern of CRISPR-Cas systems in Acetobacter genus provides insights on adaptive defense mechanisms against to invasive DNAs.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1357156}, doi = {10.3389/fmicb.2024.1357156}, pmid = {39056004}, issn = {1664-302X}, abstract = {The Acetobacter genus is primarily known for its significance in acetic acid production and its application in various industrial processes. This study aimed to shed light on the prevalence, diversity, and functional implications of CRISPR-Cas systems in the Acetobacter genus using a genome mining approach. The investigation analyzed the CRISPR-Cas architectures and components of 34 Acetobacter species, as well as the evolutionary strategies employed by these bacteria in response to phage invasion and foreign DNA. Furthermore, phylogenetic analysis based on CAS1 protein sequences was performed to gain insights into the evolutionary relationships among Acetobacter strains, with an emphasis on the potential of this protein for genotyping purposes. The results showed that 15 species had orphan, while20 species had complete CRISPR-Cas systems, resulting in an occurrence rate of 38% for complete systems in Acetobacter strains. The predicted complete CRISPR-Cas systems were categorized into I-C, I-F, I-E, and II-C subtypes, with subtype I-E being the most prevalent in Acetobacter. Additionally, spacer homology analysis revealed against such the dynamic interaction between Acetobacter strains and foreign invasive DNAs, emphasizing the pivotal role of CRISPR-Cas systems in defending against such invasions. Furthermore, the investigation of the secondary structures of CRISPR arrays revealed the conserved patterns within subtypes despite variations in repeat sequences. The exploration of protospacer adjacent motifs (PAMs) identified distinct recognition motifs in the flanking regions of protospacers. In conclusion, this research not only contributes to the growing body of knowledge on CRISPR-Cas systems but also establishes a foundation for future studies on the adaptive defense mechanisms of Acetobacter. The findings provide valuable insights into the intricate interplay between bacteria and phages, with implications for industrial applications and potential biotechnological advancements.}, }
@article {pmid39055984, year = {2024}, author = {Kumar Sachan, RS and Choudhary, A and Devgon, I and Karnwal, A and Al-Tawaha, ARMS and Malik, T}, title = {Bibliometric analysis on CRISPR/Cas: a potential Sherlock Holmes for disease detection.}, journal = {Frontiers in molecular biosciences}, volume = {11}, number = {}, pages = {1383268}, doi = {10.3389/fmolb.2024.1383268}, pmid = {39055984}, issn = {2296-889X}, abstract = {CRISPR has revolutionized illness detection by using precision gene editing to identify specific sequences in recent years. Using the Scopus database, this study performs a comprehensive bibliometric analysis, looking at academic papers on CRISPR that were published between 1992 and 2023. After screening a dataset of 1407 articles using Zotero, trends in annual publishing, citation patterns, author affiliations, and keyword co-occurrence are revealed using analysis tools such as VOSviewer, RStudio, and MS Excel. According to the report, there was only one CRISPR publication in 1992. By 2017, there were a meager 64 papers. Nonetheless, there is a notable upsurge between 2018 and 2023. Leading nations involved in CRISPR-based illness detection research include Germany, the United States, China, India, and the United Kingdom. Chongqing University Three Gorges Hospital, Chongqing University Medical University, and Chongqing University Bioengineering College are a few of the top institutions. With the greatest publication numbers (1688 and 1616) and strong total link strengths (TLS) of 42 and 77, respectively, authors Liu, C., and Li, Y., stand out. The field with the greatest citation counts as of 2023 is Broughton's 2020 study on CRISPR-based SARS-CoV-2 detection in Nature Biotechnology, with 1598 citations. Biosensors and Bioelectronics comprise 14.99% of papers. Researchers, decision-makers, and interested parties can use this thorough summary to help them make well-informed decisions about future CRISPR-based disease detection studies.}, }
@article {pmid39054353, year = {2024}, author = {Lee, CY and Kim, H and Degani, I and Lee, H and Sandoval, A and Nam, Y and Pascavis, M and Park, HG and Randall, T and Ly, A and Castro, CM and Lee, H}, title = {Empowering the on-site detection of nucleic acids by integrating CRISPR and digital signal processing.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6271}, pmid = {39054353}, issn = {2041-1723}, support = {5R25CA174650//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; }, mesh = {Humans ; *Uterine Cervical Neoplasms/diagnosis/virology/genetics ; Female ; *Papillomavirus Infections/diagnosis/virology ; CRISPR-Cas Systems/genetics ; DNA, Viral/genetics ; Papillomaviridae/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Signal Processing, Computer-Assisted ; Cervix Uteri/virology ; }, abstract = {Addressing the global disparity in cancer care necessitates the development of rapid and affordable nucleic acid (NA) testing technologies. This need is particularly critical for cervical cancer, where molecular detection of human papillomavirus (HPV) has emerged as an accurate screening method. However, implementing this transition in low- and middle-income countries has been challenging due to the high costs and centralized facilities required for current NA tests. Here, we present CreDiT (CRISPR Enhanced Digital Testing) for on-site NA detection. The CreDiT platform integrates i) a one-pot CRISPR strategy that simultaneously amplifies both target NAs and analytical signals and ii) a robust fluorescent detection based on digital communication (encoding/decoding) technology. These features enable a rapid assay (<35 minutes) in a single streamlined workflow. We demonstrate the sensitive detection of cell-derived HPV DNA targets down to single copies and accurate identification of HPV types in clinical cervical brushing specimens (n = 121).}, }
@article {pmid39052485, year = {2024}, author = {Choi, H and Shin, H and Kim, CY and Park, J and Kim, H}, title = {Highly efficient CRISPR/Cas9-RNP mediated CaPAD1 editing in protoplasts of three pepper (Capsicum annuum L.) cultivars.}, journal = {Plant signaling &amp; behavior}, volume = {19}, number = {1}, pages = {2383822}, doi = {10.1080/15592324.2024.2383822}, pmid = {39052485}, issn = {1559-2324}, mesh = {*Capsicum/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Protoplasts/metabolism ; *Ribonucleoproteins/metabolism/genetics ; Plant Proteins/genetics/metabolism ; }, abstract = {Parthenocarpy, characterized by seedless fruit development without pollination or fertilization, offers the advantage of consistent fruit formation, even under challenging conditions such as high temperatures. It can be induced by regulating auxin homeostasis; PAD1 (PARENTAL ADVICE-1) is an inducer of parthenocarpy in Solanaceae plants. However, precise editing of PAD1 is not well studied in peppers. Here, we report a highly efficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) for CaPAD1 editing in three valuable cultivars of pepper (Capsicum annuum L.): Dempsey, a gene-editable bell pepper; C15, a transformable commercial inbred line; and Younggo 4, a Korean landrace. To achieve the seedless pepper trait under high temperatures caused by unstable climate change, we designed five single guide RNAs (sgRNAs) targeting the CaPAD1 gene. We evaluated the in vitro on-target activity of the RNP complexes in three cultivars. Subsequently, we introduced five CRISPR/Cas9-RNP complexes into protoplasts isolated from three pepper leaves and compared indel frequencies and patterns through targeted deep sequencing analyses. We selected two sgRNAs, sgRNA2 and sgRNA5, which had high in vivo target efficiencies for the CaPAD1 gene across the three cultivars and were validated as potential off-targets in their genomes. These findings are expected to be valuable tools for developing new seedless pepper cultivars through precise molecular breeding of recalcitrant crops in response to climate change.}, }
@article {pmid39051216, year = {2024}, author = {Krishna, L and Prashant, A and Kumar, YH and Paneyala, S and Patil, SJ and Ramachandra, SC and Vishwanath, P}, title = {Molecular and Biochemical Therapeutic Strategies for Duchenne Muscular Dystrophy.}, journal = {Neurology international}, volume = {16}, number = {4}, pages = {731-760}, doi = {10.3390/neurolint16040055}, pmid = {39051216}, issn = {2035-8385}, abstract = {Significant progress has been achieved in understanding Duchenne muscular dystrophy (DMD) mechanisms and developing treatments to slow disease progression. This review article thoroughly assesses primary and secondary DMD therapies, focusing on innovative modalities. The primary therapy addresses the genetic abnormality causing DMD, specifically the absence or reduced expression of dystrophin. Gene replacement therapies, such as exon skipping, readthrough, and gene editing technologies, show promise in restoring dystrophin expression. Adeno-associated viruses (AAVs), a recent advancement in viral vector-based gene therapies, have shown encouraging results in preclinical and clinical studies. Secondary therapies aim to maintain muscle function and improve quality of life by mitigating DMD symptoms and complications. Glucocorticoid drugs like prednisone and deflazacort have proven effective in slowing disease progression and delaying loss of ambulation. Supportive treatments targeting calcium dysregulation, histone deacetylase, and redox imbalance are also crucial for preserving overall health and function. Additionally, the review includes a detailed table of ongoing and approved clinical trials for DMD, exploring various therapeutic approaches such as gene therapies, exon skipping drugs, utrophin modulators, anti-inflammatory agents, and novel compounds. This highlights the dynamic research field and ongoing efforts to develop effective DMD treatments.}, }
@article {pmid39051081, year = {2024}, author = {Zheng, M and Liu, Y and Liu, J and Kang, Q and Wang, T}, title = {[Effect of deletion of protein 4.1R on proliferation, apoptosis and glycolysis of hepatocyte HL-7702 cells].}, journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University}, volume = {44}, number = {7}, pages = {1355-1360}, doi = {10.12122/j.issn.1673-4254.2024.07.15}, pmid = {39051081}, issn = {1673-4254}, mesh = {*Apoptosis ; *Glycolysis ; *Cell Proliferation ; Humans ; *Hepatocytes/metabolism/cytology ; Cell Line ; AMP-Activated Protein Kinases/metabolism/genetics ; CRISPR-Cas Systems ; Glucose/metabolism ; Signal Transduction ; }, abstract = {OBJECTIVE: To explore the effects of deletion of protein 4.1R on hepatocyte proliferation, apoptosis, and glycolysis and the molecular mechanisms.<br><br>METHODS: A 4.1R[-/-] HL-7702 cell line was constructed using CRISPR/Cas9 technique, and with 4.1R[+/+]HL-7702 cells as the control, its proliferative capacity and cell apoptosis were assessed using CCK-8 assay, EdU-488 staining, flow cytometry and Annexin V-FITC/PI staining at 24, 48, 72 h of cell culture. The changes in glucose uptake, lactate secretion, ATP production and pH value of the culture supernatant of 4.1R[-/-] HL-7702 cells were determined. The mRNA expressions of the key regulatory enzymes HK2, PFKL, PKM2 and LDHA in glycolysis were detected with qRT-PCR, and the protein expressions of AMPK, p-AMPK, Raptor and p-Raptor were determined using Western blotting.<br><br>RESULTS: Western blotting and sequencing analysis both confirmed the successful construction of 4.1R[-/-] HL-7702 cell line. Compared with the wild-type cells, 4.1R[-/-] HL-7702 cells exhibited a lowered proliferative activity with increased cell apoptosis. The deletion of protein 4.1R also resulted in significantly decreased glucose uptake, lactate secretion and ATP production of the cells and increased pH value of the cell culture supernatant. qRT-PCR showed significantly decreased mRNA expressions of the key regulatory enzymes in glycolysis in 4.1R[-/-] HL-7702 cells. Compared with those in HL-7702 cells, the expression levels of AMPK and Raptor proteins were decreased while the expression levels of p-AMPK and p-Raptor proteins increased significantly in 4.1R[-/-] HL-7702 cells.<br><br>CONCLUSION: Deletion of protein 4.1R in HL-7702 cells results in reduced proliferative capacity, increased apoptosis and suppression of glycolysis, and this regulatory mechanism is closely related with the activation of the downstream AMPK-mTORC1 signaling pathway.}, }
@article {pmid39049493, year = {2024}, author = {Yao, Q and Shen, R and Shao, Y and Tian, Y and Han, P and Zhang, X and Zhu, JK and Lu, Y}, title = {Efficient and multiplex gene upregulation in plants through CRISPR/Cas-mediated knock-in of enhancers.}, journal = {Molecular plant}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.molp.2024.07.009}, pmid = {39049493}, issn = {1752-9867}, abstract = {Gene upregulation through genome editing is important for plant research and breeding. Targeted insertion of short transcriptional enhancers (STEs) into gene promoters may offer a universal solution akin to transgene-mediated overexpression, while avoiding the drawbacks associated with transgenesis. Here, we introduce an "in-locus activation" technique in rice that leverages specifically screened STEs for refined, heritable, and multiplexed gene upregulation. To address the scarcity of potent enhancers, we developed a large-scale mining approach and discovered a suite of STEs capable of enhancing gene expression in rice protoplasts. The in-locus integration of these STEs into eight rice genes resulted in substantial transcriptional enhancements, with up to 869.1-fold increases in the edited plants. Employing a variety of STEs, we achieved delicate control of gene expression, enabling the fine-tuning of key phenotypic traits such as plant height. Our approach also enabled efficient multiplexed gene upregulation, with up to four genes simultaneously activated, significantly enhancing the nicotinamide mononucleotide (NMN) metabolic pathway. Importantly, heritability studies from the T0 to T3 generations confirmed the stable and heritable nature of STE-driven gene activation. Coupled with our STE-mining technique, in-locus activation holds great promise to make gene upregulation a major application of genome editing in plant research and breeding.}, }
@article {pmid39048937, year = {2024}, author = {Aghaali, Z and Naghavi, MR}, title = {Developing benzylisoquinoline alkaloid-enriched opium poppy via CRISPR-directed genome editing: A review.}, journal = {BMC plant biology}, volume = {24}, number = {1}, pages = {700}, pmid = {39048937}, issn = {1471-2229}, mesh = {*Papaver/genetics/metabolism ; *Benzylisoquinolines/metabolism ; *Gene Editing ; *CRISPR-Cas Systems ; Metabolic Engineering/methods ; Genome, Plant ; }, abstract = {Among plant-derived secondary metabolites are benzylisoquinoline alkaloids (BIAs) that play a vital role in medicine. The most conspicuous BIAs frequently found in opium poppy are morphine, codeine, thebaine, papaverine, sanguinarine, and noscapine. BIAs have provided abundant clinically useful drugs used in the treatment of various diseases and ailments With an increasing demand for these herbal remedies, genetic improvement of poppy plants appears to be essential to live up to the expectations of the pharmaceutical industry. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9), the field of metabolic engineering has undergone a paradigm shift in its approach due to its appealing attributes, such as the transgene-free editing capability, precision, selectivity, robustness, and versatility. The potentiality of the CRISPR system for manipulating metabolic pathways in opium poppy was demonstrated, but further investigations regarding the use of CRISPR in BIA pathway engineering should be undertaken to develop opium poppy into a bioreactor synthesizing BIAs at the industrial-scale levels. In this regard, the recruitment of RNA-guided genome editing for knocking out miRNAs, flower responsible genes, genes involved in competitive pathways, and base editing are described. The approaches presented here have never been suggested or applied in opium poppy so far.}, }
@article {pmid39048715, year = {2024}, author = {Cao, Y and Li, X and Pan, Y and Wang, H and Yang, S and Hong, L and Ye, L}, title = {CRISPR-based genetic screens advance cancer immunology.}, journal = {Science China. Life sciences}, volume = {}, number = {}, pages = {}, pmid = {39048715}, issn = {1869-1889}, abstract = {CRISPR technologies have revolutionized research areas ranging from fundamental science to translational medicine. CRISPR-based genetic screens offer a powerful platform for unbiased screening in various fields, such as cancer immunology. Immune checkpoint blockade (ICB) therapy has been shown to strongly affect cancer treatment. However, the currently available ICBs are limited and do not work in all cancer patients. Pooled CRISPR screens enable the identification of previously unknown immune regulators that can regulate T-cell activation, cytotoxicity, persistence, infiltration into tumors, cytokine secretion, memory formation, T-cell metabolism, and CD4[+] T-cell differentiation. These novel targets can be developed as new immunotherapies or used with the current ICBs as new combination therapies that may yield synergistic efficacy. Here, we review the progress made in the development of CRISPR technologies, particularly technological advances in CRISPR screens and their application in novel target identification for immunotherapy.}, }
@article {pmid39048567, year = {2024}, author = {Hatanaka, F and Suzuki, K and Shojima, K and Yu, J and Takahashi, Y and Sakamoto, A and Prieto, J and Shokhirev, M and Nuñez Delicado, E and Rodriguez Esteban, C and Izpisua Belmonte, JC}, title = {Therapeutic strategy for spinal muscular atrophy by combining gene supplementation and genome editing.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {6191}, pmid = {39048567}, issn = {2041-1723}, mesh = {*Muscular Atrophy, Spinal/therapy/genetics ; Animals ; *Gene Editing/methods ; *Survival of Motor Neuron 1 Protein/genetics ; *CRISPR-Cas Systems ; Mice ; *Genetic Therapy/methods ; Disease Models, Animal ; Humans ; Motor Neurons/metabolism/pathology ; Mutation ; Male ; Female ; }, abstract = {Defect in the SMN1 gene causes spinal muscular atrophy (SMA), which shows loss of motor neurons, muscle weakness and atrophy. While current treatment strategies, including small molecules or viral vectors, have shown promise in improving motor function and survival, achieving a definitive and long-term correction of SMA's endogenous mutations and phenotypes remains highly challenging. We have previously developed a CRISPR-Cas9 based homology-independent targeted integration (HITI) strategy, enabling unidirectional DNA knock-in in both dividing and non-dividing cells in vivo. In this study, we demonstrated its utility by correcting an SMA mutation in mice. When combined with Smn1 cDNA supplementation, it exhibited long-term therapeutic benefits in SMA mice. Our observations may provide new avenues for the long-term and efficient treatment of inherited diseases.}, }
@article {pmid39047725, year = {2024}, author = {Zhang, C and Chen, F and Wang, F and Xu, H and Xue, J and Li, Z}, title = {Mechanisms for HNH-mediated target DNA cleavage in type I CRISPR-Cas systems.}, journal = {Molecular cell}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.molcel.2024.06.033}, pmid = {39047725}, issn = {1097-4164}, abstract = {The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-F[HNH] and I-E[HNH] Cascade complexes at different states are reported. In type I-F[HNH] Cascade, Cas8f[HNH] loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8f[HNH] to detach and rotate ∼150° to accommodate target ssDNA for cleavage. In type I-E[HNH] Cascade, Cas5e[HNH] domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.}, }
@article {pmid39047109, year = {2024}, author = {Liu, H and Dong, J and Duan, Z and Xia, F and Willner, I and Huang, F}, title = {Light-activated CRISPR-Cas12a for amplified imaging of microRNA in cell cycle phases at single-cell levels.}, journal = {Science advances}, volume = {10}, number = {30}, pages = {eadp6166}, doi = {10.1126/sciadv.adp6166}, pmid = {39047109}, issn = {2375-2548}, mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Cell Cycle/genetics ; *Single-Cell Analysis/methods ; Light ; MCF-7 Cells ; Microscopy, Confocal/methods ; }, abstract = {An ortho-nitrobenzyl phosphate ester-caged nucleic acid hairpin structure coupled to the CRISPR-Cas12a complex is introduced as a functional reaction module for the light-induced activation of the CRISPR-Cas12a (LAC12a) machinery toward the amplified fluorescence detection of microRNA-21 (miRNA-21). The LAC12a machinery is applied for the selective, in vitro sensing of miRNA-21 and for the intracellular imaging of miRNA-21 in different cell lines. The LAC12a system is used to image miRNA-21 in different cell cycle phases of MCF-7 cells. Moreover, the LAC12a machinery integrated in cells enables the two-photon laser confocal microscopy-assisted, light-stimulated spatiotemporal, selective activation of the CRISPR-Cas12a miRNA-21 imaging machinery at the single-cell level and the evaluation of relative expression levels of miRNA-21 at distinct cell cycle phases. The method is implemented to map the distribution of cell cycle phases in an array of single cells.}, }
@article {pmid39046587, year = {2024}, author = {Zhou, J and Chen, X and Li, SM}, title = {Construction of an expression platform for fungal secondary metabolite biosynthesis in Penicillium crustosum.}, journal = {Applied microbiology and biotechnology}, volume = {108}, number = {1}, pages = {427}, pmid = {39046587}, issn = {1432-0614}, support = {Li844/11-1//Deutsche Forschungsgemeinschaft/ ; INST 160/620-1//Deutsche Forschungsgemeinschaft/ ; 202206170029//Chinese Government Scholarship/ ; }, mesh = {*Penicillium/genetics/metabolism ; *Secondary Metabolism/genetics ; *CRISPR-Cas Systems ; Aspergillus nidulans/genetics/metabolism ; Polyketide Synthases/genetics/metabolism ; Multigene Family ; Gene Targeting/methods ; Gene Expression Regulation, Fungal ; Fungal Proteins/genetics/metabolism ; Biosynthetic Pathways/genetics ; Metabolic Engineering/methods ; Gene Expression ; }, abstract = {Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes. KEY POINTS: Construction of a highly efficient Penicillium crustosum heterologous expression host Reduction of secondary metabolite background by genetic dereplication strategy Integration of wA site to provide an alternative host besides Aspergillus nidulans.}, }
@article {pmid38969245, year = {2024}, author = {Li, Q and Yu, H and Li, Q}, title = {Dual sgRNA-directed tyrosinases knockout using CRISPR/Cas9 technology in Pacific oyster (Crassostrea gigas) reveals their roles in early shell calcification.}, journal = {Gene}, volume = {927}, number = {}, pages = {148748}, doi = {10.1016/j.gene.2024.148748}, pmid = {38969245}, issn = {1879-0038}, mesh = {Animals ; *CRISPR-Cas Systems ; *Crassostrea/genetics/growth &amp; development/metabolism ; *Animal Shells/metabolism/growth &amp; development ; *Gene Knockout Techniques/methods ; *Calcification, Physiologic/genetics ; *Calcium Carbonate/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Biomineralization/genetics ; }, abstract = {Biomineralization processes in bivalves, particularly the initial production of molecular components (such as matrix deposition and calcification) in the early stages of shell development are highly complex and well-organized. This study investigated the temporal dynamics of organic matrix and calcium carbonate (CaCO3) deposition in Pacific oysters (Crassostrea gigas) across various development stages. The shell-field initiated matrix secretion during the gastrula stage. Subsequent larval development triggered central shell-field calcification, accompanied by expansion of the calcium ring from its interior to the periphery. Notably, the expression patterns of CgTyrp-2 and CgTyr closely correlated with matrix deposition and calcification during early developmental stages, with peak expression occurring in oyster's gastrula and D-veliger stages. Subsequently, the CRISPR/Cas9 system was utilized to knock out CgTyrp-2 and CgTyr with more distinct phenotypic alterations observed when both genes were concurrently knocked out. The relative gene expression was analyzed post-knockout, indicating that the knockout of CgTyr or CgTyrp-2 led to reduced expression of CgChs1, along with increased expression of CgChit4. Furthermore, when dual-sgRNAs were employed to knockout CgTyrp-2, a large deletion (2 kb) within the CgTyrp-2 gene was identified. In summary, early shell formation in C. gigas is the result of a complex interplay of multiple molecular components with CgTyrp-2 and CgTyr playing key roles in regulating CaCO3 deposition.}, }
@article {pmid38945310, year = {2024}, author = {Wang, Y and Jiang, H and Li, M and Xu, Z and Xu, H and Chen, Y and Chen, K and Zheng, W and Lin, W and Liu, Z and Lin, Z and Zhang, M}, title = {Delivery of CRISPR/Cas9 system by AAV as vectors for gene therapy.}, journal = {Gene}, volume = {927}, number = {}, pages = {148733}, doi = {10.1016/j.gene.2024.148733}, pmid = {38945310}, issn = {1879-0038}, mesh = {*Dependovirus/genetics ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; Humans ; *Genetic Vectors/genetics ; Animals ; *Gene Editing/methods ; }, abstract = {The adeno-associated virus (AAV) is a defective single-stranded DNA virus with the simplest structure reported to date. It constitutes a capsid protein and single-stranded DNA. With its high transduction efficiency, low immunogenicity, and tissue specificity, it is the most widely used and promising gene therapy vector. The clustered regularly interspaced short palindromic sequence (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system is an emerging technology that utilizes cas9 nuclease to specifically recognize and cleave target genes under the guidance of small guide RNA and realizes gene editing through homologous directional repair and non-homologous recombination repair. In recent years, an increasing number of animal experiments and clinical studies have revealed the great potential of AAV as a vector to deliver the CRISPR/cas9 system for treating genetic diseases and viral infections. However, the immunogenicity, toxicity, low transmission efficiency in brain and ear tissues, packaging size limitations of AAV, and immunogenicity and off-target effects of Cas9 protein pose several clinical challenges. This research reviews the role, challenges, and countermeasures of the AAV-CRISPR/cas9 system in gene therapy.}, }
@article {pmid38897560, year = {2024}, author = {Li, J and Wu, T and Wang, J and Chen, Y and Zhang, W and Cai, L and Lai, S and Hu, K and Jin, W}, title = {Dual auxotrophy coupled red labeling strategy for efficient genome editing in Saccharomyces cerevisiae.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {173}, number = {}, pages = {103910}, doi = {10.1016/j.fgb.2024.103910}, pmid = {38897560}, issn = {1096-0937}, mesh = {*Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Homologous Recombination/genetics ; Genome, Fungal/genetics ; Gene Knockout Techniques/methods ; Saccharomyces cerevisiae Proteins/genetics ; }, abstract = {The homologous recombination strategy has a long history of editing Saccharomyces cerevisiae target genes. The application of CRISPR/Cas9 strategy to editing target genes in S. cerevisiae has also received a lot of attention in recent years. All findings seem to indicate that editing relevant target genes in S. cerevisiae is an extremely easy event. In this study, we systematically analyzed the advantages and disadvantages of homologous recombination (HR) strategy, CRISPR/Cas9 strategy, and CRISPR/Cas9 combined homology-mediated repair (CRISPR/Case9-HDR) strategy in knocking out BY4742 ade2. Our data showed that when the ade2 was knocked out by HR strategy, a large number of clones appeared to be off-target, and 10 %-80 % of the so-called knockout clones obtained were heteroclones. When the CRISPR/Cas9 strategy was applied, 60% of clones were off-target and the rest were all heteroclones. Interestingly, most of the cells were edited successfully, but at least 60 % of the clones were heteroclones, when the CRISPR/Cas9-HDR strategy was employed. Our results clearly showed that the emergence of heteroclone seems inevitable regardless of the strategies used for editing BY4742 ade2. Given the characteristics of BY4742 defective in ade2 showing red on the YPD plate, we attempted to build an efficient yeast gene editing strategy, in which the CRISPR/Cas9 combines homology-mediated repair template carrying an ade2 expression cassette, BY4742(ade2Δ0) as the start strain. We used this strategy to successfully achieve 100 % knockout efficiency of trp1, indicating that technical challenges of how to easily screen out pure knockout clones without color phenotype have been solved. Our data showed in this study not only establishes an efficient yeast gene knockout strategy with dual auxotrophy coupled red labeling but also provides new ideas and references for the knockout of target genes in the monokaryotic mycelium of macrofungi.}, }
@article {pmid38878988, year = {2024}, author = {Xiao, Z and Yang, W and Yang, A and Deng, L and Geng, R and Xiang, H and Kong, W and Jiang, C and Li, X and Chen, Z and Gao, Q}, title = {CRISPR/Cas9-mediated knockout of NtMYC2a gene involved in resistance to bacterial wilt in tobacco.}, journal = {Gene}, volume = {927}, number = {}, pages = {148622}, doi = {10.1016/j.gene.2024.148622}, pmid = {38878988}, issn = {1879-0038}, mesh = {*Nicotiana/genetics/microbiology ; *Disease Resistance/genetics ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology/genetics ; *Ralstonia solanacearum/pathogenicity ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Cyclopentanes/metabolism ; Signal Transduction ; Oxylipins/metabolism ; Plants, Genetically Modified/genetics ; }, abstract = {MYC2 is a class of bHLH family transcription factors and a major regulatory factor in the JA signaling pathway, and its molecular function in tobacco has not been reported. In this study, CRISPR/Cas9-mediated MYC2 gene NtMYC2a knockout mutants at tobacco was obtained and its agronomic traits, disease resistance, and chemical composition were identified. Comparing with the WT, the leaf width of the KO-NtMYC2a was narrowed, the nornicotine content and mecamylamine content increased significantly and the resistance to Ralstonia solanacearum significantly decreased. The transcriptome sequencing results showed that DEGs related to immunity, signal transduction and growth and development were enriched between KO-NtMYC2a and WT. NtJAR1 and NtCOI1 in KO-NtMYC2a were down-regulated to regulating the JA signaling pathway, result in a significant decrease in tobacco's resistance to R. solanacearum. Our research provides theoretical support for the functional research of MYC2 and the study of the mechanism of tobacco bacterial wilt resistance.}, }
@article {pmid38842342, year = {2024}, author = {McGinn, J and Wen, A and Edwards, DL and Brinkley, DM and Lamason, RL}, title = {An expanded genetic toolkit for inducible expression and targeted gene silencing in Rickettsia parkeri.}, journal = {Journal of bacteriology}, volume = {206}, number = {7}, pages = {e0009124}, pmid = {38842342}, issn = {1098-5530}, support = {R01 AI155489/AI/NIAID NIH HHS/United States ; TB200032//U.S. Department of Defense (DOD)/ ; DRG-2396-20//Damon Runyon Cancer Research Foundation (DRCRF)/ ; }, mesh = {*Rickettsia/genetics ; *CRISPR-Cas Systems ; *Gene Silencing ; *Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics/metabolism ; Promoter Regions, Genetic ; Humans ; }, abstract = {Pathogenic species within the Rickettsia genus are transmitted to humans through arthropod vectors and cause a spectrum of diseases ranging from mild to life-threatening. Despite rickettsiae posing an emerging global health risk, the genetic requirements of their infectious life cycles remain poorly understood. A major hurdle toward building this understanding has been the lack of efficient tools for genetic manipulation, owing to the technical difficulties associated with their obligate intracellular nature. To this end, we implemented the Tet-On system to enable conditional gene expression in Rickettsia parkeri. Using Tet-On, we show inducible expression of antibiotic resistance and a fluorescent reporter. We further used this inducible promoter to screen the ability of R. parkeri to express four variants of the catalytically dead Cas9 (dCas9). We demonstrate that all four dCas9 variants can be expressed in R. parkeri and used for CRISPR interference (CRISPRi)-mediated targeted gene knockdown. We show targeted knockdown of an antibiotic resistance gene as well as the endogenous virulence factor sca2. Altogether, we have developed systems for inducible gene expression and CRISPRi-mediated gene knockdown for the first time in rickettsiae, laying the groundwork for more scalable, targeted mechanistic investigations into their infectious life cycles.IMPORTANCEThe spotted fever group of Rickettsia contains vector-borne pathogenic bacteria that are neglected and emerging threats to public health. Due to the obligate intracellular nature of rickettsiae, the development of tools for genetic manipulation has been stunted, and the molecular and genetic underpinnings of their infectious lifecycle remain poorly understood. Here, we expand the genetic toolkit by introducing systems for conditional gene expression and CRISPR interference (CRISPRi)-mediated gene knockdown. These systems allow for relatively easy manipulation of rickettsial gene expression. We demonstrate the effectiveness of these tools by disrupting the intracellular life cycle using CRISPRi to deplete the sca2 virulence factor. These tools will be crucial for building a more comprehensive and detailed understanding of rickettsial biology and pathogenesis.}, }
@article {pmid38838774, year = {2024}, author = {Dey, D and Chakravarti, R and Bhattacharjee, O and Majumder, S and Chaudhuri, D and Ahmed, KT and Roy, D and Bhattacharya, B and Arya, M and Gautam, A and Singh, R and Gupta, R and Ravichandiran, V and Chattopadhyay, D and Ghosh, A and Giri, K and Roy, S and Ghosh, D}, title = {A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.}, journal = {The Journal of biological chemistry}, volume = {300}, number = {7}, pages = {107439}, pmid = {38838774}, issn = {1083-351X}, mesh = {Humans ; *CRISPR-Cas Systems ; *Base Pair Mismatch ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; DNA/chemistry/metabolism ; Molecular Dynamics Simulation ; RNA/chemistry/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry ; HEK293 Cells ; Gene Editing ; }, abstract = {The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.}, }
@article {pmid38838231, year = {2024}, author = {Mo, J and Wermeling, F and Nilsson, G and Dahlin, JS}, title = {CRISPR/Cas9-mediated gene disruption determines the roles of MITF and CITED2 in human mast cell differentiation.}, journal = {Blood advances}, volume = {8}, number = {15}, pages = {3941-3945}, doi = {10.1182/bloodadvances.2023012279}, pmid = {38838231}, issn = {2473-9537}, mesh = {Humans ; *Microphthalmia-Associated Transcription Factor/genetics/metabolism ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems ; *Mast Cells/metabolism ; Trans-Activators/genetics/metabolism ; }, }
@article {pmid38762829, year = {2024}, author = {Yousefi, Y and Nejati, R and Eslahi, A and Alizadeh, F and Farrokhi, S and Asoodeh, A and Mojarrad, M}, title = {Enhancing Temozolomide (TMZ) chemosensitivity using CRISPR-dCas9-mediated downregulation of O[6]-methylguanine DNA methyltransferase (MGMT).}, journal = {Journal of neuro-oncology}, volume = {169}, number = {1}, pages = {129-135}, pmid = {38762829}, issn = {1573-7373}, mesh = {Humans ; *Temozolomide/pharmacology ; *Antineoplastic Agents, Alkylating/pharmacology/therapeutic use ; *CRISPR-Cas Systems ; *Down-Regulation ; *Glioblastoma/genetics/drug therapy ; DNA Repair Enzymes/genetics/metabolism ; Brain Neoplasms/genetics/drug therapy/pathology ; DNA Modification Methylases/genetics/metabolism ; Tumor Suppressor Proteins/genetics/metabolism ; HEK293 Cells ; Drug Resistance, Neoplasm/genetics ; DNA Methylation/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; Cell Line, Tumor ; Promoter Regions, Genetic ; }, abstract = {PURPOSE: Glioblastoma (GBM) stands out as the most prevalent and aggressive intracranial tumor, notorious for its poor prognosis. The current standard-of-care for GBM patients involves surgical resection followed by radiotherapy, combined with concurrent and adjuvant chemotherapy using Temozolomide (TMZ). The effectiveness of TMZ primarily relies on the activity of O[6]-methylguanine DNA methyltransferase (MGMT), which removes alkyl adducts from the O[6] position of guanine at the DNA level, thereby counteracting the toxic effects of TMZ.<br><br>METHOD: In this study, we employed fusions of catalytically-inactive Cas9 (dCas9) to DNA methyltransferases (dCas9-DNMT3A) to selectively downregulation MGMT transcription by inducing methylation at MGMT promoter and K-M enhancer.<br><br>RESULT: Our findings demonstrate a significant reduction in MGMT expression, leading to intensified TMZ sensitivity in the HEK293T cell line.<br><br>CONCLUSION: This study serves as a proof of concept for the utilization of CRISPR-based gene suppression to overcome TMZ resistance and enhance the lethal effect of TMZ in glioblastoma tumor cells.}, }
@article {pmid38718928, year = {2024}, author = {Tonetto, E and Cucci, A and Follenzi, A and Bernardi, F and Pinotti, M and Balestra, D}, title = {DNA base editing corrects common hemophilia A mutations and restores factor VIII expression in in vitro and ex vivo models.}, journal = {Journal of thrombosis and haemostasis : JTH}, volume = {22}, number = {8}, pages = {2171-2183}, doi = {10.1016/j.jtha.2024.04.020}, pmid = {38718928}, issn = {1538-7836}, mesh = {*Factor VIII/genetics/metabolism ; Humans ; *Hemophilia A/genetics/therapy/blood ; *Gene Editing/methods ; HEK293 Cells ; *Genetic Therapy ; Endothelial Cells/metabolism ; Point Mutation ; Mutation ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Replacement and nonreplacement therapies effectively control bleeding in hemophilia A (HA) but imply lifelong interventions. Authorized gene addition therapy could provide a cure but still poses questions on durability. FVIIIgene correction would definitively restore factor (F)VIII production, as shown in animal models through nuclease-mediated homologous recombination (HR). However, low efficiency and potential off-target double-strand break still limit HR translatability.<br><br>OBJECTIVES: To correct common model single point mutations leading to severe HA through the recently developed double-strand break/HR-independent base editing (BE) and prime editing (PE) approaches.<br><br>METHODS: Screening for efficacy of BE/PE systems in HEK293T cells transiently expressing FVIII variants and validation at DNA (sequencing) and protein (enzyme-linked immunosorbent assay; activated partial thromboplastin time) level in stable clones. Evaluation of rescue in engineered blood outgrowth endothelial cells by lentiviral-mediated delivery of BE.<br><br>RESULTS: Transient assays identified the best-performing BE/PE systems for each variant, with the highest rescue of FVIII expression (up to 25% of wild-type recombinant FVIII) for the p.R2166&#x2217; and p.R2228Q mutations. In stable clones, we demonstrated that the mutation reversion on DNA (&#x223c;24%) was consistent with the rescue of FVIII secretion and activity of 20% to 30%. The lentiviral-mediated delivery of the selected BE systems was attempted in engineered blood outgrowth endothelial cells harboring the p.R2166&#x2217; and p.R2228Q variants, which led to an appreciable and dose-dependent rescue of secreted functional FVIII.<br><br>CONCLUSION: Overall data provide the first proof-of-concept for effective BE/PE-mediated correction of HA-causing mutations, which encourage studies in mouse models to develop a personalized cure for large cohorts of patients through a single intervention.}, }
@article {pmid38708901, year = {2024}, author = {Mahmood, M and Taufiq, I and Mazhar, S and Hafeez, F and Malik, K and Afzal, S}, title = {Revolutionizing personalized cancer treatment: the synergy of next-generation sequencing and CRISPR/Cas9.}, journal = {Personalized medicine}, volume = {21}, number = {3}, pages = {175-190}, doi = {10.1080/17410541.2024.2341610}, pmid = {38708901}, issn = {1744-828X}, mesh = {Humans ; *Precision Medicine/methods/trends ; *High-Throughput Nucleotide Sequencing/methods ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Mutation/genetics ; Gene Editing/methods ; }, abstract = {In the context of cancer heterogeneity, the synergistic action of next-generation sequencing (NGS) and CRISPR/Cas9 plays a promising role in the personalized treatment of cancer. NGS enables high-throughput genomic profiling of tumors and pinpoints specific mutations that primarily lead to cancer. Oncologists use this information obtained from NGS in the form of DNA profiling or RNA analysis to tailor precision strategies based on an individual's unique molecular signature. Furthermore, the CRISPR technique enables precise editing of cancer-specific mutations, allowing targeted gene modifications. Harnessing the potential insights of NGS and CRISPR/Cas9 heralds a remarkable frontier in cancer therapeutics with unprecedented precision, effectiveness and minimal off-target effects.}, }
@article {pmid38651662, year = {2024}, author = {Yao, Z and He, K and Wang, H and Feng, S and Ding, X and Xu, Y and Wang, Q and Xu, X and Wu, Q and Wang, L}, title = {Tuning the Dynamic Reaction Balance of CRISPR/Cas12a and RPA in One Pot: A Key to Switch Nucleic Acid Quantification.}, journal = {ACS sensors}, volume = {9}, number = {7}, pages = {3511-3519}, doi = {10.1021/acssensors.3c02485}, pmid = {38651662}, issn = {2379-3694}, mesh = {*CRISPR-Cas Systems/genetics ; *SARS-CoV-2/genetics ; *Nucleic Acid Amplification Techniques/methods ; COVID-19/diagnosis/virology ; Lactobacillus/genetics ; Humans ; CRISPR-Associated Proteins/genetics ; Recombinases/metabolism ; Endodeoxyribonucleases/genetics ; Bacterial Proteins ; }, abstract = {Excavating nucleic acid quantitative capabilities by combining clustered regularly interspaced short palindromic repeats (CRISPR) and isothermal amplification in one pot is of common interest. However, the mutual interference between CRISPR cleavage and isothermal amplification is the primary obstacle to quantitative detection. Though several works have demonstrated enhanced detection sensitivity by reducing the inhibition of CRISPR on amplification in one pot, few paid attention to the amplification process and even dynamic reaction processes between the two. Herein, we find that DNA quantification can be realized by regulating either recombinase polymerase amplification (RPA) efficiency or CRISPR/Cas12a cleaving efficiency (namely, tuning the dynamic reaction balance) in one pot. The sensitive quantification is realized by utilizing dual PAM-free crRNAs for CRISPR/Cas12a recognition. The varied RPA primer concentration with stabilized CRISPR systems significantly affects the amplification efficiency and quantitative performances. Alternatively, quantitative detection can also be achieved by stabilizing the amplification process while regulating the CRISPR/Cas12a concentration. The quantitative capability is proved by detecting DNA targets from Lactobacillus acetotolerans and SARS-CoV-2. The quantitative performance toward real samples is comparable to quantitative real-time PCR for detecting L. acetotolerans spiked in fermented food samples and SARS-CoV-2 clinical samples. We expect that the presented method will be a powerful tool for quantifying other nucleic acid targets.}, }
@article {pmid38402425, year = {2024}, author = {Qiu, L and Sun, M and Chen, L and Jiang, J and Fu, Z and Wang, Y and Bi, Y and Guo, Q and Bai, H and Chen, S and Gao, L and Chang, G}, title = {Iron-Confined CRISPR/Cas9-Ribonucleoprotein Delivery System for Redox-Responsive Gene Editing.}, journal = {Small (Weinheim an der Bergstrasse, Germany)}, volume = {20}, number = {30}, pages = {e2309431}, doi = {10.1002/smll.202309431}, pmid = {38402425}, issn = {1613-6829}, support = {22121003//National Natural Science Foundation of China/ ; 81930050//National Natural Science Foundation of China/ ; 32272921//National Natural Science Foundation of China/ ; 2019YFA0709200//National Key R&amp;D Program of China/ ; 2023ZD04053//Major Project of Agricultural Biological Breeding/ ; BE2022341//Jiangsu Provincial Key Research and Development Program/ ; CARS-41//Earmarked Fund for China Agriculture Research System/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Iron/chemistry ; *Oxidation-Reduction ; Humans ; *Ribonucleoproteins/metabolism/chemistry ; Liposomes/chemistry ; Gene Transfer Techniques ; CRISPR-Associated Protein 9/metabolism ; }, abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) is a promising gene editing tool to treat diseases at the genetic level. Nonetheless, the challenge of the safe and efficient delivery of CRISPR/Cas9 to host cells constrains its clinical applicability. In the current study, a facile, redox-responsive CRISPR/Cas9-Ribonucleoprotein (RNP) delivery system by combining iron-coordinated aggregation with liposomes (Fe-RNP@L) is reported. The Fe-RNP is formed by the coordination of Fe[3+] with amino and carboxyl groups of Cas9, which modifies the lipophilicity and surface charge of RNP and alters cellular uptake from primary endocytosis to endocytosis and cholesterol-dependent membrane fusion. RNP can be rapidly and reversibly released from Fe-RNP in response to glutathione without loss of structural integrity and enzymatic activity. In addition, iron coordination also improves the stability of RNP and substantially mitigates cytotoxicity. This construct enabled highly efficient cytoplasmic/nuclear delivery (≈90%) and gene-editing efficiency (≈70%) even at low concentrations. The high payload content, high editing efficiency, good stability, low immunogenicity, and ease of production and storage, highlight its potential for diverse genome editing and clinical applications.}, }
@article {pmid37977820, year = {2023}, author = {Davies, B and Zhang, G and Moralli, D and Alghadban, S and Biggs, D and Preece, C and Donnelly, P and Hinch, AG}, title = {Characterization of meiotic recombination intermediates through gene knockouts in founder hybrid mice.}, journal = {Genome research}, volume = {33}, number = {11}, pages = {2018-2027}, pmid = {37977820}, issn = {1549-5469}, support = {/WT_/Wellcome Trust/United Kingdom ; NC/R001014/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; }, mesh = {Animals ; *Meiosis/genetics ; Mice ; *Rad51 Recombinase/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; *Cell Cycle Proteins/genetics/metabolism ; *Mice, Knockout ; DNA Breaks, Double-Stranded ; Phosphate-Binding Proteins/genetics/metabolism ; Replication Protein A/metabolism/genetics ; Recombination, Genetic ; Male ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Female ; Homologous Recombination ; }, abstract = {Mammalian meiotic recombination proceeds via repair of hundreds of programmed DNA double-strand breaks, which requires choreographed binding of RPA, DMC1, and RAD51 to single-stranded DNA substrates. High-resolution in vivo binding maps of these proteins provide insights into the underlying molecular mechanisms. When assayed in F1-hybrid mice, these maps can distinguish the broken chromosome from the chromosome used as template for repair, revealing more mechanistic detail and enabling the structure of the recombination intermediates to be inferred. By applying CRISPR-Cas9 mutagenesis directly on F1-hybrid embryos, we have extended this approach to explore the molecular detail of recombination when a key component is knocked out. As a proof of concept, we have generated hybrid biallelic knockouts of Dmc1 and built maps of meiotic binding of RAD51 and RPA in them. DMC1 is essential for meiotic recombination, and comparison of these maps with those from wild-type mice is informative about the structure and timing of critical recombination intermediates. We observe redistribution of RAD51 binding and complete abrogation of D-loop recombination intermediates at a molecular level in Dmc1 mutants. These data provide insight on the configuration of RPA in D-loop intermediates and suggest that stable strand exchange proceeds via multiple rounds of strand invasion with template switching in mouse. Our methodology provides a high-throughput approach for characterization of gene function in meiotic recombination at low animal cost.}, }
@article {pmid35066025, year = {2022}, author = {Pan, X and Luo, Y and Liao, N and Zhang, Y and Xiao, M and Chen, P and Lu, C and Dong, Z}, title = {CRISPR/Cpf1 multiplex genome editing system increases silkworm tolerance to BmNPV.}, journal = {International journal of biological macromolecules}, volume = {200}, number = {}, pages = {566-573}, doi = {10.1016/j.ijbiomac.2022.01.109}, pmid = {35066025}, issn = {1879-0003}, mesh = {Animals ; *Bombyx/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Animals, Genetically Modified ; Nucleopolyhedroviruses/genetics ; }, abstract = {The CRISPR/Cas9 genome editing technology is now widely used in insect studies, but the use of CRISPR can be further increased to improve insect genome engineering. We established a direct mutation at multiple loci in several genes simultaneously used by CRISPR/Cpf1 multiplex genome editing technology to target the BmNPV genome. We constructed a transgenic line that can target the BmNPV ie-1, gp64, and DNApoly genes simultaneously, and hybridized this line with an FnCpf1 transgenic line to obtain an FnCpf1 × gNPVM binary hybrid expression system and to activate the FnCpf1 gene editing system. We showed that the multiple gene editing system introduced deletions, mutations, and insertions at three target sites, and that it did not affect the economic traits of transgenic silkworm lines. The antiviral response of multiplexed genome editing lines increased significantly, and viral gene transcription and replication were significantly affected in the transgenic silkworm lines. This study provides innovative resistance materials for silkworm breeding and also provides a simplified platform for efficient insect multi genome engineering and genetic operation.}, }
@article {pmid35041685, year = {2022}, author = {Rawsthorne-Manning, H and Calahorro, F and G Izquierdo, P and Tardy, P and Boulin, T and Holden-Dye, L and O'Connor, V and Dillon, J}, title = {Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes.}, journal = {PloS one}, volume = {17}, number = {1}, pages = {e0253351}, pmid = {35041685}, issn = {1932-6203}, mesh = {Animals ; *Caenorhabditis elegans/genetics ; *CRISPR-Cas Systems ; *Caenorhabditis elegans Proteins/genetics ; Gene Editing/methods ; Mutation ; Genotype ; Genetic Markers ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Phenotype ; }, abstract = {Multiple advances have been made to increase the efficiency of CRISPR/Cas9 editing using the model genetic organism Caenorhabditis elegans (C. elegans). Here we report on the use of co-CRISPR 'marker' genes: worms in which co-CRISPR events have occurred have overt, visible phenotypes which facilitates the selection of worms that harbour CRISPR events in the target gene. Mutation in the co-CRISPR gene is then removed by outcrossing to wild type but this can be challenging if the CRISPR and co-CRISPR gene are hard to segregate. However, segregating away the co-CRISPR modified gene can be less challenging if the worms selected appear wild type and are selected from a jackpot brood. These are broods in which a high proportion of the progeny of a single injected worm display the co-CRISPR phenotype suggesting high CRISPR efficiency. This can deliver worms that harbour the desired mutation in the target gene locus without the co-CRISPR mutation. We have successfully generated a discrete mutation in the C. elegans nlg-1 gene using this method. However, in the process of sequencing to authenticate editing in the nlg-1 gene we discovered genomic rearrangements that arise at the co-CRISPR gene unc-58 that by visual observation were phenotypically silent but nonetheless resulted in a significant reduction in motility scored by thrashing behaviour. This highlights that careful consideration of the hidden consequences of co-CRISPR mediated genetic changes should be taken before downstream analysis of gene function. Given this, we suggest sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection as part of the pipeline.}, }
@article {pmid34997243, year = {2022}, author = {de Bakker, V and Liu, X and Bravo, AM and Veening, JW}, title = {CRISPRi-seq for genome-wide fitness quantification in bacteria.}, journal = {Nature protocols}, volume = {17}, number = {2}, pages = {252-281}, pmid = {34997243}, issn = {1750-2799}, mesh = {*Streptococcus pneumoniae/genetics ; *Genetic Fitness ; *Genome, Bacterial/genetics ; *Gene Library ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {CRISPR interference (CRISPRi) is a powerful tool to link essential and nonessential genes to specific phenotypes and to explore their functions. Here we describe a protocol for CRISPRi screenings to assess genome-wide gene fitness in a single sequencing step (CRISPRi-seq). We demonstrate the use of the protocol in Streptococcus pneumoniae, an important human pathogen; however, the protocol can easily be adapted for use in other organisms. The protocol includes a pipeline for single-guide RNA library design, workflows for pooled CRISPRi library construction, growth assays and sequencing steps, a read analysis tool (2FAST2Q) and instructions for fitness quantification. We describe how to make an IPTG-inducible system with small libraries that are easy to handle and cost-effective and overcome bottleneck issues, which can be a problem when using similar, transposon mutagenesis-based methods. Ultimately, the procedure yields a fitness score per single-guide RNA target for any given growth condition. A genome-wide screening can be finished in 1 week with a constructed library. Data analysis and follow-up confirmation experiments can be completed in another 2-3 weeks.}, }
@article {pmid34939826, year = {2022}, author = {Johansen, KH}, title = {How CRISPR/Cas9 Gene Editing Is Revolutionizing T Cell Research.}, journal = {DNA and cell biology}, volume = {41}, number = {1}, pages = {53-57}, pmid = {34939826}, issn = {1557-7430}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *T-Lymphocytes/immunology/metabolism ; Humans ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows for precise gene targeting in mammalian cells, including T cells, allowing scientists to disrupt or edit specific genes of interest. This has enabled immunologists to investigate T cell functions as well as opened the path for novel therapeutics involving gene editing of T cells ex vivo before transferring these back to patients to increase T cell efficacy. This review outlines how CRISPR/Cas9 has transformed T cell research allowing immunologists to rapidly probe the roles of genes in T cells thus paving the way for novel therapeutics. Furthermore, this review describes how these tools reduce the requirement for genetic mouse models, while increasing the translational potential of T cell research.}, }
@article {pmid34902367, year = {2022}, author = {Lucero, D and Dikilitas, O and Mendelson, MM and Aligabi, Z and Islam, P and Neufeld, EB and Bansal, AT and Freeman, LA and Vaisman, B and Tang, J and Combs, CA and Li, Y and Voros, S and Kullo, IJ and Remaley, AT}, title = {Transgelin: a new gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen.}, journal = {Journal of lipid research}, volume = {63}, number = {1}, pages = {100160}, pmid = {34902367}, issn = {1539-7262}, support = {K99 HL136875/HL/NHLBI NIH HHS/United States ; }, mesh = {Humans ; *Endocytosis ; Hep G2 Cells ; *CRISPR-Cas Systems/genetics ; *Microfilament Proteins/genetics/metabolism ; *Muscle Proteins/genetics/metabolism ; Receptors, LDL/genetics/metabolism ; Lipoproteins, LDL/metabolism ; Sterol Regulatory Element Binding Protein 2/metabolism/genetics ; Genome-Wide Association Study ; }, abstract = {A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were significantly associated with elevated plasma lipids (triglycerides, total cholesterol, and LDL-C) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. In biochemical studies, TAGLN-KO HepG2 cells showed a reduction in cellular LDL uptake, as measured by flow cytometry. In confocal microscopy imaging, TAGLN-KO cells had disrupted actin filaments as well as an accumulation of LDL receptor on their surface because of decreased receptor internalization. Furthermore, TAGLN-KO cells exhibited a reduction in total and free cholesterol content, activation of SREBP2, and a compensatory increase in cholesterol biosynthesis. TAGLN deficiency also disrupted the uptake of VLDL and transferrin, other known cargoes for receptors that depend upon clathrin-mediated endocytosis. Our data suggest that TAGLN is a novel factor involved in the actin-dependent phase of clathrin-mediated endocytosis of LDL. The identification of novel genes involved in the endocytic uptake of LDL may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease.}, }
@article {pmid34849910, year = {2021}, author = {Christesen, D and Yang, YT and Chen, W and Batterham, P and Perry, T}, title = {Loss of the Dβ1 nicotinic acetylcholine receptor subunit disrupts bursicon-driven wing expansion and diminishes adult viability in Drosophila melanogaster.}, journal = {Genetics}, volume = {219}, number = {1}, pages = {}, pmid = {34849910}, issn = {1943-2631}, mesh = {Animals ; *Drosophila melanogaster/genetics/metabolism ; *Receptors, Nicotinic/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Wings, Animal/metabolism/growth &amp; development ; *Invertebrate Hormones/metabolism/genetics ; Male ; CRISPR-Cas Systems ; Neurons/metabolism ; Female ; Gene Editing ; Longevity/genetics ; }, abstract = {Cholinergic signaling dominates the insect central nervous system, contributing to numerous fundamental pathways and behavioral circuits. However, we are only just beginning to uncover the diverse roles different cholinergic receptors may play. Historically, insect nicotinic acetylcholine receptors have received attention due to several subunits being key insecticide targets. More recently, there has been a focus on teasing apart the roles of these receptors, and their constituent subunits, in native signaling pathways. In this study, we use CRISPR-Cas9 genome editing to generate germline and somatic deletions of the Dβ1 nicotinic acetylcholine receptor subunit and investigate the consequences of loss of function in Drosophila melanogaster. Severe impacts on movement, male courtship, longevity, and wing expansion were found. Loss of Dβ1 was also associated with a reduction in transcript levels for the wing expansion hormone bursicon. Neuron-specific somatic deletion of Dβ1 in bursicon-producing neurons (CCAP-GAL4) was sufficient to disrupt wing expansion. Furthermore, CCAP-GAL4-specific expression of Dβ1 in a germline deletion background was sufficient to rescue the wing phenotype, pinpointing CCAP neurons as the neuronal subset requiring Dβ1 for the wing expansion pathway. Dβ1 is a known target of multiple commercially important insecticides, and the fitness costs exposed here explain why field-isolated target-site resistance has only been reported for amino acid replacements and not loss of function. This work reveals the importance of Dβ1-containing nicotinic acetylcholine receptors in CCAP neurons for robust bursicon-driven wing expansion.}, }
@article {pmid34791245, year = {2022}, author = {Medley, JC and Hebbar, S and Sydzyik, JT and Zinovyeva, AY}, title = {Single nucleotide substitutions effectively block Cas9 and allow for scarless genome editing in Caenorhabditis elegans.}, journal = {Genetics}, volume = {220}, number = {1}, pages = {}, pmid = {34791245}, issn = {1943-2631}, support = {P20 GM103418/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; R35 GM124828/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Genome, Helminth ; }, abstract = {In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize "off-target" sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts postinjection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.}, }
@article {pmid34791226, year = {2022}, author = {Huang, MY and Joshi, MB and Boucher, MJ and Lee, S and Loza, LC and Gaylord, EA and Doering, TL and Madhani, HD}, title = {Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging.}, journal = {Genetics}, volume = {220}, number = {1}, pages = {}, pmid = {34791226}, issn = {1943-2631}, support = {F31 AI150194/AI/NIAID NIH HHS/United States ; T32 AI060537/AI/NIAID NIH HHS/United States ; F32 AI152270/AI/NIAID NIH HHS/United States ; T32 HL007185/HL/NHLBI NIH HHS/United States ; R01 AI100272/AI/NIAID NIH HHS/United States ; R01 AI135012/AI/NIAID NIH HHS/United States ; R01 AI027243/AI/NIAID NIH HHS/United States ; R01 AI087794/AI/NIAID NIH HHS/United States ; T32 GM007067/GM/NIGMS NIH HHS/United States ; }, mesh = {*Cryptococcus neoformans/genetics/pathogenicity ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Deletion ; Recombinational DNA Repair ; CRISPR-Associated Protein 9/genetics/metabolism ; Homologous Recombination ; Animals ; Fungal Proteins/genetics/metabolism ; }, abstract = {Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized (Cno) Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (Cno CAS9 or "CnoCAS9") reliably enabled genome editing in the widely used KN99α C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.}, }
@article {pmid34791209, year = {2022}, author = {Lutz, S and Van Dyke, K and Feraru, MA and Albert, FW}, title = {Multiple epistatic DNA variants in a single gene affect gene expression in trans.}, journal = {Genetics}, volume = {220}, number = {1}, pages = {}, pmid = {34791209}, issn = {1943-2631}, support = {R35 GM124676/GM/NIGMS NIH HHS/United States ; }, mesh = {*Saccharomyces cerevisiae/genetics ; *Epistasis, Genetic ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; *Gene Expression Regulation, Fungal ; Genetic Variation ; CRISPR-Cas Systems ; }, abstract = {DNA variants that alter gene expression in trans are important sources of phenotypic variation. Nevertheless, the identity of trans-acting variants remains poorly understood. Single causal variants in several genes have been reported to affect the expression of numerous distant genes in trans. Whether these simple molecular architectures are representative of trans-acting variation is unknown. Here, we studied the large RAS signaling regulator gene IRA2, which contains variants with extensive trans-acting effects on gene expression in the yeast Saccharomyces cerevisiae. We used systematic CRISPR-based genome engineering and a sensitive phenotyping strategy to dissect causal variants to the nucleotide level. In contrast to the simple molecular architectures known so far, IRA2 contained at least seven causal nonsynonymous variants. The effects of these variants were modulated by nonadditive, epistatic interactions. Two variants at the 5'-end affected gene expression and growth only when combined with a third variant that also had no effect in isolation. Our findings indicate that the molecular basis of trans-acting genetic variation may be considerably more complex than previously appreciated.}, }
@article {pmid34788305, year = {2021}, author = {Ahmed, S and Roy, MC and Al Baki, MA and Jung, JK and Lee, D and Kim, Y}, title = {CRISPR/Cas9 mutagenesis against sex pheromone biosynthesis leads to loss of female attractiveness in Spodoptera exigua, an insect pestt.}, journal = {PloS one}, volume = {16}, number = {11}, pages = {e0259322}, pmid = {34788305}, issn = {1932-6203}, mesh = {Animals ; *Sex Attractants/metabolism/biosynthesis ; Female ; *Spodoptera/physiology/genetics ; *CRISPR-Cas Systems ; Male ; Sexual Behavior, Animal ; Mutagenesis ; Fatty Acid Desaturases/genetics/metabolism ; }, abstract = {Virgin female moths are known to release sex pheromones to attract conspecific males. Accurate sex pheromones are required for their chemical communication. Sex pheromones of Spodoptera exigua, a lepidopteran insect, contain unsaturated fatty acid derivatives having a double bond at the 12th carbon position. A desaturase of S. exigua (SexiDES5) was proposed to have dual functions by forming double bonds at the 11th and 12th carbons to synthesize Z9,E12-tetradecedienoic acid, which could be acetylated to be a main sex pheromone component Z9,E12-tetradecenoic acetate (Z9E12-14:Ac). A deletion of SexiDES5 using CRISPR/Cas9 was generated and inbred to obtain homozygotes. Mutant females could not produce Z9E12-14:Ac along with Z9-14:Ac and Z11-14:Ac. Subsequently, pheromone extract of mutant females did not induce a sensory signal in male antennae. They failed to induce male mating behavior including hair pencil erection and orientation. In the field, these mutant females did not attract any males while control females attracted males. These results indicate that SexiDES5 can catalyze the desaturation at the 11th and 12th positions to produce sex pheromone components in S. exigua. This study also suggests an application of the genome editing technology to insect pest control by generating non-attractive female moths.}, }
@article {pmid34562094, year = {2021}, author = {Hasebe, F and Yuba, H and Hashimoto, T and Saito, K and Funa, N and Shoji, T}, title = {CRISPR/Cas9-mediated disruption of the PYRROLIDINE KETIDE SYNTHASE gene reduces the accumulation of tropane alkaloids in Atropa belladonna hairy roots.}, journal = {Bioscience, biotechnology, and biochemistry}, volume = {85}, number = {12}, pages = {2404-2409}, doi = {10.1093/bbb/zbab165}, pmid = {34562094}, issn = {1347-6947}, support = {19H05652//Japan Society for the Promotion of Science/ ; }, mesh = {*Plant Roots/metabolism/genetics ; *CRISPR-Cas Systems ; *Tropanes/metabolism ; *Atropa belladonna/metabolism/genetics/enzymology ; Gene Editing ; Pyrrolidines/metabolism ; }, abstract = {Tropane alkaloids, including clinically important hyoscyamine and scopolamine, are produced in the roots of medicinal plant species, such as Atropa belladonna, from the Solanaceae family. Recent molecular and genomic approaches have advanced our understanding of the metabolic enzymes involved in tropane alkaloid biosynthesis. A noncanonical type III polyketide synthase, pyrrolidine ketide synthase (PYKS) catalyzes a two-step decarboxylative reaction, which involves imine-ketide condensation indispensable to tropane skeleton construction. In this study, we generated pyks mutant A. belladonna hairy roots via CRISPR/Cas9-mediated genome editing and analyzed the metabolic consequences of the loss of PYKS activity on tropane alkaloids, providing insights into a crucial role of the scaffold-forming reaction in the biosynthetic pathway.}, }
@article {pmid34531148, year = {2021}, author = {Dai, Z and Li, R and Hou, Y and Li, Q and Zhao, K and Li, T and Li, MJ and Wu, X}, title = {Inducible CRISPRa screen identifies putative enhancers.}, journal = {Journal of genetics and genomics = Yi chuan xue bao}, volume = {48}, number = {10}, pages = {917-927}, doi = {10.1016/j.jgg.2021.06.012}, pmid = {34531148}, issn = {1673-8527}, mesh = {Animals ; *Enhancer Elements, Genetic/genetics ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; Mouse Embryonic Stem Cells/metabolism/cytology ; Doxycycline/pharmacology ; Cell Differentiation/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Chromatin/genetics/metabolism ; }, abstract = {Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates. However, the identification of enhancers in native cellular contexts still remains a challenge. Here, we develop an inducible CRISPR activation (CRISPRa) system by transgenic expression of doxycycline (Dox)-inducible dCas9-VPR in mouse embryonic stem cells (iVPR ESC). With this line, a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible, reversible, and Dox concentration-dependent manner. Taking advantage of this system, we induce tiled CRISPRa across genomic regions (105 kilobases) surrounding T (Brachyury), one of the key mesodermal development regulator genes. Moreover, we identify several CRISPRa-responsive elements with chromatin features of putative enhancers, including a region the homologous sequence in which humans harbors a body height risk variant. Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation. Therefore, our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers.}, }
@article {pmid34508260, year = {2021}, author = {Aregger, M and Xing, K and Gonatopoulos-Pournatzis, T}, title = {Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening.}, journal = {Nature protocols}, volume = {16}, number = {10}, pages = {4722-4765}, pmid = {34508260}, issn = {1750-2799}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Gene Deletion ; Endonucleases/metabolism/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Endodeoxyribonucleases/genetics/metabolism ; Animals ; Bacterial Proteins/genetics/metabolism ; }, abstract = {CRISPR-based forward genetic screening represents a powerful approach for the systematic characterization of gene function. Recent efforts have been directed toward establishing CRISPR-based tools for the programmable delivery of combinatorial genetic perturbations, most of which are mediated by a single nuclease and the expression of structurally identical guide backbones from two promoters. In contrast, we have developed CHyMErA (Cas hybrid for multiplexed editing and screening applications), which is based on the co-expression of Cas9 and Cas12a nucleases in conjunction with a hybrid guide RNA (hgRNA) engineered by the fusion of Cas9 and Cas12a guides and expressed from a single U6 promoter. CHyMErA is suitable for the high-throughput deletion of genetic segments including the excision of individual exons. Furthermore, CHyMErA enables the concomitant targeting of two or more genes and can thus be used for the systematic mapping of genetic interactions in mammalian cells. CHyMErA can also be applied for the perturbation of paralogous gene pairs, thereby allowing the capturing of phenotypic roles that would otherwise be masked because of genetic redundancy. Here, we provide instructions for the cloning of hgRNA screening libraries and individual hgRNA constructs and offer guidelines for designing and performing combinatorial pooled genetic screens using CHyMErA. Starting with the generation of Cas9- and Cas12a-expressing cell lines, CHyMErA screening can be implemented within 15-20 weeks.}, }
@article {pmid34508259, year = {2021}, author = {Ward, HN and Aregger, M and Gonatopoulos-Pournatzis, T and Billmann, M and Ohsumi, TK and Brown, KR and Blencowe, BJ and Moffat, J and Myers, CL}, title = {Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline.}, journal = {Nature protocols}, volume = {16}, number = {10}, pages = {4766-4798}, pmid = {34508259}, issn = {1750-2799}, support = {R01 HG005084/HG/NHGRI NIH HHS/United States ; R01 HG005853/HG/NHGRI NIH HHS/United States ; MOP-142375//CIHR/Canada ; FDN-148434//CIHR/Canada ; }, mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Software ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computational Biology/methods ; Gene Editing/methods ; Animals ; }, abstract = {The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.}, }
@article {pmid34411150, year = {2021}, author = {Waldmann, L and Leyhr, J and Zhang, H and Öhman-Mägi, C and Allalou, A and Haitina, T}, title = {The broad role of Nkx3.2 in the development of the zebrafish axial skeleton.}, journal = {PloS one}, volume = {16}, number = {8}, pages = {e0255953}, pmid = {34411150}, issn = {1932-6203}, mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Homeodomain Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Bone Development/genetics ; Gene Expression Regulation, Developmental ; Phenotype ; Bone and Bones/metabolism ; }, abstract = {The transcription factor Nkx3.2 (Bapx1) is an important chondrocyte maturation inhibitor. Previous Nkx3.2 knockdown and overexpression studies in non-mammalian gnathostomes have focused on its role in primary jaw joint development, while the function of this gene in broader skeletal development is not fully described. We generated a mutant allele of nkx3.2 in zebrafish with CRISPR/Cas9 and applied a range of techniques to characterize skeletal phenotypes at developmental stages from larva to adult, revealing loss of the jaw joint, fusions in bones of the occiput, morphological changes in the Weberian apparatus, and the loss or deformation of bony elements derived from basiventral cartilages of the vertebrae. Axial phenotypes are reminiscent of Nkx3.2 knockout in mammals, suggesting that the function of this gene in axial skeletal development is ancestral to osteichthyans. Our results highlight the broad role of nkx3.2 in zebrafish skeletal development and its context-specific functions in different skeletal elements.}, }
@article {pmid34390914, year = {2021}, author = {Shin, U and Nakhro, K and Oh, CK and Carrington, B and Song, H and Varshney, GK and Kim, Y and Song, H and Jeon, S and Robbins, G and Kim, S and Yoon, S and Choi, YJ and Kim, YJ and Burgess, S and Kang, S and Sood, R and Lee, Y and Myung, K}, title = {Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes.}, journal = {DNA repair}, volume = {107}, number = {}, pages = {103173}, doi = {10.1016/j.dnarep.2021.103173}, pmid = {34390914}, issn = {1568-7856}, mesh = {*Zebrafish/genetics ; Animals ; *DNA Repair ; *CRISPR-Cas Systems ; *Phenotype ; *Zebrafish Proteins/genetics/metabolism ; *Mutation ; DNA Damage ; Gene Editing ; }, abstract = {A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental approaches using animal model systems. Zebrafish has become a powerful vertebrate genetic model system with availability due to the ease of genome editing and large-scale phenotype screening. Here, we generated zebrafish mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. Large-scale phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.}, }
@article {pmid34362681, year = {2021}, author = {Li, Y and Li, W and Li, J}, title = {The CRISPR/Cas9 revolution continues: From base editing to prime editing in plant science.}, journal = {Journal of genetics and genomics = Yi chuan xue bao}, volume = {48}, number = {8}, pages = {661-670}, doi = {10.1016/j.jgg.2021.05.001}, pmid = {34362681}, issn = {1673-8527}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plants/genetics ; *Genome, Plant/genetics ; }, abstract = {The ability to precisely inactivate or modify genes in model organisms helps us understand the mysteries of life. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a revolutionary technology that could generate targeted mutants, has facilitated notable advances in plant science. Genome editing with CRISPR/Cas9 has gained great popularity and enabled several technical breakthroughs. Herein, we briefly introduce the CRISPR/Cas9, with a focus on the latest breakthroughs in precise genome editing (e.g., base editing and prime editing), and we summarize various platforms that developed to increase the editing efficiency, expand the targeting scope, and improve the specificity of base editing in plants. In addition, we emphasize the recent applications of these technologies to plants. Finally, we predict that CRISPR/Cas9 and CRISPR/Cas9-based genome editing will continue to revolutionize plant science and provide technical support for sustainable agricultural development.}, }
@article {pmid34215255, year = {2021}, author = {Bensalel, J and Xu, H and Lu, ML and Capobianco, E and Wei, J}, title = {RNA-seq analysis reveals significant transcriptome changes in huntingtin-null human neuroblastoma cells.}, journal = {BMC medical genomics}, volume = {14}, number = {1}, pages = {176}, pmid = {34215255}, issn = {1755-8794}, support = {R01 EB025819/EB/NIBIB NIH HHS/United States ; R21 NS111202/NS/NINDS NIH HHS/United States ; }, mesh = {Humans ; *Huntingtin Protein/genetics ; *Neuroblastoma/genetics/pathology/metabolism ; *Transcriptome ; Cell Line, Tumor ; RNA-Seq ; Gene Expression Profiling ; Sequence Analysis, RNA ; CRISPR-Cas Systems ; Gene Knockout Techniques ; }, abstract = {BACKGROUND: Huntingtin (Htt) protein is the product of the gene mutated in Huntington's disease (HD), a fatal, autosomal dominant, neurodegenerative disorder. Normal Htt is essential for early embryogenesis and the development of the central nervous system. However, the role of Htt in adult tissues is less defined. Following the recent promising clinical trial in which both normal and mutant Htt mRNA were knocked down in HD patients, there is an urgent need to fully understand the molecular consequences of knocking out/down Htt in adult tissues. Htt has been identified as an important transcriptional regulator. Unbiased investigations of transcriptome changes with RNA-sequencing (RNA-Seq) have been done in multiple cell types in HD, further confirming that transcriptional dysregulation is a central pathogenic mechanism in HD. However, there is lack of direct understanding of the transcriptional regulation by normal Htt.<br><br>METHODS: To investigate the transcriptional role of normal Htt, we first knocked out Htt in the human neuroblastoma SH-SY5Y cell line using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) gene editing approach. We then performed RNA-seq analysis on Htt-null and wild type SH-SY5Y cells to probe the global transcriptome changes induced by Htt deletion.<br><br>RESULTS: In general, Htt has a widespread effect on gene transcription. Functional analysis of the differentially expressed genes (DEGs) using various bioinformatic tools revealed irregularities in pathways related to cell communication and signaling, and more specifically those related to neuron development, neurotransmission and synaptic signaling. We further examined the transcription factors that may regulate these DEGs. Consistent with the disrupted pathways associated with cellular development, we showed that Htt-null cells exhibited slower cell proliferation than wild type cells. We finally validated some of the top DEGS with quantitative RT-PCR.<br><br>CONCLUSIONS: The widespread transcriptome changes in Htt-null cells could be directly caused by the loss of Htt-mediated transcriptional regulation or due to the secondary consequences of disruption in the gene regulatory network. Our study therefore provides valuable information about key genes associated with Htt-mediated transcription and improves our understanding of the molecular mechanisms underlying the cellular functions of normal and mutant Htt.}, }
@article {pmid34046943, year = {2021}, author = {Shen, J and Lu, Z and Wang, J and Hao, Q and Ji, W and Wu, Y and Peng, H and Zhao, R and Yang, J and Li, Y and Shi, Z and Zhang, X}, title = {Traceable Nano-Biohybrid Complexes by One-Step Synthesis as CRISPR-Chem Vectors for Neurodegenerative Diseases Synergistic Treatment.}, journal = {Advanced materials (Deerfield Beach, Fla.)}, volume = {33}, number = {27}, pages = {e2101993}, doi = {10.1002/adma.202101993}, pmid = {34046943}, issn = {1521-4095}, support = {52073287//National Natural Science Foundation of China/ ; L172046//Beijing Natural Science Foundation/ ; 2192057//Beijing Natural Science Foundation/ ; 31771095//National Natural Science Foundation of China/ ; 21875254//National Natural Science Foundation of China/ ; 21905283//National Natural Science Foundation of China/ ; 22075289//National Natural Science Foundation of China/ ; 32071391//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; Neurodegenerative Diseases/therapy ; Amyloid beta-Peptides/metabolism/chemistry ; Alzheimer Disease/therapy ; Gene Editing/methods ; Humans ; Brain/metabolism/pathology ; Plasmids/genetics/metabolism ; Mice, Transgenic ; Nanoparticles/chemistry ; }, abstract = {Abnormal protein aggregations are essential pathological features of neurodegenerative diseases. Eliminating while inhibiting the regeneration of these protein aggregates is considered an effective treatment strategy. Herein, the CRISPR/Cas9 gene-editing tool is employed to inhibit the regeneration of disease-related proteins, while chemical drugs are applied to eliminate the proteins that are produced. To efficiently deliver CRISPR-chem drugs into brain lesions, traceable nano-biohybrid complexes (F-TBIO) are constructed by one-step synthesis and CRISPR/Cas9 plasmids (CF-TBIO) are loaded in a controllable manner. CF-TBIO can knock out the BACE1 gene and reduce the burden of amyloid-β, and thereby significantly improve the cognitive abilities of 2xTg-AD mice. In particular, by prolonging the dosing interval, the pathological damage and behavioral abilities of 2xTg-AD mice are still significantly improved. During the therapeutic process, CF-TBIO with a high relaxation rate provides accurate imaging signals in the complex brain physiological environment. The finding shows that CF-TBIO has great potential to serve as a CRISPR-chem drug-delivery platform for neurodegenerative diseases therapy.}, }
@article {pmid33967119, year = {2021}, author = {Nishida, K and Tsuchiya, K and Obinata, H and Onodera, S and Honda, Y and Lai, YC and Haruta, N and Sugimoto, A}, title = {Expression Patterns and Levels of All Tubulin Isotypes Analyzed in GFP Knock-In C. elegans Strains.}, journal = {Cell structure and function}, volume = {46}, number = {1}, pages = {51-64}, pmid = {33967119}, issn = {1347-3700}, support = {P40 OD010440/OD/NIH HHS/United States ; }, mesh = {Animals ; *Caenorhabditis elegans/metabolism/genetics ; *Tubulin/metabolism/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Protein Isoforms/metabolism/genetics ; Caenorhabditis elegans Proteins/metabolism/genetics ; Microtubules/metabolism ; Gene Knock-In Techniques ; CRISPR-Cas Systems/genetics ; }, abstract = {Most organisms have multiple α- and β-tubulin isotypes that likely contribute to the diversity of microtubule (MT) functions. To understand the functional differences of tubulin isotypes in Caenorhabditis elegans, which has nine α-tubulin isotypes and six β-tubulin isotypes, we systematically constructed null mutants and GFP-fusion strains for all tubulin isotypes with the CRISPR/Cas9 system and analyzed their expression patterns and levels in adult hermaphrodites. Four isotypes-α-tubulins TBA-1 and TBA-2 and β-tubulins TBB-1 and TBB-2-were expressed in virtually all tissues, with a distinct tissue-specific spectrum. Other isotypes were expressed in specific tissues or cell types at significantly lower levels than the broadly expressed isotypes. Four isotypes (TBA-5, TBA-6, TBA-9, and TBB-4) were expressed in different subsets of ciliated sensory neurons, and TBB-4 was inefficiently incorporated into mitotic spindle MTs. Taken together, we propose that MTs in C. elegans are mainly composed of four broadly expressed tubulin isotypes and that incorporation of a small amount of tissue-specific isotypes may contribute to tissue-specific MT properties. These newly constructed strains will be useful for further elucidating the distinct roles of tubulin isotypes.Key words: tubulin isotypes, microtubules, C. elegans.}, }
@article {pmid33939982, year = {2021}, author = {Green, CD and Weigel, C and Oyeniran, C and James, BN and Davis, D and Mahawar, U and Newton, J and Wattenberg, BW and Maceyka, M and Spiegel, S}, title = {CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism.}, journal = {Journal of lipid research}, volume = {62}, number = {}, pages = {100082}, pmid = {33939982}, issn = {1539-7262}, support = {R01 AI125433/AI/NIAID NIH HHS/United States ; R01 HL131340/HL/NHLBI NIH HHS/United States ; P30 CA016059/CA/NCI NIH HHS/United States ; R25 GM090084/GM/NIGMS NIH HHS/United States ; K99 HD096117/HD/NICHD NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; *Sphingolipids/metabolism ; Humans ; *Membrane Proteins/genetics/metabolism/deficiency ; Gene Deletion ; Ceramides/metabolism ; Animals ; Serine C-Palmitoyltransferase/genetics/metabolism ; }, abstract = {The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. In addition, ORMDL-TKO but not ORMDL3-KO dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids.}, }
@article {pmid33850267, year = {2021}, author = {Choi, M and Yun, JY and Kim, JH and Kim, JS and Kim, ST}, title = {The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana.}, journal = {Scientific reports}, volume = {11}, number = {1}, pages = {8087}, pmid = {33850267}, issn = {2045-2322}, mesh = {*Arabidopsis/genetics ; *Gene Editing/methods ; *Promoter Regions, Genetic ; *CRISPR-Cas Systems ; *Cytosine/metabolism ; Plants, Genetically Modified/genetics ; Arabidopsis Proteins/genetics ; Animals ; Ribosomal Proteins/genetics ; Rats ; }, abstract = {CRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strateg