@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.},
}

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

@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.},
}

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

@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.},
}

*Antidotes/pharmacology
Humans
*CRISPR-Cas Systems
*Computer Simulation
Drug Evaluation, Preclinical/methods
Animals

@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.},
}

*Solanum lycopersicum/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Transcriptional Activation/genetics
Genome, Plant/genetics
Plants, Genetically Modified/genetics

@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.},
}

Humans
*Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics
*Deubiquitinating Enzymes/metabolism/genetics
Mutagenesis
CRISPR-Cas Systems
HEK293 Cells
Signal Transduction
Ubiquitination

@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.},
}

*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

@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 & 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.

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.

RESULTS: The sensitivity of the ERA-CRISPR/Cas12a fluorescence and lateral flow systems was 9 copies/μL and 90 copies/μL, 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.

DISCUSSION: The ERA-CRISPR/Cas12a system offers a rapid, sensitive, and specific method that presents a novel approach to laboratory diagnosis of M. tuberculosis.},
}

*Mycobacterium tuberculosis/genetics/isolation & 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

@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.},
}

*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

@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.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
Epigenome
Animals
Transcription Activator-Like Effector Nucleases/genetics/metabolism
DNA Breaks, Double-Stranded
Genetic Therapy/methods

@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 & 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.},
}

*Mycoplasma pneumoniae/genetics/isolation & purification
Humans
*CRISPR-Cas Systems/genetics
Nucleic Acid Amplification Techniques/methods
Pneumonia, Mycoplasma/diagnosis/microbiology

@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.},
}

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

@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.},
}

*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

@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.},
}

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

@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.},
}

*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

@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.},
}

Animals
Male
Mice
*Spermatogenesis/genetics
*Meiosis/genetics
*Azoospermia/genetics/pathology
Infertility, Male/genetics
Sequence Deletion
Humans
Testis/metabolism/pathology
CRISPR-Cas Systems

@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.},
}

*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

@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.},
}

Humans
*Genetic Therapy/methods/ethics
*CRISPR-Cas Systems
Genetic Diseases, Inborn/therapy

@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.},
}

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

@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.

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.

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.},
}

*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

@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.},
}

*Neurospora crassa/genetics
*CRISPR-Cas Systems
*Mutagenesis
*Gene Editing/methods
Fungal Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Fungal

@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.},
}

*Fusarium/metabolism/genetics
*Betacyanins/metabolism/chemistry
*Fungal Proteins/genetics/metabolism
*Fermentation
Metabolic Engineering
CRISPR-Cas Systems
Food Coloring Agents/metabolism/chemistry

@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.},
}

Animals
*Histones/metabolism/genetics
*Drosophila melanogaster/genetics
*Drosophila Proteins/genetics/metabolism
Gene Editing/methods
Multigene Family
CRISPR-Cas Systems

@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 & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI156203//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI124302//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI158076//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI124402//U.S. Department of Health & 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.},
}

*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

@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.},
}

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

@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.},
}

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

@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.},
}

*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

@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 & 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.},
}

*Allelopathy
*Crops, Agricultural/genetics/metabolism
Biotechnology
Crop Production/methods
CRISPR-Cas Systems
Weed Control/methods

@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.},
}

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

@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.},
}

*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

@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.},
}

*CRISPR-Cas Systems
*Integrases/genetics/metabolism
Animals
*Zebrafish/genetics
*Genotype
Gene Transfer Techniques
Genotyping Techniques
Transgenes
Genes, Reporter
Gene Editing/methods
Animals, Genetically Modified

@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.},
}

*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

@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 & 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.},
}

*Glycine max/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Genome, Plant
Genomics/methods
Plant Breeding/methods
Polymorphism, Single Nucleotide
Genome-Wide Association Study

@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.},
}

*Gene Editing
*Edible Grain/genetics
*Stress, Physiological/genetics
*CRISPR-Cas Systems
Climate Change
Genome, Plant

@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.},
}

*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

@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 & 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.},
}

*Saccharum/virology
*CRISPR-Cas Systems/genetics
*Plant Diseases/virology
*Nucleic Acid Amplification Techniques/methods
*Luteoviridae/genetics/isolation & purification
*Plant Leaves/virology
Molecular Diagnostic Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@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.},
}

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

@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 ; },
}

*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.},
}

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

@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.

METHODS: Thus, we generate a 4-gene (GGTA1, CMAH, β4GalNT2, and CIITA) knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously.

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 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.

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.},
}

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

@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 ; },
}

*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.},
}

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

@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.},
}

Humans
*Gene Editing/methods
*T-Lymphocytes/immunology/metabolism
CRISPR-Cas Systems
Receptors, Antigen, T-Cell/genetics/immunology

@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.},
}

*Aedes/genetics
Animals
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems/genetics
Animals, Genetically Modified/genetics

@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.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Animals
*RNA, Guide, CRISPR-Cas Systems/genetics
*Culicidae/genetics

@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.},
}

*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

@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.

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 fM-500 pM and a limit-of-detection (LOD) of 5.3 fM.

SIGNIFICANCE: The Cas12a-based ECL imaging approach offers high sensitivity and a broad detection range, making it highly promising for nucleic acid detection applications.},
}

*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

@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.},
}

Humans
*Calcium/metabolism
*Phospholipid Transfer Proteins/metabolism/genetics
*Phospholipids/metabolism
HEK293 Cells
Ion Channels/metabolism/genetics
Animals
CRISPR-Cas Systems

@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 & 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.},
}

*Metapneumovirus/genetics/isolation & 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

@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.},
}

*CRISPR-Cas Systems/genetics
*Genome, Bacterial/genetics
*Salmonella/genetics
Paper
Lactuca/microbiology/genetics
Animals
Milk/microbiology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@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.},
}

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

@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.

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.

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.},
}

Humans
*Synthetic Lethal Mutations
K562 Cells
Computational Biology/methods
CRISPR-Cas Systems
Algorithms
Jurkat Cells
Gene Knockout Techniques
Neoplasms/genetics

@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.

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.

RESULTS: OCT imaging demonstrated an outer retinal degeneration in Lmln[-/-] mice (P = 7.3 × 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 × 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).

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 & 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.},
}

*Gene Editing/methods
*Crops, Agricultural/genetics/growth & development
Europe
*Plants, Genetically Modified/genetics
*CRISPR-Cas Systems
Genome, Plant
Humans

@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.},
}

Animals
*Y Chromosome/genetics
Swine/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Male

@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.},
}

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

@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 & 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.},
}

*Photosynthesis/genetics
*Gene Editing/methods
*Crops, Agricultural/genetics/growth & development
CRISPR-Cas Systems
Genetic Engineering

@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.},
}

*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

@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.},
}

*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

@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.},
}

*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

@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.},
}

Animals
*CRISPR-Cas Systems
Chick Embryo
*Genetic Vectors/genetics
*Chickens/genetics
*Adenoviridae/genetics
*Aorta/metabolism
Gene Editing/methods
Male

@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 > 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.},
}

*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

@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.},
}

*Schizosaccharomyces/genetics/metabolism
*Meiosis/genetics
*CRISPR-Cas Systems/genetics
*Schizosaccharomyces pombe Proteins/genetics/metabolism
Recombination, Genetic
DNA, Fungal/genetics/metabolism
Gene Editing/methods

@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.},
}

*Retinitis Pigmentosa/therapy/genetics
Humans
*Genetic Therapy/methods
*Gene Editing/methods
Animals
Mutation
Genetic Vectors/genetics
Eye Proteins/genetics

@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 & 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.},
}

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 & inhibitors
Female

@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 & 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.},
}

*DNA (Cytosine-5-)-Methyltransferase 1/metabolism/antagonists & 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

@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.},
}

*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

@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, MÂB 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 & 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.},
}

*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

@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.},
}

*CRISPR-Cas Systems
*Saccharomycetales/genetics
Oligonucleotides/genetics
Genetic Engineering/methods
Gene Deletion
Genetic Vectors/genetics
DNA End-Joining Repair
Gene Editing/methods

@article {pmid39177875,
year = {2024},
author = {Beraza-Millor, M and Rodríguez-Castejón, J and Del Pozo-Rodríguez, A and Rodríguez-Gascón, A and Solinís, MÁ},
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, Innovación 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 & 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).

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.

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.

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 one recently published study was CRISPR/Cas9 applied to treat Fabry disease. Specific tissue targeting, availability of relevant 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.

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.},
}

Humans
*Lysosomal Storage Diseases/therapy/genetics
*Genetic Therapy/methods
Animals
*Nanoparticles
RNA, Small Interfering/administration & dosage/therapeutic use
CRISPR-Cas Systems
Gene Silencing
Drug Delivery Systems
Liposomes

@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.},
}

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

@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 & bioelectronics},
volume = {264},
number = {},
pages = {116678},
doi = {10.1016/j.bios.2024.116678},
pmid = {39154508},
issn = {1873-4235},
mesh = {*Pseudomonas aeruginosa/isolation & 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.},
}

*Pseudomonas aeruginosa/isolation & 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

@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 & 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.},
}

Humans
*MicroRNAs/genetics/analysis
*CRISPR-Cas Systems
*Lung Neoplasms/genetics/diagnosis
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods

@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 & 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.},
}

*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

@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.},
}

Animals
*Coturnix
Male
Female
*Germ Cells/physiology/cytology
*DEAD-box RNA Helicases/metabolism/genetics
Sex Characteristics
Cell Proliferation/physiology
CRISPR-Cas Systems

@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.},
}

Animals
*Anopheles/parasitology/genetics
Malaria/transmission/parasitology
Insect Proteins/genetics/metabolism
Mice
CRISPR-Cas Systems/genetics
Female
Mosquito Vectors/parasitology/genetics

@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.},
}

*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

@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.},
}

*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