@article {pmid37742320,
year = {2023},
author = {Terradas, G and Macias, VM and Peterson, H and McKeand, S and Krawczyk, G and Rasgon, JL},
title = {The development and expansion of in vivo germline editing technologies in arthropods: Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and beyond.},
journal = {Integrative and comparative biology},
volume = {},
number = {},
pages = {},
doi = {10.1093/icb/icad123},
pmid = {37742320},
issn = {1557-7023},
abstract = {In the past 20 years, sequencing technologies have led to easy access to genomic data from non-model organisms in all biological realms. Insect genetic manipulation, however, continues to be a challenge due to various factors, including technical and cost-related issues. Traditional techniques such as microinjection of gene editing vectors into early-stage embryos have been used for arthropod transgenesis and the discovery of CRISPR-Cas technologies allowed for targeted mutagenesis and the creation of knock-outs or knock-ins in arthropods. Receptor-Mediated Ovary transduction of Cargo (ReMOT Control) acts as an alternative to embryonic microinjections, which requires expensive equipment and extensive hands-on training. ReMOT Control's main advantage is its ease of use coupled with the ability to hypothetically target any vitellogenic species, as injections are administered to the egg-laying adult rather than embryos. After its initial application in the mosquito Aedes aegypti, ReMOT Control has successfully produced mutants not only for mosquitoes but for multiple arthropod species from diverse orders, such as ticks, mites, wasps, beetles, and true bugs, and is being extended to crustaceans, demonstrating the versatility of the technique. In this review, we discuss the current state of ReMOT Control from its proof-of-concept to the advances and challenges on the application across species after 5 years since its development, including novel extensions of the technique such as DIPA-CRISPR.},
}
@article {pmid37741886,
year = {2023},
author = {Yu, Q and Van Minsel, P and Galle, E and Thienpont, B},
title = {GiRAFR improves gRNA detection and annotation in single-cell CRISPR screens.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {975},
pmid = {37741886},
issn = {2399-3642},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Down-Regulation ; *RNA, Guide, CRISPR-Cas Systems ; Research Personnel ; Software ; },
abstract = {Novel methods that combine single cell RNA-seq with CRISPR screens enable high-throughput characterization of transcriptional changes caused by genetic perturbations. Dedicated software is however lacking to annotate CRISPR guide RNA (gRNA) libraries and associate them with single cell transcriptomes. Here, we describe a CRISPR droplet sequencing (CROP-seq) dataset. During analysis, we observed that the most commonly used method fails to detect mutant gRNAs. We therefore developed a python tool to identify and characterize intact and mutant gRNAs, called GiRAFR. We show that mutant gRNAs are dysfunctional, and failure to detect and annotate them leads to an inflated estimate of the number of untransformed cells, attenuated downregulation of target genes, as well as an underestimated multiplet frequency. These findings are mirrored in publicly available datasets, where we find that up to 35% of cells are transduced with a mutant gRNA. Applying GiRAFR hence stands to improve the annotation and quality of single cell CRISPR screens.},
}
@article {pmid37741566,
year = {2023},
author = {Zhou, X and Zhao, Y and Ni, P and Ni, Z and Sun, Q and Zong, Y},
title = {CRISPR-mediated acceleration of wheat improvement: advances and perspectives.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2023.09.007},
pmid = {37741566},
issn = {1673-8527},
abstract = {Common wheat (Triticum aestivum) is one of the most widely cultivated and consumed crops globally. In the face of limited arable land and climate changes, it is a great challenge to maintain current and increased future wheat production. Enhancing agronomic traits in wheat by introducing mutations across all three homoeologous copies of each gene has proven to be a difficult task due to its large genome with high repetition. However, CRISPR/Cas genome editing technologies offer a powerful means of precisely manipulating the genomes of crop species, thereby opening up new possibilities for biotechnology and breeding. In this review, we first focus on the development and optimization of current CRISPR-based genome editing tools in wheat, emphasizing recent breakthroughs in precise and multiplex genome editing. We then describe the general procedure of wheat genome editing and highlight different methods to deliver the necessary genome editing reagents into wheat cells. Furthermore, we summarize the recent applications and advancements of CRISPR/Cas technologies for wheat improvement. Lastly, we discuss the remaining challenges specific to wheat genome editing and its future prospects.},
}
@article {pmid37741424,
year = {2023},
author = {Lv, X and Li, Y and Xiu, X and Liao, C and Xu, Y and Liu, Y and Li, J and Du, G and Liu, L},
title = {CRISPR genetic toolkits of classical food microorganisms: Current state and future prospects.},
journal = {Biotechnology advances},
volume = {69},
number = {},
pages = {108261},
doi = {10.1016/j.biotechadv.2023.108261},
pmid = {37741424},
issn = {1873-1899},
abstract = {Production of food-related products using microorganisms in an environmentally friendly manner is a crucial solution to global food safety and environmental pollution issues. Traditional microbial modification methods rely on artificial selection or natural mutations, which require time for repeated screening and reproduction, leading to unstable results. Therefore, it is imperative to develop rapid, efficient, and precise microbial modification technologies. This review summarizes recent advances in the construction of gene editing and metabolic regulation toolkits based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) systems and their applications in reconstructing food microorganism metabolic networks. The development and application of gene editing toolkits from single-site gene editing to multi-site and genome-scale gene editing was also introduced. Moreover, it presented a detailed introduction to CRISPR interference, CRISPR activation, and logic circuit toolkits for metabolic network regulation. Moreover, the current challenges and future prospects for developing CRISPR genetic toolkits were also discussed.},
}
@article {pmid37739561,
year = {2023},
author = {Paul, SS},
title = {Phage engineering for development of diagnostic tools.},
journal = {Progress in molecular biology and translational science},
volume = {200},
number = {},
pages = {91-101},
doi = {10.1016/bs.pmbts.2023.04.004},
pmid = {37739561},
issn = {1878-0814},
mesh = {Humans ; *Technology ; *Bacteriophages/genetics ; Biological Assay ; Gene Editing ; },
abstract = {The bacteriophages rely on the host cell to provide energy and resources for their own replication. Antibody-based diagnostic tests rely on the antibody and the biomarker interactions. Since, most of these diagnostic tools employ the use of antibodies; hence, they require intensive storage protocols at cold conditions and incur high time and capital cost due to their production and purification process. Phage-based diagnostics can overcome this limitation. Bacteriophages, have been used as emerging tools for the detection of various pathogens. Rapid phage-mediated detection assays have become commercial diagnostic tools. Conventional method and new cloning approaches have been followed to specifically detect a disease- causing microbial strains. This review discusses use of Phage typing as diagnostic tools, phage-based detection methods, and their usage for signal amplification. Design rules for reporter phage engineering are also discussed followed by different engineering platforms for phage genome editing. We also discuss recent examples of how phage research is influencing the recent advances in the development of phage-based diagnostics for ultra-sensitive detection of various bio-species, outlining the advantages and limitations of detection technology of phage-based assays.},
}
@article {pmid37736272,
year = {2023},
author = {Yin, G and Wang, XH and Sun, Y},
title = {Recent advances in CRISPR-Cas system for the treatment of genetic hearing loss.},
journal = {American journal of stem cells},
volume = {12},
number = {3},
pages = {37-50},
pmid = {37736272},
issn = {2160-4150},
abstract = {Genetic hearing loss has emerged as a significant public health concern that demands attention. Among the various treatment strategies, gene therapy based on gene editing technology is considered the most promising approach for addressing genetic hearing loss by repairing or eliminating mutated genes. The advent of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has revolutionized gene therapy through its remarkable gene editing capabilities. This system has been extensively employed in mammalian gene editing and is currently being evaluated through clinical trials. Against this backdrop, this review aims to provide an overview of recent advances in utilizing the CRISPR-Cas system to treat genetic hearing loss. Additionally, we delve into the primary challenges and prospects associated with the current application of this system in addressing genetic hearing loss.},
}
@article {pmid37733197,
year = {2023},
author = {Raudstein, M and Kjærner-Semb, E and Barvik, M and Broll, S and Straume, AH and Edvardsen, RB},
title = {In vivo CRISPR/LbCas12a-mediated knock-in and knock-out in Atlantic salmon (Salmo salar L.).},
journal = {Transgenic research},
volume = {},
number = {},
pages = {},
pmid = {37733197},
issn = {1573-9368},
support = {301602//Norges Forskningsråd/ ; 301602//Norges Forskningsråd/ ; },
abstract = {Genome editing using the CRISPR/Cas system offers the potential to enhance current breeding programs and introduce desirable genetic traits, including disease resistance, in salmon aquaculture. Several nucleases are available using this system, displaying differences regarding structure, cleavage, and PAM requirement. Cas9 is well established in Atlantic salmon, but Cas12a has yet to be tested in vivo in this species. In the present work, we microinjected salmon embryos with LbCas12a ribonucleoprotein complexes targeting the pigmentation gene solute carrier family 45 member 2 (slc45a2). Using CRISPR/LbCas12a, we were able to knock-out slc45a2 and knock-in a FLAG sequence element by providing single-stranded DNA templates. High-throughput sequencing revealed perfect HDR rates up to 34.3% and 54.9% in individual larvae using either target or non-target strand template design, respectively. In this work, we demonstrate the in vivo application of CRISPR/LbCas12a in Atlantic salmon, expanding the toolbox for editing the genome of this important aquaculture species.},
}
@article {pmid37723277,
year = {2023},
author = {Ledford, H},
title = {Super-precise CRISPR tool enters US clinical trials for the first time.},
journal = {Nature},
volume = {621},
number = {7980},
pages = {667-668},
pmid = {37723277},
issn = {1476-4687},
mesh = {United States ; *Clinical Trials as Topic ; *Gene Editing/methods/standards ; *CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods/standards ; Leukemia/therapy ; Humans ; },
}
@article {pmid37494933,
year = {2023},
author = {Roberts, MA and Deol, KK and Mathiowetz, AJ and Lange, M and Leto, DE and Stevenson, J and Hashemi, SH and Morgens, DW and Easter, E and Heydari, K and Nalls, MA and Bassik, MC and Kampmann, M and Kopito, RR and Faghri, F and Olzmann, JA},
title = {Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation.},
journal = {Developmental cell},
volume = {58},
number = {18},
pages = {1782-1800.e10},
pmid = {37494933},
issn = {1878-1551},
support = {F32 GM113370/GM/NIGMS NIH HHS/United States ; R01 DK128099/DK/NIDDK NIH HHS/United States ; F31 DK121477/DK/NIDDK NIH HHS/United States ; R01 GM074874/GM/NIGMS NIH HHS/United States ; R01 GM112948/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Perilipin-2/genetics/metabolism ; *Lipid Droplets/metabolism ; *CRISPR-Cas Systems/genetics ; Lipid Metabolism/genetics ; Cell Line ; },
abstract = {Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism.},
}
@article {pmid37731160,
year = {2023},
author = {Hussain, A and Akram, S and Ahmad, D and Rehman, M and Ahmed, A and Ali, SA},
title = {Molecular Assessment and Validation of the Selected Enterococcal Strains as Probiotics.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {37731160},
issn = {1867-1314},
support = {NRPU-20-1339/R&D/09//Higher Education Commision, Pakistan/ ; },
abstract = {Probiotics are live microorganisms which confer health benefits to the host. Lactic acid bacteria (LAB) are used as probiotics since decades. Enterococci being the member of LAB have proven probiotic strains; therefore, this study was aimed at finding out the potential probiotic candidates from the pool of locally isolated strains. For initial screening, one hundred and twenty-two strains were selected and subjected to different confirmatory and phenotypic tests to choose the best strains that have potential probiotic criteria, i.e., no potential virulence traits, antibiotic resistance, and having tolerance properties. Keeping this criterion, only eleven strains (n = 11) were selected for further assessment. All virulence traits such as production of hemolysin, gelatinase, biofilm, and DNase were performed and not found in the tested strains. The molecular assessment indicates the presence of few virulence-associated genes in Enterococcus faecalis strains with variable frequency. The phenotypic and genotypic assessments of antibiotic resistance profile indicate that the selected strain was susceptible to ten commonly used antibiotics, and there were no transferrable antibiotic resistance genes. The presence of CRISPR-Cas genes also confirmed the absence of antibiotic resistance genes. Various enterocin-producing genes like EntP, EntB, EntA, and EntQ were also identified in the selected strains which make them promising probiotic lead strains. Different tolerance assays like acid, NaCl, and gastric juice tolerance that mimic host conditions was also evaluated by providing artificial conditions. Cellular adhesion and aggregation properties like auto- and co-aggregation were also checked and their results reflect all in the favor of lead probiotic strains.},
}
@article {pmid37696181,
year = {2023},
author = {Liu, Y and Fan, R and Yi, J and Cui, Q and Cui, C},
title = {A fusion framework of deep learning and machine learning for predicting sgRNA cleavage efficiency.},
journal = {Computers in biology and medicine},
volume = {165},
number = {},
pages = {107476},
doi = {10.1016/j.compbiomed.2023.107476},
pmid = {37696181},
issn = {1879-0534},
mesh = {*Deep Learning ; RNA, Guide, CRISPR-Cas Systems ; Machine Learning ; Neural Networks, Computer ; },
abstract = {CRISPR/Cas9 system is a powerful tool for genome editing. Numerous studies have shown that sgRNAs can strongly affect the efficiency of editing. However, it is still not clear what rules should be followed for designing sgRNA with high cleavage efficiency. At present, several machine learning or deep learning methods have been developed to predict the cleavage efficiency of sgRNAs, however, the prediction accuracy of these tools is still not satisfactory. Here we propose a fusion framework of deep learning and machine learning, which first deals with the primary sequence and secondary structure features of the sgRNAs using both convolutional neural network (CNN) and recurrent neural network (RNN), and then uses the features extracted by the deep neural network to train a conventional machine learning model with LGBM. As a result, the new approach overwhelmed previous methods. The Spearman's correlation coefficient between predicted and measured sgRNA cleavage efficiency of our model (0.917) is improved by over 5% compared with the most advanced method (0.865), and the mean square error reduces from 7.89 × 10[-3] to 4.75 × 10[-3]. Finally, we developed an online tool, CRISep (http://www.cuilab.cn/CRISep), to evaluate the availability of sgRNAs based on our models.},
}
@article {pmid37669145,
year = {2023},
author = {Wang, X and Li, Y and Wang, X and Sandoval, DM and He, Z and A, S and Sáez, IL and Wang, W},
title = {Guanidyl-Rich Poly(β Amino Ester)s for Universal Functional Cytosolic Protein Delivery and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 Ribonucleoprotein Based Gene Editing.},
journal = {ACS nano},
volume = {17},
number = {18},
pages = {17799-17810},
doi = {10.1021/acsnano.3c03269},
pmid = {37669145},
issn = {1936-086X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; HeLa Cells ; Green Fluorescent Proteins ; Esters ; Guanidine ; Guanidines ; },
abstract = {Protein therapeutics are highly promising for complex disease treatment. However, the lack of ideal delivery vectors impedes their clinical use, especially the carriers for in vivo delivery of functional cytosolic protein. In this study, we modified poly(β amino ester)s (PAEs) with a phenyl guanidine (PG) group to enhance their suitability for cytosolic protein delivery. The effects of the PG group on protein binding, cell internalization, protein function protection, and endo/lysosomal escape were systematically evaluated. Compared to the unmodified PAEs (L3), guanidyl rich PAEs (L3PG) presented superior efficiency of protein binding and protein internalization, mainly via clathrin-mediated endocytosis. In addition, both PAEs showed robust capabilities to deliver cytosolic proteins with different molecular weight (ranging from 30 to 464 kDa) and isoelectric points (ranging from 4.3 to 9), which were significantly improved in comparison with the commercial reagents of PULsin and Pierce Protein Transection Reagent. Moreover, L3PG successfully delivered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 ribonucleoprotein (RNP) into HeLa cells expressing green fluorescent protein (GFP) and achieved more than 80% GFP expression knockout. These results demonstrated that guanidyl modification on PAEs can enhance its capabilities for intracellular delivery of cytosolic functional proteins and CRISPR/Cas9 ribonucleoprotein. The guanidyl-rich PAEs are promising nonviral vectors for functional protein delivery and potential use in protein and nuclease-based gene editing therapies.},
}
@article {pmid37593879,
year = {2023},
author = {Zhai, Y and Yang, L and Zheng, W and Wang, Q and Zhu, Z and Han, F and Hao, Y and Ma, S and Cheng, G},
title = {A precise design strategy for a cell-derived extracellular matrix based on CRISPR/Cas9 for regulating neural stem cell function.},
journal = {Biomaterials science},
volume = {11},
number = {19},
pages = {6537-6544},
doi = {10.1039/d2bm01466a},
pmid = {37593879},
issn = {2047-4849},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Fibroblasts/metabolism ; Extracellular Matrix/metabolism ; Heparan Sulfate Proteoglycans ; Heparitin Sulfate/metabolism ; *Neural Stem Cells/metabolism ; },
abstract = {The extracellular matrix (ECM) is a natural microenvironment pivotal for stem cell survival, as well as proliferation, differentiation and metastasis, composed of a variety of biological molecular complexes secreted by resident cells in tissues and organs. Heparan sulfate proteoglycan (HSPG) is a type of ECM protein that contains one or more covalently attached heparan sulfate chains. Heparan sulphate chains have high affinity with growth factors, chemokines and morphogens, acting as cytokine-binding domains of great importance in development and normal physiology. Herein, we constructed endogenous HSPG2 overexpression in mouse embryonic fibroblasts based on the CRISPR/Cas9 synergistic activation mediator system and then fabricated a cell-derived HSPG2 functional ECM (ECM[HSPG2]). The ECM[HSPG2] is capable of enriching basic fibroblast growth factor (bFGF), which binds more strongly than the negative control ECM. With a growing bFGF concentration, ECM[HSPG2] could better maintain neural stem cell (NSCs) stemness and promote NSC proliferation and differentiation in culture. These findings provide a precise design strategy for producing a specific cell-derived ECM for biomaterials in research and regenerative medicine.},
}
@article {pmid37562129,
year = {2023},
author = {Zou, K and Wang, F and Zhang, Z and Zhou, Y and Li, P and Wang, D and Zhu, M and Jia, C and Wei, Z},
title = {Optimized CRISPR/Cas9 system for gene knockout in chicken DF1 cells.},
journal = {Poultry science},
volume = {102},
number = {10},
pages = {102970},
pmid = {37562129},
issn = {1525-3171},
mesh = {Animals ; Gene Knockout Techniques/veterinary ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Chickens/genetics ; Endonucleases/genetics ; },
abstract = {The editing efficiency primarily hinders the utility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in poultry. For a better understanding of the factors that influence the efficiency of gene knockout mediated by CRISPR/Cas9 in chicken DF1 cells, the single or dual single guide RNA (sgRNA) targeted exon regions of genes (taking anti-Müllerian hormone, TGF-beta receptor type-2 and Peroxisome proliferator-activated receptor gamma as examples) were designed. The sgRNA-CRISPR/Cas9 vectors with corresponding reporter vectors were transfected into DF1 cells. T7 endonuclease 1 (T7E1) and amplicon sequencing assay were compared for evaluating genome editing efficiency and the indel profiles were analyzed based on the data of amplicon sequencing. Meanwhile, to evaluate the precision of Cas9 cleavage, we also analyzed the homology of small insertion with the nucleotides of upstream and downstream of cleave sties. The surrogate reporter systems showed strong enrichment function, and the indel percentages were increased after puromycin selection. The indel ratios of T7E1 assay were lower than amplicon sequencing assay, which indicated T7E1 isn't fit to be used as the sole evaluation criterion for the targeting efficiency of CRISPR/Cas9. Based on the amplicon sequencing analysis, the editing efficiency showed noticeable differences among cells treated with different sgRNAs. However, the variety of indel efficiencies was not related to the GC content of sgRNA or chromosome types of targeted genes. The results showed that the dual sgRNA might not raise the indel ratios compared with individual sgRNA, but they could increase the ratios of the fragment deletions. The present study suggested that the surrogate reporter was an effective method to promote the editing efficiencies of CRISPR/Cas9 in chicken cells. The dual sgRNA could increase the fragment deletions, and the sensitivity of amplicon sequencing to detect cleavage was higher than the T7 endonuclease 1 assay. These results are essential to improve the application of CRISPR/Cas9 technology in chicken cells.},
}
@article {pmid37494991,
year = {2023},
author = {Huang, T and Li, L and Li, J and Li, X and Li, S and Wang, X and Zhang, N and Yu, Y and Zhang, X and Zhao, Z and Guo, Y and Cao, L and Gong, P},
title = {Rapid, sensitive, and visual detection of Clonorchis sinensis with an RPA-CRISPR/Cas12a-based dual readout portable platform.},
journal = {International journal of biological macromolecules},
volume = {249},
number = {},
pages = {125967},
doi = {10.1016/j.ijbiomac.2023.125967},
pmid = {37494991},
issn = {1879-0003},
mesh = {Animals ; Humans ; *Clonorchis sinensis/genetics ; CRISPR-Cas Systems/genetics ; Cross Reactions ; Food ; Liver ; },
abstract = {Clonorchis sinensis is a food-borne parasite that parasitizes the liver and bile ducts of humans and many animals. This parasite exerts a high burden due to diverse hepatobiliary morbidities (e.g., cholangitis, cholecystitis, cholelithiasis, and cholangiocarcinoma), and an effective detection strategy is urgently needed. CRISPR/Cas12a exhibits nonspecific trans-cleavage activity upon binding to its specific target and has been widely used for nucleic acid detection. In this study, an RPA-CRISPR/Cas12a-based dual readout portable detection platform was established, which shows high sensitivity (one copy/μl) and specificity (no cross-reactivity with common pathogens) by rapid preamplification and combines lateral flow strips and visual fluorescence for visualization of results by the naked eye within 1 h. Moreover, 50 human fecal swabs and 50 fish flesh samples were detected by this platform and nested PCR. The CRISPR/Cas12a-based dual readout portable platform showed 10.0 % (5/50) C. sinensis-positive samples in human fecal swabs and 28.0 % (14/50) in fish flesh, which was consistent with the results of nested PCR. The results demonstrate that our portable platform has the advantages of stability, sensitivity, accuracy, and low equipment requirements. Furthermore, we provide novel point-of-care testing (POCT) for clinical use in remote rural and resource-constrained areas.},
}
@article {pmid37485817,
year = {2023},
author = {Beha, MJ and Kim, JC and Im, SH and Kim, Y and Yang, S and Lee, J and Nam, YR and Lee, H and Park, HS and Chung, HJ},
title = {Bioorthogonal CRISPR/Cas9-Drug Conjugate: A Combinatorial Nanomedicine Platform.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {10},
number = {27},
pages = {e2302253},
pmid = {37485817},
issn = {2198-3844},
support = {2021R1A2C2011763//National Research Foundation of Korea/ ; 2021M3E5E3080383//National Research Foundation of Korea/ ; 2022R1A4A5028131//National Research Foundation of Korea/ ; 2023R1A2C1005945//National Research Foundation of Korea/ ; HI22C2010//Ministry of Health and Welfare/ ; SSTF-BA1702-09//Samsung Science and Technology Foundation/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; Pharmaceutical Preparations ; Nanomedicine ; Gene Editing ; },
abstract = {Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.},
}
@article {pmid37452759,
year = {2023},
author = {Chikmagalur Nagaraja, B and Karuppannasamy, A and Ramasamy, A and Cholenahalli Narayanappa, A and Chalapathi, P and Maligeppagol, M},
title = {CRISPR/Cas9-mediated mutagenesis of Sex lethal (Sxl) gene impacts fertility of the Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).},
journal = {Archives of insect biochemistry and physiology},
volume = {114},
number = {2},
pages = {1-15},
doi = {10.1002/arch.22035},
pmid = {37452759},
issn = {1520-6327},
support = {//ICAR CABin project/ ; },
mesh = {Male ; Animals ; Spodoptera/genetics ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Fertility/genetics ; Mutagenesis ; Larva ; Zea mays ; },
abstract = {Identification of novel approaches for managing the global pest, the Fall armyworm, Spodoptera frugiperda, is the need of the hour, as it defies many management strategies including synthetic chemicals, Bt transgenics, and so on. Recently CRISPR/Cas9-based genome editing opened up newer avenues to design novel pest management strategies such as precision-guided sterile insect technique (pgSIT). In this regard, genes governing sex determination, egg reproduction, and spermatogenesis could be the prime targets for genome editing. This requires validation of the target genes, preferably by a nontransgenic DNA-free editing, before the final application. One such important gene regulating sex determination in Drosophila is the Sex lethal (Sxl). However, the function of Sxl is not highly conserved in other insects and, in particular, we are beginning to comprehend its role in Lepidoptera with only one reference available in Spodoptera litura till date. In the present study, we have edited the sxl gene of S. frugiperda through the delivery of ribonucleoprotein complex (sgRNA + Cas9) at G0 stage embryo, targeting the conserved region of all the documented five splice variants. Results clearly showed that editing of sxl gene impacted the overall fecundity and hatching rate. Therefore, Sxl could be one of the target genes for developing pgSIT approach for the management of S. frugiperda.},
}
@article {pmid37519013,
year = {2023},
author = {Balmas, E and Sozza, F and Bottini, S and Ratto, ML and Savorè, G and Becca, S and Snijders, KE and Bertero, A},
title = {Manipulating and studying gene function in human pluripotent stem cell models.},
journal = {FEBS letters},
volume = {597},
number = {18},
pages = {2250-2287},
doi = {10.1002/1873-3468.14709},
pmid = {37519013},
issn = {1873-3468},
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Pluripotent Stem Cells ; Transfection ; Biomarkers/metabolism ; },
abstract = {Human pluripotent stem cells (hPSCs) are uniquely suited to study human development and disease and promise to revolutionize regenerative medicine. These applications rely on robust methods to manipulate gene function in hPSC models. This comprehensive review aims to both empower scientists approaching the field and update experienced stem cell biologists. We begin by highlighting challenges with manipulating gene expression in hPSCs and their differentiated derivatives, and relevant solutions (transfection, transduction, transposition, and genomic safe harbor editing). We then outline how to perform robust constitutive or inducible loss-, gain-, and change-of-function experiments in hPSCs models, both using historical methods (RNA interference, transgenesis, and homologous recombination) and modern programmable nucleases (particularly CRISPR/Cas9 and its derivatives, i.e., CRISPR interference, activation, base editing, and prime editing). We further describe extension of these approaches for arrayed or pooled functional studies, including emerging single-cell genomic methods, and the related design and analytical bioinformatic tools. Finally, we suggest some directions for future advancements in all of these areas. Mastering the combination of these transformative technologies will empower unprecedented advances in human biology and medicine.},
}
@article {pmid37730702,
year = {2023},
author = {Deng, X and Osikpa, E and Yang, J and Oladeji, SJ and Smith, J and Gao, X and Gao, Y},
title = {Structural basis for the activation of a compact CRISPR-Cas13 nuclease.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5845},
pmid = {37730702},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Dependovirus/genetics ; Endonucleases/genetics ; RNA ; },
abstract = {The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 effectors and their non-specific RNA cleavage upon target activation limit the adeno-associated virus based delivery of Cas13 systems for therapeutic applications. Herein, we report detailed biochemical and structural characterizations of a compact Cas13 (Cas13bt3) suitable for adeno-associated virus delivery. Distinct from many other Cas13 systems, Cas13bt3 cleaves the target and other nonspecific RNA at internal "UC" sites and is activated in a target length-dependent manner. The cryo-electron microscope structure of Cas13bt3 in a fully active state illustrates the structural basis of Cas13bt3 activation. Guided by the structure, we obtain engineered Cas13bt3 variants with minimal off-target cleavage yet maintained target cleavage activities. In conclusion, our biochemical and structural data illustrate a distinct mechanism for Cas13bt3 activation and guide the engineering of Cas13bt3 applications.},
}
@article {pmid37729124,
year = {2023},
author = {Liu, M and Li, Z and Chen, J and Lin, J and Lu, Q and Ye, Y and Zhang, H and Zhang, B and Ouyang, S},
title = {Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage.},
journal = {PLoS genetics},
volume = {19},
number = {9},
pages = {e1010930},
pmid = {37729124},
issn = {1553-7404},
mesh = {RNA Cleavage ; *RNA, Guide, CRISPR-Cas Systems ; *Endonucleases/genetics ; Endoribonucleases ; RNA/genetics ; },
abstract = {Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing.},
}
@article {pmid37728624,
year = {2023},
author = {Huang, Y and Chen, M and Hu, G and Wu, B and He, M},
title = {Elimination of editing plasmid mediated by theophylline riboswitch in Zymomonas mobilis.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
pmid = {37728624},
issn = {1432-0614},
abstract = {Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. KEY POINTS: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.},
}
@article {pmid37728251,
year = {2023},
author = {Sparmann, A and Vogel, J},
title = {RNA-based medicine: from molecular mechanisms to therapy.},
journal = {The EMBO journal},
volume = {},
number = {},
pages = {e114760},
doi = {10.15252/embj.2023114760},
pmid = {37728251},
issn = {1460-2075},
support = {//Elite Network Bavaria/Elitenetzwerk Bayern/ ; },
abstract = {RNA-based therapeutics have the potential to revolutionize the treatment and prevention of human diseases. While early research faced setbacks, it established the basis for breakthroughs in RNA-based drug design that culminated in the extraordinarily fast development of mRNA vaccines to combat the COVID-19 pandemic. We have now reached a pivotal moment where RNA medicines are poised to make a broad impact in the clinic. In this review, we present an overview of different RNA-based strategies to generate novel therapeutics, including antisense and RNAi-based mechanisms, mRNA-based approaches, and CRISPR-Cas-mediated genome editing. Using three rare genetic diseases as examples, we highlight the opportunities, but also the challenges to wide-ranging applications of this class of drugs.},
}
@article {pmid37727048,
year = {2023},
author = {Zhou, S and Zhu, S and Huang, Z and Chen, J and Li, J and Yang, M and Jin, L and Huo, D and Hou, C},
title = {Target-mediated rolling circle transcription coupling with CRISPR/Cas12a-Cas13a for simultaneous detection of HPV16 and HPV18.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3cc04223e},
pmid = {37727048},
issn = {1364-548X},
abstract = {Simultaneous detection of multiple targets can provide important data support for clinical diagnosis and treatment. Here, we report a facile isothermal assay based on target-mediated rolling circle transcription coupling with CRISPR/Cas12a-Cas13a (TM-RCT/Cas12a-Cas13a). Through facile one-step amplification (TM-RCT), two target DNAs are converted to RNA amplified products. The simultaneous detection of HPV16 and HPV18 is then achieved by combining two CRISPR/Cas systems. This system shows excellent sensing performance and provides a universal method for simultaneous detection.},
}
@article {pmid37726790,
year = {2023},
author = {Danti, L and Lundin, K and Sepponen, K and Yohannes, DA and Kere, J and Tuuri, T and Tapanainen, JS},
title = {CRISPR/Cas9-mediated activation of NR5A1 steers female human embryonic stem cell-derived bipotential gonadal-like cells towards a steroidogenic cell fate.},
journal = {Journal of ovarian research},
volume = {16},
number = {1},
pages = {194},
pmid = {37726790},
issn = {1757-2215},
support = {813707//European Union's Horizon 2020 research and innovation programme under Marie Sklodowska-Curie/ ; },
mesh = {Animals ; Humans ; Female ; Male ; *Human Embryonic Stem Cells ; CRISPR-Cas Systems ; Steroidogenic Factor 1/genetics ; Cell Differentiation/genetics ; Cytochrome P450 Family 17 ; },
abstract = {The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms' tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.},
}
@article {pmid37572348,
year = {2023},
author = {Cachera, P and Olsson, H and Coumou, H and Jensen, ML and Sánchez, BJ and Strucko, T and van den Broek, M and Daran, JM and Jensen, MK and Sonnenschein, N and Lisby, M and Mortensen, UH},
title = {CRI-SPA: a high-throughput method for systematic genetic editing of yeast libraries.},
journal = {Nucleic acids research},
volume = {51},
number = {17},
pages = {e91},
pmid = {37572348},
issn = {1362-4962},
support = {//European Union's Horizon 2020/ ; 722287//Marie Skłodowska-Curie/ ; 087-2012-3//Danish National Advanced Technology Foundation/ ; DNRF115//Danish National Research Foundation/ ; NNF19SA0035438//Novo Nordisk Foundation Bioscience Ph.D. Programme/ ; NNF17SA0031362//Fermentation-based Biomanufacturing Initiative/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; Betaxanthins ; Reproducibility of Results ; Gene Editing/methods ; Genetic Engineering/methods ; },
abstract = {Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and new research tools allowing simple and rapid mapping of sequence to function are desirable. Here, we describe CRI-SPA, a method allowing the transfer of chromosomal genetic features from a CRI-SPA Donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion, and Selective Ploidy Ablation. CRI-SPA can be massively parallelized with automation and can be executed within a week. We demonstrate the power of CRI-SPA by transferring four genes that enable betaxanthin production into each strain of the yeast knockout collection (≈4800 strains). Using this setup, we show that CRI-SPA is highly efficient and reproducible, and even allows marker-free transfer of genetic features. Moreover, we validate a set of CRI-SPA hits by showing that their phenotypes correlate strongly with the phenotypes of the corresponding mutant strains recreated by reverse genetic engineering. Hence, our results provide a genome-wide overview of the genetic requirements for betaxanthin production. We envision that the simplicity, speed, and reliability offered by CRI-SPA will make it a versatile tool to forward systems-level understanding of biological processes.},
}
@article {pmid37568030,
year = {2023},
author = {Gruber, K and Melton, L},
title = {CRISPR upgrades insect proteins for feed.},
journal = {Nature biotechnology},
volume = {41},
number = {8},
pages = {1038-1040},
doi = {10.1038/s41587-023-01902-9},
pmid = {37568030},
issn = {1546-1696},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; *Insect Proteins/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37560931,
year = {2023},
author = {Marsic, T and Gundra, SR and Wang, Q and Aman, R and Mahas, A and Mahfouz, MM},
title = {Programmable site-specific DNA double-strand breaks via PNA-assisted prokaryotic Argonautes.},
journal = {Nucleic acids research},
volume = {51},
number = {17},
pages = {9491-9506},
pmid = {37560931},
issn = {1362-4962},
support = {//KAUST Smart Health Initiative (KSHI)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; *Peptide Nucleic Acids/genetics ; Gene Editing ; Genome ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Programmable site-specific nucleases promise to unlock myriad applications in basic biology research, biotechnology and gene therapy. Gene-editing systems have revolutionized our ability to engineer genomes across diverse eukaryotic species. However, key challenges, including delivery, specificity and targeting organellar genomes, pose barriers to translational applications. Here, we use peptide nucleic acids (PNAs) to facilitate precise DNA strand invasion and unwinding, enabling prokaryotic Argonaute (pAgo) proteins to specifically bind displaced single-stranded DNA and introduce site-specific double-strand breaks (DSBs) independent of the target sequence. We named this technology PNA-assisted pAgo editing (PNP editing) and determined key parameters for designing PNP editors to efficiently generate programable site-specific DSBs. Our design allows the simultaneous use of multiple PNP editors to generate multiple site-specific DSBs, thereby informing design considerations for potential in vitro and in vivo applications, including genome editing.},
}
@article {pmid37500264,
year = {2023},
author = {Igarashi, T and Katayama, T and Maruyama, JI},
title = {CRISPR/Cas9 genome editing for comparative genetic analysis related to soy sauce brewing in Aspergillus sojae industrial strains.},
journal = {Bioscience, biotechnology, and biochemistry},
volume = {87},
number = {10},
pages = {1236-1248},
doi = {10.1093/bbb/zbad101},
pmid = {37500264},
issn = {1347-6947},
support = {23H04547//Grant-in-Aid for Transformative Research Areas/ ; //Japan Society for the Promotion of Science/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Soy Foods ; Aspergillus/genetics ; },
abstract = {Aspergillus sojae has traditionally been used in soy sauce brewing. Genetic modification techniques have been established in A. sojae, but it is difficult to apply them to various industrial strains. Although we have previously developed a CRISPR/Cpf1 system for genetic modification of A. sojae, another genome editing system was required for versatile modification. In addition, repetitive genetic modification using the CRISPR system has not been established in A. sojae. In this study, we demonstrated mutagenesis, gene deletion/integration, and large deletion of a chromosomal region in A. sojae using the CRISPR/Cas9 system. We also successfully performed repetitive genetic modification using a method that involved forced recycling of genome-editing plasmids. Moreover, we demonstrated that the effects of genetic modification related to soy sauce brewing differed among A. sojae industrial strains. These results showed that our technique of using the CRISPR/Cas9 system is a powerful tool for genetic modification in A. sojae.},
}
@article {pmid36931733,
year = {2023},
author = {Sun, R and Raban, R and Akbari, OS},
title = {Generating Aedes aegypti Mutant Strains with Transgenic Cas9.},
journal = {Cold Spring Harbor protocols},
volume = {2023},
number = {9},
pages = {671-678},
doi = {10.1101/pdb.prot108085},
pmid = {36931733},
issn = {1559-6095},
support = {R01 AI151004/AI/NIAID NIH HHS/United States ; R01 GM132825/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Aedes/genetics ; Animals, Genetically Modified ; Recombinational DNA Repair ; },
abstract = {Here, we provide a protocol for generating Aedes aegypti mutant strains via end-joining (EJ) or homology-directed repair (HDR) mechanisms using genetically encoded Cas9.},
}
@article {pmid35637909,
year = {2022},
author = {Shirai, Y and Piulachs, MD and Belles, X and Daimon, T},
title = {DIPA-CRISPR is a simple and accessible method for insect gene editing.},
journal = {Cell reports methods},
volume = {2},
number = {5},
pages = {100215},
pmid = {35637909},
issn = {2667-2375},
mesh = {Animals ; Female ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Insecta/genetics ; },
abstract = {Current approaches for insect gene editing require microinjection of materials into early embryos. This severely limits the application of gene editing to a great number of insect species, especially to those whose reproduction systems preclude access to early embryos for injection. To overcome these limitations, we report a simple and accessible method for insect gene editing, termed "direct parental" CRISPR (DIPA-CRISPR). We show that injection of Cas9 ribonucleoproteins (RNPs) into the haemocoel of adult females efficiently introduces heritable mutations in developing oocytes. Importantly, commercially available standard Cas9 protein can be directly used for DIPA-CRISPR, which makes this approach highly practical and feasible. DIPA-CRISPR enables highly efficient gene editing in the cockroaches, on which conventional approaches cannot be applied, and in the model beetle Tribolium castaneum. Due to its simplicity and accessibility, DIPA-CRISPR will greatly extend the application of gene editing technology to a wide variety of insects.},
}
@article {pmid37725519,
year = {2023},
author = {Saini, H and Thakur, R and Gill, R and Tyagi, K and Goswami, M},
title = {CRISPR/Cas9-gene editing approaches in plant breeding.},
journal = {GM crops & food},
volume = {14},
number = {1},
pages = {1-17},
pmid = {37725519},
issn = {2164-5701},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Plant Breeding ; Agriculture ; Blotting, Southern ; },
abstract = {CRISPR/Cas9 gene editing system is recently developed robust genome editing technology for accelerating plant breeding. Various modifications of this editing system have been established for adaptability in plant varieties as well as for its improved efficiency and portability. This review provides an in-depth look at the various strategies for synthesizing gRNAs for efficient delivery in plant cells, including chemical synthesis and in vitro transcription. It also covers traditional analytical tools and emerging developments in detection methods to analyze CRISPR/Cas9 mediated mutation in plant breeding. Additionally, the review outlines the various analytical tools which are used to detect and analyze CRISPR/Cas9 mediated mutations, such as next-generation sequencing, restriction enzyme analysis, and southern blotting. Finally, the review discusses emerging detection methods, including digital PCR and qPCR. Hence, CRISPR/Cas9 has great potential for transforming agriculture and opening avenues for new advancements in the system for gene editing in plants.},
}
@article {pmid37723839,
year = {2023},
author = {Whitley, JA and Cai, H},
title = {Engineering extracellular vesicles to deliver CRISPR ribonucleoprotein for gene editing.},
journal = {Journal of extracellular vesicles},
volume = {12},
number = {9},
pages = {e12343},
pmid = {37723839},
issn = {2001-3078},
support = {U01 CA225784-01/CA/NCI NIH HHS/United States ; R21 AI157831/AI/NIAID NIH HHS/United States ; R21 AI171944/AI/NIAID NIH HHS/United States ; },
mesh = {*Extracellular Vesicles ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; Cell Membrane ; Genetic Therapy ; },
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR) is a gene editing tool with tremendous therapeutic potential. Recently, ribonucleoprotein (RNP) complex-based CRISPR systems have gained momentum due to their reduction of off-target editing. This has coincided with the emergence of extracellular vesicles (EVs) as a therapeutic delivery vehicle due to its low immunogenicity and high capacity for manipulation. EVs are cell-derived membranous nanoparticles which mediate the intercellular transfer of molecular components. Current technologies achieve CRISPR RNP encapsulation into EVs through EVs biogenesis, thereby avoiding unnecessary physical, chemical or biological manipulations to the vesicles directly. Herein, we identify sixteen EVs-based CRISPR RNP encapsulation strategies, each with distinct genetic features to encapsulate CRISPR RNP. According to the molecular mechanism facilitating the encapsulation process, there are six strategies of encapsulating Cas9 RNP into virus-like particles based on genetic fusion, seven into EVs based on protein tethering, and three based on sgRNA-coupled encapsulation. Additionally, the incorporation of a targeting moiety to the EVs membrane surface through EVs biogenesis confers tropism and increases delivery efficiency to specific cell types. The targeting moieties include viral envelope proteins, recombinant proteins containing a ligand peptide, single-chain fragment variable (scFv) antibodies, and integrins. However, current strategies still have a number of limitations which prevent their use in clinical trials. Among those, the incorporation of viral proteins for encapsulation of Cas9 RNP have raised issues of biocompatibility due to host immune response. Future studies should focus on genetically engineering the EVs without viral proteins, enhancing EVs delivery specificity, and promoting EVs-based homology directed repair. Nevertheless, the integration of CRISPR RNP encapsulation and tropism technologies will provide strategies for the EVs-based delivery of CRISPR RNP in gene therapy and disease treatment.},
}
@article {pmid37723612,
year = {2023},
author = {Rashid, SJ and Nale, JY and Millard, AD and Clokie, MRJ},
title = {Novel ribotype/sequence type associations and diverse CRISPR-Cas systems in environmental Clostridioides difficile strains from Northern Iraq.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnad091},
pmid = {37723612},
issn = {1574-6968},
abstract = {The environment is a natural reservoir of Clostridioides difficile and here, we aimed to isolate the pathogen from seven locations in Northern Iraq. Four of the sites yielded thirty-one isolates (ten from soils, twenty-one from sediments) which together represent ribotypes 001 (five), 010 (five), 011 (two), 035 (two), 091 (eight) and 604 (nine). Twenty-five of the isolates (∼81%) are non-toxigenic while 6 (∼19%) encode the toxin A and B genes. The genomes of eleven selected isolates represent six sequence types: ST-3 (two), ST-15 (one), ST-107 (five), ST-137 (one), ST-177 (one) and ST-181 (one). Five novel ribotype/sequence type (RT/ST) associations: RT011/ST-137, RT035/ST-107, RT091/ST-107, RT604/ST-177 and RT604/ST-181 were identified and the first three are linked to ribotypes previously uncharacterised by multilocus sequence typing (MLST). Nine of the genomes belong to Clade 1 and two are closely related to the cryptic C-I clade. Diverse multiple prophages and CRISPR-Cas systems (class 1 subtype I-B1 and class 2 type V CRISPR-Cas systems) with spacers identical to other C. difficile phages and plasmids were detected in the genomes. Our data show the broader diversity that exists within environmental C. difficile strains from a much less studied location and their potential role in evolution and emergence of new strains.},
}
@article {pmid37722021,
year = {2023},
author = {Zhu, F and Zhao, Q},
title = {CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c02834},
pmid = {37722021},
issn = {1520-6882},
abstract = {CRISPR/Cas systems are powerful tools for sensitive nucleic acid molecular diagnosis due to their specific nucleic acid recognition and high trans-cleavage activity and have also allowed for quantification of non-nucleic acid targets, relying on a strategy to convert the target detection to analysis of nucleic acids. Here, we describe a CRISPR/Cas12a-powered immunosorbent assay for sensitive small-molecule detection by using the antibody coated on the microplate to recognize the target and the small molecule-labeled active DNA (acDNA) to trigger the activity of CRISPR/Cas12a. In the absence of small-molecule targets, acDNA probes are captured by the antibody on the microplate and then activate Cas12a in catalytic trans-cleavage of fluorescent DNA reporters, generating strong fluorescence. The presence of small-molecule targets displaces the acDNA probes from the antibody, causing a decrease of acDNA probes on the microplate and reduction of activated Cas12a, so the fluorescence signal decreases, and small molecules can be detected by monitoring the fluorescence change. After systematically optimizing experimental conditions (e.g., Cas12a reaction), the proposed method achieved the detection of three model small molecules, biotin, digoxin, and folic acid, with low detection limits, and a flexible detection concentration range was obtained by simply changing the amount of acDNA probes and immobilized antibodies. The assay showed high selectivity and good applicability in complex media. The integration of the CRISPR/Cas12a system improves the analytical performance of immunoassay, broadening and facilitating its applications in rapid, simple, and sensitive small molecule analysis.},
}
@article {pmid37720722,
year = {2023},
author = {Menchaca, A},
title = {Assisted Reproductive Technologies (ART) and genome editing to support a sustainable livestock.},
journal = {Animal reproduction},
volume = {20},
number = {2},
pages = {e20230074},
pmid = {37720722},
issn = {1984-3143},
abstract = {This article provides an overview of assisted reproductive technologies (ART) and genome engineering to improve livestock production systems for the contribution of global sustainability. Most ruminant production systems are conducted on grassland conditions, as is the case of South American countries that are leaders in meat and milk production worldwide with a well-established grass-feed livestock. These systems have many strengths from an environmental perspective and consumer preferences but requires certain improvements to enhance resource efficiency. Reproductive performance is one of the main challenges particularly in cow-calf operations that usually are conducted under adverse conditions and thus ART can make a great contribution. Fixed-time artificial insemination is applied in South America in large scale programs as 20 to 30% of cows receive this technology every year in each country, with greater calving rate and significant herd genetic gain occurred in this region. Sexed semen has also been increasingly implemented, enhancing resource efficiency by a) obtaining desired female replacement and improving animal welfare by avoiding newborn male sacrifice in dairy industry, or b) alternatively producing male calves for beef industry. In vitro embryo production has been massively applied, with this region showing the greatest number of embryos produced worldwide leading to significant improvement in herd genetics and productivity. Although the contribution of these technologies is considerable, further improvements will be required for a significant livestock transformation and novel biotechnologies such as genome editing are already available. Through the CRISPR/Cas-based system it is possible to enhance food yield and quality, avoid animal welfare concerns, overcome animal health threats, and control pests and invasive species harming food production. In summary, a significant enhancement in livestock productivity and resource efficiency can be made through reproductive technologies and genome editing, improving at the same time profitability for farmers, and global food security and sustainability.},
}
@article {pmid37720320,
year = {2023},
author = {Li, X and Zhu, S and Zhang, X and Ren, Y and He, J and Zhou, J and Yin, L and Wang, G and Zhong, T and Wang, L and Xiao, Y and Zhu, C and Yin, C and Yu, X},
title = {Advances in the application of recombinase-aided amplification combined with CRISPR-Cas technology in quick detection of pathogenic microbes.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1215466},
pmid = {37720320},
issn = {2296-4185},
abstract = {The rapid diagnosis of pathogenic infections plays a vital role in disease prevention, control, and public health safety. Recombinase-aided amplification (RAA) is an innovative isothermal nucleic acid amplification technology capable of fast DNA or RNA amplification at low temperatures. RAA offers advantages such as simplicity, speed, precision, energy efficiency, and convenient operation. This technology relies on four essential components: recombinase, single-stranded DNA-binding protein (SSB), DNA polymerase, and deoxyribonucleoside triphosphates, which collectively replace the laborious thermal cycling process of traditional polymerase chain reaction (PCR). In recent years, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) system, a groundbreaking genome engineering tool, has garnered widespread attention across biotechnology, agriculture, and medicine. Increasingly, researchers have integrated the recombinase polymerase amplification system (or RAA system) with CRISPR technology, enabling more convenient and intuitive determination of detection results. This integration has significantly expanded the application of RAA in pathogen detection. The step-by-step operation of these two systems has been successfully employed for molecular diagnosis of pathogenic microbes, while the single-tube one-step method holds promise for efficient pathogen detection. This paper provides a comprehensive review of RAA combined with CRISPR-Cas and its applications in pathogen detection, aiming to serve as a valuable reference for further research in related fields.},
}
@article {pmid37670689,
year = {2023},
author = {Frion, J and Meller, A and Marbach, G and Lévesque, D and Roucou, X and Boisvert, FM},
title = {CRISPR/Cas9-mediated knockout of the ubiquitin variant UbKEKS reveals a role in regulating nucleolar structures and composition.},
journal = {Biology open},
volume = {12},
number = {9},
pages = {},
doi = {10.1242/bio.059984},
pmid = {37670689},
issn = {2046-6390},
support = {398925/CAPMC/CIHR/Canada ; },
mesh = {*Ubiquitin/genetics ; *CRISPR-Cas Systems ; Ubiquitins ; Ubiquitination ; Apoptosis ; },
abstract = {Ubiquitination is a post-translational modification responsible for one of the most complex multilayered communication and regulation systems in the cell. Over the past decades, new ubiquitin variants and ubiquitin-like proteins arose to further enrich this mechanism. Recently discovered ubiquitin variant UbKEKS can specifically target several proteins and yet, functional consequences of this new modification remain unknown. Depletion of UbKEKS induces accumulation of lamin A in the nucleoli, highlighting the need for deeper investigations about protein composition and functions regulation of this highly dynamic and membrane-less compartment. Using data-independent acquisition mass spectrometry and microscopy, we show that despite not impacting protein stability, UbKEKS is required to maintain a normal nucleolar organization. The absence of UbKEKS increases nucleoli's size and accentuate their circularity while disrupting dense fibrillar component and fibrillar centre structures. Moreover, depletion of UbKEKS leads to distinct changes in nucleolar composition. Lack of UbKEKS favours nucleolar sequestration of known apoptotic regulators such as IFI16 or p14ARF, resulting in an increase of apoptosis observed by flow cytometry and real-time monitoring. Overall, these results identify the first cellular functions of the UbKEKS variant and lay the foundation stone to establish UbKEKS as a new universal layer of regulation in the ubiquitination system.},
}
@article {pmid37336378,
year = {2023},
author = {Ma, W and Yang, J and Ding, J and Duan, C and Zhao, W and Peng, YL and Bhadauria, V},
title = {CRISPR/Cas9-mediated deletion of large chromosomal segments identifies a minichromosome modulating the Colletotrichum graminicola virulence on maize.},
journal = {International journal of biological macromolecules},
volume = {245},
number = {},
pages = {125462},
doi = {10.1016/j.ijbiomac.2023.125462},
pmid = {37336378},
issn = {1879-0003},
mesh = {Virulence/genetics ; *Zea mays/genetics/microbiology ; *CRISPR-Cas Systems/genetics ; DNA ; },
abstract = {Colletotrichum graminicola causes anthracnose on maize, an economically significant disease worldwide. To decipher how the pathogen controls its virulence/pathogenicity on maize at the minichromosomal level, we sequenced the genome and transcriptome of the C. graminicola strain T1-3-3. The 61.91 Mb genome contains three transcriptionally repressed, full-length strain-specific minichromosomes (<1 Mb; Chr11 through Chr13). A CRISPR/Cas9-based system was developed to knock out large chromosomal segments; it involved the generation of multiple simultaneous DNA double-strand breaks across a targeted genomic region, followed by homology-directed replacement thereof with a donor DNA template carrying the selectable marker hygromycin phosphotransferase gene flanked by homologous sequence arms of the targeted region. Using this system, we obtained distinct mutants functionally nullisomic for individual minichromosomes. Only the ΔChr12 mutant lacking the 498.44 Kb genomic region carrying all of the 31 genes of Chr12 exhibited attenuated virulence on maize and was indistinguishable from T1-3-3 in fungal growth and conidiation, indicating that Chr12 is a conditionally dispensable minichromosome and imparts full virulence to C. graminicola on maize. The CRISPR/Cas9-mediated genome editing system developed in this study will enable the determination of the biological functions of minichromosomes or large chromosomal segments in fungal plant pathogens.},
}
@article {pmid37719218,
year = {2023},
author = {Muthusamy, M and Son, S and Park, SR and Lee, SI},
title = {Heat shock factor binding protein BrHSBP1 regulates seed and pod development in Brassica rapa.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1232736},
pmid = {37719218},
issn = {1664-462X},
abstract = {Plant heat shock factor binding proteins (HSBPs) are well known for their implication in the negative regulation of heat stress response (HSR) pathways. Herein, we report on the hitherto unknown functions of HSBP1 in Brassica rapa (BrHSBP1). BrHBSP1 was found to be predominant in flower buds and young leaves, while its segmental duplicate, BrHSBP1-like, was abundant in green siliques. Exposure to abiotic stress conditions, such as heat, drought, cold, and H2O2, and to phytohormones was found to differentially regulate BrHSBP1. The activity of BrHSBP1-GFP fusion proteins revealed their cellular localization in nuclei and cytosols. Transgenic overexpression of BrHSBP1 (BrHSBP1[OX]) improved pod and seed sizes, while CRISPR-Cas BrHSBP1 knock-out mutants (Brhsbp1_KO) were associated with aborted seed and pod development. The transcriptomic signatures of BrHSBP1[OX] and Brhsbp1_KO lines revealed that 360 and 2381 genes, respectively, were differentially expressed (Log2FC≥2, padj<0.05) expressed relative to control lines. In particular, developmental processes, including plant reproductive structure development (RSD)-related genes, were relatively downregulated in Brhsbp1_KO. Furthermore, yeast two-hybrid assays confirmed that BrHSBP1 can physically bind to RSD and other genes. Taking the findings together, it is clear that BrHSBP1 is involved in seed development via the modulation of RSD genes. Our findings represent the addition of a new regulatory player in seed and pod development in B. rapa.},
}
@article {pmid37718766,
year = {2023},
author = {Yousefi-Najafabadi, Z and Mehmandoostli, Z and Asgari, Y and Kaboli, S and Falak, R and Kardar, GA},
title = {Reversing T Cell Exhaustion by Converting Membrane PD-1 to Its Soluble form in Jurkat Cells; Applying The CRISPR/Cas9 Exon Skipping Strategy.},
journal = {Cell journal},
volume = {25},
number = {9},
pages = {633-644},
doi = {10.22074/cellj.2023.1999548.1269},
pmid = {37718766},
issn = {2228-5806},
abstract = {OBJECTIVE: T-cells express two functional forms of the programmed cell death protein 1 (PD-1): membrane (mPD-1) and soluble (sPD-1). The binding of mPD-1 and its ligand (PD-L1) on tumor cells could lead activated lymphocytes toward exhaustion. Selective deletion of the transmembrane domain via alternative splicing of exon-3 in PD-1 mRNA could generate sPD-1. Overexpression of sPD-1 could disrupt the mPD-1/PD-L1 interaction in tumor-specific T cells. We investigated the effect of secreted sPD-1 from pooled engineered and non-engineered T cell supernatant on survival and proliferation of lymphocytes in the tumor microenvironment (TME).<br><br>MATERIALS AND METHODS: In this experimental study, we designed two sgRNA sequences upstream and downstream of exon-3 in the PDCD1 gene. The lentiCRISPRv2 puro vector was used to clone the dual sgRNAs and produce lentiviral particles to transduce Jurkat T cells. Analysis assays were used to clarify the change in PD-1 expression pattern in the pooled (engineered and non-engineered) Jurkat cells. Co-culture conditions were established with PD-L1+ cancer cells and lymphocytes.<br><br>RESULTS: CRISPR/Cas9 could delete exon-3 of the PDCD1 gene in the engineered cells based on the tracking of indels by decomposition (TIDE) and interference of CRISPR edit (ICE) sequencing analysis reports. Our results showed a 12% reduction in mPD-1 positive cell population after CRISPR manipulation and increment in sPD-1 concentration in the supernatant. The increased sPD-1 confirmed its positive effect on proliferation of lymphocytes co-cultured with PDL1+ cancer cells. The survival percent of lymphocytes co-cultured with the pooled cells supernatant was 12.5% more than the control.<br><br>CONCLUSION: The CRISPR/Cas9 exon skipping approach could be used in adoptive cell immunotherapies to change PD-1 expression patterns and overcome exhaustion.},
}
@article {pmid37717422,
year = {2023},
author = {Dong, J and Wu, X and Hu, Q and Sun, C and Li, J and Song, P and Su, Y and Zhou, L},
title = {An immobilization-free electrochemical biosensor based on CRISPR/Cas13a and FAM-RNA-MB for simultaneous detection of multiple pathogens.},
journal = {Biosensors &amp; bioelectronics},
volume = {241},
number = {},
pages = {115673},
doi = {10.1016/j.bios.2023.115673},
pmid = {37717422},
issn = {1873-4235},
abstract = {To better respond to biosecurity issues, we need to build good technology and material reserves for pathogenic microorganism screening. Here, we designed an electrochemical/optical signal probe with a common fluorophore and an electrochemically active group, breaking the previous perception that the signal probe is composed of a fluorophore and a quenching group and realizing the response of three signals: electrochemistry, fluorescence, and direct observation. Then, we proposed a homogeneous electrochemical nucleic acid detection system based on CRISPR/Cas named "HELEN-CR" by integrating free electrochemical/optical signal probes and Cas13a cleavage, achieving a limit of detection of 1 pM within 25 min. To improve the detection sensitivity, we applied recombinase polymerase amplification to amplify the target nucleic acid, achieving a limit of detection of 30 zM within 45 min. Complemented by our self-developed multi-chamber microfluidic chip and portable electrochemical instrument, simultaneous detection of multiple pathogens can be achieved within 50 min, facilitating minimally trained personnel to obtain detection results quickly in a difficult environment. This study proposes a simple, scalable, and general idea and solution for the rapid detection of pathogenic microorganisms and biosecurity monitoring.},
}
@article {pmid37716509,
year = {2023},
author = {Saha, K and Subramenium Ganapathy, A and Wang, A and Arumugam, P and Michael Morris, N and Harris, L and Yochum, G and Koltun, W and Perdew, GH and Nighot, M and Ma, T and Nighot, P},
title = {Alpha-tocopherylquinone-mediated activation of the Aryl Hydrocarbon Receptor regulates the production of inflammation-inducing cytokines and ameliorates intestinal inflammation.},
journal = {Mucosal immunology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.mucimm.2023.09.003},
pmid = {37716509},
issn = {1935-3456},
abstract = {This study investigated the role of Alpha-tocopherylquinone (TQ) in regulating the intestinal immune system and the underlying mechanisms. In the experimental dextran sodium sulfate and T cell-mediated colitis models, TQ significantly reduced the mRNA levels of IL-6, IL-1β, IL17A, IL-23, and TNF-α, and the abundance of pro-inflammatory macrophages, Th17 cells and ILC3s in the colons of WT mice. TQ also prevented LPS-induced activation of NFκB and Stat-3 pathway in the human macrophage U937 cells. Pharmacological inhibition or CRISPR-Cas-9-mediated knockout of Aryl hydrocarbon Receptor (AHR) prevented the anti-inflammatory effects of TQ in the LPS-treated U937 cells. Furthermore, TQ reduced the mRNA levels of the LPS-induced proinflammatory cytokines in the WT but not Ahr[-/-] mice splenocytes. TQ also reduced IL-6R protein levels and IL-6-induced Stat-3 activation in Jurkat cells and in-vitro differentiation of Th17 cells from WT, but not Ahr[-/-] mice naïve T cells. Additionally, TQ prevented the pro-inflammatory effects of LPS on macrophages and stimulation of T cells in human PBMCs and significantly reduced the abundance of TNF-α, IL-1β, and IL-6[hi] inflammatory macrophages and Th17 cells in surgically resected Crohn's disease (CD) tissue. Our study shows that TQ is a naturally occurring, non-toxic, and effective immune modulator that activates AhR and suppresses the Stat-3-NFkB signaling.},
}
@article {pmid37693568,
year = {2023},
author = {Nemudraia, A and Nemudryi, A and Wiedenheft, B},
title = {Repair of CRISPR-guided RNA breaks enables site-specific RNA editing in human cells.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37693568},
abstract = {UNLABELLED: Genome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. Here, we show that site-specific RNA breaks generated with RNA-targeting CRISPR complexes are repaired in human cells, and this repair can be used for programmable deletions in human transcripts that restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.<br><br>ONE-SENTENCE SUMMARY: CRISPR-guided RNA breaks are repaired in human cells, and this RNA repair can be used for programmable editing of human transcriptomes.},
}
@article {pmid37672770,
year = {2023},
author = {Naveed, M and Aqib Shabbir, M and Aziz, T and Hurraira, HM and Fatima Zaidi, S and Athar, R and Chattha, HA and Alharbi, M and Alshammari, A and Alasmari, AF},
title = {CRISPR-Cas9 guided rna based model for the treatment of Amyotrophic Lateral Sclerosis: A progressive neurodegenerative disorder.},
journal = {Acta biochimica Polonica},
volume = {70},
number = {3},
pages = {643-653},
doi = {10.18388/abp.2020_6789},
pmid = {37672770},
issn = {1734-154X},
mesh = {Humans ; *Amyotrophic Lateral Sclerosis/genetics/therapy ; CRISPR-Cas Systems/genetics ; Gene Editing ; Muscles ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that leads to the degeneration of motor neurons and the weakening of muscles. Despite extensive research efforts, there is currently no cure for ALS and existing treatments only address its symptoms. To address this unmet medical need, genome editing technologies, such as CRISPR-Cas9, have emerged as a promising solution for the development of new treatments for ALS. Studies have shown that CRISPR-Cas9-guided RNAs have the potential to provide accurate and effective silencing in the genetic disease of ALS. Results have demonstrated a 67% on-target score and a 98% off-target score with GC content within the range of 40-60%. This is further validated by the correlation between the gRNA's structural accuracy and the minimum free energy. The use of CRISPR-Cas9 provides a unique opportunity to target this disease at the molecular level, offering hope for the development of a more effective treatment. In silico and computational therapeutic approaches for ALS suggest that the CRISPR-Cas9 protein holds promise as a future treatment candidate. The CRISPR mechanism and the specificity of gRNA provide a novel therapeutic approach for this genetic disease, offering new hope to those affected by ALS. This study highlights the potential of CRISPR-Cas9 as a promising solution for the development of new treatments for ALS. Further research is required to validate these findings in preclinical and clinical trials and to establish the safety and efficacy of this approach in the treatment of ALS.},
}
@article {pmid37658843,
year = {2023},
author = {Duarte, F and Vachey, G and Caron, NS and Sipion, M and Rey, M and Perrier, AL and Hayden, MR and Déglon, N},
title = {Limitations of Dual-Single Guide RNA CRISPR Strategies for the Treatment of Central Nervous System Genetic Disorders.},
journal = {Human gene therapy},
volume = {34},
number = {17-18},
pages = {958-974},
doi = {10.1089/hum.2023.109},
pmid = {37658843},
issn = {1557-7422},
mesh = {Humans ; Animals ; Mice ; RNA, Guide, CRISPR-Cas Systems ; HEK293 Cells ; Exons/genetics ; Alleles ; *Central Nervous System Diseases ; *Huntington Disease/genetics/therapy ; Central Nervous System ; },
abstract = {Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a toxic gain-of-function CAG expansion in the first exon of the huntingtin (HTT) gene. The monogenic nature of HD makes mutant HTT (mHTT) inactivation a promising therapeutic strategy. Single nucleotide polymorphisms frequently associated with CAG expansion have been explored to selectively inactivate mHTT allele using the CRISPR/Cas9 system. One of such allele-selective approaches consists of excising a region flanking the first exon of mHTT by inducing simultaneous double-strand breaks at upstream and downstream positions of the mHTT exon 1. The removal of the first exon of mHTT deletes the CAG expansion and important transcription regulatory sites, leading to mHTT inactivation. However, the frequency of deletion events is yet to be quantified either in vitro or in vivo. Here, we developed accurate quantitative digital polymerase chain reaction-based assays to assess HTT exon 1 deletion in vitro and in fully humanized HU97/18 mice. Our results demonstrate that dual-single guide RNA (sgRNA) strategies are efficient and that 67% of HTT editing events are leading to exon 1 deletion in HEK293T cells. In contrast, these sgRNA actively cleaved HTT in HU97/18 mice, but most editing events do not lead to exon 1 deletion (10% exon 1 deletion). We also showed that the in vivo editing pattern is not affected by CAG expansion but may potentially be due to the presence of multiple copies of wildtype (wt)/mHTT genes HU97/18 mice as well as the slow kinetics of AAV-mediated CRISPR/Cas9 delivery.},
}
@article {pmid37254814,
year = {2023},
author = {Eroglu, M and Yu, B and Derry, WB},
title = {Efficient CRISPR/Cas9 mediated large insertions using long single-stranded oligonucleotide donors in C. elegans.},
journal = {The FEBS journal},
volume = {290},
number = {18},
pages = {4429-4439},
doi = {10.1111/febs.16876},
pmid = {37254814},
issn = {1742-4658},
support = {PJT 165837//CIHR/Canada ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Oligonucleotides/genetics ; DNA ; DNA, Single-Stranded/genetics ; },
abstract = {Highly efficient generation of deletions, substitutions, and small insertions (up to ~ 150 bp) into the Caenorhabditis elegans genome by CRISPR/Cas9 has been facilitated by the use of single-stranded oligonucleotide donors as repair templates. However, insertion of larger sequences such as fluorescent markers and other functional domains remains challenging due to uncertainty of optimal performance between single-stranded or double-stranded repair templates and labor-intensive as well as inefficient protocols for their preparations. Here, we simplify the generation of long ssDNA as donors in CRISPR/Cas9. High yields of ssDNA can be rapidly generated using a standard PCR followed by a single enzymatic digest with lambda exonuclease. Comparison of long ssDNA donors obtained using this method to dsDNA demonstrates orders of magnitude increased insertion frequency for ssDNA donors. This can be leveraged to simultaneously generate multiple large insertions as well as successful edits without the use of selection or co-conversion (co-CRISPR) markers when necessary. Our approach complements the CRISPR/Cas9 toolkit for C. elegans to enable highly efficient insertion of longer sequences with a simple, standardized, and labor-minimal protocol.},
}
@article {pmid37226388,
year = {2023},
author = {Chu, J and Romero, A and Taulbee, J and Aran, K},
title = {Development of Single Molecule Techniques for Sensing and Manipulation of CRISPR and Polymerase Enzymes.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {19},
number = {38},
pages = {e2300328},
doi = {10.1002/smll.202300328},
pmid = {37226388},
issn = {1613-6829},
support = {1R01HL161361-01/NH/NIH HHS/United States ; 1R01HL161361-01/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; Biotechnology ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and polymerases are powerful enzymes and their diverse applications in genomics, proteomics, and transcriptomics have revolutionized the biotechnology industry today. CRISPR has been widely adopted for genomic editing applications and Polymerases can efficiently amplify genomic transcripts via polymerase chain reaction (PCR). Further investigations into these enzymes can reveal specific details about their mechanisms that greatly expand their use. Single-molecule techniques are an effective way to probe enzymatic mechanisms because they may resolve intermediary conformations and states with greater detail than ensemble or bulk biosensing techniques. This review discusses various techniques for sensing and manipulation of single biomolecules that can help facilitate and expedite these discoveries. Each platform is categorized as optical, mechanical, or electronic. The methods, operating principles, outputs, and utility of each technique are briefly introduced, followed by a discussion of their applications to monitor and control CRISPR and Polymerases at the single molecule level, and closing with a brief overview of their limitations and future prospects.},
}
@article {pmid37715442,
year = {2023},
author = {Vasques Raposo, J and Rodrigues Carvalho Barros, L and Ribas Torres, L and Barbosa da Silva Pinto, R and De Oliveira Lopes, A and Mello de Souza, E and Hernan Bonamino, M and Salete de Paula, V},
title = {CRISPR/Cas-9 vector system: targets UL-39 and inhibits Simplexvirus humanalpha1 (HSV-1) replication in vitro.},
journal = {Cellular and molecular biology (Noisy-le-Grand, France)},
volume = {69},
number = {7},
pages = {19-23},
doi = {10.14715/cmb/2023.69.7.3},
pmid = {37715442},
issn = {1165-158X},
mesh = {Chlorocebus aethiops ; Animals ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Vero Cells ; *Herpesvirus 1, Human/genetics ; Biological Assay ; },
abstract = {Simplexvirus humanalpha1 (HSV-1) affects approximately 67% of the world's population. Here, we sought to use the CRISPR / Cas9 system with the UL39 target, essential for virus replication. The sgRNA sequence was inserted into the plasmid (PX459-UL39). Vero cells were transfected with PX459-UL39, and inhibition of viral replication was assessed 24 and 48 hours later using plaque assays and fluorescence and qPCR. Fluorescence analyses revealed the presence of anti-HSV-1 CRISPR/Cas9 within Vero cells, and qPCR showed that the viral load decreased by> 95% of cells transfected with anti-HSV-1 CRISPR / Cas 9. Our data demonstrate the usefulness of the PX459-UL39 to inhibit HSV-1 infection.},
}
@article {pmid37715317,
year = {2023},
author = {Chan, WT and Garcillán-Barcia, MP and Yeo, CC and Espinosa, M},
title = {Type II bacterial toxin-antitoxins: hypotheses, facts, and the newfound plethora of the PezAT system.},
journal = {FEMS microbiology reviews},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsre/fuad052},
pmid = {37715317},
issn = {1574-6976},
abstract = {Toxin-antitoxin (TA) systems are entities found in the prokaryotic genomes, with eight reported types. Type II, the best characterised, is comprised of two genes organised as an operon. Whereas toxins impair growth, the cognate antitoxin neutralises its activity. TAs appeared to be involved in plasmid maintenance, persistence, virulence, and defence against bacteriophages. Most Type II toxins target the bacterial translational machinery. They seem to be antecessors of Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) RNases, minimal nucleotidyltransferase domains, or CRISPR-Cas systems. Four TAs encoded by Streptococcus pneumoniae, RelBE, YefMYoeB, Phd-Doc, and HicAB, belong to HEPN-RNases. The fifth is represented by PezAT/Epsilon-Zeta. PezT/Zeta toxins phosphorylate the peptidoglycan precursors, thereby blocking cell wall synthesis. We explore the body of knowledge (facts) and hypotheses procured for Type II TAs and analyse the data accumulated on the PezAT family. Bioinformatics analyses showed that homologues of PezT/Zeta toxin are abundantly distributed among 14 bacterial phyla mostly in Proteobacteria (48%), Firmicutes (27%), and Actinobacteria (18%), showing the widespread distribution of this TA. The pezAT locus was found to be mainly chromosomally encoded whereas its homologue, the tripartite omega-epsilon-zeta locus, was found mostly on plasmids. We found several orphan pezT/zeta toxins, unaccompanied by a cognate antitoxin.},
}
@article {pmid37715312,
year = {2023},
author = {Xi, Y and Zhao, J and Zhang, J and Jin, Y and Yang, H and Duan, G and Chen, S and Long, J},
title = {Analysis of the features of 105 confirmed CRISPR loci in 487 Klebsiella variicola.},
journal = {Letters in applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/lambio/ovad108},
pmid = {37715312},
issn = {1472-765X},
abstract = {Klebsiella variicola (K. variicola), an emerging human pathogen, poses a threat to public health. The horizontal gene transfer (HGT) of plasmids is an important driver of the emergence of multiple antibiotic-resistant K. variicola. Clustered regularly interspersed short palindromic repeats (CRISPR) coupled with CRISPR-associated genes (CRISPR/Cas) constitute an adaptive immune system in bacteria, and can provide acquired immunity against HGT. However, the information about the CRISPR/Cas system in K. variicola is still limited. In this study, 487 genomes of K. variicola obtained from the National Center for Biotechnology Information database were used to analyze the characteristics of CRISPR/Cas systems. Approximately 21.56% of genomes (105/487) harbor at least one confirmed CRISPR array. Three types of CRISPR/Cas systems, namely, the types I-E, I-E*, and IV-A systems, were identified among 105 strains. Spacer origin analysis further revealed that approximately one-third of spacers significantly match plasmids or phages, which demonstrates the implication of CRISPR/Cas systems in controlling HGT. Moreover, spacers in K. variicola tend to target mobile genetic elements from K. pneumoniae. This finding provides new evidence of the interaction of K. variicola and K. pneumoniae during their evolution. Collectively, our results provide valuable insights into the role of CRISPR/Cas systems in K. variicola.},
}
@article {pmid37714365,
year = {2023},
author = {Fadaie, M and Dianat-Moghadam, H and Ghafouri, E and Naderi, S and Darvishali, MH and Ghovvati, M and Khanahmad, H and Boshtam, M and Makvandi, P},
title = {Unraveling the potential of M13 phages in biomedicine: Advancing drug nanodelivery and gene therapy.},
journal = {Environmental research},
volume = {},
number = {},
pages = {117132},
doi = {10.1016/j.envres.2023.117132},
pmid = {37714365},
issn = {1096-0953},
abstract = {M13 phages possessing filamentous phage genomes offer the benefits of selective display of molecular moieties and delivery of therapeutic agent payloads with a tolerable safety profile. M13 phage-displayed technology for resembling antigen portions led to the discovery of mimetic epitopes that applied to antibody-based therapy and could be useful in the design of anticancer vaccines. To date, the excremental experiences have engaged the M13 phage in the development of innovative biosensors for detecting biospecies, biomolecules, and human cells with an acceptable limit of detection. Addressing the emergence of antibiotic-resistant bacteria, M13 phages are potent for packaging the programmed gene editing tools, such as CRISPR/Cas, to target multiple antimicrobial genes. Moreover, their display potential in combination with nanoparticles inspires new approaches for engineering targeted theragnostic platforms targeting multiple cellular biomarkers in vivo. In this review, we present the available data on optimizing the use of bacteriophages with a focus on the to date experiences with M13 phages, either as monoagent or as part of combination regimens in the practices of biosensors, vaccines, bactericidal, modeling of specific antigen epitopes, and phage-guided nanoparticles for drug delivery systems. Despite increasing research interest, a deep understanding of the underlying biological and genetic behaviors of M13 phages is needed to enable the full potential of these bioagents in biomedicine, as discussed here. We also discuss some of the challenges that have thus far limited the development and practical marketing of M13 phages.},
}
@article {pmid37713428,
year = {2023},
author = {Watson, BNJ and Pursey, E and Gandon, S and Westra, ER},
title = {Transient eco-evolutionary dynamics early in a phage epidemic have strong and lasting impact on the long-term evolution of bacterial defences.},
journal = {PLoS biology},
volume = {21},
number = {9},
pages = {e3002122},
doi = {10.1371/journal.pbio.3002122},
pmid = {37713428},
issn = {1545-7885},
abstract = {Organisms have evolved a range of constitutive (always active) and inducible (elicited by parasites) defence mechanisms, but we have limited understanding of what drives the evolution of these orthogonal defence strategies. Bacteria and their phages offer a tractable system to study this: Bacteria can acquire constitutive resistance by mutation of the phage receptor (surface mutation, sm) or induced resistance through their CRISPR-Cas-adaptive immune system. Using a combination of theory and experiments, we demonstrate that the mechanism that establishes first has a strong advantage because it weakens selection for the alternative resistance mechanism. As a consequence, ecological factors that alter the relative frequencies at which the different resistances are acquired have a strong and lasting impact: High growth conditions promote the evolution of sm resistance by increasing the influx of receptor mutation events during the early stages of the epidemic, whereas a high infection risk during this stage of the epidemic promotes the evolution of CRISPR immunity, since it fuels the (infection-dependent) acquisition of CRISPR immunity. This work highlights the strong and lasting impact of the transient evolutionary dynamics during the early stages of an epidemic on the long-term evolution of constitutive and induced defences, which may be leveraged to manipulate phage resistance evolution in clinical and applied settings.},
}
@article {pmid37676272,
year = {2023},
author = {Luo, P and Huang, X and Luo, F and Chen, Z and Chen, Y and Lin, C and Wang, J and Qiu, B and Lin, Z},
title = {Low-Background Signal-On Homogeneous Electrochemiluminescence Biosensor for Hepatitis B Virus Detection Based on the Regulation of the Length of DNA Modified on the Nanoparticles by CRISPR/Cas12a and Hybridization Chain Reaction.},
journal = {Analytical chemistry},
volume = {95},
number = {37},
pages = {14127-14134},
doi = {10.1021/acs.analchem.3c03141},
pmid = {37676272},
issn = {1520-6882},
mesh = {Hepatitis B virus/genetics ; CRISPR-Cas Systems ; Reproducibility of Results ; *Ruthenium ; Silicon Dioxide ; DNA ; *Nanoparticles ; },
abstract = {In this work, combined with the high amplification efficiency of hybridization chain reaction (HCR), high specificity of the CRISPR/Cas12a system, and convenience of the homogeneous electrochemiluminescence (ECL) assay based on the regulation of negative charge on the reporting probes, a sensitive ECL biosensor for hepatitis B virus DNA (chosen as a model target) had been developed. The initiator chain trigger DNA that can induce HCR amplification is modified on the surface of ruthenium bipyridine-doped silica nanoparticles (Ru@SiO2 NPs) first, and large amounts of negative charges modified on the particles were achieved through the HCR amplification reaction. The efficiency of the nanoparticles reaching the negatively charged working electrode can be regulated and realize the change of the ECL signal. In addition, long DNA on the surface of the luminescent body may prevent the coreactant from entering the pore to react with ruthenium bipyridine. These factors combine to produce a low-background system. The presence of the target can activate the CRISPR/Cas12a system and make trigger DNA disappear from the nanoparticle surface, and strong ECL can be detected. The sensor does not require a complex electrode modification; therefore, it has better reproducibility. Additionally, due to dual signal amplification, the sensor has a high sensitivity. In the range of 10 fM to 10 nM, the ECL intensity exhibits a strong linear relationship with the logarithm of the target concentration, and the detection limit is 7.41 fM. This sensor has shown high accuracy in detecting clinical samples, which holds significant potential for application in clinical testing.},
}
@article {pmid37668549,
year = {2023},
author = {Jia, Z and Zhang, Y and Zhang, C and Wei, X and Zhang, M},
title = {Biosensing Intestinal Alkaline Phosphatase by Pregnancy Test Strips Based on Target-Triggered CRISPR-Cas12a Activity to Monitor Intestinal Inflammation.},
journal = {Analytical chemistry},
volume = {95},
number = {37},
pages = {14111-14118},
doi = {10.1021/acs.analchem.3c03099},
pmid = {37668549},
issn = {1520-6882},
mesh = {Female ; Pregnancy ; Animals ; Mice ; Alkaline Phosphatase ; CRISPR-Cas Systems ; *Inflammatory Bowel Diseases/diagnosis ; *Colitis ; DNA, Single-Stranded ; *Pregnancy Tests ; Inflammation/diagnosis ; },
abstract = {With an increasing incidence worldwide, inflammatory bowel disease (IBD) is a chronic inflammatory disease affecting the gastrointestinal tract, which impairs the life quality of patients. Therefore, it is of great significance to construct a sensitive, simple, and convenient biosensor to analyze IBD-associated biomarkers for an auxiliary diagnosis of IBD. Intestinal alkaline phosphatase (IAP), expressed by the intestinal epithelium, is an endogenous protein that is thought to play a vital role in maintaining intestinal homeostasis and is considered a potential biomarker for IBD. Here, an IAP detection method was developed using pregnancy test strips by dephosphorylation. Initially, a double-stranded DNA (dsDNA) was designed to respond to IAP and acted as an activator of Cas12a. In the presence of IAP, the designed dsDNA was not digested by lambda exonuclease (λ exo), which hybridized to the Cas12a-crRNA duplex and resulted in the activation of the trans-cleavage of Cas12a. Further, the activated Cas12a cleaved the single-strand DNA (ssDNA) linker in the MBs-ssDNA-hCG probe, triggering the release of hCG. With magnetic separation, the released hCG could be quantitatively detected by pregnancy test strips. IAP levels were analyzed in feces from colitis and healthy mice by pregnancy test strips. The results showed that the IAP level of colitis mice (3.89 ± 1.92 U/L) was much lower than that of healthy mice (39.64 ± 24.93 U/L), indicating the correlation between IAP and intestinal inflammation. Taken together, a sensitive, user-friendly detection assay based on pregnancy test strips was constructed to monitor IAP and used as an auxiliary diagnostic approach for IBD in a clinical scene.},
}
@article {pmid37712284,
year = {2023},
author = {Huang, D and Zhao, Y and Fang, M and Shen, P and Xu, H and He, Y and Chen, S and Si, Z and Xu, Z},
title = {Magnetofluid-integrated biosensors based on DNase-dead Cas12a for visual point-of-care testing of HIV-1 by an up and down chip.},
journal = {Lab on a chip},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3lc00558e},
pmid = {37712284},
issn = {1473-0189},
abstract = {The CRISPR Cas system, as a novel nucleic acid detection tool, is often hindered by cumbersome experimental procedures, complicated reagent transfer processes, and associated aerosol pollution risks. In this study, an integrated nucleic acid detection platform named "up and down chip" was developed, which combined RT-RAA technology for nucleic acid amplification, DNase-dead Cas12a-modified magnetic beads for specific recognition of target nucleic acid, and HRP-TMB chromogenic reaction for signal output in different chambers of a single microfluidic chip. The magnetic beads were migrated in an up-and-down manner between different chambers through magnetic driving, achieving a "sample-in, result-out" detection mode. By introducing a homemade heating box for temperature control during the reaction and using the naked eye or a smartphone APP for color-based signal reading, no professional or precise instruments were required in this platform. Using this platform, highly sensitive detection of the HIV-1 genome as low as 250 copies (CPs) per mL was achieved within 100 min while maintaining good detection performance against common variants as well as excellent specificity and anti-interference ability. In addition, compared with qRT-PCR, it also exhibited good accuracy for 56 spiked plasma samples, indicating its promising potential for clinical application.},
}
@article {pmid37711452,
year = {2023},
author = {Xiao, J and Li, J and Quan, S and Wang, Y and Jiang, G and Wang, Y and Huang, H and Jiao, W and Shen, A},
title = {Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of Mycobacterium tuberculosis complex.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1233353},
pmid = {37711452},
issn = {2296-4185},
abstract = {Since the onset of the COVID-19 pandemic in 2020, global efforts towards tuberculosis (TB) control have encountered unprecedented challenges. There is an urgent demand for efficient and cost-effective diagnostic technologies for TB. Recent advancements in CRISPR-Cas technologies have improved our capacity to detect pathogens. The present study established a CRISPR-Cas12a-based multiplex detection (designated as MCMD) that simultaneously targets two conserved insertion sequences (IS6110 and IS1081) to detect Mycobacterium tuberculosis complex (MTBC). The MCMD integrated a graphene oxide-assisted multiplex recombinase polymerase amplification (RPA) assay with a Cas12a-based trans-cleavage assay identified with fluorescent or lateral flow biosensor (LFB). The process can be performed at a constant temperature of around 37°C and completed within 1 h. The limit of detection (LoD) was 4 copies μL[-1], and no cross-reaction was observed with non-MTBC bacteria strains. This MCMD showed 74.8% sensitivity and 100% specificity in clinical samples from 107 patients with pulmonary TB and 40 non-TB patients compared to Xpert MTB/RIF assay (63.6%, 100%). In this study, we have developed a straightforward, rapid, highly sensitive, specific, and cost-effective assay for the multiplex detection of MTBC. Our assay showed superior diagnostic performance when compared to the widely used Xpert assay. The novel approach employed in this study makes a substantial contribution to the detection of strains with low or no copies of IS6110 and facilitates point-of-care (POC) testing for MTBC in resource-limited countries.},
}
@article {pmid37711183,
year = {2023},
author = {Deng, B and Xue, J},
title = {HIV infection detection using CRISPR/Cas systems: Present and future prospects.},
journal = {Computational and structural biotechnology journal},
volume = {21},
number = {},
pages = {4409-4423},
pmid = {37711183},
issn = {2001-0370},
abstract = {Human immunodeficiency virus (HIV) infection poses substantial medical risks to global public health. An essential strategy to combat the HIV epidemic is timely and effective virus testing. CRISPR-based assays combine the highly compatible CRISPR system with different elements, yielding portability, digitization capabilities, low economic burden and low operational thresholds. The application of CRISPR-based assays has demonstrated rapid, accurate, and accessible means of pathogen testing, suggesting great potential as point-of-care (POC) assays. This review outlines the different types of CRISPR/Cas systems based on Cas proteins and their applications for the detection of HIV. Additionally, we also offer an overview of future perspectives on CRISPR-based methods for HIV detection, including advances in nucleic acid amplification-free testing, improved personal testing, and refined testing for HIV genotypes and drug-resistant strains.},
}
@article {pmid37710076,
year = {2023},
author = {He, Q and Lei, X and Liu, Y and Wang, X and Ji, N and Yin, H and Wang, H and Zhang, H and Yu, G},
title = {Nucleic Acid Detection through RNA-Guided Protease Activity in Type III-E CRISPR-Cas Systems.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {},
number = {},
pages = {e202300401},
doi = {10.1002/cbic.202300401},
pmid = {37710076},
issn = {1439-7633},
abstract = {RNA-guided protease activity was recently discovered in the type III-E CRISPR-Cas systems (Craspase), providing a novel platform for engineering a protein probe instead of the commonly used nucleic acid probe in the nucleic acid detection assays. Here, by adapting a fluorescence readout technique using the affinity- and fluorescent protein dual-tagged Csx30 protein substrate, we established an assay monitoring Csx30 cleavage by target ssRNA-activated Craspase. Four Craspase-based nucleic acid detection systems for genes from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), norovirus, and the influenza virus (IFV) were reconstituted with demonstrated specificity. The assay could reliably detect target ssRNAs with concentrations down to 25 pM, which could be further improved by approximately 15,000-fold (~ 2 fM) through incorporating the recombinase polymerase isothermal preamplification step. Importantly, the species-specific substrate cleavage specificity of Craspase enabled multiplexed diagnosis, as demonstrated by the reconstituted composite systems for simultaneous detection of two genes from the same virus (SARS-CoV-2, spike and nsp12) or two types of viruses (SARS-CoV-2 and IFV). The assay could be further expanded by diversifying the fluorescent tags in the substrate and including Craspase systems of various species, potentially providing an easily adaptable platform for clinical diagnosis.},
}
@article {pmid37709482,
year = {2023},
author = {Liu, Y and Liu, H and Yu, G and Sun, W and Aizaz, M and Yang, G and Chen, L},
title = {One-tube RPA-CRISPR Cas12a/Cas13a rapid detection of methicillin-resistant Staphylococcus aureus.},
journal = {Analytica chimica acta},
volume = {1278},
number = {},
pages = {341757},
doi = {10.1016/j.aca.2023.341757},
pmid = {37709482},
issn = {1873-4324},
mesh = {Humans ; *Methicillin-Resistant Staphylococcus aureus/genetics ; CRISPR-Cas Systems ; Environmental Pollution ; Escherichia coli ; Point-of-Care Testing ; },
abstract = {At present, methicillin-resistant Staphylococcus aureus (MRSA) has caused a serious impact on a global scale. The infection and carrier rate of MRSA in the community is increasing year by year, but there is still no convenient detection system for on-site rapid detection. It is very important to select a rapid detection system to accurately and quickly detect patients infected with MRSA. We have developed a high-efficient single-tube detection platform based on RPA and CRISPR reaction system to detect the genes of mecA and clfA of MRSA. Using this detection platform, visual MRSA detection could be achieved in 30 min. It was observed that this detection platform was capable to successfully detect the target genomic as low as 5 copies μL[-1], and the reaction was completed in one step without opening the lid. This detection platform could only detect MRSA, but not other common clinical pathogenic bacteria, such as Salmonella, Pseudomonas aeruginosa, Staphylococcus xylosus, Aeromonas hydrophila, Escherichia coli and Staphylococcus warneri, indicated its satisfactory selectivity for MRSA without interference from other bacteria. The results of clinical samples show that the platform has outstanding advantages in sensitivity, specificity and identification of methicillin resistance. The entire reaction can be completed in one step in the handheld instrument without opening the cover, avoiding aerosol pollution during the reaction. The detection platform combined with handheld instruments will have great application potential in point-of-care testing.},
}
@article {pmid37709481,
year = {2023},
author = {Shi, K and Chen, J and Li, Y and Li, Q and Song, J and Yi, Z and Li, D and Zhang, J},
title = {Hg[2+]-triggered cascade strand displacement assisted CRISPR-Cas12a for Hg[2+] quantitative detection using a portable glucose meter.},
journal = {Analytica chimica acta},
volume = {1278},
number = {},
pages = {341756},
doi = {10.1016/j.aca.2023.341756},
pmid = {37709481},
issn = {1873-4324},
mesh = {*Glucose ; CRISPR-Cas Systems ; Catalysis ; *Mercury ; Sucrase ; },
abstract = {CRISPR-Cas12a is a powerful and programmable tool that has revolutionized the field of biosensing. However, the construction of a CRISPR-Cas12a-mediated portable system for on-site and quantitative detection of mercury ion (Hg[2+]) has yet to be explored. By integrating a target-triggered cascade toehold-mediated strand displacement reaction (TSDR) and CRISPR-Cas12a, we herein construct a portable on-site biosensor for the quantitative, sensitive, and selective detection of Hg[2+] with a glucose meter. The Hg[2+] initiates two cascade TSDRs through the T-Hg[2+]-T interaction to produce multiple double-stranded DNAs that can activate Cas12a's trans-cleavage activity. The Cas12a cleaves the sucrase-modified DNA on the electrode, resulting in the liberation of sucrase into the solution. The freed sucrase can catalyze sucrose to generate glucose, which can be quantitatively monitored by a glucometer. The developed portable biosensor provides a dynamic range of 5 orders of magnitude with a detection limit of 40 fM. This biosensor also displays excellent selectivity and stability for detecting Hg[2+]. Moreover, environmental water samples are utilized to further verify the robustness and effectiveness of the developed biosensor, highlighting its potential application in environmental monitoring and food safety analysis.},
}
@article {pmid37709435,
year = {2023},
author = {Ren, D and Chen, Q and Xia, X and Xu, G and Wei, F and Yang, J and Hu, Q and Cen, Y},
title = {CRISPR/Cas12a-based fluorescence aptasensor integrated with two-dimensional cobalt oxyhydroxide nanosheets for IFN-γ detection.},
journal = {Analytica chimica acta},
volume = {1278},
number = {},
pages = {341750},
doi = {10.1016/j.aca.2023.341750},
pmid = {37709435},
issn = {1873-4324},
mesh = {*Interferon-gamma ; *CRISPR-Cas Systems ; DNA, Complementary ; Cytokines ; Oligonucleotides ; },
abstract = {Cytokine storm (CS) is a risky immune overreaction accompanied by significant elevations of pro-inflammatory cytokines including interferon-γ (IFN-γ), interleukin and tumor necrosis factor. Sensitive detection of cytokine is conducive to studying CS progress and diagnosing infectious diseases. In this study, we developed a tandem system combining aptamer, strand displacement amplification (SDA), CRISPR/Cas12a, and cobalt oxyhydroxide nanosheets (termed Apt-SCN tandem system) as a signal-amplified platform for IFN-γ detection. Owing to the stronger affinity, target IFN-γ bound specifically to the aptamer from aptamer-complementary DNA (Apt-cDNA) duplex. The cDNA released from the Apt-cDNA duplex initiated SDA, resulting in the generation of double-stranded DNA products that could activate the trans-cleavage activity of CRISPR/Cas12a. The activated CRISPR/Cas12a further cleaved FAM-labeled single-stranded DNA probe, preventing it from adhering to the cobalt oxyhydroxide nanosheets and recovering the fluorescence signal. Sensitive fluorometric analysis of IFN-γ was successfully performed with detection limit as low as 0.37 nM. Unlike traditional protein analysis methods, Apt-SCN tandem system incorporates multiple signal amplification techniques and may also be applicable for other cytokines assay. This study was the initial study to utilize SDA and CRISPR/Cas12a to detect IFN-γ, showing great potential for cytokines clinical assay and CS prevention.},
}
@article {pmid37708031,
year = {2023},
author = {Li, M and Li, D and Lin, L and Wang, P and Zhao, W},
title = {Precise Interference of RNA-Protein Interaction by CRISPR-Cas13-Mediated Peptide Competition.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.3c00287},
pmid = {37708031},
issn = {2161-5063},
abstract = {RNA-protein interactions are essential nodes of cellular regulatory circuits and play critical roles in normal physiology and disease. However, the precise roles of individual RNA-protein interactions remain elusive. Here we report a method for precise interference of endogenous RNA interacting with the RNA binding protein (RBP). TTP is an RBP that recognizes the AU-rich element (ARE) of mRNA via the binding domain TZF and represses gene expression. We engineer Cas13b, a class 2 type VI CRISPR-Cas endonuclease that exclusively targets RNA, to direct the peptide of TZF to the binding site and compete with endogenous TTP. We show that this tool specifically interferes with TTP interacting with the PIM1 and IL-2 3' UTR under the guidance of the gRNA specific for the AREs. Further, precise interference with the TTP-PIM1 interaction exerts a distinct effect on cell proliferation compared to transcriptome-wide interference. Thus, our work establishes a tool for deep understanding of RNA-RBP interactions.},
}
@article {pmid37707449,
year = {2023},
author = {Farooq, A and Lee, M and Han, S and Jung, GY and Kim, SJ and Jung, MY},
title = {Kinetic, genomic, and physiological analysis reveals diversity in the ecological adaptation and metabolic potential of Brachybacterium equifaecis sp. nov. isolated from horse feces.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0504822},
doi = {10.1128/spectrum.05048-22},
pmid = {37707449},
issn = {2165-0497},
abstract = {Brachybacterium species have been identified in various ecological niches and belong to the family Dermabacteriaceae within the phylum Actinobacteria. In this study, we isolated a novel Brachybacterium equifaecis JHP9 strain from horse feces and compared its kinetic, biochemical, and genomic features with those of other Brachybacterium strains. Moreover, comparative genomic analysis using publicly available Brachybacterium genomes was performed to determine the properties involved in their ecological adaptation and metabolic potential. Novel species delineation was determined phylogenetically through 16S rRNA gene similarity (up to 97.9%), average nucleotide identity (79.5-82.5%), average amino acid identity (66.7-75.8%), and in silico DNA-DNA hybridization (23.7-27.9) using closely related strains. This study also presents the first report of the kinetic properties of Brachybacterium species. Most of the Brachybacterium strains displayed high oxygen (K m(app) =1.6-24.2 µM) and glucose (K m(app) =0.73-1.22 µM) affinities, which may manifest niche adaptations. Various carbohydrate metabolisms under aerobic and anaerobic conditions, antibiotic resistance, mobile genetic elements, carbohydrate-active enzymes, lactic acid production, and the clustered regularly interspaced short palindromic repeats-Cas and bacteriophage exclusion systems were observed in the genotypic and/or phenotypic properties of Brachybacterium species, suggesting their genome flexibility, defense mechanisms, and adaptability. Our study contributes to the knowledge of the kinetic, physiological, and genomic properties of Brachybacterium species, including the novel JHP9 strain, which advocates for their tolerant and thriving nature in various environments, leading to their ecological adaptation. IMPORTANCE Basic physiological and genomic properties of most of the Brachybacterium isolates have been studied; however, the ability of this bacterium to adapt to diverse environments, which may demonstrate its role in niche differentiation, is to be identified yet. Therefore, here, we explored cellular kinetics, metabolic diversity, and ecological adaptation/defensive properties of the novel Brachybacterium strain through physiological and comparative genomic analysis. In addition, we presented the first report examining Brachybacterium kinetics, indicating that all strains of Brachybacterium, including the novel one, have high oxygen and glucose affinity. Furthermore, the comparative genomic analysis also revealed that the novel bacterium contains versatile genomic properties, which provide the novel bacterium with significant competitive advantages. Thus, in-depth genotypic and phenotypic analysis with kinetic properties at the species level of this genus is beneficial in clarifying its differential characteristics, conferring the ability to inhabit diverse ecological niches.},
}
@article {pmid37643152,
year = {2023},
author = {Teng, Y and Wang, J and Jiang, T and Zou, Y and Yan, Y},
title = {Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria.},
journal = {ACS synthetic biology},
volume = {12},
number = {9},
pages = {2764-2772},
doi = {10.1021/acssynbio.3c00366},
pmid = {37643152},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Streptococcus pyogenes/genetics ; CRISPR-Associated Protein 9 ; Gene Expression Regulation, Bacterial ; Gene Editing ; },
abstract = {The RNA-guided Cas9 endonucleases have revolutionized gene editing and regulation, but their targeting scope is limited by the protospacer adjacent motif (PAM) requirement. The most extensively used SpCas9 from Streptococcus pyogenes recognizes the NGG PAM via an RxR PAM-binding motif within its PAM-interaction (PI) domain. To overcome the strict PAM requirement, we identified and characterized a Cas9 ortholog from Streptococcus equinus HC5 (SeHCas9) that shows high sequence identity with SpCas9 but harbors a different RxQ PAM-binding motif. Complete PAM profiling revealed that SeHCas9 recognized an NAG PAM and accommodated NKG and NAW PAMs. We investigated the PAM interaction mechanism by identifying the crucial role of R1336 within the RxQ motif in determining PAM specificity, as well as the essentiality of two conserved residues (R1152 and Q1229) across Cas9 orthologs bearing the RxQ motif for PAM recognition. Further protein engineering created two variants, SeHdCas9-Q1229R and SeHdCas9-RR, that showed robust repression across an NNG and NNN PAM range, respectively. Our work proposes a novel Cas9 PAM interaction mechanism and establishes PAM-free Cas9 variants for bacterial gene control with almost no targeting restriction.},
}
@article {pmid37602730,
year = {2023},
author = {Gussak, A and Ferrando, ML and Schrama, M and van Baarlen, P and Wells, JM},
title = {Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology.},
journal = {ACS synthetic biology},
volume = {12},
number = {9},
pages = {2546-2560},
doi = {10.1021/acssynbio.3c00110},
pmid = {37602730},
issn = {2161-5063},
mesh = {Humans ; Animals ; Swine ; *Streptococcus suis/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Technology ; Anti-Bacterial Agents ; },
abstract = {Streptococcussuis is an important zoonotic pathogen that causes severe invasive disease in pigs and humans. Current methods for genome engineering of S. suis rely on the insertion of antibiotic resistance markers, which is time-consuming and labor-intensive and does not allow the precise introduction of small genomic mutations. Here we developed a system for CRISPR-based genome editing in S. suis, utilizing linear DNA fragments for homologous recombination (HR) and a plasmid-based negative selection system for bacteria not edited by HR. To enable the use of this system in other bacteria, we engineered a broad-host-range replicon in the CRISPR plasmid. We demonstrated the utility of this system to rapidly introduce multiple gene deletions in successive rounds of genome editing and to make precise nucleotide changes in essential genes. Furthermore, we characterized a mechanism by which S. suis can escape killing by a targeted Cas9-sgRNA complex in the absence of HR. A characteristic of this new mechanism is the presence of very slow-growing colonies in a persister-like state that may allow for DNA repair or the introduction of mutations, alleviating Cas9 pressure. This does not impact the utility of CRISPR-based genome editing because the escape colonies are easily distinguished from genetically edited clones due to their small colony size. Our CRISPR-based editing system is a valuable addition to the genetic toolbox for engineering of S. suis, as it accelerates the process of mutant construction and simplifies the removal of antibiotic markers between successive rounds of genome editing.},
}
@article {pmid37584634,
year = {2023},
author = {Dykstra, CB and Pyne, ME and Martin, VJJ},
title = {CRAPS: Chromosomal-Repair-Assisted Pathway Shuffling in Yeast.},
journal = {ACS synthetic biology},
volume = {12},
number = {9},
pages = {2578-2587},
doi = {10.1021/acssynbio.3c00170},
pmid = {37584634},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Carotenoids/metabolism ; Metabolic Engineering/methods ; CRISPR-Cas Systems ; },
abstract = {A fundamental challenge of metabolic engineering involves assembling and screening vast combinations of orthologous enzymes across a multistep biochemical pathway. Current pathway assembly workflows involve combining genetic parts ex vivo and assembling one pathway configuration per tube or well. Here, we present CRAPS, Chromosomal-Repair-Assisted Pathway Shuffling, an in vivo pathway engineering technique that enables the self-assembly of one pathway configuration per cell. CRAPS leverages the yeast chromosomal repair pathway and utilizes a pool of inactive, chromosomally integrated orthologous gene variants corresponding to a target multistep pathway. Supplying gRNAs to the CRAPS host activates the expression of one gene variant per pathway step, resulting in a unique pathway configuration in each cell. We deployed CRAPS to build more than 1000 theoretical combinations of a four-step carotenoid biosynthesis network. Sampling the CRAPS pathway space yielded strains with distinct color phenotypes and carotenoid product profiles. We anticipate that CRAPS will expedite strain engineering campaigns by enabling the generation and sampling of vast biochemical spaces.},
}
@article {pmid37399127,
year = {2023},
author = {Shi, L and Li, X and Xue, L and Zhang, J and Huang, B and Sun, Z and Zhang, Z and Dai, X and Han, S and Dong, W and Zhang, X},
title = {Creation of herbicide-resistance in allotetraploid peanut using CRISPR/Cas9-meditated cytosine base-editing.},
journal = {Plant biotechnology journal},
volume = {21},
number = {10},
pages = {1923-1925},
pmid = {37399127},
issn = {1467-7652},
support = {201300111000//the Key Project of Science and Technology of Henan Province/ ; 221100110300//the Key Project of Science and Technology of Henan Province/ ; CARS-13//China Agriculture Research System of MOF and MARA/ ; 2022YFD1200400//National Key Research and Development Program of China/ ; S2012-5//the Henan Province Agriculture Research System/ ; 222301420026//the Joint Funds of Natural Science Foundation of Henan Province/ ; 212102110256//the Programs for Science and Technology Development of Henan Province/ ; },
mesh = {Arachis/genetics ; CRISPR-Cas Systems/genetics ; Cytosine ; *Fabaceae ; *Herbicides ; Gene Editing ; },
}
@article {pmid37390886,
year = {2023},
author = {Cui, J and Wen, D and Wang, L and Meng, C and Wang, Y and Zhao, Z and Wu, C},
title = {CRISPR/Cas9-induced asap1a and asap1b co-knockout mutant zebrafish displayed abnormal embryonic development and impaired neutrophil migration.},
journal = {Gene expression patterns : GEP},
volume = {49},
number = {},
pages = {119331},
doi = {10.1016/j.gep.2023.119331},
pmid = {37390886},
issn = {1872-7298},
mesh = {Animals ; Humans ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; Neutrophils ; Protein Isoforms/genetics ; Embryonic Development ; },
abstract = {ASAP1 (Arf-GAP with SH3 domain, the ankyrin repeat and the PH domain) is the GTPase activating protein of the small G protein Arf. To understand more about the physiological functions of ASAP1 in vivo, we chose to use the zebrafish as an animal model, and analyzed the characterization of asap1 using loss-of-function studies. Here, two isoforms in zebrafish, asap1a and asap1b, were found to be homologous to human ASAP1, and the gene knockout zebrafish lines for asap1a and asap1b were established using the CRISPR/Cas9 technique with different insertions and deletions of bases. Zebrafish with asap1a and asap1b co-knockout showed a significant reduction in survival and hatching rates, as well as an increase in malformation rates during the early stages of development, while the asap1a or asap1b single knockout mutants did not affect the growth and development of individual zebrafish. Exploring the gene expression compensation between asap1a and asap1b using qRT-PCR, we found that asap1b had increased expression when asap1a was knocked out, showing a clear compensatory effect against asap1a knockout; In turn, asap1a did not have detectable compensating expression after asap1b knockout. Furthermore, the co-knockout homozygous mutants displayed impaired neutrophil migration to Mycobacterium marinum infection, and showed an increased bacterial load. Together, these are the first inherited asap1a and/or asap1b mutant zebrafish lines by the CRISPR/Cas9 gene editing approach, and by serving as useful models, they can significantly contribute to better annotation and follow-up physiological studies of human ASAP1.},
}
@article {pmid35357269,
year = {2023},
author = {Deb, R and Chaudhary, P and De, S},
title = {CRISPR/cas9 cassette targeting Escherichia coli [bla]CTX-M specific gene of mastitis cow milk origin can alter the antibiotic resistant phenotype for cefotaxime.},
journal = {Animal biotechnology},
volume = {34},
number = {5},
pages = {1849-1854},
doi = {10.1080/10495398.2022.2053695},
pmid = {35357269},
issn = {1532-2378},
mesh = {Cattle ; Female ; Animals ; Anti-Bacterial Agents/pharmacology ; Cefotaxime/pharmacology ; Escherichia coli/genetics/metabolism ; *Escherichia coli Infections/veterinary/epidemiology/genetics ; Milk/metabolism ; CRISPR-Cas Systems/genetics ; Phenotype ; beta-Lactamases/genetics/metabolism ; beta-Lactams ; *Mastitis/genetics ; *Cattle Diseases/genetics ; },
abstract = {CTX-M beta-lactamases are one of the most important extended spectrum beta-lactamase (ESBL) resistance enzymes found in E. coli. In the present study, 59% of E. coli isolates from mastitis cow milk were reported to be positive for ESBL types. The prevalence of beta-lactam (β-lactam) antibiotic resistance was reported to be 84%, 72.7%, 52.27%, 50%, and 45.4% for cefotaxime, cefepime, cefuroxime, oxacillin, and cephalexine, respectively. The [bla]CTX-M gene was found in 65% (n = 17) of the E. coli isolates when they were genotyped. Further, the use of a CRISPR/cas9 cassette to target the E. coli [bla]CTX-M gene revealed changes in antibiotic phenotypes for cefotaxime.},
}
@article {pmid37704762,
year = {2023},
author = {Li, C and Fleck, JS and Martins-Costa, C and Burkard, TR and Themann, J and Stuempflen, M and Peer, AM and Vertesy, &#xc1; and Littleboy, JB and Esk, C and Elling, U and Kasprian, G and Corsini, NS and Treutlein, B and Knoblich, JA},
title = {Single-cell brain organoid screening identifies developmental defects in autism.},
journal = {Nature},
volume = {621},
number = {7978},
pages = {373-380},
pmid = {37704762},
issn = {1476-4687},
mesh = {Humans ; *Autism Spectrum Disorder/complications/genetics/pathology ; Autistic Disorder/complications/genetics/pathology ; *Brain/cytology/metabolism ; Cell Lineage/genetics ; Chromatin/genetics ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; *Developmental Disabilities/complications/genetics/pathology ; Gene Editing ; Loss of Function Mutation ; Mosaicism ; Neurons/metabolism/pathology ; *Organoids/cytology/metabolism ; RNA, Guide, CRISPR-Cas Systems ; *Single-Cell Gene Expression Analysis ; Transcription, Genetic ; },
abstract = {The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders[1-4]. Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR-human organoids-single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR-Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.},
}
@article {pmid37703922,
year = {2023},
author = {Nami, Y and Rostampour, M and Panahi, B},
title = {CRISPR-Cas systems and diversity of targeting phages in Lactobacillus johnsonii strains; insights from genome mining approach.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {114},
number = {},
pages = {105500},
doi = {10.1016/j.meegid.2023.105500},
pmid = {37703922},
issn = {1567-7257},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes make up bacteria's adaptive immune system. These genes protect bacteria from being eaten by bacteriophages. In this study, CRISPR-Cas systems were characterized using a genomic approach. For this purpose, genome sequences of Lactobacillus johnsonii strains were retrieved, and the diversity, occurrence, and evolution of the CRISPR-Cas systems were analyzed. Then, homology analyses of spacer sequences in identified CRISPR arrays were performed to analyze and characterize the diversity of target phages and plasmids. Finally, the evolutionary paths of spaceromes in each subtype of CRISPR arrays were performed using acquisition and deletion events surveyed under the selective pressure of foreign plasmids and phages. Results showed that 138 strains contain valid CRISPR-Cas structures (CRISPR loci together with the Cas genes) in their genomes, which accounted for about 17% of the L. johnsonii studied strains belonging to subtypes II-A, I-E, and I-C. Moreover, results indicated that some specific groups of plasmids were targeted with specific CRISPR array systems. Homology analysis of spacer sequences with phage genomes also revealed that spacers of strains that harbored only CRISPR-Cas subtype-II targeted a greater diversity of foreign phages. In conclusion, the current study indicates that there is great diversity between the CRISPR-Cas systems identified in L. johnsonii strains. Such diverse CRISPR-Cas systems indicate that these systems are naturally active and important in terms of adaptive immunity and evolutionary relationships.},
}
@article {pmid37699682,
year = {2022},
author = {Yang, C and Dong, X and Zhang, X and Bi, C},
title = {[Application of genome editing technology in industrial microorganisms: current status and perspectives].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {38},
number = {11},
pages = {4132-4145},
doi = {10.13345/j.cjb.220566},
pmid = {37699682},
issn = {1872-2075},
mesh = {*Gene Editing ; *Biotechnology ; Endonucleases ; Escherichia coli/genetics ; Saccharomyces cerevisiae/genetics ; },
abstract = {Precise and efficient manipulation of gene expression or rewriting genome sequence is the research hotspots of genome editing, and it is also the core enabling technology contributing to the rapid development of industrial biotechnology. Genome editing technology has experienced three stages of development, from zinc finger nuclease (ZFNs), to transcription activator like effector nuclease (TALEN) and Cas nuclease. Currently, vigorous development of CRISPR/Cas has enabled researchers establish a series of first-generation and second-generation Cas-based genome editing technologies. This contributed to the establishment and optimization for prokaryotic chassis such as Escherichia coli or eukaryotic chassis such as Saccharomyces cerevisiae. This paper summarizes the current development and application of industrial biotechnology using conventional chassis cells, and prospects future development trend with the aim to facilitate researchers to optimize industrial biotechnology and its potential applications.},
}
@article {pmid37699034,
year = {2023},
author = {Sheng, H and Wu, S and Xue, Y and Zhao, W and Caplan, AB and Hovde, CJ and Minnich, SA},
title = {Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance.},
journal = {PloS one},
volume = {18},
number = {9},
pages = {e0291520},
pmid = {37699034},
issn = {1932-6203},
support = {P20 GM103408/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Mice ; CRISPR-Cas Systems/genetics ; Engineering ; *Gastroenteritis ; *Enterohemorrhagic Escherichia coli/genetics ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Microbial ; *Escherichia coli O157 ; },
abstract = {Pathogenic Escherichia coli and Salmonella enterica pose serious public health threats due to their ability to cause severe gastroenteritis and life-threatening sequela, particularly in young children. Moreover, the emergence and dissemination of antibiotic resistance in these bacteria have complicated control of infections. Alternative strategies that effectively target these enteric pathogens and negate or reduce the need of antibiotics are urgently needed. Such an alternative is the CRISPR-Cas9 system because it can generate sequence-specific lethal double stranded DNA breaks. In this study, two self-transmissible broad host range conjugative plasmids, pRK24 and pBP136, were engineered to deliver multiplexed CRSIPR-Cas9 systems that specifically target Enterohemorrhagic and Enteropathogenic strains of E. coli (EHEC and EPEC), S. enterica, and blaCMY-2 antibiotic resistance plasmids. Using in vitro mating assays, we show that the conjugative delivery of pRK24-CRISPR-Cas9 carrying guide RNAs to the EPEC/EHEC eae (intimin) gene can selectively kill enterohemorrhagic E. coli O157 eae+ cells (3 log kill at 6 h) but does not kill the isogenic Δeae mutant (P<0.001). Similar results were also obtained with a pBP136 derivative, pTF16, carrying multiplexed guide RNAs targeting E. coli eae and the S. enterica ssaN gene coding for the type III secretion ATPase. Another pBP136 derivative, TF18, carries guide RNAs targeting S. enterica ssaN and the antibiotic resistance gene, blaCMY-2, carried on the multi-drug resistant pAR06302. Introduction of pTF18 into bacteria harboring pAR06302 showed plasmids were cured at an efficiency of 53% (P<0.05). Using a murine neonate EPEC infection model, pTF16 was delivered by a murine derived E. coli strain to EPEC infected mice and showed significant reductions of intestinal EPEC (P<0.05). These results suggest that establishing conjugative CRISPR-Cas9 antimicrobials in the intestinal microbiome may provide protection from enteric pathogens and reduce antibiotic resistance without disrupting the normal microbiota.},
}
@article {pmid37698688,
year = {2023},
author = {Shakoor, S and Rao, AQ and Ajmal, S and Yasmeen, A and Khan, MAU and Sadaqat, S and Ashraf, NM and Wolter, F and Pacher, M and Husnain, T},
title = {Multiplex Cas9-based excision of CLCuV betasatellite and DNA-A revealed reduction of viral load with asymptomatic cotton plants.},
journal = {Planta},
volume = {258},
number = {4},
pages = {79},
pmid = {37698688},
issn = {1432-2048},
mesh = {Viral Load ; *Gossypium/genetics ; *CRISPR-Cas Systems/genetics ; Proteomics ; DNA ; },
abstract = {Multiplexed Cas9-based genome editing of cotton resulted in reduction of viral load with asymptomatic cotton plants. In depth imaging of proteomic dynamics of resulting CLCuV betasatellite and DNA-A protein was also performed. The notorious cotton leaf curl virus (CLCuV), which is transmitted by the sap-sucking insect whitefly, continuously damages cotton crops. Although the application of various toxins and RNAi has shown some promise, sustained control has not been achieved. Consequently, CRISPR_Cas9 was applied by designing multiplex targets against DNA-A (AC2 and AC3) and betasatellite (βC1) of CLCuV using CRISPR direct and ligating into the destination vector of the plant using gateway ligation method. The successful ligation of targets into the destination vector was confirmed by the amplification of 1049 bp using a primer created from the promoter and target, while restriction digestion using the AflII and Asc1 enzymes determined how compact the plasmid developed and the nucleotide specificity of the plasmid was achieved through Sanger sequencing. PCR confirmed the successful introduction of plasmid into CKC-1 cotton variety. Through Sanger sequencing and correlation with the mRNA expression of DNA-A and betasatellite in genome-edited cotton plants subjected to agroinfiltration of CLCuV infectious clone, the effectiveness of knockout was established. The genome-edited cotton plants demonstrated edited efficacy of 72% for AC2 and AC3 and 90% for the (βC1) through amplicon sequencing, Molecular dynamics (MD) simulations were used to further validate the results. Higher RMSD values for the edited βC1 and AC3 proteins indicated functional loss caused by denaturation. Thus, CRISPR_Cas9 constructs can be rationally designed using high-throughput MD simulation technique. The confidence in using this technology to control plant virus and its vector was determined by the knockout efficiency and the virus inoculation assay.},
}
@article {pmid37697159,
year = {2023},
author = {Singh, A and Pandey, H and Pandey, S and Lal, D and Chauhan, D and Aparna, and Antre, SH and B, S and Kumar, A},
title = {Drought stress in maize: stress perception to molecular response and strategies for its improvement.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {4},
pages = {296},
pmid = {37697159},
issn = {1438-7948},
mesh = {*Droughts ; *Zea mays/genetics ; Plant Breeding ; Drought Resistance ; Crops, Agricultural/genetics ; Perception ; },
abstract = {Given the future demand for food crops, increasing crop productivity in drought-prone rainfed areas has become essential. Drought-tolerant varieties are warranted to solve this problem in major crops, with drought tolerance as a high-priority trait for future research. Maize is one such crop affected by drought stress, which limits production, resulting in substantial economic losses. It became a more serious issue due to global climate change. The most drought sensitive among all stages of maize is the reproductive stages and the most important for overall maize production. The exact molecular basis of reproductive drought sensitivity remains unclear due to genes' complex regulation of drought stress. Understanding the molecular biology and signaling of the unexplored area of reproductive drought tolerance will provide an opportunity to develop climate-smart drought-tolerant next-generation maize cultivars. In recent decades, significant progress has been made in maize to understand the drought tolerance mechanism. However, improving maize drought tolerance through breeding is ineffective due to the complex nature and multigenic control of drought traits. With the help of advanced breeding techniques, molecular genetics, and a precision genome editing approach like CRISPR-Cas, candidate genes for drought-tolerant maize can be identified and targeted. This review summarizes the effects of drought stress on each growth stage of maize, potential genes, and transcription factors that determine drought tolerance. In addition, we discussed drought stress sensing, its molecular mechanisms, different approaches to developing drought-resistant maize varieties, and how molecular breeding and genome editing will help with the current unpredictable climate change.},
}
@article {pmid37696879,
year = {2023},
author = {Przybyszewska-Podstawka, A and Czapiński, J and Kałafut, J and Rivero-Müller, A},
title = {Synthetic circuits based on split Cas9 to detect cellular events.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {14988},
pmid = {37696879},
issn = {2045-2322},
support = {DEC-2015/17/B/NZ1/01777//Narodowe Centrum Nauki/ ; DEC-2017/25/B/NZ4/02364//Narodowym Centrum Nauki/ ; },
mesh = {*Epithelial-Mesenchymal Transition/genetics ; *CRISPR-Cas Systems ; Epithelial Cells ; Giant Cells ; Acclimatization ; },
abstract = {Synthetic biology involves the engineering of logic circuit gates that process different inputs to produce specific outputs, enabling the creation or control of biological functions. While CRISPR has become the tool of choice in molecular biology due to its RNA-guided targetability to other nucleic acids, it has not been frequently applied to logic gates beyond those controlling the guide RNA (gRNA). In this study, we present an adaptation of split Cas9 to generate logic gates capable of sensing biological events, leveraging a Cas9 reporter (EGxxFP) to detect occurrences such as cancer cell origin, epithelial to mesenchymal transition (EMT), and cell-cell fusion. First, we positioned the complementing halves of split Cas9 under different promoters-one specific to cancer cells of epithelial origin (phCEA) and the other a universal promoter. The use of self-assembling inteins facilitated the reconstitution of the Cas9 halves. Consequently, only cancer cells with an epithelial origin activated the reporter, exhibiting green fluorescence. Subsequently, we explored whether this system could detect biological processes such as epithelial to mesenchymal transition (EMT). To achieve this, we designed a logic gate where one half of Cas9 is expressed under the phCEA, while the other is activated by TWIST1. The results showed that cells undergoing EMT effectively activated the reporter. Next, we combined the two inputs (epithelial origin and EMT) to create a new logic gate, where only cancer epithelial cells undergoing EMT activated the reporter. Lastly, we applied the split-Cas9 logic gate as a sensor of cell-cell fusion, both in induced and naturally occurring scenarios. Each cell type expressed one half of split Cas9, and the induction of fusion resulted in the appearance of multinucleated syncytia and the fluorescent reporter. The simplicity of the split Cas9 system presented here allows for its integration into various cellular processes, not only as a sensor but also as an actuator.},
}
@article {pmid37696787,
year = {2023},
author = {Auradkar, A and Guichard, A and Kaduwal, S and Sneider, M and Bier, E},
title = {tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5587},
pmid = {37696787},
issn = {2041-1723},
support = {R01 GM117321/GM/NIGMS NIH HHS/United States ; R01 AI162911/AI/NIAID NIH HHS/United States ; R01 GM144608/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Drosophila melanogaster/genetics ; Gene Knockdown Techniques ; CRISPR-Associated Protein 9/genetics ; DNA Cleavage ; Mammals ; },
abstract = {CRISPR-interference (CRISPRi), a highly effective method for silencing genes in mammalian cells, employs an enzymatically dead form of Cas9 (dCas9) complexed with one or more guide RNAs (gRNAs) with 20 nucleotides (nt) of complementarity to transcription initiation sites of target genes. Such gRNA/dCas9 complexes bind to DNA, impeding transcription of the targeted locus. Here, we present an alternative gene-suppression strategy using active Cas9 complexed with truncated gRNAs (tgRNAs). Cas9/tgRNA complexes bind to specific target sites without triggering DNA cleavage. When targeted near transcriptional start sites, these short 14-15 nts tgRNAs efficiently repress expression of several target genes throughout somatic tissues in Drosophila melanogaster without generating any detectable target site mutations. tgRNAs also can activate target gene expression when complexed with a Cas9-VPR fusion protein or modulate enhancer activity, and can be incorporated into a gene-drive, wherein a traditional gRNA sustains drive while a tgRNA inhibits target gene expression.},
}
@article {pmid37695079,
year = {2023},
author = {Jiang, T and Liu, R and Shen, J},
title = {CRISPR dual enzyme cleavage triggers DNA and RNA substrate cleavage for SARS-CoV-2 dual gene detection.},
journal = {Journal of medical virology},
volume = {95},
number = {9},
pages = {e29090},
doi = {10.1002/jmv.29090},
pmid = {37695079},
issn = {1096-9071},
support = {2022zhyx-C61//Research Fund of Anhui Institute of Translational Medicine/ ; },
mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis ; DNA ; Nucleotidyltransferases ; RNA ; },
abstract = {The widespread dissemination of coronavirus 2019 imposes a significant burden on society. Therefore, rapid detection facilitates the reduction of transmission risk. In this study, we proposed a multiplex diagnostic platform for the rapid, ultrasensitive, visual, and simultaneous detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 1ab (ORF1ab) and N genes. A visual diagnostic method was developed using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a/Cas13a dual-enzyme digestion system integrated with multiplex reverse transcriptase-recombinase polymerase amplification (RT-RPA). Two CRISPR-Cas proteins (Cas12a and Cas13a) were introduced into the system to recognize and cleave the N gene and ORF1ab gene, respectively. We used fluorescent or CRISPR double digestion test strips to detect the digested products, with the N gene corresponding to the FAM channel in the PCR instrument or the T1 line on the test strip, and the ORF1ab gene corresponding to the ROX channel in the PCR instrument or the T2 line on the test strip. The analysis can be completed in less than 20 min. Meanwhile, we assessed the application of the platform and determined a sensitivity of up to 200 copies/mL. Additionally, dual gene validation in 105 clinical nasopharyngeal swab samples showed a 100% positive predictive value agreement and a 95.7% negative predictive value agreement between our method and quantitative reverse transcription-polymerase chain reaction. Overall, our method offered a novel insight into the rapid diagnosis of SARS-CoV-2.},
}
@article {pmid37694285,
year = {2023},
author = {Wang, J and Zhao, YT and Zhang, L and Dubielecka, PM and Qin, G and Chin, YE and Gower, AC and Zhuang, S and Liu, PY and Zhao, TC},
title = {Irisin deficiency exacerbates diet-induced insulin resistance and cardiac dysfunction in type II diabetes in mice.},
journal = {American journal of physiology. Cell physiology},
volume = {},
number = {},
pages = {},
doi = {10.1152/ajpcell.00232.2023},
pmid = {37694285},
issn = {1522-1563},
support = {R01 HL115265/HL/NHLBI NIH HHS/United States ; },
abstract = {Myokine Irisin is involved in the regulation of a variety of physiological conditions, metabolism, and survival. We and others have demonstrated that recombinant irisin contributes critically to modulation of insulin resistance and the improvement of cardiac function. However, whether deletion of irisin will regulates cardiac function and insulin sensitivity in type II diabetes remains unclear. We utilized the CRISPR/Cas-9 genome-editing system to delete irisin globally in mice and high fat diet (HFD)-induced type II diabetes model. We found that irisin deficiency did not result in developmental abnormality during adult stage, which illustrates normal cardiac function and insulin sensitivity in the absence of stress. The ultrastructural analysis of the transmission electronic microscope (TEM) indicated that deletion of irisin did not change the morphology of mitochondria in myocardium. Gene expression profiling showed that several key signaling pathways related to integrin signaling, extracellular matrix and insulin-like growth factors signaling were coordinately downregulated by deletion of irisin. When mice were fed with a HFD for sixteen weeks, ablation of irisin in mice exposed to HFD resulted in much severer insulin resistance, metabolic derangements, profound cardiac dysfunction and hypertrophic response and remodeling as compared with wild type control. Taken together, our results indicate that the loss of irisin exacerbates insulin resistance, metabolic disorders, and cardiac dysfunction in response to a high fat diet and promoted myocardial remodeling and hypertrophic response. This evidence reveals the molecular evidence and the critical role of irisin in modulating insulin resistance and cardiac function in type II diabetes.},
}
@article {pmid37694158,
year = {2023},
author = {Davis, DJ and McNew, JF and Maresca-Fichter, H and Chen, K and Telugu, BP and Bryda, EC},
title = {Efficient DNA knock-in using AAV-mediated delivery with 2-cell embryo CRISPR-Cas9 electroporation.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1256451},
pmid = {37694158},
issn = {2673-3439},
abstract = {Recent advances in CRISPR-Cas genome editing technology have been instrumental in improving the efficiency to produce genetically modified animal models. In this study we have combined four very promising approaches to come up with a highly effective pipeline to produce knock-in mouse and rat models. The four combined methods include: AAV-mediated DNA delivery, single-stranded DNA donor templates, 2-cell embryo modification, and CRISPR-Cas ribonucleoprotein (RNP) electroporation. Using this new combined approach, we were able to produce successfully targeted knock-in rat models containing either Cre or Flp recombinase sequences with knock-in efficiencies over 90%. Furthermore, we were able to produce a knock-in mouse model containing a Cre recombinase targeted insertion with over 50% knock-in efficiency directly comparing efficiencies to other commonly used approaches. Our modified AAV-mediated DNA delivery with 2-cell embryo CRISPR-Cas9 RNP electroporation technique has proven to be highly effective for generating both knock-in mouse and knock-in rat models.},
}
@article {pmid37669587,
year = {2023},
author = {Wang, H and Sun, Y and Zhou, Y and Liu, Y and Chen, S and Sun, W and Zhang, Z and Guo, J and Yang, C and Li, Z and Chen, L},
title = {Unamplified system for sensitive and typing detection of ASFV by the cascade platform that CRISPR-Cas12a combined with graphene field-effect transistor.},
journal = {Biosensors &amp; bioelectronics},
volume = {240},
number = {},
pages = {115637},
doi = {10.1016/j.bios.2023.115637},
pmid = {37669587},
issn = {1873-4235},
mesh = {CRISPR-Cas Systems/genetics ; *Graphite ; *Biosensing Techniques ; Electricity ; Real-Time Polymerase Chain Reaction ; },
abstract = {At present, the 100% case fatality and the cross-infection of virus strains make the ASFV 's harm to society continue to expand. The absence of an effective commercial vaccine poses early detection remains the most effective means of curbing ASFV infection. Here, we report a cascaded detection platform based on the CRISPR-Cas12a system combined with graphene field-effect transistor sensors. The cascade platform could detect ASFV as low as 0.5 aM within 30 min and achieve typing of wild and vaccine strains of ASFV in a single detection system. The evaluation of 16 clinical samples proved that, compared with the gold standard Real-time PCR method, this platform has outstanding advantages in sensitivity, specificity and typing. Combining CRISPR-Cas12a's high specificity with the bipolar electric field effect of graphene field-effect transistor, the cascade platform is expected to achieve clinical application in the field of DNA disease detection, and provides a new direction for multi-strain disease typing.},
}
@article {pmid37648854,
year = {2023},
author = {Mamedov, MR and Vedova, S and Freimer, JW and Sahu, AD and Ramesh, A and Arce, MM and Meringa, AD and Ota, M and Chen, PA and Hanspers, K and Nguyen, VQ and Takeshima, KA and Rios, AC and Pritchard, JK and Kuball, J and Sebestyen, Z and Adams, EJ and Marson, A},
title = {CRISPR screens decode cancer cell pathways that trigger γδ T cell detection.},
journal = {Nature},
volume = {621},
number = {7977},
pages = {188-195},
pmid = {37648854},
issn = {1476-4687},
mesh = {Humans ; AMP-Activated Protein Kinases/genetics/metabolism ; Cell Line ; Cell Membrane/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; *Neoplasms/genetics/immunology/metabolism ; *Receptors, Antigen, T-Cell, gamma-delta/immunology/metabolism ; *T-Lymphocytes/immunology/metabolism ; },
abstract = {γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background[1-5]. γδ T cells can sense conserved cell stress signals prevalent in transformed cells[2,3], although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells-the most abundant subset of human γδ T cells[4]-recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. [6-8]), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1-BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity.},
}
@article {pmid37625413,
year = {2023},
author = {Kita, Y and Okuzaki, Y and Naoe, Y and Lee, J and Bang, U and Okawa, N and Ichiki, A and Jonouchi, T and Sakurai, H and Kojima, Y and Hotta, A},
title = {Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD patient-derived iPSCs.},
journal = {Stem cell reports},
volume = {18},
number = {9},
pages = {1753-1765},
doi = {10.1016/j.stemcr.2023.07.007},
pmid = {37625413},
issn = {2213-6711},
mesh = {Humans ; *Dystrophin/genetics ; CRISPR-Cas Systems/genetics ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Binding Sites ; Exons/genetics ; },
abstract = {To restore dystrophin protein in various mutation patterns of Duchenne muscular dystrophy (DMD), the multi-exon skipping (MES) approach has been investigated. However, only limited techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases. Here, we utilized the CRISPR-Cas3 system for MES induction and showed that dual crRNAs could induce a large deletion at the dystrophin exon 45-55 region (∼340 kb), which can be applied to various types of DMD patients. We developed a two-color SSA-based reporter system for Cas3 to enrich the genome-edited cell population and demonstrated that MES induction restored dystrophin protein in DMD-iPSCs with three distinct mutations. Whole-genome sequencing and distance analysis detected no significant off-target deletion near the putative crRNA binding sites. Altogether, dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via MES induction.},
}
@article {pmid37622284,
year = {2023},
author = {Xu, H and Pan, R and Huang, W and Zhu, X},
title = {Label-free dual-mode sensing platform based on target-regulated CRISPR-Cas12a activity for ochratoxin A in Morinda officinalis.},
journal = {Analytical methods : advancing methods and applications},
volume = {15},
number = {35},
pages = {4518-4523},
doi = {10.1039/d3ay01025b},
pmid = {37622284},
issn = {1759-9679},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Morinda ; *Ochratoxins ; *Mycotoxins ; *Rubiaceae ; Oligonucleotides ; },
abstract = {Many traditional Chinese herbs are susceptible to ochratoxin A (OTA), a potent mycotoxin, which causes serious effects on the quality of the herb and on people's health. The development of methods to detect OTA is extremely important. Most methods for detecting OTA are based on a single-signal output mode, which might be easily influenced by complex environmental conditions. In this research, by taking advantage of the cleavage of DNA by target-induced CRISPR-Cas12a activity and the difference in electrostatic force of DNA to different charge electrochemiluminescent (ECL) and electrochemical (EC) probes, a biosensor is developed for the detection of OTA. First, the CRISPR-Cas12a system consists of a well-designed crRNA, its complementary strand (also as an aptamer for OTA), and Cas12a. Without the target, this CRISPR-Cas12a system is in the "activated stage", which digests hairpin DNA on the electrode, resulting in a weak ECL signal and strong current response. With the introduction of OTA bound with the aptamer, CRISPR-Cas12a activity is inhibited ("locked stage"). Thus, hairpin DNA remained intact on the electrode, resulting in recovery of the ECL signal and attenuation of the current intensity. As a result, this label-free dual-mode sensing platform realizes an assay for OTA in Morinda officinalis. This target-regulated CRISPR-Cas12a activity-sensing platform with dual-mode output not only provides high sensitivity (due to the CRISPR-Cas12a system), but also has good anti-interference ability against complex substrates (due to dual-mode output), and exhibits a broad range of prospects for application.},
}
@article {pmid37598442,
year = {2024},
author = {Gong, S and Song, K and Zhang, S and Zhou, P and Pan, W and Li, N and Tang, B},
title = {CRISPR-Cas12a-mediated dual-enzyme cascade amplification for sensitive colorimetric detection of HPV-16 target and ATP.},
journal = {Talanta},
volume = {266},
number = {Pt 2},
pages = {125050},
doi = {10.1016/j.talanta.2023.125050},
pmid = {37598442},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; Colorimetry ; Human papillomavirus 16/genetics ; Catalysis ; Glucose Oxidase ; *Nucleic Acids ; Adenosine Triphosphate ; },
abstract = {The establishment of sensitive and facile colorimetric platform based on the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is of great significance for in vitro diagnosis. Herein, we develop a dual-enzyme cascade amplification strategy based on CRISPR-Cas12a and glucose oxidase (GOx) for instrument-free and sensitive detection of target analytes. HPV-16 DNA as the model nucleic acid target directly initiated CRISPR-Cas12a-based signal transduction, resulting in the enzymatic cleavage of ssDNA linker and the release of GOx from magnetic nanoparticles 1 (MNPs1). Following simple magnetic separation, the supernatant containing GOx was taken out and used to catalyze the substrate, resulting in a visually detectable color change. The detection limit (LOD) of HPV-16 DNA was as low as 1 pM, and the entire process could be completed within 70 min without the need for expensive equipment. Notably, the dual-enzyme cascade amplification strategy was successfully applied to the detection of non-nucleic acid targets, such as ATP, via a simple signal transduction process. The visual LOD for ATP detection reaches 2.5 μM. The approach provides a robust, sensitive and reliable point-of-care biosensing platform for the detection of target analytes.},
}
@article {pmid37579676,
year = {2024},
author = {Wu, Y and Pei, J and Li, Y and Wang, G and Li, L and Liu, J and Tian, G},
title = {High-sensitive and rapid electrochemical detection of miRNA-31 in saliva using Cas12a-based 3D nano-harvester with improved trans-cleavage efficiency.},
journal = {Talanta},
volume = {266},
number = {Pt 2},
pages = {125066},
doi = {10.1016/j.talanta.2023.125066},
pmid = {37579676},
issn = {1873-3573},
mesh = {Humans ; Saliva ; CRISPR-Cas Systems ; *Carcinoma, Squamous Cell ; *Mouth Neoplasms/diagnosis/genetics ; Squamous Cell Carcinoma of Head and Neck ; *Head and Neck Neoplasms ; *MicroRNAs ; *Biosensing Techniques ; },
abstract = {Salivary miRNA-31 is a reliable diagnostic marker for early-stage oral squamous cell carcinoma (OSCC), but accurate detection of miRNA-31 in saliva samples is a challenge because of its low level and high sequence homology. The CRISPR/Cas12a system has the exceptional potential to enable simple nucleic acid analysis but suffers from low speed and sensitivity. To achieve rapid and high-sensitive detection of miRNA-31 using the CRISPR/Cas12a system, a Cas12a-based nano-harvester activated by a polymerase-driven DNA walker, named as dual 3D nanorobots, was developed. The target walked rapidly on the surface of DNA hairpin-modified magnetic nanoparticles driven by DNA polymerase, generating numerous double-strand DNA (dsDNA). Then, the Cas12a bound to the generated dsDNA for activating its trans-cleavage activity, forming 3D nano-harvester. Subsequently, the harvester cut and released methylene blue-labeled DNA hairpins immobilized on the sensing interface, leading to the change in electrochemical signal. We found that the trans-cleavage activity of the harvester was higher than the conventional CRISPR/Cas12a system. The developed dual 3D nanorobots could enable rapid (detection time within 60 min), high-sensitive (detection limit of femtomolar), and specific analysis of miRNA-31 in saliva samples. Thus, our established electrochemical biosensing strategy has great potential for early diagnosis of OSCC.},
}
@article {pmid37567119,
year = {2024},
author = {Peng, Y and Xue, P and Wang, R and Shang, H and Yao, B and Zheng, Z and Yan, C and Chen, W and Xu, J},
title = {Engineering of an adaptive tandem CRISPR/Cas12a molecular amplifier permits robust analysis of Vibrio parahaemolyticus.},
journal = {Talanta},
volume = {266},
number = {Pt 2},
pages = {125061},
doi = {10.1016/j.talanta.2023.125061},
pmid = {37567119},
issn = {1873-3573},
mesh = {Humans ; *Vibrio parahaemolyticus/genetics ; CRISPR-Cas Systems/genetics ; Coloring Agents ; DNA Nucleotidylexotransferase ; Food Safety ; Recombinases ; Nucleic Acid Amplification Techniques ; },
abstract = {Seeking new molecular diagnostic method for pathogenic bacteria detection is of utmost importance for ensuring food safety and protecting human health. Herein, we have engineered an adaptive tandem CRISPR/Cas12a molecular amplifier specifically designed for robust analysis of vibrio parahaemolyticus (V. parahaemolyticus), one of the most harmful pathogens. Our strategy involves the integration of three crucial processes: recombinase polymerase amplification (RPA) for copy number amplification, terminal deoxynucleotidyl transferase (TdT) for template-free strand elongation, and CRISPR/Cas12a-mediated trans-cleavage of a reporter molecule. By combining these processes, the target genomic DNA extracted from V. parahaemolyticus is able to activate many CRISPR/Cas12a units (CRISPR/Cas12a[n]) simultaneously, resulting in a greatly amplified target signal to indicate the presence and concentration of V. parahaemolyticus. This unique model offers more advantages compared to traditional amplification models that use one RPA amplicon to activate one CRISPR/Cas12a unit. Under optimized conditions, our method enables the detection of target V. parahaemolyticus within a linear range of 1 × 10[2]-1 × 10[7] CFU/mL, with an impressive limit of detection as low as 12.4 CFU/mL. It is conceivable that the adaptive tandem CRISPR/Cas12a molecular amplifier could be adapted as routine diagnostic kits in future for in-field detection of pathogens.},
}
@article {pmid37428871,
year = {2023},
author = {Li, BE and Li, GY and Cai, W and Zhu, Q and Seruggia, D and Fujiwara, Y and Vakoc, CR and Orkin, SH},
title = {In vivo CRISPR/Cas9 screening identifies Pbrm1 as a regulator of myeloid leukemia development in mice.},
journal = {Blood advances},
volume = {7},
number = {18},
pages = {5281-5293},
doi = {10.1182/bloodadvances.2022009455},
pmid = {37428871},
issn = {2473-9537},
mesh = {Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; Disease Progression ; Gene Editing ; *Leukemia, Myeloid/genetics ; Mutation ; *DNA-Binding Proteins/genetics ; *Transcription Factors/genetics ; },
abstract = {CRISPR/Cas9 screening approaches are powerful tool for identifying in vivo cancer dependencies. Hematopoietic malignancies are genetically complex disorders in which the sequential acquisition of somatic mutations generates clonal diversity. Over time, additional cooperating mutations may drive disease progression. Using an in vivo pooled gene editing screen of epigenetic factors in primary murine hematopoietic stem and progenitor cells (HSPCs), we sought to uncover unrecognized genes that contribute to leukemia progression. We, first, modeled myeloid leukemia in mice by functionally abrogating both Tet2 and Tet3 in HSPCs, followed by transplantation. We, then, performed pooled CRISPR/Cas9 editing of genes encoding epigenetic factors and identified Pbrm1/Baf180, a subunit of the polybromo BRG1/BRM-associated factor SWItch/Sucrose Non-Fermenting chromatin-remodeling complex, as a negative driver of disease progression. We found that Pbrm1 loss promoted leukemogenesis with a significantly shortened latency. Pbrm1-deficient leukemia cells were less immunogenic and were characterized by attenuated interferon signaling and reduced major histocompatibility complex class II (MHC II) expression. We explored the potential relevance to human leukemia by assessing the involvement of PBRM1 in the control of interferon pathway components and found that PBRM1 binds to the promoters of a subset of these genes, most notably IRF1, which in turn regulates MHC II expression. Our findings revealed a novel role for Pbrm1 in leukemia progression. More generally, CRISPR/Cas9 screening coupled with phenotypic readouts in vivo has helped identify a pathway by which transcriptional control of interferon signaling influences leukemia cell interactions with the immune system.},
}
@article {pmid37376803,
year = {2023},
author = {Qing, D and Chen, W and Huang, S and Li, J and Pan, Y and Zhou, W and Liang, Q and Yuan, J and Gan, D and Chen, L and Chen, L and Huang, J and Zhou, Y and Dai, G and Deng, G},
title = {Editing of rice (Oryza sativa L.) OsMKK3 gene using CRISPR/Cas9 decreases grain length by modulating the expression of photosystem components.},
journal = {Proteomics},
volume = {23},
number = {18},
pages = {e2200538},
doi = {10.1002/pmic.202200538},
pmid = {37376803},
issn = {1615-9861},
support = {U20A2032//National Natural Science Foundation of China/ ; 2023GXNSFDA026060//Guangxi Natural Science Foundation/ ; 2021GXNSFAA220083//Guangxi Natural Science Foundation/ ; },
mesh = {*Oryza/metabolism ; Proteomics/methods ; CRISPR-Cas Systems/genetics ; Edible Grain/metabolism ; Photosynthesis/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; },
abstract = {Grain size is one of the most important agronomic traits for grain yield determination in rice. To better understand the proteins that are regulated by the grain size regulatory gene OsMKK3, this gene was knocked out using the CRISPR/Cas9 system, and tandem mass tag (TMT) labeling combined with liquid chromatograph-tandem mass spectrometry analysis was performed to study the regulation of proteins in the panicle. Quantitative proteomic screening revealed a total of 106 differentially expressed proteins (DEPs) via comparison of the OsMKK3 mutant line to the wild-type YexiangB, including 15 and 91 up-regulated and down-regulated DEPs, respectively. Pathway analysis revealed that DEPs were enriched in metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and photosynthesis. Strong interactions were detected among seven down-regulated proteins related to photosystem components in the protein-protein interaction network, and photosynthetic rate was decreased in mutant plants. The results of the liquid chromatography-parallel reaction monitoring/mass spectromery analysis and western blot analysis were consistent with the results of the proteomic analysis, and the results of the quantitative reverse transcription polymerase chain reaction analysis revealed that the expression levels of most candidate genes were consistent with protein levels. Overall, OsMKK3 controls grain size by regulating the protein content in cells. Our findings provide new candidate genes that will aid the study of grain size regulatory mechanisms associated with the mitogen-activated protein kinase (MAPK) signaling pathway.},
}
@article {pmid37693076,
year = {2022},
author = {Jinka, C and Sainath, C and Babu, S and Chennupati, AC and Muppidi, LP and Krishnan, M and Sekar, G and Chinnaiyan, M and Andugula, SK},
title = {CRISPR-Cas9 gene editing and human diseases.},
journal = {Bioinformation},
volume = {18},
number = {11},
pages = {1081-1086},
pmid = {37693076},
issn = {0973-2063},
abstract = {CRISPR/Cas-9 mediated genome editing has recently emerged as a potential and innovative technology in therapeutic development and biomedical research. Several recent studies have been performed to understand gene modification techniques in obtaining effective ex vivo results. Generally, the disease targets for gene correction will be in specific organs, so understanding the complete potential of genomic treatment methods is essential. From such a perspective, the present review revealed the significant importance of the CRISPR/ Cas9 delivery system. Both the promising gene-editing delivery systems, such as synthetic (non-viral) and viral vector systems are discussed in this review. In addition, this paper attempted to summarize the tissue-specific and organ-specific mRNA delivery systems that provide possible research information for future researchers. Further, the major challenges of the CRISPR/Cas9 system, such as off-target delivery, immunogenicity, and limited packaging, were also elucidated. Accordingly, this review illustrated a wide range of clinical applications associated with the efficient delivery of CRISPR/ Cas9 gene-editing. Moreover, this article emphasizes the role of the CRISPR/Cas9 system in treating Intra Cerebral haemorrhage (ICH), thereby suggesting future researchers to adopt more clinical trials on this breakthrough delivery system.},
}
@article {pmid37691844,
year = {2023},
author = {Hegde, S and Rauch, HE and Hughes, GL and Shariat, N},
title = {Identification and characterization of two CRISPR/Cas systems associated with the mosquito microbiome.},
journal = {Access microbiology},
volume = {5},
number = {8},
pages = {},
pmid = {37691844},
issn = {2516-8290},
abstract = {The microbiome profoundly influences many traits in medically relevant vectors such as mosquitoes, and a greater functional understanding of host-microbe interactions may be exploited for novel microbial-based approaches to control mosquito-borne disease. Here, we characterized two novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems in Serratia sp. Ag1, which was isolated from the gut of an Anopheles gambiae mosquito. Two distinct CRISPR/Cas systems were identified in Serratia Ag1, CRISPR1 and CRISPR2. Based on cas gene composition, CRISPR1 is classified as a type I-E CRISPR/Cas system and has a single array, CRISPR1. CRISPR2 is a type I-F system with two arrays, CRISPR2.1 and CRISPR2.2. RT-PCR analyses show that all cas genes from both systems are expressed during logarithmic growth in culture media. The direct repeat sequences of CRISPRs 2.1 and 2.2 are identical and found in the arrays of other Serratia spp., including S. marcescens and S. fonticola , whereas CRISPR1 is not. We searched for potential spacer targets and revealed an interesting difference between the two systems: only 9 % of CRISPR1 (type I-E) targets are in phage sequences and 91 % are in plasmid sequences. Conversely, ~66 % of CRISPR2 (type I-F) targets are found within phage genomes. Our results highlight the presence of CRISPR loci in gut-associated bacteria of mosquitoes and indicate interplay between symbionts and invasive mobile genetic elements over evolutionary time.},
}
@article {pmid37691410,
year = {2023},
author = {Qiao, J and Zhao, Z and Li, Y and Lu, M and Man, S and Ye, S and Zhang, Q and Ma, L},
title = {Recent advances of food safety detection by nucleic acid isothermal amplification integrated with CRISPR/Cas.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-22},
doi = {10.1080/10408398.2023.2246558},
pmid = {37691410},
issn = {1549-7852},
abstract = {Food safety problems have become one of the most important public health issues worldwide. Therefore, the development of rapid, effective and robust detection is of great importance. Amongst a range of methods, nucleic acid isothermal amplification (NAIA) plays a great role in food safety detection. However, the widespread application remains limited due to a few shortcomings. CRISPR/Cas system has emerged as a powerful tool in nucleic acid detection, which could be readily integrated with NAIA to improve the detection sensitivity, specificity, adaptability versatility and dependability. However, currently there was a lack of a comprehensive summary regarding the integration of NAIA and CRISPR/Cas in the field of food safety detection. In this review, the recent advances in food safety detection based on CRISPR/Cas-integrated NAIA were comprehensively reviewed. To begin with, the development of NAIA was summarized. Then, the types and working principles of CRISPR/Cas were introduced. The applications of the integration of NAIA and CRISPR/Cas for food safety were mainly introduced and objectively discussed. Lastly, current challenges and future opportunities were proposed. In summary, this technology is expected to become an important approach for food safety detection, leading to a safer and more reliable food industry.},
}
@article {pmid37689895,
year = {2023},
author = {Shangpliang, HNJ and Tamang, JP},
title = {Metagenomics and metagenome-assembled genomes mining of health benefits in jalebi batter, a naturally fermented cereal-based food of India.},
journal = {Food research international (Ottawa, Ont.)},
volume = {172},
number = {},
pages = {113130},
doi = {10.1016/j.foodres.2023.113130},
pmid = {37689895},
issn = {1873-7145},
mesh = {*Metagenome ; *Edible Grain ; Metagenomics ; India ; },
abstract = {Jalebi is one of the oldest Indian traditional fermented wheat-based confectioneries. Since jalebi is prepared by natural fermentation, diverse microbial community is expected to play bio-functional activities. Due to limited studies, information on microbial community structure in jalebi is unknown. Hence, the present study is aimed to profile the microbial community in jalebi by shotgun metagenomics and also to predict putative probiotic and functional genes by metagenome-assembled genome (MAG). Bacteria were the most abundant domain (91.91%) under which Bacillota was the most abundant phylum (82%). The most abundant species was Lapidilactobacillus dextrinicus followed by several species of lactic acid bacteria, acetic acid bacteria including few yeasts. Lap. dextrinicus was also significantly abundant in jalebi when compared to similar fermented wheat-based sourdough. Additionally, Lap. bayanensis, Pediococcus stilesii, and yeast- Candida glabrata, Gluconobacter japonicus, Pichia kudriavzevii, Wickerhamomyces anomalus were only detected in jalebi, which are not detected in sourdough. Few viruses and archaea were detected with < 1 % abundance. In silico screening of genes from the abundant species was mined using both KEGG and EggNOG database for putative health beneficial attributes. Circular genomes of five high-quality MAGs, identified as Lapidilactobacillus dextrinicus, Enterococcus hirae, Pediococcus stilesii, Acetobacter indonesiensis and Acetobacter cibinongensis, were constructed separately and putative genes were mapped and annotated. The CRISPR/Cas gene clusters in the genomes of four MAGs except Acetobacter cibinongensis were detected. MAGs also showed several secondary metabolites. Since, the identified MAGs have different putative genes for bio-functional properties, this may pave the way to selectively culture the uncultivated putative microbes for jalebi production. We believe this is the first report on metagenomic and MAGs of jalebi.},
}
@article {pmid37658470,
year = {2023},
author = {Xiang, X and Xing, G and Liu, Y and Wen, Q and Wei, Y and Lu, J and Chen, Y and Ji, Y and Chen, S and Liu, T and Shang, Y},
title = {Immunomagnetic Separation Combined with RCA-CRISPR/Cas12a for the Detection of Salmonella typhimurium on a Figure-Actuated Microfluidic Biosensor.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {36},
pages = {13518-13526},
doi = {10.1021/acs.jafc.3c03799},
pmid = {37658470},
issn = {1520-5118},
mesh = {*Immunomagnetic Separation ; *Salmonella typhimurium/genetics ; CRISPR-Cas Systems ; Microfluidics ; Antibodies ; },
abstract = {A figure-actuated microfluidic biosensor was developed for the rapid and sensitive detection of Salmonella typhimurium using immunomagnetic separation to separate target bacteria and rolling circle amplification (RCA) combined with CRISPR/Cas12a to amplify the detection signal. The magnetic nanoparticles (MNPs) modified with the capture antibodies (MNPs@Ab1) and RCA primer linked with recognized antibodies (primer@Ab2) were first used to react with S. typhimurium, resulting in the formation of MNPs@Ab1-S. typhimurium-primer@Ab2 complexes. Then, the RCA and CRISPR/Cas12a reagents were successively pumped into the chamber and incubated at the appropriate conditions. With the help of a 3D-printed signal detector, the fluorescence signal was collected and analyzed using the smartphone APP for the determination of bacterial concentration. This biosensor exhibited a wide linear range for the detection of S. typhimurium with a low limit of detection of 1.93 × 10[2] CFU/mL and a mean recovery of about 106% in the spiked milk sample.},
}
@article {pmid37646615,
year = {2023},
author = {Liu, X and Cao, Z and Wang, W and Zou, C and Wang, Y and Pan, L and Jia, B and Zhang, K and Zhang, W and Li, W and Hao, Q and Zhang, Y and Zhang, W and Xue, X and Lin, W and Li, M and Gu, J},
title = {Engineered Extracellular Vesicle-Delivered CRISPR/Cas9 for Radiotherapy Sensitization of Glioblastoma.},
journal = {ACS nano},
volume = {17},
number = {17},
pages = {16432-16447},
doi = {10.1021/acsnano.2c12857},
pmid = {37646615},
issn = {1936-086X},
mesh = {Animals ; Mice ; *Glioblastoma/genetics/radiotherapy ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Glioma ; *Extracellular Vesicles/genetics ; Glutathione ; },
abstract = {Radiotherapy is a mainstay of glioblastoma (GBM) treatment; however, the development of therapeutic resistance has hampered the efficacy of radiotherapy, suggesting that additional treatment strategies are needed. Here, an in vivo loss-of-function genome-wide CRISPR screen was carried out in orthotopic tumors in mice subjected to radiation treatment to identify synthetic lethal genes associated with radiotherapy. Using functional screening and transcriptome analyses, glutathione synthetase (GSS) was found to be a potential regulator of radioresistance through ferroptosis. High GSS levels were closely related to poor prognosis and relapse in patients with glioma. Mechanistic studies demonstrated that GSS was associated with the suppression of radiotherapy-induced ferroptosis in glioma cells. The depletion of GSS resulted in the disruption of glutathione (GSH) synthesis, thereby causing the inactivation of GPX4 and iron accumulation, thus enhancing the induction of ferroptosis upon radiotherapy treatment. Moreover, to overcome the obstacles to broad therapeutic translation of CRISPR editing, we report a previously unidentified genome editing delivery system, in which Cas9 protein/sgRNA complex was loaded into Angiopep-2 (Ang) and the trans-activator of the transcription (TAT) peptide dual-modified extracellular vesicle (EV), which not only targeted the blood-brain barrier (BBB) and GBM but also permeated the BBB and penetrated the tumor. Our encapsulating EVs showed encouraging signs of GBM tissue targeting, which resulted in high GSS gene editing efficiency in GBM (up to 67.2%) with negligible off-target gene editing. These results demonstrate that a combination of unbiased genetic screens, and CRISPR-Cas9-based gene therapy is feasible for identifying potential synthetic lethal genes and, by extension, therapeutic targets.},
}
@article {pmid37595030,
year = {2023},
author = {Wang, T and Chen, G and Zhang, S and Li, D and Wei, G and Zhao, X and Liu, Y and Ding, D and Zhang, X},
title = {Steerable Microneedles Enabling Deep Delivery of Photosensitizers and CRISPR/Cas9 Systems for Effective Combination Cancer Therapy.},
journal = {Nano letters},
volume = {23},
number = {17},
pages = {7990-7999},
doi = {10.1021/acs.nanolett.3c01914},
pmid = {37595030},
issn = {1530-6992},
mesh = {*Photosensitizing Agents ; CRISPR-Cas Systems/genetics ; Combined Modality Therapy ; Phototherapy ; Lysosomes ; *Neoplasms ; },
abstract = {Although gene therapy has shown prospects in treating triple-negative breast cancer, it is insufficient to treat such a malignant tumor. Herein, nanoparticles (NPs)-embedded dissolving microneedles (IR780-PL/pFBXO44@MNs) with steerable and flectional property were developed to achieve the codelivery of FBXO44-targeted CRISPR/Cas9 plasmids (pFBXO44) and hydrophobic photosensitizers. For improved NP penetration in tumor tissue, collagenase@MNs were preapplied to degrade the tumor matrix. Under light irradiation, IR780 exhibited remarkable phototherapy, while the escape efficiency of NPs from lysosomes was improved. pFBXO44 was subsequently released in tumor cell cytoplasm via reducing the disulfide bonds of NPs, which could specifically knock out the FBXO44 gene to inhibit the migration and invasion of tumor cells. As a result, tumor cells were eradicated, and lung metastasis was effectively suppressed. This micelle-incorporated microneedle platform broadens the potential of combining gene editing and photo synergistic cancer therapy.},
}
@article {pmid37584415,
year = {2023},
author = {Sun, Y and Xu, X and Chen, L and Chew, WL and Ping, Y and Miserez, A},
title = {Redox-Responsive Phase-Separating Peptide as a Universal Delivery Vehicle for CRISPR/Cas9 Genome Editing Machinery.},
journal = {ACS nano},
volume = {17},
number = {17},
pages = {16597-16606},
doi = {10.1021/acsnano.3c02669},
pmid = {37584415},
issn = {1936-086X},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Reproducibility of Results ; Peptides/genetics ; Oxidation-Reduction ; },
abstract = {CRISPR/Cas9-based genome editing tools have enormous potential for the development of various therapeutic treatments due to their reliability and broad applicability. A central requirement of CRISPR/Cas9 is the efficient intracellular delivery of the editing machinery, which remains a well-recognized challenge, notably to deliver Cas9 in its native protein form. Herein, a phase-separating peptide with intracellular redox-triggered release properties is employed to encapsulate and deliver all three forms of CRISRP-Cas9 editing machinery, namely, pDNA, mRNA/sgRNA, and the ribonucleoprotein complex. These modalities are readily recruited within peptide coacervates during liquid-liquid phase separation by simple mixing and exhibit higher transfection and editing efficiency compared to highly optimized commercially available transfection reagents currently used for genome editing.},
}
@article {pmid37582231,
year = {2023},
author = {Fletcher, RB and Stokes, LD and Kelly, IB and Henderson, KM and Vallecillo-Viejo, IC and Colazo, JM and Wong, BV and Yu, F and d'Arcy, R and Struthers, MN and Evans, BC and Ayers, J and Castanon, M and Weirich, MJ and Reilly, SK and Patel, SS and Ivanova, YI and Silvera Batista, CA and Weiss, SM and Gersbach, CA and Brunger, JM and Duvall, CL},
title = {Nonviral In Vivo Delivery of CRISPR-Cas9 Using Protein-Agnostic, High-Loading Porous Silicon and Polymer Nanoparticles.},
journal = {ACS nano},
volume = {17},
number = {17},
pages = {16412-16431},
doi = {10.1021/acsnano.2c12261},
pmid = {37582231},
issn = {1936-086X},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; Silicon ; Porosity ; Polymers ; *Nanoparticles ; },
abstract = {The complexity of CRISPR machinery is a challenge to its application for nonviral in vivo therapeutic gene editing. Here, we demonstrate that proteins, regardless of size or charge, efficiently load into porous silicon nanoparticles (PSiNPs). Optimizing the loading strategy yields formulations that are ultrahigh loading─>40% cargo by volume─and highly active. Further tuning of a polymeric coating on the loaded PSiNPs yields nanocomposites that achieve colloidal stability under cryopreservation, endosome escape, and gene editing efficiencies twice that of the commercial standard Lipofectamine CRISPRMAX. In a mouse model of arthritis, PSiNPs edit cells in both the cartilage and synovium of knee joints, and achieve 60% reduction in expression of the therapeutically relevant MMP13 gene. Administered intramuscularly, they are active over a broad dose range, with the highest tested dose yielding nearly 100% muscle fiber editing at the injection site. The nanocomposite PSiNPs are also amenable to systemic delivery. Administered intravenously in a model that mimics muscular dystrophy, they edit sites of inflamed muscle. Collectively, the results demonstrate that the PSiNP nanocomposites are a versatile system that can achieve high loading of diverse cargoes and can be applied for gene editing in both local and systemic delivery applications.},
}
@article {pmid37087571,
year = {2023},
author = {Wu, Y and Xiao, N and Cai, Y and Yang, Q and Yu, L and Chen, Z and Shi, W and Liu, J and Pan, C and Li, Y and Zhang, X and Zhou, C and Huang, N and Ji, H and Zhu, S and Li, A},
title = {CRISPR-Cas9-mediated editing of the OsHPPD 3' UTR confers enhanced resistance to HPPD-inhibiting herbicides in rice.},
journal = {Plant communications},
volume = {4},
number = {5},
pages = {100605},
doi = {10.1016/j.xplc.2023.100605},
pmid = {37087571},
issn = {2590-3462},
mesh = {CRISPR-Cas Systems/genetics ; *Oryza/genetics ; 3' Untranslated Regions ; *Herbicides/pharmacology ; },
abstract = {This study reports the creation of herbicide-resistant rice lines via CRISPR-Cas9-mediated editing of the 3' UTR of OsHPPD. Resistance index calculations revealed that two resistant lines, TS8-2[#]-10 and TS8-8[#]-6, exhibited 4.8-fold and 3.7-fold greater resistance to HPPD-inhibiting herbicides compared with the wild type, YG3012.},
}
@article {pmid36702901,
year = {2023},
author = {},
title = {CLASH enables large-scale parallel knock-in for cell engineering.},
journal = {Nature biotechnology},
volume = {41},
number = {9},
pages = {1202-1203},
pmid = {36702901},
issn = {1546-1696},
mesh = {*Cell Engineering ; Gene Knock-In Techniques ; *Genetic Engineering ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid36702900,
year = {2023},
author = {Dai, X and Park, JJ and Du, Y and Na, Z and Lam, SZ and Chow, RD and Renauer, PA and Gu, J and Xin, S and Chu, Z and Liao, C and Clark, P and Zhao, H and Slavoff, S and Chen, S},
title = {Massively parallel knock-in engineering of human T cells.},
journal = {Nature biotechnology},
volume = {41},
number = {9},
pages = {1239-1255},
pmid = {36702900},
issn = {1546-1696},
support = {R01GM122984//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DP2CA238295//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01CA231112//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R33CA225498//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1RF1DA048811//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; U54CA209992//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; W81XWH-21-1-0514//U.S. Department of Defense (United States Department of Defense)/ ; },
mesh = {Humans ; *Bacterial Proteins/genetics ; *Gene Editing/methods ; CD4-Positive T-Lymphocytes/metabolism ; RNA ; CRISPR-Cas Systems/genetics ; },
abstract = {The efficiency of targeted knock-in for cell therapeutic applications is generally low, and the scale is limited. In this study, we developed CLASH, a system that enables high-efficiency, high-throughput knock-in engineering. In CLASH, Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses mediate simultaneous gene editing and precise transgene knock-in using massively parallel homology-directed repair, thereby producing a pool of stably integrated mutant variants each with targeted gene editing. We applied this technology in primary human T cells and performed time-coursed CLASH experiments in blood cancer and solid tumor models using CD3, CD8 and CD4 T cells, enabling pooled generation and unbiased selection of favorable CAR-T variants. Emerging from CLASH experiments, a unique CRISPR RNA (crRNA) generates an exon3 skip mutant of PRDM1 in CAR-Ts, which leads to increased proliferation, stem-like properties, central memory and longevity in these cells, resulting in higher efficacy in vivo across multiple cancer models, including a solid tumor model. The versatility of CLASH makes it broadly applicable to diverse cellular and therapeutic engineering applications.},
}
@article {pmid36693989,
year = {2023},
author = {},
title = {CRISPR-Csm for eukaryotic RNA knockdown and imaging without toxicity.},
journal = {Nature biotechnology},
volume = {41},
number = {9},
pages = {1204-1205},
pmid = {36693989},
issn = {1546-1696},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats ; RNA ; Eukaryota/genetics ; *CRISPR-Associated Proteins/metabolism ; RNA, Bacterial/genetics ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37689118,
year = {2023},
author = {Zhou, J and Liu, S and Xie, B and Wang, W and Xu, N and Xu, A and Dong, W and Jiang, M},
title = {Enhancing rhamnolipid production through a two-stage fermentation control strategy based on metabolic engineering and nitrate feeding.},
journal = {Bioresource technology},
volume = {388},
number = {},
pages = {129716},
doi = {10.1016/j.biortech.2023.129716},
pmid = {37689118},
issn = {1873-2976},
abstract = {Nitrate plays a crucial role in the high-efficient fermentation production of rhamnolipids (RLs). However, the underlying mechanism remains unclear. Firstly, by knocking out the restriction endonuclease PaeKI and utilizatiing the endogenous CRISPR-Cas-mediated single-plasmid recombineering system, a genome editing system for P. aeruginosa KT1115 has been established. Secondly, an engineered strain KT1115ΔpaeKIΔnirS was obtained with a 87% of reduction in nitric oxide (NO) accumulation and a 93% of reduction in RLs production, revealing the crucial role of NO signaling molecule produced from nitrate metabolism in RLs production. Finally, by combining metabolic engineering of the nitrate metabolism pathway with nitrogen feeding, a new two-stage fermentation process was developed. The fermentation production period was reduced from 168 h to 120 h while achieving a high yield of 0.8 g/g, and the average productivity increased by 55%. In all, this study provides a novel insights in the RLs biosynthesis and fermentation control strategy.},
}
@article {pmid37688677,
year = {2023},
author = {Shaheen, N and Ahmad, S and Alghamdi, SS and Rehman, HM and Javed, MA and Tabassum, J and Shao, G},
title = {CRISPR-Cas System, a Possible "Savior" of Rice Threatened by Climate Change: An Updated Review.},
journal = {Rice (New York, N.Y.)},
volume = {16},
number = {1},
pages = {39},
pmid = {37688677},
issn = {1939-8425},
support = {2021PE0AC05//Intelligent technology and platform development for rice breeding/ ; },
abstract = {Climate change has significantly affected agriculture production, particularly the rice crop that is consumed by almost half of the world's population and contributes significantly to global food security. Rice is vulnerable to several abiotic and biotic stresses such as drought, heat, salinity, heavy metals, rice blast, and bacterial blight that cause huge yield losses in rice, thus threatening food security worldwide. In this regard, several plant breeding and biotechnological techniques have been used to raise such rice varieties that could tackle climate changes. Nowadays, gene editing (GE) technology has revolutionized crop improvement. Among GE technology, CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) system has emerged as one of the most convenient, robust, cost-effective, and less labor-intensive system due to which it has got more popularity among plant researchers, especially rice breeders and geneticists. Since 2013 (the year of first application of CRISPR/Cas-based GE system in rice), several trait-specific climate-resilient rice lines have been developed using CRISPR/Cas-based GE tools. Earlier, several reports have been published confirming the successful application of GE tools for rice improvement. However, this review particularly aims to provide an updated and well-synthesized brief discussion based on the recent studies (from 2020 to present) on the applications of GE tools, particularly CRISPR-based systems for developing CRISPR rice to tackle the current alarming situation of climate change, worldwide. Moreover, potential limitations and technical bottlenecks in the development of CRISPR rice, and prospects are also discussed.},
}
@article {pmid37687368,
year = {2023},
author = {Tang, Q and Wang, X and Jin, X and Peng, J and Zhang, H and Wang, Y},
title = {CRISPR/Cas Technology Revolutionizes Crop Breeding.},
journal = {Plants (Basel, Switzerland)},
volume = {12},
number = {17},
pages = {},
pmid = {37687368},
issn = {2223-7747},
abstract = {Crop breeding is an important global strategy to meet sustainable food demand. CRISPR/Cas is a most promising gene-editing technology for rapid and precise generation of novel germplasm and promoting the development of a series of new breeding techniques, which will certainly lead to the transformation of agricultural innovation. In this review, we summarize recent advances of CRISPR/Cas technology in gene function analyses and the generation of new germplasms with increased yield, improved product quality, and enhanced resistance to biotic and abiotic stress. We highlight their applications and breakthroughs in agriculture, including crop de novo domestication, decoupling the gene pleiotropy tradeoff, crop hybrid seed conventional production, hybrid rice asexual reproduction, and double haploid breeding; the continuous development and application of these technologies will undoubtedly usher in a new era for crop breeding. Moreover, the challenges and development of CRISPR/Cas technology in crops are also discussed.},
}
@article {pmid37624847,
year = {2023},
author = {Jirawannaporn, S and Limothai, U and Tachaboon, S and Dinhuzen, J and Kiatamornrak, P and Chaisuriyong, W and Srisawat, N},
title = {The combination of RPA-CRISPR/Cas12a and Leptospira IgM RDT enhances the early detection of leptospirosis.},
journal = {PLoS neglected tropical diseases},
volume = {17},
number = {8},
pages = {e0011596},
pmid = {37624847},
issn = {1935-2735},
mesh = {Humans ; Recombinases ; *Leptospira/genetics ; CRISPR-Cas Systems ; *Leptospirosis/diagnosis ; Antibodies, Bacterial ; Fever ; Immunoglobulin M ; },
abstract = {BACKGROUND: Lack of available sensitive point-of-care testing is one of the primary obstacles to the rapid diagnosis of leptospirosis. The purpose of this study was to test the performance of two point-of-care tests, a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) fluorescence-based diagnostic assay (FBDA), a Leptospira immunoglobulin M (IgM) rapid diagnostic test (RDT), and the two tests combined.<br><br>For the diagnosis of 171 clinical samples, a recombinase polymerase amplification (RPA)-CRISPR/Cas12a FBDA for whole blood and Leptospira IgM RDT (Medical Science Public Health, Thailand) for serum were used. The confirmed cases were determined by using any positive qPCR, microscopic agglutination test (MAT), and culture results. Diagnostic accuracy was assessed on the first day of enrollment and stratified by the day after symptom onset. The overall sensitivity of the Leptospira IgM RDT and RPA-CRISPR/Cas12a FBDA was 55.66% and 60.38%, respectively. When the two tests were combined, the sensitivity rose to 84.91%. The specificity of each test was 63.08% and 100%, respectively, and 63.08% when combined. The sensitivity of the Leptospira IgM RDT rose on days 4-6 after the onset of fever, while the RPA-CRISPR/Cas12a FBDA continued to decrease. When the two tests were combined, the sensitivity was over 80% at different days post-onset of fever.<br><br>CONCLUSIONS/SIGNIFICANCE: The combination of Leptospira IgM RDT and RPA-CRISPR/Cas12 FBDA exhibited significant sensitivity for the detection of leptospires at various days after the onset of fever, thereby reducing the likelihood of misdiagnosis. The combination of these assays may be suitable for early leptospirosis screening in situations with limited resources.},
}
@article {pmid37623910,
year = {2023},
author = {Wang, H and Li, YL and Fan, YJ and Dong, JX and Ren, X and Ma, H and Wu, D and Gao, ZF and Wei, Q and Xia, F},
title = {DNA Tile and Invading Stacking Primer-Assisted CRISPR-Cas12a Multiple Amplification System for Entropy-Driven Electrochemical Detection of MicroRNA with Tunable Sensitivity.},
journal = {Analytical chemistry},
volume = {95},
number = {36},
pages = {13659-13667},
doi = {10.1021/acs.analchem.3c02603},
pmid = {37623910},
issn = {1520-6882},
mesh = {Entropy ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Electrodes ; *MicroRNAs/genetics ; },
abstract = {Conventional electrochemical detection of microRNA (miRNA) encounters issues of poor sensitivity and fixed dynamic range. Here, we report a DNA tile and invading stacking primer-assisted CRISPR-Cas12a multiple amplification strategy to construct an entropy-controlled electrochemical biosensor for the detection of miRNA with tunable sensitivity and dynamic range. To amplify the signal, a cascade amplification of the CRISPR-Cas12a system along with invading stacking primer signal amplification (ISPSA) was designed to detect trace amounts of miRNA-31 (miR-31). The target miR-31 could activate ISPSA and produce numerous DNAs, triggering the cleavage of the single-stranded linker probe (LP) that connects a methylene blue-labeled DNA tile with a DNA tetrahedron to form a Y-shaped DNA scaffold on the electrode. Based on the decrease of current, miR-31 can be accurately and efficiently detected. Impressively, by changing the loop length of the LP, it is possible to finely tune the entropic contribution while keeping the enthalpic contribution constant. This strategy has shown a tunable limit of detection for miRNA from 0.31 fM to 0.56 pM, as well as a dynamic range from ∼2200-fold to ∼270,000-fold. Moreover, it demonstrated satisfactory results in identifying cancer cells with a high expression of miR-31. Our strategy broadens the application of conventional electrochemical biosensing and provides a tunable strategy for detecting miRNAs at varying concentrations.},
}
@article {pmid37529831,
year = {2023},
author = {Liang, Z and Wei, S and Wu, Y and Guo, Y and Zhang, B and Yang, H},
title = {Temporally gene knockout using heat shock-inducible genome-editing system in plants.},
journal = {The plant genome},
volume = {16},
number = {3},
pages = {e20376},
doi = {10.1002/tpg2.20376},
pmid = {37529831},
issn = {1940-3372},
support = {32170410//National Natural Science Foundation of China/ ; 202204051001019//Science and Technology Innovation Young Talent Team of Shanxi Province/ ; 2022YFF1002802//National Key Research and Development Program of China/ ; },
mesh = {Gene Knockout Techniques ; *CRISPR-Cas Systems ; *Genome, Plant ; Heat-Shock Response ; Immunoglobulin E/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) has emerged as a powerful tool to generate targeted loss-of-function mutations for functional genomic studies. As a next step, tools to generate genome modifications in a spatially and temporally precise manner will enable researchers to further dissect gene function. Here, we present two heat shock-inducible genome-editing (IGE) systems that efficiently edit target genes when the system is induced, thus allowing us to target specific developmental stages. For this conditional editing system, we chose the natural heat-inducible promoter from heat-shock protein 18.2 (HSP18.2) from Arabidopsis thaliana and the synthetic heat-inducible promoter heat shock-response element HSE-COR15A to drive the expression of Cas9. We tested these two IGE systems in Arabidopsis using cyclic or continuous heat-shock treatments at the seedling and bolting stages. A real-time quantitative polymerase chain reaction analysis revealed that the HSP18.2 IGE system exhibited higher Cas9 expression levels than the HSE-COR15A IGE system upon both cyclic and continuous treatments. By targeting brassinosteroid-insensitive 1 (BRI1) and phytoene desaturase (PDS), we demonstrate that both cyclic and continuous heat inductions successfully activated the HSP18.2 IGE system at the two developmental stages, resulting in highly efficient targeted mutagenesis and clear phenotypic outcomes. By contrast, the HSE-COR15A IGE system was only induced at the seedling stage and was less effective than the HSP18.2 IGE system in terms of mutagenesis frequencies. The presented heat shock-IGE systems can be conditionally induced to efficiently inactivate genes at any developmental stage and are uniquely suited for the dissection and systematic characterization of essential genes.},
}
@article {pmid37686249,
year = {2023},
author = {Huang, S and Dai, R and Zhang, Z and Zhang, H and Zhang, M and Li, Z and Zhao, K and Xiong, W and Cheng, S and Wang, B and Wan, Y},
title = {CRISPR/Cas-Based Techniques for Live-Cell Imaging and Bioanalysis.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37686249},
issn = {1422-0067},
support = {322MS032//Hainan Provincial Natural Science Foundation of China/ ; ZY2022HN01//The Central Government Guides Local Science and Technology Development Projects/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Diagnostic Imaging ; Gene Editing ; },
abstract = {CRISPR/Cas systems have found widespread applications in gene editing due to their high accuracy, high programmability, ease of use, and affordability. Benefiting from the cleavage properties (trans- or cis-) of Cas enzymes, the scope of CRISPR/Cas systems has expanded beyond gene editing and they have been utilized in various fields, particularly in live-cell imaging and bioanalysis. In this review, we summarize some fundamental working mechanisms and concepts of the CRISPR/Cas systems, describe the recent advances and design principles of CRISPR/Cas mediated techniques employed in live-cell imaging and bioanalysis, highlight the main applications in the imaging and biosensing of a wide range of molecular targets, and discuss the challenges and prospects of CRISPR/Cas systems in live-cell imaging and biosensing. By illustrating the imaging and bio-sensing processes, we hope this review will guide the best use of the CRISPR/Cas in imaging and quantifying biological and clinical elements and inspire new ideas for better tool design in live-cell imaging and bioanalysis.},
}
@article {pmid37686137,
year = {2023},
author = {Xiong, Y and Xi, X and Xiang, Y and Li, S and Liu, H and Su, Y and He, R and Xiong, C and Xu, B and Wang, X and Fu, L and Zhao, C and Han, X and Li, X and Xie, S and Ruan, J},
title = {CRISPR-Cas9-Mediated Cytosine Base Editing Screen for the Functional Assessment of CALR Intron Variants in Japanese Encephalitis Virus Replication.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37686137},
issn = {1422-0067},
support = {No. 32072685//National Natural Science Foundation of China/ ; },
mesh = {Humans ; Animals ; Swine ; *Encephalitis Virus, Japanese/genetics ; Introns/genetics ; RNA, Guide, CRISPR-Cas Systems ; Calreticulin ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Encephalitis Viruses, Japanese ; Mutation ; Antiviral Agents ; Cytosine ; Mammals ; },
abstract = {The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of JEV replication and its potential antiviral approaches. However, information on exon and intron mutations associated with JEV replication is still scanty. CRISPR-Cas9-mediated cytosine base editing can efficiently generate C: G-to-T: A conversion in the genome of living cells. One intriguing application of base editing is to screen pivotal variants for gene function that is yet to be achieved in pigs. Here, we illustrate that CRISPR-Cas9-mediated cytosine base editor, known as AncBE4max, can be used for the functional analysis of calreticulin (CALR) variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of CALR to identify loss-of-function variants involved in JEV replication. We unexpectedly uncovered that two enriched sgRNAs targeted the same site in intron-2 of the CALR gene. We found that mutating four consecutive G bases in the intron-2 of the CALR gene to four A bases significantly inhibited JEV replication. Thus, we established a CRISPR-Cas9-mediated cytosine-base-editing point mutation screening technique in pigs. Our results suggest that CRISPR-mediated base editing is a powerful tool for identifying the antiviral functions of variants in the coding and noncoding regions of the CALR gene.},
}
@article {pmid37686051,
year = {2023},
author = {Huang, W and Zheng, A and Huang, H and Chen, Z and Ma, J and Li, X and Liang, Q and Li, L and Liu, R and Huang, Z and Qin, Y and Tang, Y and Li, H and Zhang, F and Wang, Q and Sun, B},
title = {Effects of sgRNAs, Promoters, and Explants on the Gene Editing Efficiency of the CRISPR/Cas9 System in Chinese Kale.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37686051},
issn = {1422-0067},
support = {32072586, 31500247, 32172593//National Natural Science Foundation of China/ ; 2022NSFSC1689//Natural Science Foundation of Sichuan Province/ ; sccxtd-2023-05//Project of New Varieties Breeding of Sichuan Vegetable Innovation Team/ ; },
mesh = {*Brassica/genetics ; Gene Editing ; *RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The CRISPR/Cas9 system is extensively used for plant gene editing. This study developed an efficient CRISPR/Cas9 system for Chinese kale using multiple sgRNAs and two promoters to create various CRISPR/Cas9 vectors. These vectors targeted BoaZDS and BoaCRTISO in Chinese kale protoplasts and cotyledons. Transient transformation of Chinese kale protoplasts was assessed for editing efficiency at three BoaZDS sites. Notably, sgRNA: Z2 achieved the highest efficiency (90%). Efficiency reached 100% when two sgRNAs targeted BoaZDS with a deletion of a large fragment (576 bp) between them. However, simultaneous targeting of BoaZDS and BoaCRTISO yielded lower efficiency. Transformation of cotyledons led to Chinese kale mutants with albino phenotypes for boazds mutants and orange-mottled phenotypes for boacrtiso mutants. The mutation efficiency of 35S-CRISPR/Cas9 (92.59%) exceeded YAO-CRISPR/Cas9 (70.97%) in protoplasts, and YAO-CRISPR/Cas9 (96.49%) surpassed 35S-CRISPR/Cas9 (58%) in cotyledons. These findings introduce a strategy for enhancing CRISPR/Cas9 editing efficiency in Chinese kale.},
}
@article {pmid37686014,
year = {2023},
author = {Schneider, P and Wander, P and Arentsen-Peters, STCJM and Vrenken, KS and Rockx-Brouwer, D and Adriaanse, FRS and Hoeve, V and Paassen, I and Drost, J and Pieters, R and Stam, RW},
title = {CRISPR-Cas9 Library Screening Identifies Novel Molecular Vulnerabilities in KMT2A-Rearranged Acute Lymphoblastic Leukemia.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37686014},
issn = {1422-0067},
support = {Not applicable//Foundation KiKa/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Library ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics ; Transcription Factors ; Cell Line ; Antigens, Neoplasm ; Neoplasm Proteins ; },
abstract = {In acute lymphoblastic leukemia (ALL), chromosomal translocations involving the KMT2A gene represent highly unfavorable prognostic factors and most commonly occur in patients less than 1 year of age. Rearrangements of the KMT2A gene drive epigenetic changes that lead to aberrant gene expression profiles that strongly favor leukemia development. Apart from this genetic lesion, the mutational landscape of KMT2A-rearranged ALL is remarkably silent, providing limited insights for the development of targeted therapy. Consequently, identifying potential therapeutic targets often relies on differential gene expression, yet the inhibition of these genes has rarely translated into successful therapeutic strategies. Therefore, we performed CRISPR-Cas9 knock-out screens to search for genetic dependencies in KMT2A-rearranged ALL. We utilized small-guide RNA libraries directed against the entire human epigenome and kinome in various KMT2A-rearranged ALL, as well as wild-type KMT2A ALL cell line models. This screening approach led to the discovery of the epigenetic regulators ARID4B and MBD3, as well as the receptor kinase BMPR2 as novel molecular vulnerabilities and attractive therapeutic targets in KMT2A-rearranged ALL.},
}
@article {pmid37686009,
year = {2023},
author = {Lu, X and Zhang, M and Li, G and Zhang, S and Zhang, J and Fu, X and Sun, F},
title = {Applications and Research Advances in the Delivery of CRISPR/Cas9 Systems for the Treatment of Inherited Diseases.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37686009},
issn = {1422-0067},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Medicine ; *Cystic Fibrosis ; Endonucleases ; *Nucleic Acids ; },
abstract = {The rapid advancements in gene therapy have opened up new possibilities for treating genetic disorders, including Duchenne muscular dystrophy, thalassemia, cystic fibrosis, hemophilia, and familial hypercholesterolemia. The utilization of the clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has revolutionized the field of gene therapy by enabling precise targeting of genes. In recent years, CRISPR/Cas9 has demonstrated remarkable efficacy in treating cancer and genetic diseases. However, the susceptibility of nucleic acid drugs to degradation by nucleic acid endonucleases necessitates the development of functional vectors capable of protecting the nucleic acids from enzymatic degradation while ensuring safety and effectiveness. This review explores the biomedical potential of non-viral vector-based CRISPR/Cas9 systems for treating genetic diseases. Furthermore, it provides a comprehensive overview of recent advances in viral and non-viral vector-based gene therapy for genetic disorders, including preclinical and clinical study insights. Additionally, the review analyzes the current limitations of these delivery systems and proposes avenues for developing novel nano-delivery platforms.},
}
@article {pmid37685921,
year = {2023},
author = {Lee, YR and Ko, KS and Lee, HE and Lee, ES and Han, K and Yoo, JY and Vu, BN and Choi, HN and Lee, YN and Hong, JC and Lee, KO and Kim, DS},
title = {CRISPR/Cas9-Mediated HY5 Gene Editing Reduces Growth Inhibition in Chinese Cabbage (Brassica rapa) under ER Stress.},
journal = {International journal of molecular sciences},
volume = {24},
number = {17},
pages = {},
pmid = {37685921},
issn = {1422-0067},
support = {PJ01626602//Cooperative Research Program for Agriculture Science and Technology Development/ ; },
mesh = {*Brassica rapa/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Hypocotyl ; RNA, Guide, CRISPR-Cas Systems ; Reactive Oxygen Species ; Crops, Agricultural ; Tunicamycin ; },
abstract = {Various stresses can affect the quality and yield of crops, including vegetables. In this study, CRISPR/Cas9 technology was employed to examine the role of the ELONGATED HYPOCOTYL 5 (HY5) gene in influencing the growth of Chinese cabbage (Brassica rapa). Single guide RNAs (sgRNAs) were designed to target the HY5 gene, and deep-sequencing analysis confirmed the induction of mutations in the bZIP domain of the gene. To investigate the response of Chinese cabbage to endoplasmic reticulum (ER) stress, plants were treated with tunicamycin (TM). Both wild-type and hy5 mutant plants showed increased growth inhibition with increasing TM concentration. However, the hy5 mutant plants displayed less severe growth inhibition compared to the wild type. Using nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) staining methods, we determined the amount of reactive oxygen species (ROS) produced under ER stress conditions, and found that the hy5 mutant plants generated lower levels of ROS compared to the wild type. Under ER stress conditions, the hy5 mutant plants exhibited lower expression levels of UPR- and cell death-related genes than the wild type. These results indicate that CRISPR/Cas9-mediated editing of the HY5 gene can mitigate growth inhibition in Chinese cabbage under stresses, improving the quality and yield of crops.},
}
@article {pmid37684555,
year = {2023},
author = {Zhao, J and Xi, Y and Zhang, J and Jin, Y and Yang, H and Duan, G and Chen, S and Long, J},
title = {Characterization and diversity of CRISPR/Cas systems in Klebsiella oxytoca.},
journal = {Molecular genetics and genomics : MGG},
volume = {},
number = {},
pages = {},
pmid = {37684555},
issn = {1617-4623},
support = {2022M712859//China Postdoctoral Science Foundation/ ; 2018ZX10301407//National Science and Technology Specific Projects/ ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) system is a crucial adaptive immune system for bacteria to resist foreign DNA infection. In this study, we investigated the prevalence and diversity of CRISPR/Cas systems in 175 Klebsiella oxytoca (K. oxytoca) strains. Specifically, 58.86% (103/175) of these strains possessed at least one confirmed CRISPR locus. Two CRISPR/Cas system types, I-F and IV-A3, were identified in 69 strains. Type I-F system was the most prevalent in this species, which correlated well with MLST. Differently, type IV-A3 system was randomly distributed. Moreover, the type IV-A3 system was separated into two subgroups, with subgroup-specific cas genes and repeat sequences. In addition, spacer origin analysis revealed that approximately one-fifth of type I-F spacers and one-third of type IV-A3 spacers had a significant match to MGEs. The phage tail tape measure protein and conjunctive transfer system protein were important targets of type I-F and IV-A3 systems in K. oxytoca, respectively. PAM sequences were inferred to be 5'-NCC-3' for type I-F, 5'-AAG-3' for subgroup IV-A3-a, and 5'-AAN-3' for subgroup IV-A3-b. Collectively, our findings will shed light on the prevalence, diversity, and functional effects of the CRISPR/Cas system in K. oxytoca.},
}
@article {pmid37684258,
year = {2023},
author = {Brockman, QR and Scherer, A and McGivney, GR and Gutierrez, WR and Rytlewski, J and Sheehan, A and Warrier, A and Laverty, EA and Roughton, G and Carnevale, NC and Knepper-Adrian, V and Dodd, RD},
title = {Discrepancies in indel software resolution with somatic CRISPR/Cas9 tumorigenesis models.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {14798},
pmid = {37684258},
issn = {2045-2322},
support = {T32 GM0677954/NH/NIH HHS/United States ; T32 HL07734/NH/NIH HHS/United States ; R01 NS119322/NH/NIH HHS/United States ; P30 CA086862/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Carcinogenesis/genetics ; Cell Transformation, Neoplastic ; Cell Line ; Software ; },
abstract = {CRISPR/Cas9 gene editing has evolved from a simple laboratory tool to a powerful method of in vivo genomic engineering. As the applications of CRISPR/Cas9 technology have grown, the need to characterize the breadth and depth of indels generated by editing has expanded. Traditionally, investigators use one of several publicly-available platforms to determine CRISPR/Cas9-induced indels in an edited sample. However, to our knowledge, there has not been a cross-platform comparison of available indel analysis software in samples generated from somatic in vivo mouse models. Our group has pioneered using CRISPR/Cas9 to generate somatic primary mouse models of malignant peripheral nerve sheath tumors (MPNSTs) through genetic editing of Nf1. Here, we used sequencing data from the in vivo editing of the Nf1 gene in our CRISPR/Cas9 tumorigenesis model to directly compare results across four different software platforms. By analyzing the same genetic target across a wide panel of cell lines with the same sequence file, we are able to draw systematic conclusions about the differences in these software programs for analysis of in vivo-generated indels. Surprisingly, we report high variability in the reported number, size, and frequency of indels across each software platform. These data highlight the importance of selecting indel analysis platforms specific to the context that the gene editing approach is being applied. Taken together, this analysis shows that different software platforms can report widely divergent indel data from the same sample, particularly if larger indels are present, which are common in somatic, in vivo CRISPR/Cas9 tumor models.},
}
@article {pmid37683609,
year = {2023},
author = {Patinios, C and Beisel, CL},
title = {For the CRISPR Fan(zor)atics: RNA-guided DNA endonucleases discovered in eukaryotes.},
journal = {Molecular cell},
volume = {83},
number = {17},
pages = {3046-3048},
doi = {10.1016/j.molcel.2023.08.019},
pmid = {37683609},
issn = {1097-4164},
mesh = {*Eukaryota/genetics ; *Deoxyribonuclease I ; Endonucleases/genetics ; RNA ; DNA/genetics ; },
abstract = {RNA-guided DNA endonucleases such as those from CRISPR-Cas systems were considered limited to prokaryotes. Saito et al.[1] reveal that distant eukaryotic relatives of Cas nucleases, called Fanzors, also function as RNA-guided DNA endonucleases and can be harnessed for genome editing.},
}
@article {pmid37682467,
year = {2023},
author = {Pan, C and Qi, Y},
title = {Targeted Activation of Arabidopsis Genes by a Potent CRISPR-Act3.0 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2698},
number = {},
pages = {27-40},
pmid = {37682467},
issn = {1940-6029},
mesh = {*Arabidopsis/genetics ; RNA, Guide, CRISPR-Cas Systems ; Agrobacterium ; CRISPR-Cas Systems/genetics ; RNA ; },
abstract = {The CRISPR/Cas system has emerged as a versatile platform for sequence-specific genome engineering in plants. Beyond genome editing, CRISPR/Cas systems, based on nuclease-deficient Cas9 (dCas9), have been repurposed as an RNA-guided platform for transcriptional regulation. CRISPR activation (CRISPRa) represents a novel gain-of-function (GOF) strategy, conferring robust over-expression of the target gene within its native chromosomal context. The CRISPRa systems enable precise, scalable, and robust RNA-guided transcription activation, holding great potential for a variety of fundamental and translational research. In this chapter, we provide a step-by-step guide for efficient gene activation in Arabidopsis based on a highly robust CRISPRa system, CRISPR-Act3.0. We present detailed procedures on the sgRNA design, CRISPR-Act3.0 system construction, Agrobacterium-mediated transformation of Arabidopsis using the floral dip method, and identification of desired transgenic plants.},
}
@article {pmid37679413,
year = {2023},
author = {Nakamura, A and Yano, T and Mitsuda, N and Furubayashi, M and Ito, S and Sugano, SS and Terakawa, T},
title = {The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {14205},
pmid = {37679413},
issn = {2045-2322},
mesh = {Animals ; *Oryza/genetics ; CRISPR-Cas Systems ; Gene Editing ; Sonication ; Vibrissae ; Plant Breeding ; *Callosities ; Ribonucleoproteins/genetics ; },
abstract = {CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated OsLCYβ genome-edited lines generated by RNPs. The calli and regenerated shoot of the OsLCYβ mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.},
}
@article {pmid37679324,
year = {2023},
author = {Ham, DT and Browne, TS and Banglorewala, PN and Wilson, TL and Michael, RK and Gloor, GB and Edgell, DR},
title = {A generalizable Cas9/sgRNA prediction model using machine transfer learning with small high-quality datasets.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5514},
pmid = {37679324},
issn = {2041-1723},
support = {PJT-159708//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Data Accuracy ; Endonucleases ; Machine Learning ; },
abstract = {The CRISPR/Cas9 nuclease from Streptococcus pyogenes (SpCas9) can be used with single guide RNAs (sgRNAs) as a sequence-specific antimicrobial agent and as a genome-engineering tool. However, current bacterial sgRNA activity models struggle with accurate predictions and do not generalize well, possibly because the underlying datasets used to train the models do not accurately measure SpCas9/sgRNA activity and cannot distinguish on-target cleavage from toxicity. Here, we solve this problem by using a two-plasmid positive selection system to generate high-quality data that more accurately reports on SpCas9/sgRNA cleavage and that separates activity from toxicity. We develop a machine learning architecture (crisprHAL) that can be trained on existing datasets, that shows marked improvements in sgRNA activity prediction accuracy when transfer learning is used with small amounts of high-quality data, and that can generalize predictions to different bacteria. The crisprHAL model recapitulates known SpCas9/sgRNA-target DNA interactions and provides a pathway to a generalizable sgRNA bacterial activity prediction tool that will enable accurate antimicrobial and genome engineering applications.},
}
@article {pmid37678882,
year = {2023},
author = {Haldrup, J and Andersen, S and Labial, ARL and Wolff, JH and Frandsen, FP and Skov, TW and Rovsing, AB and Nielsen, I and Jakobsen, TS and Askou, AL and Thomsen, MK and Corydon, TJ and Thomsen, EA and Mikkelsen, JG},
title = {Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad676},
pmid = {37678882},
issn = {1362-4962},
support = {R324-2019-1832//Lundbeck Foundation/ ; 8056-00010A//Innovation Fund/ ; //Synoptik Foundation/ ; 38189//Velux Foundation/ ; },
abstract = {Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.},
}
@article {pmid37677025,
year = {2023},
author = {Montiel-Manriquez, R and Castro-Hernández, C and Arriaga-Canon, C and Herrera, LA},
title = {Antisense Oligonucleotides as a Tool for Prolonged Knockdown of Nuclear lncRNAs in Human Cell Lines.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {199},
pages = {},
doi = {10.3791/65124},
pmid = {37677025},
issn = {1940-087X},
mesh = {Male ; Humans ; *Oligonucleotides, Antisense/genetics ; *RNA, Long Noncoding/genetics ; Cell Nucleus ; Oligonucleotides ; Cell Line ; },
abstract = {Long noncoding RNAs (lncRNAs) play key regulatory roles in gene expression at the transcriptional level. Experimental evidence has established that a substantial fraction of lncRNA preferentially accumulates in the nucleus. For analysis of the function of nuclear lncRNAs, it is important to achieve efficient knockdown of these transcripts inside the nucleus. In contrast to the use of RNA interference, a technology that depends on the cytoplasmic silencing machinery, an antisense oligonucleotide (ASO) technology can achieve RNA knockdown by recruiting RNase H to the RNA-DNA duplexes for nuclear RNA cleavage. Unlike the use of CRISPR-Cas tools for genome engineering, where possible alterations in the chromatin state can occur, ASOs allow the efficient knockdown of nuclear transcripts without modifying the genome. Nevertheless, one of the major obstacles to ASO-mediated knockdown is its transitory effect. For the study of long-lasting effects of lncRNA silencing, maintaining efficient knockdown for a long time is needed. In this study, a protocol was developed to achieve a knockdown effect for over 21 days. The purpose was to evaluate the cis-regulatory effects of lncRNA knockdown on the adjacent coding gene RFC4, which is related to chromosomal instability, a condition that is observed only through time and cell aging. Two different human cell lines were used: PrEC, normal primary prostate epithelial cells, and HCT116, an epithelial cell line isolated from colorectal carcinoma, achieving successful knockdown in the assayed cell lines.},
}
@article {pmid37595863,
year = {2023},
author = {Wang, Y and Yang, T and Liu, G and Xie, L and Guo, J and Xiong, W},
title = {Application of CRISPR/Cas12a in the rapid detection of pathogens.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {548},
number = {},
pages = {117520},
doi = {10.1016/j.cca.2023.117520},
pmid = {37595863},
issn = {1873-3492},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Fluorescent Dyes ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The combination of clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas protein is an effective gene-editing instrument. Among them, the CRISPR-Cas12a system forms a DNA-cleavage-capable complex with crRNA and exerts its trans-cleavage activity by recognising the PAM site on the target pathogen's gene. After amplifying the pathogenic gene, display materials such as fluorescent probes are added to the detection system, along with the advantages of rapid detection and high sensitivity of the CRISPR system, so that pathogenic bacteria can be diagnosed with greater speed and precision. This article reviews the mechanism of CRISPR-Cas12a in rapid detection, as well as its progress in the rapid detection of pathogenic bacteria in conjunction with various molecular biology techniques, in order to provide a foundation for the future development of a more effective detection platform.},
}
@article {pmid37591461,
year = {2023},
author = {Lin, YQ and Feng, KK and Lu, JY and Le, JQ and Li, WL and Zhang, BC and Li, CL and Song, XH and Tong, LW and Shao, JW},
title = {CRISPR/Cas9-based application for cancer therapy: Challenges and solutions for non-viral delivery.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {361},
number = {},
pages = {727-749},
doi = {10.1016/j.jconrel.2023.08.028},
pmid = {37591461},
issn = {1873-4995},
mesh = {*CRISPR-Cas Systems ; Gene Editing ; Technology ; *Neoplasms/genetics/therapy ; },
abstract = {CRISPR/Cas9 genome editing is a promising therapeutic technique, which makes precise and rapid gene editing technology possible on account of its high sensitivity and efficiency. CRISPR/Cas9 system has been proved to able to effectively disrupt and modify genes, which shows great potential for cancer treatment. Current researches proves that virus vectors are capable of effectively delivering the CRISPR/Cas9 system, but immunogenicity and carcinogenicity caused by virus transmission still trigger serious consequences. Therefore, the greatest challenge of CRISPR/Cas9 for cancer therapy lies on how to deliver it to the target tumor site safely and effectively. Non-viral delivery systems with specific targeting, high loading capacity, and low immune toxicity are more suitable than viral vectors, which limited by uncontrollable side effects. Their medical advances and applications have been widely concerned. Herein, we present the molecule mechanism and different construction strategies of CRISPR/Cas9 system for editing genes at the beginning of this research. Subsequently, several common CRISPR/Cas9 non-viral deliveries for cancer treatment are introduced. Lastly, based on the main factors limiting the delivery efficiency of non-viral vectors proposed in the existing researches and literature, we summarize and discuss the main methods to solve these limitations in the existing tumor treatment system, aiming to introduce further optimization and innovation of the CRISPR/Cas9 non-viral delivery system suitable for cancer treatment.},
}
@article {pmid37522352,
year = {2023},
author = {Singh, J and Liu, KG and Allen, A and Jiang, W and Qin, PZ},
title = {A DNA unwinding equilibrium serves as a checkpoint for CRISPR-Cas12a target discrimination.},
journal = {Nucleic acids research},
volume = {51},
number = {16},
pages = {8730-8743},
pmid = {37522352},
issn = {1362-4962},
support = {T32 GM118289/GM/NIGMS NIH HHS/United States ; R01GM124413/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism ; RNA/genetics ; },
abstract = {CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The finding sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.},
}
@article {pmid37503841,
year = {2023},
author = {Olsen, NS and Nielsen, TK and Cui, L and Dedon, P and Neve, H and Hansen, LH and Kot, W},
title = {A novel Queuovirinae lineage of Pseudomonas aeruginosa phages encode dPreQ0 DNA modifications with a single GA motif that provide restriction and CRISPR Cas9 protection in vitro.},
journal = {Nucleic acids research},
volume = {51},
number = {16},
pages = {8663-8676},
pmid = {37503841},
issn = {1362-4962},
support = {RGP0024/2018//Human Frontier Science Program/ ; 17595//Villum Experiment Grant/ ; //National Research Foundation of Singapore/ ; //Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance Interdisciplinary Research Group/ ; },
mesh = {Humans ; *Pseudomonas Phages/genetics ; Pseudomonas aeruginosa ; CRISPR-Cas Systems ; DNA/genetics ; *Bacteriophages/genetics ; },
abstract = {Deazaguanine DNA modifications are widespread in phages, particularly in those with pathogenic hosts. Pseudomonas phage iggy substitutes ∼16.5% of its genomic 2'-deoxyguanosine (G) with dPreQ0, and the iggy deazaguanine transglycosylase (DpdA) is unique in having a strict GA target motif, not observed previously. The iggy PreQ0 modification is shown to provide protection against both restriction endonucleases and Cas9 (when present in PAM), thus expanding our understanding of the deazaguanine modification system, its potential, and diversity. Phage iggy represents a new genus of Pseudomonas phages within the Queuovirinae subfamily; which have very little in common with other published phage genomes in terms of nucleotide similarity (<10%) and common proteins (<2%). Interestingly, shared similarity is concentrated in dpdA and preQ0 biosynthesis genes. TEM imaging confirmed a siphovirus morphology with a prolate icosahedral head and a non-contractile flexible tail with one long central tail spike. The observed protective effect of the deazaguanine modification on the iggy DNA may contribute to its broad within-species host range. Phage iggy was isolated on Pseudomonas aeruginosa PAO1, but also infects PDO300, PAK, PA14, as well as 10 of 27 tested environmental isolates and 13 of 20 tested clinical isolates of P. aeruginosa from patients with cystic fibrosis.},
}
@article {pmid36822625,
year = {2023},
author = {Li, G and Zhu, X and Wang, Y and Ma, H and Wang, Y and Wu, H and Li, X and Wang, Y and Gao, J and Chen, X and Huang, X and Yao, Y and Hu, X},
title = {Transcription-wide impact by RESCUE-induced off-target single-nucleotide variants in mammalian cells.},
journal = {Journal of molecular cell biology},
volume = {15},
number = {2},
pages = {},
pmid = {36822625},
issn = {1759-4685},
support = {2016ZX08009003-006//Ministry of Agri-culture of China/ ; 2020SKLAB6-24//National Key Laboratory Open Fund Project/ ; 02020200//Zhejiang University/ ; 32071347//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mutation ; *MicroRNAs/genetics ; RNA, Messenger ; Nucleotides ; CRISPR-Cas Systems ; Mammals ; },
abstract = {RNA base editing is a promising tool in precise molecular therapy. Currently, there are two widely used RNA base editors, REPAIR and RESCUE. REPAIR only facilitates A-to-I conversions, while RESCUE performs both A-to-I and C-to-U conversions. Thus, RESCUE can generate twice the number of mutations compared to REPAIR. However, transcription-wide impact due to RESCUE-induced off-target single-nucleotide variants (SNVs) is not fully appreciated. Therefore, to determine the off-target effects of RESCUE-mediated editing, we employed transcription-wide sequencing on cells edited by RESCUE. The SNVs showed different off-target effects on mRNA, circRNA, lncRNA, and miRNA expression patterns and their interacting networks. Our results illustrate the transcription-wide impact of RESCUE-induced off-target SNVs and highlight the need for careful characterization of the off-target impact by this editing platform.},
}
@article {pmid37676518,
year = {2022},
author = {Gentzel, IN and Ohlson, EW and Redinbaugh, MG and Wang, GL},
title = {VIGE: virus-induced genome editing for improving abiotic and biotic stress traits in plants.},
journal = {Stress biology},
volume = {2},
number = {1},
pages = {2},
pmid = {37676518},
issn = {2731-0450},
abstract = {Agricultural production is hampered by disease, pests, and environmental stresses. To minimize yield loss, it is important to develop crop cultivars with resistance or tolerance to their respective biotic and abiotic constraints. Transformation techniques are not optimized for many species and desirable cultivars may not be amenable to genetic transformation, necessitating inferior cultivar usage and time-consuming introgression through backcrossing to the preferred variety. Overcoming these limitations will greatly facilitate the development of disease, insect, and abiotic stress tolerant crops. One such avenue for rapid crop improvement is the development of viral systems to deliver CRISPR/Cas-based genome editing technology to plants to generate targeted beneficial mutations. Viral delivery of genomic editing constructs can theoretically be applied to span the entire host range of the virus utilized, circumventing the challenges associated with traditional transformation and breeding techniques. Here we explore the types of viruses that have been optimized for CRISPR/Cas9 delivery, the phenotypic outcomes achieved in recent studies, and discuss the future potential of this rapidly advancing technology.},
}
@article {pmid37674114,
year = {2023},
author = {Wang, M and Chen, M and Wu, X and Huang, X and Yu, B},
title = {CRISPR applications in cancer diagnosis and treatment.},
journal = {Cellular &amp; molecular biology letters},
volume = {28},
number = {1},
pages = {73},
pmid = {37674114},
issn = {1689-1392},
support = {SZSM201812059//Shenzhen Sanming Project/ ; SZXK040//Shenzhen Key Medical Discipline Construction Fund/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Neoplasms/diagnosis/genetics/therapy ; },
abstract = {Cancer remains a significant global health challenge, necessitating the exploration of novel and more precise therapeutic options beyond conventional treatments. In this regard, clustered regularly interspaced short palindromic repeats (CRISPR) systems have emerged as highly promising tools for clinical gene editing applications. The CRISPR family encompasses diverse CRISPR-associated (Cas) proteins that possess the ability to recognize specific target sequences. The initial CRISPR system consisted of the Cas9 protein and a single-guide RNA, which guide Cas9 to the desired target sequence, facilitating precise double-stranded cleavage. In addition to the traditional cis-cleavage activity, the more recently discovered Cas12 and Cas13 proteins exhibit trans-cleavage activity, which expands their potential applications in cancer diagnosis. In this review, we provide an overview of the functional characteristics of Cas9, Cas12, and Cas13. Furthermore, we highlight the latest advancements and applications of these CRISPR systems in cancer gene therapy and molecular diagnosis. We also emphasize the importance of understanding the strengths and limitations of each CRISPR system to maximize their clinical utility. By providing a comprehensive overview of the current state of CRISPR technology in cancer research, we aim to inspire further exploration and innovation in this rapidly evolving field.},
}
@article {pmid37673883,
year = {2023},
author = {Bestas, B and Wimberger, S and Degtev, D and Madsen, A and Rottner, AK and Karlsson, F and Naumenko, S and Callahan, M and Touza, JL and Francescatto, M and Möller, CI and Badertscher, L and Li, S and Cerboni, S and Selfjord, N and Ericson, E and Gordon, E and Firth, M and Chylinski, K and Taheri-Ghahfarokhi, A and Bohlooly-Y, M and Snowden, M and Pangalos, M and Nuttall, B and Akcakaya, P and Sienski, G and Maresca, M},
title = {A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5474},
pmid = {37673883},
issn = {2041-1723},
mesh = {Animals ; Mice ; *Translocation, Genetic ; *Proprotein Convertase 9/genetics ; CRISPR-Cas Systems/genetics ; Mutation ; Endonucleases/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9's off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.},
}
@article {pmid37673355,
year = {2023},
author = {Yao, S and Wu, X and Li, Y and Song, Y and Wang, C and Zhang, G and Feng, J},
title = {Harnessing the Native Type I-F CRISPR-Cas System of Acinetobacter baumannii for Genome Editing and Gene Repression.},
journal = {International journal of antimicrobial agents},
volume = {},
number = {},
pages = {106962},
doi = {10.1016/j.ijantimicag.2023.106962},
pmid = {37673355},
issn = {1872-7913},
abstract = {The rapid emergence of infections caused by MDR Acinetobacter baumannii poses a serious threat to global public health. Therefore, it has become important to obtain a deeper understanding of the mechanisms of multidrug resistance and pathogenesis of A. baumannii. However, there are still relatively few genetic engineering tools for A. baumannii. Although A. baumannii possesses Type I-F CRISPR-Cas systems, they have not yet been used for genetic modifications. Here, we developed a single plasmid-mediated native Type I-F CRISPR-Cas system for gene editing and gene regulation in A. baumannii. The protospacer adjacent motif (PAM) sequence was identified as 5'-NCC-3' by analysis of the CRISPR array. Through introduction of the RecAb homologous recombination system, we significantly increased the knockout efficiency of the oxyR gene from 12.5% to 75.0% in A. baumannii. To investigate transcriptional inhibition by the Type I-F CRISPR system, we deleted the gene encoding its Cas2-3 nuclease and repurposed the native Type I-F Cascade effector to regulate transcription of alcohol dehydrogenase gene adh4. The level of adh4 transcription was inhibited by up to 900-fold compared to the control. The Cascade transcriptional module was also successfully applied in a clinical Klebsiella pneumoniae isolate. This study proposed a tool for future exploration of the genetic characteristics of A. baumannii or other clinical strains.},
}
@article {pmid37671884,
year = {2023},
author = {Misra, CS and Pandey, N and Appukuttan, D and Rath, D},
title = {Effective gene silencing using type I-E CRISPR system in the multiploid, radiation-resistant bacterium Deinococcus radiodurans.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0520422},
doi = {10.1128/spectrum.05204-22},
pmid = {37671884},
issn = {2165-0497},
abstract = {The extremely radiation-resistant bacterium, Deinococcus radiodurans, is a microbe of importance, both, for studying stress tolerance mechanisms and as a chassis for industrial biotechnology. However, the molecular tools available for use in this organism continue to be limiting, with its multiploid genome presenting an additional challenge. In view of this, the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas tools provide a large repertoire of applications for gene manipulation. We show the utility of the type I-E Cascade system for knocking down gene expression in this organism. A single-vector system was designed for the expression of the Cascade components as well as the crRNA. The type I-E Cascade system was better tolerated than the type II-A dCas9 system in D. radiodurans. An assayable acid phosphatase gene, phoN integrated into the genome of this organism could be knocked down to 10% of its activity using the Cascade system. Cascade-based knockdown of ssb, a gene important for radiation resistance resulted in poor recovery post-irradiation. Targeting the Radiation and Desiccation Response Motif (RDRM), upstream of the ssb, prevented de-repression of its expression upon radiation exposure. In addition to this, multi-locus targeting was demonstrated on the deinococcal genome, by knocking down both phoN and ssb expression simultaneously. The programmable CRISPR interference tool developed in this study will facilitate the study of essential genes, hypothetical genes, and cis-elements involved in radiation response as well as enable metabolic engineering in this organism. Further, the tool can be extended for implementing high-throughput approaches in such studies. IMPORTANCE Deinococcus radiodurans is a microbe that exhibits a very high degree of radiation resistance. In addition, it is also identified as an organism of industrial importance. We report the development of a gene-knockdown system in this organism by engineering a type I-E clustered regularly interspaced short palindromic repeat (CRISPR)-Cascade system. We used this system to silence an assayable acid phosphatase gene, phoN to 10% of its activity. The study further shows the application of the Cascade system to target an essential gene ssb, that caused poor recovery from radiation. We demonstrate the utility of CRISPR-Cascade to study the role of a regulatory cis-element in radiation response as well as for multi-gene silencing. This easy-to-implement CRISPR interference system would provide an effective tool for better understanding of complex phenomena such as radiation response in D. radiodurans and may also enhance the potential of this microbe for industrial application.},
}
@article {pmid37671018,
year = {2023},
author = {Nievergelt, AP and Diener, DR and Bogdanova, A and Brown, T and Pigino, G},
title = {Efficient precision editing of endogenous Chlamydomonas reinhardtii genes with CRISPR-Cas.},
journal = {Cell reports methods},
volume = {3},
number = {8},
pages = {100562},
pmid = {37671018},
issn = {2667-2375},
mesh = {*Chlamydomonas reinhardtii ; CRISPR-Cas Systems ; *Chlamydomonas ; Culture ; Electroporation ; },
abstract = {CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until now been primarily applied to targeted gene disruption, whereas scarless knockin transgenesis has generally been considered difficult in practice. We have developed an efficient homology-directed method for knockin mutagenesis in Chlamydomonas by delivering CRISPR-Cas ribonucleoproteins and a linear double-stranded DNA (dsDNA) donor into cells by electroporation. Our method allows scarless integration of fusion tags and sequence modifications of proteins without the need for a preceding mutant line. We also present methods for high-throughput crossing of transformants and a custom quantitative PCR (qPCR)-based high-throughput screening of mutants as well as meiotic progeny. We demonstrate how to use this pipeline to facilitate the generation of mutant lines without residual selectable markers by co-targeted insertion. Finally, we describe how insertional cassettes can be erroneously mutated during insertion and suggest strategies to select for lines that are modified as designed.},
}
@article {pmid37669948,
year = {2023},
author = {Rananaware, SR and Vesco, EK and Shoemaker, GM and Anekar, SS and Sandoval, LSW and Meister, KS and Macaluso, NC and Nguyen, LT and Jain, PK},
title = {Programmable RNA detection with CRISPR-Cas12a.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5409},
pmid = {37669948},
issn = {2041-1723},
support = {R21 AI168795/AI/NIAID NIH HHS/United States ; R35 GM147788/GM/NIGMS NIH HHS/United States ; R21 AI156321/AI/NIAID NIH HHS/United States ; },
mesh = {*RNA ; *CRISPR-Cas Systems ; Point Mutation ; RNA, Guide, CRISPR-Cas Systems ; Recombination, Genetic ; },
abstract = {Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.},
}
@article {pmid37668921,
year = {2024},
author = {Shiraki, T and Kawakami, K},
title = {Generation of Transgenic Fish Harboring CRISPR/Cas9-Mediated Somatic Mutations Via a tRNA-Based Multiplex sgRNA Expression.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2707},
number = {},
pages = {305-318},
pmid = {37668921},
issn = {1940-6029},
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems ; *Zebrafish/genetics ; CRISPR-Cas Systems/genetics ; Animals, Genetically Modified/genetics ; Frameshift Mutation ; },
abstract = {The controlled expression of Cas9 and/or sgRNA in transgenic zebrafish made it possible to knock out a gene in a spatially and/or temporally controlled manner. This transgenic approach can be more useful if multiple sgRNAs are efficiently expressed since we can improve the biallelic frame-shift mutation rate and circumvent the functional redundancy of genes and genetic compensation. We developed the tRNA-based system to express multiple functional sgRNAs from a single transcript in zebrafish and found that it is applicable to the transgenic expression of multiple sgRNAs. In this chapter, we describe a procedure for the generation of plasmids containing multiple sgRNAs flanked by tRNAs and a method to induce multiple CRISPR/Cas9-mediated genome modifications in transgenic zebrafish.},
}
@article {pmid37668219,
year = {2023},
author = {Kang, YJ and Kim, JH and Lee, GH and Ha, HJ and Park, YH and Hong, E and Park, HH},
title = {The structure of AcrIC9 revealing the putative inhibitory mechanism of AcrIC9 against the type IC CRISPR-Cas system.},
journal = {IUCrJ},
volume = {10},
number = {Pt 5},
pages = {624-634},
pmid = {37668219},
issn = {2052-2525},
support = {2018R1A4A1023822//National Research Foundation of Korea/ ; 2021R1A2C3003331//National Research Foundation of Korea/ ; 20020237//Ministry of Trade, Industry and Energy, Korea Evaluation Institute of Industrial Technology/ ; },
mesh = {CRISPR-Cas Systems/genetics ; *Bacteriophages ; Polymers ; Protein Domains ; *Rhodobacter capsulatus/genetics ; },
abstract = {CRISPR-Cas systems are known to be part of the bacterial adaptive immune system that provides resistance against intruders such as viruses, phages and other mobile genetic elements. To combat this bacterial defense mechanism, phages encode inhibitors called Acrs (anti-CRISPR proteins) that can suppress them. AcrIC9 is the most recently identified member of the AcrIC family that inhibits the type IC CRISPR-Cas system. Here, the crystal structure of AcrIC9 from Rhodobacter capsulatus is reported, which comprises a novel fold made of three central antiparallel β-strands surrounded by three α-helixes, a structure that has not been detected before. It is also shown that AcrIC9 can form a dimer via disulfide bonds generated by the Cys69 residue. Finally, it is revealed that AcrIC9 directly binds to the type IC cascade. Analysis and comparison of its structure with structural homologs indicate that AcrIC9 belongs to DNA-mimic Acrs that directly bind to the cascade complex and hinder the target DNA from binding to the cascade.},
}
@article {pmid37666438,
year = {2023},
author = {Misra, G and Qaisar, S and Singh, P},
title = {CRISPR based therapeutic targeting of signaling pathways in breast cancer.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {},
number = {},
pages = {166872},
doi = {10.1016/j.bbadis.2023.166872},
pmid = {37666438},
issn = {1879-260X},
abstract = {Breast cancer represents the prevailing malignancy in females and contributing towards mortality on a global scale. There are innumerable anomalous genes that are responsible in the multi factorial carcinogenesis pathway. Although several disease-causing mutations have been detected, therapy frequently focuses on attenuating the manifestation of the disease rather than harmonizing the mutation in the target area. However, the emergence of the CRISPR-Cas system as a DNA modifying tool has proved to be a promising gene editing tool for breast cancer therapeutics. Henceforth, in this review, we have discussed different signaling pathways that have been targeted by the CRISPR-Cas system and explored their role in breast cancer therapies. We have also tried to comprehensively cover CRISPR-Cas9 mediated gene knockout in breast cancer. In this review many alterations in breast cancer cells that affect cell signaling pathways have also been described. This review will be a valuable addition to providing comprehensive knowledge of CRISPR-Cas mediated therapeutic targeting in breast cancer.},
}
@article {pmid37666372,
year = {2023},
author = {Ahmar, S and Hensel, G and Gruszka, D},
title = {CRISPR/Cas9-mediated genome editing techniques and new breeding strategies in cereals - current status, improvements, and perspectives.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108248},
doi = {10.1016/j.biotechadv.2023.108248},
pmid = {37666372},
issn = {1873-1899},
abstract = {Cereal crops, including triticeae species (barley, wheat, rye), as well as edible cereals (wheat, corn, rice, oat, rye, sorghum), are significant suppliers for human consumption, livestock feed, and breweries. Over the past half-century, modern varieties of cereal crops with increased yields have contributed to global food security. However, presently cultivated elite crop varieties were developed mainly for optimal environmental conditions. Thus, it has become evident that taking into account the ongoing climate changes, currently a priority should be given to developing new stress-tolerant cereal cultivars. It is necessary to enhance the accuracy of methods and time required to generate new cereal cultivars with the desired features to adapt to climate change and keep up with the world population expansion. The CRISPR/Cas9 system has been developed as a powerful and versatile genome editing tool to achieve desirable traits, such as developing high-yielding, stress-tolerant, and disease-resistant transgene-free lines in major cereals. Despite recent advances, the CRISPR/Cas9 application in cereals faces several challenges, including a significant amount of time required to develop transgene-free lines, laboriousness, and a limited number of genotypes that may be used for the transformation and in vitro regeneration. Additionally, developing elite lines through genome editing has been restricted in many countries, especially Europe and New Zealand, due to a lack of flexibility in GMO regulations. This review provides a comprehensive update to researchers interested in improving cereals using gene-editing technologies, such as CRISPR/Cas9. We will review some critical and recent studies on crop improvements and their contributing factors to superior cereals through gene-editing technologies.},
}
@article {pmid37651830,
year = {2023},
author = {Rivera-Sánchez, P and Søndergaard, L and Wathikthinnakon, M and B D Magnusson, H and Frederiksen, HR and Aabæk Hammer, F and Taleb, R and Christian Cassidy, C and Tranholm Bruun, M and Tümer, Z and Holst, B and Brasch-Andersen, C and Møller, RS and Freude, K and Chandrasekaran, A},
title = {Generation of eight hiPSCs lines from two pathogenic variants in CACNA1A using the CRISPR-Cas9 gene editing technology.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103193},
doi = {10.1016/j.scr.2023.103193},
pmid = {37651830},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; *Induced Pluripotent Stem Cells ; Calcium ; Cell Differentiation ; Calcium Channels ; },
abstract = {Developmental and epileptic encephalopathies (DEEs) are rare severe neurodevelopmental disorders with a cumulative incidence of 1:6.000 live births. Many epileptic conditions arise from single nucleotide variants in CACNA1A (calcium voltage-gated channel subunit alpha1 A), encoding the CaV2.1 calcium channel subunit. Human induced pluripotent stem cells (hiPSCs) are an optimal choice for modeling DEEs, as they can be differentiated in vitro into diverse neuronal subpopulations. Here, we report the generation of hiPSC lines with two pathogenic CACNA1A variants c.1767C > T, p. (Arg589Cys), referred to as R589C and c. 2139G > A, p.(Ala713Thr), referred to as A713T, previously associated with epilepsy. The variants were introduced into a hiPSC line from a healthy individual via CRISPR-Cas9 gene editing technology.},
}
@article {pmid37540965,
year = {2023},
author = {Schuurmans, IME and Wu, KM and van Karnebeek, CDM and Nadif Kasri, N and Garanto, A},
title = {Generation of an induced pluripotent stem cell line carrying biallelic deletions (SCTCi019-B) in ALDH7A1 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103173},
doi = {10.1016/j.scr.2023.103173},
pmid = {37540965},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Aldehyde Dehydrogenase/genetics/metabolism ; *Epilepsy/genetics ; Mutation ; },
abstract = {Biallelic pathogenic variants in ALDH7A1 are associated with pyridoxine-dependent epilepsy (PDE). ALDH7A1 encodes for the third enzyme of the lysine catabolism pathway. In this study a human isogenic ALDH7A1 knock-out iPSC line was created using CRISPR/Cas9 technology. One clone (SCTCi019-B) with biallelic deletions in ALDH7A1 was obtained and fully characterized, showing expression of pluripotency markers, a normal karyotype and no off-targets. Human-based models derived from this iPSC line will contribute to gain insights in the molecular mechanism of disease underlying PDE.},
}
@article {pmid37495752,
year = {2023},
author = {Rosello, M and Serafini, M and Concordet, JP and Del Bene, F},
title = {Precise mutagenesis in zebrafish using cytosine base editors.},
journal = {Nature protocols},
volume = {18},
number = {9},
pages = {2794-2813},
pmid = {37495752},
issn = {1750-2799},
support = {ANR-18-IAHU-0001//Agence Nationale de la Recherche (French National Research Agency)/ ; ANR-20-CE17-0020-02//Agence Nationale de la Recherche (French National Research Agency)/ ; ANR-11-INBS-0014-TEFOR//Agence Nationale de la Recherche (French National Research Agency)/ ; MND202003011485//Fondation pour la Recherche M&#xe9;dicale (Foundation for Medical Research in France)/ ; ECO20170637481//Fondation pour la Recherche M&#xe9;dicale (Foundation for Medical Research in France)/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Cytosine ; Mutagenesis ; },
abstract = {Base editing is a powerful CRISPR-based technology for introducing precise substitutions into the genome. This technology greatly advances mutagenesis possibilities in vivo, particularly in zebrafish, for which the generation of precise point mutations is still challenging. Zebrafish have emerged as an important model for genetic studies and in vivo disease modeling. With the development of different base editor variants that recognize protospacer-adjacent motifs (PAMs) other than the classical 5'-NGG-3' PAM, it is now possible to design and test several guide RNAs to find the most efficient way to precisely introduce the desired substitution. Here, we describe the experimental design strategies and protocols for cytosine base editing in zebrafish, from guide RNA design and selection of base editor variants to generation of the zebrafish mutant line carrying the substitution of interest. By using co-selection by introducing a loss-of-function mutation in genes necessary for the formation of pigments, injected embryos with highly efficient base editing can be directly analyzed to determine the phenotypic impact of the targeted substitution. The generation of mutant embryos after base editor injections in zebrafish can be completed within 2 weeks.},
}
@article {pmid37481965,
year = {2023},
author = {Jeong, J and Lee, D and Park, BC and Lee, TH and Park, SW},
title = {Establishment of a TNFRSF11B knock-out human induced pluripotent stem cell line (KSCBi002-B-2) via CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103167},
doi = {10.1016/j.scr.2023.103167},
pmid = {37481965},
issn = {1876-7753},
mesh = {Humans ; Osteoprotegerin/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; *Osteitis Deformans/genetics/metabolism ; },
abstract = {A TNFRSF11B (TNF Receptor Superfamily Member 11b) gene encodes a soluble decoy receptor, osteoprotegerin (OPG), which has a key role in repressing osteoclast differentiation. In this report, we generated a biallelic knock-out hiPSC line for the TNFRSF11B gene via CRISPR/Cas9. When TNFRSF11B Knock-out hiPSCs were differentiated into mesenchymal progenitor cells (MPCs), the expression level of OPG was significantly decreased compared to normal hiPSC-derived MPCs. This knock-out hiPSCs will provide a chance to study Paget disease of bone 5 (juvenile Paget disease).},
}
@article {pmid37474806,
year = {2023},
author = {Riesenberg, S and Kanis, P and Macak, D and Wollny, D and Düsterhöft, D and Kowalewski, J and Helmbrecht, N and Maricic, T and Pääbo, S},
title = {Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.},
journal = {Nature methods},
volume = {20},
number = {9},
pages = {1388-1399},
pmid = {37474806},
issn = {1548-7105},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Recombinational DNA Repair ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA ; },
abstract = {Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.},
}
@article {pmid37473460,
year = {2023},
author = {Ben Yacoub, T and Letellier, C and Wohlschlegel, J and Condroyer, C and Slembrouck-Brec, A and Goureau, O and Zeitz, C and Audo, I},
title = {Generation of gene corrected human isogenic iPSC lines (IDVi003-A_CR13, IDVi003-A_CR21, IDVi003-A_CR24) from an inherited retinal dystrophy patient-derived IPSC line ITM2B-5286-3 (IDVi003-A) carrying the ITM2B c.782A &gt; C variant using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103166},
doi = {10.1016/j.scr.2023.103166},
pmid = {37473460},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; *Retinal Dystrophies/genetics/metabolism ; Cell Differentiation ; Mutation ; Adaptor Proteins, Signal Transducing/genetics ; },
abstract = {The ITM2B-related retinal dystrophy (ITM2B-RD) was identified within patients carrying the autosomal dominant variant [c.782A > C, p.(Glu261Ala)] in ITM2B from whom induced pluripotent stem cell (IPSC) lines were previously generated. Here, we report the generation of three isogenic control iPSC lines from the derived affected subject cell line (ITM2B-5286-3) using CRISPR/Cas9 engineering. The three generated lines express pluripotency markers, can be differentiated into the three germ layers and present a normal karyotype. The generated iPSC lines can be used to study the implications of ITM2B-RD variant in vitro.},
}
@article {pmid37429070,
year = {2023},
author = {Lu, F and Gao, Y and Li, E},
title = {Generation of a FLNA knockout hESC line (WAe009-A-P) to model cardiac valvular dysplasia using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103162},
doi = {10.1016/j.scr.2023.103162},
pmid = {37429070},
issn = {1876-7753},
mesh = {Humans ; Female ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism ; Cell Line ; Mutation ; Heart Valves ; },
abstract = {The FLNA gene encodes the cytoskeletal protein filamin A which plays a key role in the structure and function of the cardiac valves. Truncating FLNA mutations are associated with cardiac valvular dysplasia. To further understand the exact role of FLNA in this disease, we have generated a human FLNA knockout cell line from H9 using CRISPR/Cas9 technology in this study. This cell line WAe009-A-P has a 2 bp deletion in the exon 2 of FLNA gene which resulted in a frameshift in the translation of FLNA and no FLNA protein was detected in this cell line. Moreover, WAe009-A-P also expressed pluripotency markers, had a normal female karyotype (46XX) and maintained the ability to differentiate into the three germ layers in vitro.},
}
@article {pmid37406498,
year = {2023},
author = {Alowaysi, M and Al-Shehri, M and Baadhaim, M and AlZahrani, H and Aboalola, D and Daghestani, M and Hashem, H and Aljahdali, R and Salem, R and Alharbi, A and Muharraq, M and Alghamdi, K and Alsobiy, F and Zia, A and Lehmann, R and Tegner, J and Alsayegh, K},
title = {Generation of myoglobin (MB)-knockout human embryonic stem cell (hESC) line (KAIMRCe002-A-1S) using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103158},
doi = {10.1016/j.scr.2023.103158},
pmid = {37406498},
issn = {1876-7753},
mesh = {Humans ; *Myoglobin/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Technology ; },
abstract = {Myoglobin (MB) is a cytoplasmic hemoprotein that is predominantly expressed in the heart and oxidative myofibers of skeletal muscle. It has been demonstrated that MB binds to oxygen and promotes its diffusion for energy production in the mitochondria. Recently, MB was found to be expressed in different forms of malignant tumors and cancer cell lines. Further studies using gene disruption technology will enhance the understanding of MB's role in human cardiovascular biology and cancers. Here, we describe the generation of a homozygous MB knockout in human embryonic stem cells (hESC-MB[-/-]) via CRISPR/Cas9 to study MB function in human biology and diseases.},
}
@article {pmid37393721,
year = {2023},
author = {Amin, G and Castañeda, SL and Zabalegui, F and Belli, C and Atorrasagasti, C and Miriuka, SG and Moro, LN},
title = {Generation of two edited iPSCs lines by CRISPR/Cas9 with point mutations in PKP2 gene for arrhythmogenic cardiomyopathy in vitro modeling.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103157},
doi = {10.1016/j.scr.2023.103157},
pmid = {37393721},
issn = {1876-7753},
mesh = {Humans ; Point Mutation ; *Induced Pluripotent Stem Cells/metabolism ; *Arrhythmogenic Right Ventricular Dysplasia/genetics ; CRISPR-Cas Systems/genetics ; *Cardiomyopathies/genetics ; Mutation/genetics ; Plakophilins/genetics/metabolism ; },
abstract = {The arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease characterized by the progressive replacement of contractile myocardium by fibro-fatty adipose tissue, that generates ventricular arrhythmias and sudden death in patients. The ACM has a genetic origin with alterations in desmosomal genes with the most commonly mutated being the PKP2 gene. We generated two CRISPR/Cas9 edited iPSCs lines, one iPSC line with a point mutation in PKP2 reported in patients with ACM and another iPSC line with a premature stop codon to knock-out the same gene.},
}
@article {pmid37343428,
year = {2023},
author = {Thongsin, N and Suwanpitak, S and Wattanapanitch, M},
title = {CRISPR-Cas9-mediated disruption of B2M and CIITA genes eliminates HLA class I and II expression in human induced pluripotent stem cells (MUSIi001-A-2).},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103138},
doi = {10.1016/j.scr.2023.103138},
pmid = {37343428},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; CRISPR-Cas Systems/genetics ; Graft Rejection ; Cell Line ; },
abstract = {Cell-based therapy offers great promise for treating degenerative diseases. Although autologous cell-based therapy is ideal, it may be impractical due to the high manufacturing cost and long production time. Allogeneic cell-based therapy offers a more cost-effective alternative; however, the risk of graft rejection is a major concern. Here, we generated HLA class-I and -II null iPSC line by knocking out CIITA gene in the B2M-knockout MUSIi001-A-1 cell line using CRISPR/Cas9 system. The MUSIi001-A-2 line provides a valuable model for studying immunological responses against allogeneic T cells and serves as a prototype for developing specific cell types for future cell-based therapy.},
}
@article {pmid37331109,
year = {2023},
author = {Boissart, C and Chatrousse, L and Poullion, T and El-Kassar, L and Giraud-Triboult, K and Benchoua, A},
title = {CRISPR/Cas9-mediated generation of human embryonic stem cell sub-lines with HPRT1 gene knockout to model Lesch Nyhan disease.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103144},
doi = {10.1016/j.scr.2023.103144},
pmid = {37331109},
issn = {1876-7753},
mesh = {Humans ; Male ; *Lesch-Nyhan Syndrome/genetics/metabolism ; Hypoxanthine Phosphoribosyltransferase/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Human Embryonic Stem Cells/metabolism ; },
abstract = {Lesch-Nyhan disease (LND) is a X-linked genetic disease affecting boys characterized by complex neurological and neuropsychiatric symptoms. LND is caused by loss of function mutations in the HPRT1 gene leading to decrease activity of hypoxanthine-guanine phosphoribosyl transferase enzyme (HGPRT) and altered purine salvage pathway (Lesch and Nyhan, 1964). This study describes the generation of isogenic clones with deletions in HPRT1 produced from one male human embryonic stem cell line using CRISPR/Cas9 strategy. Differentiation of these cells into different neuronal subtypes will help elucidating the neurodevelopmental events leading to LND and develop therapeutic strategies for this devastating neurodevelopmental disorder.},
}
@article {pmid37327621,
year = {2023},
author = {Jiang, M and Tang, C and Luo, X and Zhou, X and Chen, M and Chi, Y and Lai, L and Zou, Q},
title = {Generation of a homozygous RANGRF knockout hiPSC line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {71},
number = {},
pages = {103136},
doi = {10.1016/j.scr.2023.103136},
pmid = {37327621},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Homozygote ; Mutation ; Gene Editing ; },
abstract = {The RAN Guanine Nucleotide Release Factor (RANGRF) gene encodes the protein MOG1, which binds to Nav1.5 and facilitates its transport to the cell membrane. Nav1.5 mutations have been linked to various cardiac arrhythmias and cardiomyopathy. To investigate the role of RANGRF in this process, we utilized the CRISPR/Cas9 gene editing system to generate a homozygous RANGRF knockout hiPSC line. The availability of the cell line will prove to be an invaluable asset in the study of disease mechanisms and the testing of gene therapies for cardiomyopathy.},
}
@article {pmid36997812,
year = {2023},
author = {Paenkaew, S and Jaito, N and Pradit, W and Chomdej, S and Nganvongpanit, K and Siengdee, P and Buddhachat, K},
title = {RPA/CRISPR-cas12a as a specific, sensitive and rapid method for diagnosing Ehrlichia canis and Anaplasma platys in dogs in Thailand.},
journal = {Veterinary research communications},
volume = {47},
number = {3},
pages = {1601-1613},
pmid = {36997812},
issn = {1573-7446},
support = {N42A650332//National Research Council of Thailand/ ; R2565A060//Faculty of Science, Naresuan University, Thailand/ ; R2566C051//Global and Frontier Research University Fund, Naresuan University/ ; },
mesh = {Dogs ; Animals ; Ehrlichia canis/genetics ; *Anaplasmosis/diagnosis/epidemiology/genetics ; CRISPR-Cas Systems ; Recombinases/genetics ; Thailand ; RNA, Ribosomal, 16S/genetics ; *Ehrlichiosis/diagnosis/veterinary/genetics ; *Dog Diseases/diagnosis/epidemiology ; },
abstract = {Rickettsial pathogens including Ehrlichia canis and Anaplasma platys are bacteria that cause parasitic infections in dogs such as canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT), respectively affecting mortality and morbidity worldwide. An accurate, sensitive, and rapid method to diagnose these agents is essential for effective treatment. In this study, a recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a methods was established to detect E. canis and A. platys infection in dogs based on the 16S rRNA. The optimal condition for DNA amplification by RPA was 37 °C for 20 min, followed by CRISPR-Cas12a digestion at 37 °C for one hour. A combination of RPA and the cas12a detection method did not react with other pathogens and demonstrated strong sensitivity, detecting as low as 100 copies of both E. canis and A. platys. This simultaneous detection method was significantly more sensitive than conventional PCR. The RPA-assisted cas12a assay provides specific, sensitive, rapid, simple and appropriate detection of rickettsial agents in canine blood at the point-of-care for diagnostics, disease prevention and surveillance.},
}
@article {pmid37663930,
year = {2023},
author = {Tang, D and Jia, T and Luo, Y and Mou, B and Cheng, J and Qi, S and Yao, S and Su, Z and Yu, Y and Chen, Q},
title = {DnaQ mediates directional spacer acquisition in the CRISPR-Cas system by a time-dependent mechanism.},
journal = {Innovation (Cambridge (Mass.))},
volume = {4},
number = {5},
pages = {100495},
pmid = {37663930},
issn = {2666-6758},
abstract = {In the spacer acquisition stage of CRISPR-Cas immunity, spacer orientation and protospacer adjacent motif (PAM) removal are two prerequisites for functional spacer integration. Cas4 has been implicated in both processing the prespacer and determining the spacer orientation. In Cas4-lacking systems, host 3'-5' DnaQ family exonucleases were recently reported to play a Cas4-like role. However, the molecular details of DnaQ functions remain elusive. Here, we characterized the spacer acquisition of the adaptation module of the Streptococcus thermophilus type I-E system, in which a DnaQ domain naturally fuses with Cas2. We presented X-ray crystal structures and cryo-electron microscopy structures of this adaptation module. Our biochemical data showed that DnaQ trimmed PAM-containing and PAM-deficient overhangs with different efficiencies. Based on these results, we proposed a time-dependent model for DnaQ-mediated spacer acquisition to elucidate PAM removal and spacer orientation determination in Cas4-lacking CRISPR-Cas systems.},
}
@article {pmid37662968,
year = {2023},
author = {Han, H and Yang, Y and Jiao, Y and Qi, H and Han, Z and Wang, L and Dong, L and Tian, J and Vanhaesebroeck, B and Li, X and Liu, J and Ma, G and Lei, H},
title = {Leverage of nuclease-deficient CasX for preventing pathological angiogenesis.},
journal = {Molecular therapy. Nucleic acids},
volume = {33},
number = {},
pages = {738-748},
pmid = {37662968},
issn = {2162-2531},
abstract = {Gene editing with a CRISPR/Cas system is a novel potential strategy for treating human diseases. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) δ suppresses retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Here we show that an innovative system of adeno-associated virus (AAV)-mediated CRISPR/nuclease-deficient (d)CasX fused with the Krueppel-associated box (KRAB) domain is leveraged to block (81.2% ± 6.5%) in vitro expression of p110δ, the catalytic subunit of PI3Kδ, encoded by Pik3cd. This CRISPR/dCasX-KRAB (4, 269 bp) system is small enough to be fit into a single AAV vector. We then document that recombinant AAV serotype (rAAV)1 efficiently transduces vascular endothelial cells from pathologic retinal vessels, which show high expression of p110δ; furthermore, we demonstrate that blockade of retinal p110δ expression by intravitreally injected rAAV1-CRISPR/dCasX-KRAB targeting the Pik3cd promoter prevents (32.1% ± 5.3%) retinal p110δ expression as well as pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. These data establish a strong foundation for treating pathological angiogenesis by AAV-mediated CRISPR interference with p110δ expression.},
}
@article {pmid37662220,
year = {2023},
author = {Szekely, O and Rangadurai, AK and Gu, S and Manghrani, A and Guseva, S and Al-Hashimi, HM},
title = {NMR measurements of transient low-populated tautomeric and anionic Watson-Crick-like G·T/U in RNA:DNA hybrids: Implications for the fidelity of transcription and CRISPR/Cas9 gene editing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.08.24.554670},
pmid = {37662220},
abstract = {Many biochemical processes use the Watson-Crick geometry to distinguish correct from incorrect base pairing. However, on rare occasions, mismatches such as G•T/U can transiently adopt Watson-Crick-like conformations through tautomerization or ionization of the bases, giving rise to replicative and translational errors. The propensities to form Watson-Crick-like mismatches in RNA:DNA hybrids remain unknown, making it unclear whether they can also contribute to errors during processes such as transcription and CRISPR/Cas editing. Here, using NMR R 1ρ experiments, we show that dG•rU and dT•rG mismatches in two RNA:DNA hybrids transiently form tautomeric (G [enol] •T/U ⇄G•T [enol] /U [enol]) and anionic (G•T [-] /U [-]) Watson-Crick-like conformations. The tautomerization dynamics were like those measured in A-RNA and B-DNA duplexes. However, anionic dG•rU [-] formed with a ten-fold higher propensity relative to dT [-] •rG and dG•dT [-] and this could be attributed to the lower pK a (Δ pK a ∼0.4-0.9) of U versus T. Our findings suggest plausible roles for Watson-Crick-like G•T/U mismatches in transcriptional errors and CRISPR/Cas9 off-target gene editing, uncover a crucial difference between the chemical dynamics of G•U versus G•T, and indicate that anionic Watson-Crick-like G•U [-] could play a significant role evading Watson-Crick fidelity checkpoints in RNA:DNA hybrids and RNA duplexes.},
}
@article {pmid37624706,
year = {2023},
author = {Wang, Y and Xia, X and Wu, M and Sun, Q and Zhang, W and Qiu, Y and Deng, R and Luo, A},
title = {Species-Level Monitoring of Key Bacteria in Fermentation Processes Using Single-Nucleotide Resolved Nucleic Acid Assays Based on CRISPR/Cas12.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {35},
pages = {13147-13155},
doi = {10.1021/acs.jafc.3c04775},
pmid = {37624706},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; Fermentation ; Bacteria ; *Bacillus ; *Bacillus amyloliquefaciens ; Nucleotides ; *Nucleic Acids ; },
abstract = {Microorganisms can determine the flavor and quality of fermented food, such as Baijiu, which is produced via Daqu fermentation. Therefore, monitoring key microorganisms during fermentation is important for ensuring high-quality fermented food. Here, we report a single-nucleotide resolved nucleic acid assay based on the CRISPR/Cas12 system, enabling the quantification of Bacillus amyloliquefaciens, a key microorganism in Daqu fermentation at the species level. The assay employs an amplification-refractory mutation system derived from PCR to analyze minor genetic differences between different Bacillus species. The utilization of CRISPR/Cas12 further guaranties the specificity of identifying the PCR amplicon and enables the quantification of Bacillus amyloliquefaciens via end-measurement fluorescence. Compared to conventional qPCR, the assay allows for species-level detection of bacteria, thus enabling the precise detection of the Bacillus strain that yields high-level 2,3,5,6-tetramethylpyrazine. The assay promises the precise monitoring of bacterial growth and contribution to flavor during Daqu fermentation, thus facilitating fermented food quality control.},
}
@article {pmid37579713,
year = {2023},
author = {Wu, Z and Sun, DW and Pu, H},
title = {CRISPR/Cas12a and G-quadruplex DNAzyme-driven multimodal biosensor for visual detection of Aflatoxin B1.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {302},
number = {},
pages = {123121},
doi = {10.1016/j.saa.2023.123121},
pmid = {37579713},
issn = {1873-3557},
mesh = {Humans ; Aflatoxin B1/analysis ; DNA, Complementary/chemistry/genetics ; *DNA, Catalytic ; CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry ; Food Contamination/analysis ; Limit of Detection ; *Biosensing Techniques/methods ; Coloring Agents ; },
abstract = {Aflatoxin B1 (AFB1) contamination severely threatens human and animal health, it is thus critical to construct a strategy for its rapid, accurate, and visual detection. Herein, a multimodal biosensor was proposed based on CRISPR/Cas12a cleaved G-quadruplex (G4) for AFB1 detection. Briefly, specific binding of AFB1 to the aptamer occupied the binding site of the complementary DNA (cDNA), and cDNA then activated Cas12a to cleave G4 into fragments. Meanwhile, the intact G4-DNAzyme could catalyze 3, 3', 5, 5'-tetramethylbenzidine (TMB) to form colourimetric/SERS/fluorescent signal-enhanced TMBox, and the yellow solution produced by TMBox under acidic conditions could be integrated with a smartphone application for visual detection. The colourimetric/SERS/fluorescent biosensor yielded detection limits of 0.85, 0.79, and 1.65 pg·mL[-1], respectively, and was applied for detecting AFB1 in peanut, maize, and badam samples. The method is suitable for visual detection in naturally contaminated peanut samples and has prospective applications in the food industry.},
}
@article {pmid37662134,
year = {2023},
author = {Kannadasan, AB and Sumantran, VN and Vaidyanathan, R},
title = {A Global Comprehensive Study of the Distribution of Type I-E and Type I-E* CRISPR-Cas Systems in Klebsiella pneumoniae.},
journal = {Indian journal of community medicine : official publication of Indian Association of Preventive &amp; Social Medicine},
volume = {48},
number = {4},
pages = {567-572},
pmid = {37662134},
issn = {0970-0218},
abstract = {BACKGROUND: The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems are the short DNA sequences and RNA-dependent nuclease involved in the adaptive immunity in bacteria and archaea. The type of CRISPR-Cas system influences antibiotic susceptibility in Klebsiella pneumoniae. Here, our objective was to study the diversity of CRISPR-Cas system in the genome of K. pneumoniae from the available whole genome sequencing (WGS) data.<br><br>MATERIAL AND METHODS: We identified the CRISPR-Cas systems of K. pneumoniae using the CRISPR-CasFinder database. The complete genome sequence and its submission details were obtained from the National Center for Biotechnology Information (NCBI) database.<br><br>RESULTS: A total of 1607 K. pneumoniae whole genome sequences were analyzed. The major contributors of WGS data of K. pneumoniae were China (26.6%), United States (21.5%), Australia (10%), South Korea (8%), India (5.5%), and United Kingdom (4.9%). Out of 1607 genomes analyzed, almost one-fourth were CRISPR-Cas positive (403/1607) and three-fourth were CRISPR-Cas negative (1204/1607). Among CRISPR-Cas positive strains, 220 belonged to type I-E* and 183 were type I-E. Furthermore, type I-E* CRISPR-Cas systems were significantly higher in Asia (P < 0.001), whereas type I-E were significantly higher in Europe (P < 0.01). Among countries, typically, type I-E* strains were found to be higher in China (P < 0.01) and India (P < 0.01), whereas type I-E strains were higher in Germany (P < 0.01).<br><br>CONCLUSION: Hence, it is important to know the type of CRISPR-Cas systems in K. pneumoniae strains across the countries and it can help to understand the diversity of CRISPR-Cas systems worldwide.},
}
@article {pmid37662004,
year = {2023},
author = {Qian, Y and Zhou, D and Li, M and Zhao, Y and Liu, H and Yang, L and Ying, Z and Huang, G},
title = {Application of CRISPR-Cas system in the diagnosis and therapy of ESKAPE infections.},
journal = {Frontiers in cellular and infection microbiology},
volume = {13},
number = {},
pages = {1223696},
pmid = {37662004},
issn = {2235-2988},
mesh = {Humans ; CRISPR-Cas Systems ; *Acinetobacter baumannii/genetics ; Anti-Bacterial Agents/therapeutic use ; *Cross Infection/diagnosis ; *Enterococcus faecium ; },
abstract = {Antimicrobial-resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens represent a global threat to human health. ESKAPE pathogens are the most common opportunistic pathogens in nosocomial infections, and a considerable number of their clinical isolates are not susceptible to conventional antimicrobial therapy. Therefore, innovative therapeutic strategies that can effectively deal with ESKAPE pathogens will bring huge social and economic benefits and ease the suffering of tens of thousands of patients. Among these strategies, CRISPR (clustered regularly interspaced short palindromic repeats) system has received extra attention due to its high specificity. Regrettably, there is currently no direct CRISPR-system-based anti-infective treatment. This paper reviews the applications of CRISPR-Cas system in the study of ESKAPE pathogens, aiming to provide directions for the research of ideal new drugs and provide a reference for solving a series of problems caused by multidrug-resistant bacteria (MDR) in the post-antibiotic era. However, most research is still far from clinical application.},
}
@article {pmid37659410,
year = {2023},
author = {Liu, C and Wang, R and Li, J and Cheng, F and Shu, X and Zhao, H and Xue, Q and Yu, H and Wu, A and Wang, L and Hu, S and Zhang, Y and Yang, J and Xiang, H and Li, M},
title = {Widespread RNA-based cas regulation monitors crRNA abundance and anti-CRISPR proteins.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2023.08.005},
pmid = {37659410},
issn = {1934-6069},
abstract = {CRISPR RNAs (crRNAs) and Cas proteins work together to provide prokaryotes with adaptive immunity against genetic invaders like bacteriophages and plasmids. However, the coordination of crRNA production and cas expression remains poorly understood. Here, we demonstrate that widespread modulatory mini-CRISPRs encode cas-regulating RNAs (CreRs) that mediate autorepression of type I-B, I-E, and V-A Cas proteins, based on their limited complementarity to cas promoters. This autorepression not only reduces autoimmune risks but also responds to changes in the abundance of canonical crRNAs that compete with CreR for Cas proteins. Furthermore, the CreR-guided autorepression of Cas proteins can be alleviated or even subverted by diverse bacteriophage anti-CRISPR (Acr) proteins that inhibit Cas effectors, which, in turn, promotes the generation of new Cas proteins. Our findings reveal a general RNA-guided autorepression paradigm for diverse Cas effectors, shedding light on the intricate self-coordination of CRISPR-Cas and its transcriptional counterstrategy against Acr proteins.},
}
@article {pmid37657665,
year = {2023},
author = {Redhead, C and Taye, N and Hubmacher, D},
title = {En route towards a personalized medicine approach: Innovative therapeutic modalities for connective tissue disorders.},
journal = {Matrix biology : journal of the International Society for Matrix Biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.matbio.2023.08.005},
pmid = {37657665},
issn = {1569-1802},
abstract = {Connective tissue disorders can be caused by pathogenic variants (mutations) in genes encoding extracellular matrix (ECM) proteins. Such disorders typically manifest during development or postnatal growth and result in significant morbidity and mortality. The development of curative treatments for connective tissue disorders is hampered in part by the inability of many mature connective tissues to efficiently regenerate. To be most effective, therapeutic strategies designed to preserve or restore tissue function will likely need to be initiated during phases of significant endogenous connective tissue remodeling and organ sculpting postnatally and directly target the underlying ECM protein mutations. With recent advances in whole exome sequencing, in-vitro and in-vivo disease modeling, and the development of mutation-specific molecular therapeutic modalities, it is now feasible to directly correct disease-causing mutations underlying connective tissue disorders and ameliorate their pathogenic consequences. These technological advances may lead to potentially curative personalized medicine approaches for connective tissue disorders that have previously been considered incurable. In this review, we highlight innovative therapeutic modalities including gene replacement, exon skipping, DNA/mRNA editing, and pharmacological approaches that were used to preserve or restore tissue function in the context of connective tissue disorders. Inherent to a successful application of these approaches is the need to further the understanding of mechanisms that regulate ECM formation and homeostasis, and to decipher how individual mutations in ECM proteins compromise ECM and connective tissue development and function.},
}
@article {pmid37611446,
year = {2023},
author = {Lin, C and Chen, F and Huang, D and Li, W and He, C and Tang, Y and Li, X and Liu, C and Han, L and Yang, Y and Zhu, Y and Chen, R and Shi, Y and Xia, C and Yan, Z and Du, H and Huang, L},
title = {A universal all-in-one RPA-Cas12a strategy with de novo autodesigner and its application in on-site ultrasensitive detection of DNA and RNA viruses.},
journal = {Biosensors &amp; bioelectronics},
volume = {239},
number = {},
pages = {115609},
doi = {10.1016/j.bios.2023.115609},
pmid = {37611446},
issn = {1873-4235},
mesh = {Humans ; CRISPR-Cas Systems ; *COVID-19/diagnosis ; SARS-CoV-2 ; *Biosensing Techniques ; DNA ; *RNA Viruses ; },
abstract = {Revolutionary all-in-one RPA-CRISPR assays are rapidly becoming the most sought-after tools for point-of-care testing (POCT) due to their high sensitivity and ease of use. Despite the availability of one-pot methods for specific targets, the development of more efficient methods for new targets remains a significant challenge. In this study, we present a rapid and universal approach to establishing an all-in-one RPA-Cas12a method CORDSv2 based on rational balancing amplification and Cas12a cleavage, which achieves ultrasensitive detection of several targets, including SARS-CoV-2, ASFV, HPV16, and HPV18. CORDSv2 demonstrates a limit of detection (LOD) of 0.6 cp/μL and 100% sensitivity for SARS-CoV-2, comparable to qPCR. Combining with our portable device(hippo-CORDS), it has a visual detection LOD of 6 cp/μL and a sensitivity up to 100% for SARS-CoV-2 and 97% for Ct<35 ASFV samples, surpassing most one-pot visual methods. To simplify and accelerate the process for new targets, we also develop a de novo autodesigner by which the optimal couples of primers and crRNA can be selected rapidly. As a universal all-in-one RPA-CRISPR method for on-site testing, CORDSv2 becomes an attractive choice for rapid and accurate diagnosis in resource-limited settings.},
}
@article {pmid37597500,
year = {2023},
author = {Hu, H and Dong, K and Yan, B and Mu, Y and Liao, Y and Zhang, L and Guo, S and Xiao, X and Wang, X},
title = {Highly-sensitive and homogenous detection of 8-oxoguanine based DNA oxidative damage by a CRISPR-enhanced structure-switching aptamer assay.},
journal = {Biosensors &amp; bioelectronics},
volume = {239},
number = {},
pages = {115588},
doi = {10.1016/j.bios.2023.115588},
pmid = {37597500},
issn = {1873-4235},
mesh = {Male ; Humans ; *Biosensing Techniques ; Semen ; Biological Assay ; Oligonucleotides ; Oxidative Stress ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {8-oxoguanine (8-oxoG) based DNA damage is the most common type of DNA damage which greatly affect gene expression. Therefore, accurate quantification of 8-oxoG based DNA damage is of high clinical significance. However, current methods for 8-oxoG detection struggle to balance convenience, low cost, and sensitivity. Herein, we have proposed and investigated the shortened crRNA mode of CRISPR-Cas12a system and greatly enhanced its signal-to-noise ratio. Taking advantages of the shortened crRNA mode, we further developed a CRISPR-enhanced structure-switching aptamer assay (CESA) for 8-oxoG. The analytical performance of CESA was thoroughly investigated via detecting free 8-oxoG and 8-oxoG on gDNA. The CESA displayed impressive sensitivity for free 8-oxoG, with detection and quantification limits of 32.3 pM and 0.107 nM. These limits modestly rose to 64.5 pM and 0.215 nM when examining 8-oxoG on gDNA. To demonstrate the clinical practicability and significance of the CESA system, we further applied it to measuring 8-oxoG levels in 7 plasma samples (Cervical carcinoma, 11.87 ± 0.69 nM VS. Healthy control, 2.66 ± 0.42 nM), 24 seminal plasma samples (Asthenospermia, 22.29 ± 7.48 nM VS. Normal sperm, 9.75 ± 3.59 nM), 10 breast-tissue gDNA samples (Breast cancer, 2.77 ± 0.63 nM/μg VS. Healthy control, 0.41 ± 0.09 nM/μg), and 24 sperm gDNA samples (Asthenospermia, 28.62 ± 4.84 VS. Normal sperm, 16.67 ± 3.31). This work not only proposes a novel design paradigm of shortened crRNA for developing CRISPR-Cas12a based biosensors but also offers a powerful tool for detecting 8-oxoG based DNA damage.},
}
@article {pmid37589291,
year = {2023},
author = {Johnson, CJ and Kulkarni, A and Buxton, WJ and Hui, TY and Kayastha, A and Khoja, AA and Leandre, J and Mehta, VV and Ostrowski, L and Pareizs, EG and Scotto, RL and Vargas, V and Vellingiri, RM and Verzino, G and Vohra, R and Wakade, SC and Winkeljohn, VM and Winkeljohn, VM and Rotterman, TM and Stolfi, A},
title = {Using CRISPR/Cas9 to identify genes required for mechanosensory neuron development and function.},
journal = {Biology open},
volume = {12},
number = {9},
pages = {},
doi = {10.1242/bio.060002},
pmid = {37589291},
issn = {2046-6390},
support = {K99 NS126576/NS/NINDS NIH HHS/United States ; R01 GM143326/GM/NIGMS NIH HHS/United States ; R01GM143326/NH/NIH HHS/United States ; K99NS126576/NH/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Ciona ; Larva/genetics ; Metamorphosis, Biological/genetics ; Sensory Receptor Cells ; },
abstract = {Tunicates are marine, non-vertebrate chordates that comprise the sister group to the vertebrates. Most tunicates have a biphasic lifecycle that alternates between a swimming larva and a sessile adult. Recent advances have shed light on the neural basis for the tunicate larva's ability to sense a proper substrate for settlement and initiate metamorphosis. Work in the highly tractable laboratory model tunicate Ciona robusta suggests that sensory neurons embedded in the anterior papillae transduce mechanosensory stimuli to trigger larval tail retraction and initiate the process of metamorphosis. Here, we take advantage of the low-cost and simplicity of Ciona by using tissue-specific CRISPR/Cas9-mediated mutagenesis to screen for genes potentially involved in mechanosensation and metamorphosis, in the context of an undergraduate 'capstone' research course. This small screen revealed at least one gene, Vamp1/2/3, which appears crucial for the ability of the papillae to trigger metamorphosis. We also provide step-by-step protocols and tutorials associated with this course, in the hope that it might be replicated in similar CRISPR-based laboratory courses wherever Ciona are available.},
}
@article {pmid37552860,
year = {2023},
author = {Li, Y and Cooper, BH and Liu, Y and Wu, D and Zhang, X and Rohs, R and Qin, PZ},
title = {CRISPR-Cas9 Activities with Truncated 16-Nucleotide RNA Guides Are Tuned by Target Duplex Stability Beyond the RNA/DNA Hybrid.},
journal = {Biochemistry},
volume = {62},
number = {17},
pages = {2541-2548},
doi = {10.1021/acs.biochem.3c00250},
pmid = {37552860},
issn = {1520-4995},
mesh = {*RNA/genetics ; *CRISPR-Cas Systems ; Nucleotides ; DNA/genetics ; Gene Editing/methods ; },
abstract = {CRISPR-Cas9 has been adapted as a readily programmable genome manipulation agent, and continuing technological advances rely on an in-depth mechanistic understanding of Cas9 target discrimination. Cas9 interrogates a target by unwinding the DNA duplex to form an R-loop, where the RNA guide hybridizes with one of the DNA strands. It has been shown that RNA guides shorter than the normal length of 20-nucleotide (-nt) support Cas9 cleavage activity by enabling partial unwinding beyond the RNA/DNA hybrid. To investigate whether DNA segment beyond the RNA/DNA hybrid can impact Cas9 target discrimination with truncated guides, Cas9 double-stranded DNA cleavage rates (kcat) were measured with 16-nt guides on targets with varying sequences at +17 to +20 positions distal to the protospacer-adjacent-motif (PAM). The data reveal a log-linear inverse correlation between kcat and the PAM+(17-20) DNA duplex dissociation free energy (ΔGNN(17-20)[0]), with sequences having smaller ΔGNN(17-20)[0] showing faster cleavage and a higher degree of unwinding. The results indicate that, with a 16-nt guide, "peripheral" DNA sequences beyond the RNA/DNA hybrid contribute to target discrimination by tuning the cleavage reaction transition state through the modulation of PAM-distal unwinding. The finding provides mechanistic insights for the further development of strategies that use RNA guide truncation to enhance Cas9 specificity.},
}
@article {pmid37356052,
year = {2023},
author = {Zheng, R and Zhang, L and Parvin, R and Su, L and Chi, J and Shi, K and Ye, F and Huang, X},
title = {Progress and Perspective of CRISPR-Cas9 Technology in Translational Medicine.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {10},
number = {25},
pages = {e2300195},
pmid = {37356052},
issn = {2198-3844},
support = {2022C03007//Key Research and Development Plan of Zhejiang Province "Spearhead"/ ; 2020ZY0036//Wenzhou Major Science and Technology Innovation Tackling Project/ ; LGF22C100003//Zhejiang Provincial Natural Science Foundation of China/ ; WIUCASQD2021012//Wenzhou high-level innovation team : Development and application team of functional liver cancer-on-a-chip program of Wenzhou Institute, University of Chinese Academy of Sciences/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Translational Science, Biomedical ; Gene Editing/methods ; Genetic Therapy/methods ; Genomics ; },
abstract = {Translational medicine aims to improve human health by exploring potential treatment methods developed during basic scientific research and applying them to the treatment of patients in clinical settings. The advanced perceptions of gene functions have remarkably revolutionized clinical treatment strategies for target agents. However, the progress in gene editing therapy has been hindered due to the severe off-target effects and limited editing sites. Fortunately, the development in the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system has renewed hope for gene therapy field. The CRISPR-Cas9 system can fulfill various simple or complex purposes, including gene knockout, knock-in, activation, interference, base editing, and sequence detection. Accordingly, the CRISPR-Cas9 system is adaptable to translational medicine, which calls for the alteration of genomic sequences. This review aims to present the latest CRISPR-Cas9 technology achievements and prospect to translational medicine advances. The principle and characterization of the CRISPR-Cas9 system are firstly introduced. The authors then focus on recent pre-clinical and clinical research directions, including the construction of disease models, disease-related gene screening and regulation, and disease treatment and diagnosis for multiple refractory diseases. Finally, some clinical challenges including off-target effects, in vivo vectors, and ethical problems, and future perspective are also discussed.},
}
@article {pmid37288647,
year = {2023},
author = {Meng, W and Qiao, K and Liu, F and Gao, X and Hu, X and Liu, J and Gao, Y and Zhu, J},
title = {Construction and application of a new CRISPR/Cas12a system in Stenotrophomonas AGS-1 from aerobic granular sludge.},
journal = {Biotechnology journal},
volume = {18},
number = {9},
pages = {e2200596},
doi = {10.1002/biot.202200596},
pmid = {37288647},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems/genetics ; *Sewage ; Gene Editing/methods ; Plasmids ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Aerobic granular sludge (AGS) is a microbial aggregate with a biofilm structure. Thus, investigating AGS in the aspect of biofilm and microbial attachment at the genetic level would help to reveal the mechanism of granule biofilm formation. In this work, a two-plasmid clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas)12a genome editing system was constructed to identify attachment genes for the first time in Stenotrophomonas AGS-1 from AGS. One plasmid contained a Cas12a cassette driven by an arabinose-inducible promoter, and another contained the specific crRNA and homologous arms (HAs). Acidaminococcus sp. Cas12a (AsCas12a) was adopted and proven to have mild toxicity (compared to Cas9) and strong cleavage activity for AGS-1. CRISPR/Cas12a-mediated rmlA knockout decreased attachment ability by 38.26%. Overexpression of rmlA in AGS-1 resulted in an increase of 30.33% in attachment ability. These results showed that the modulation of rmlA was an important factor for the biofilm formation of AGS-1. Moreover, two other genes (xanB and rpfF) were knocked out by CRISPR/Cas12a and identified as attachment-related genes in AGS-1. Also, this system could achieve point mutations. These data indicated that the CRISPR/Cas12a system could be an effective molecular platform for attachment gene function identification, which would be useful for the development of AGS in wastewater treatment.},
}
@article {pmid37657377,
year = {2023},
author = {Long, K and Cao, G and Qiu, Y and Yang, N and Chen, J and Yang, M and Hou, C and Huo, D},
title = {Hybridization chain reaction circuit controller: CRISPR/Cas12a conversion amplifier for miRNA-21 sensitive detection.},
journal = {Talanta},
volume = {266},
number = {Pt 2},
pages = {125130},
doi = {10.1016/j.talanta.2023.125130},
pmid = {37657377},
issn = {1873-3573},
abstract = {MicroRNA (miRNA) is crucial to the diagnose of various diseases. However, the accurate detection of miRNA has been challenging due to its short length and low abundance. Here, we designed a hybridization chain reaction (HCR) circuit controller to initiate the CRISPR/Cas12a conversion amplifier (HCR-Cas12a controller) for sensitive detection of miRNA-21 (miR-21). In the HCR, pre-crRNA was encapsulated in a hairpin structure until the miR-21 was present. Afterward, Cas12a fully exerted its RNase activity to self-mature pre-crRNA. Then, the trans-cleavage activity of Cas12a was initiated by activator. This results in the conversion of biological signals to fluorescent signal. During HCR-Cas12a controller, the circuit formed quickly, while the Cas12a system worked in a short time. The miR-21 was ultra-sensitively detected with the wide detection range of 1 fM - 100 nM, and the calculated limit of detection was 75.4 aM. The sensitivity was an order of magnitude lower than the standard method. The formation of HCR at room temperature does not require a thermal cycler. Additionally, Cas12a can work without the need for precise or expensive instruments. Therefore, our proposed method was suitable for low-resource settings, and provided a technical basis for sensitive detection of miRNA in low concentration range.},
}
@article {pmid37462508,
year = {2023},
author = {Son, W and Chung, KW},
title = {Targeted recombination of homologous chromosomes using CRISPR-Cas9.},
journal = {FEBS open bio},
volume = {13},
number = {9},
pages = {1658-1666},
pmid = {37462508},
issn = {2211-5463},
support = {NRF-2019R1A2C1087547//National Research Foundation of Korea/ ; NRF-2021R1A4A2001389//National Research Foundation of Korea/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Homologous Recombination/genetics ; Mutagenesis ; Drosophila/genetics ; Chromosomes ; },
abstract = {CRISPR mutagenesis is an efficient way to disrupt specific target genes in many model organisms. We previously devised a targeted CRISPR recombination method to generate intragenic recombinants of alleles in Drosophila. Here, we assessed the applicability of CRISPR targeting-induced recombination to different genetic loci. We compared the ectopic recombination rates in the male germline by CRISPR targeting at two neighboring genetic loci within the genomic region that consists of the repressed chromatin domain of the Lobe gene, and the transcriptionally active domain of PRAS40. Targeting around the transcription initiation of PRAS40 resulted in higher recombination rates of homologous chromosomes than targeting at the Lobe intron. Based on the efficient homologous recombination by CRISPR targeting observed around transcriptionally active loci, we further investigated targeted recombination between P-elements that are inserted at different genomic locations. Male recombination by CRISPR targeting of P-elements located proximally and distally to the ebony gene produced recombinants deficient for the intervening region of ebony transcription. Taken together, we suggest that targeted homologous recombination by CRISPR targeting may have specific genetic applications, such as generation of allelic combinations or chromosomal variations.},
}
@article {pmid37381714,
year = {2023},
author = {Fu, D and Yan, J and Zhang, Z and Liu, Y and Ma, X and Ding, J and Yang, S and Zhao, R and Chang, A and Gao, C and Liu, J and Zhao, T and Wang, X and Huang, C and Gao, S and Ma, Y and Tang, B and Feng, Y and Wang, H and Hao, J},
title = {Nuclear PLD1 combined with NPM1 induces gemcitabine resistance through tumorigenic IL7R in pancreatic adenocarcinoma.},
journal = {Cancer biology &amp; medicine},
volume = {20},
number = {8},
pages = {599-626},
pmid = {37381714},
issn = {2095-3941},
mesh = {Humans ; Gemcitabine ; *Pancreatic Neoplasms/drug therapy/genetics ; STAT5 Transcription Factor/metabolism/pharmacology ; Deoxycytidine/pharmacology/therapeutic use ; Receptors, Interleukin-7/metabolism ; *Adenocarcinoma/drug therapy/genetics ; RNA, Guide, CRISPR-Cas Systems ; Antimetabolites, Antineoplastic/pharmacology/therapeutic use ; Drug Resistance, Neoplasm/genetics ; *Carcinoma, Pancreatic Ductal/drug therapy/genetics/pathology ; Nuclear Proteins/genetics/metabolism ; Proto-Oncogene Proteins c-bcl-2/metabolism ; },
abstract = {OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant gastrointestinal cancer with a 5-year survival rate of only 9%. Of PDAC patients, 15%-20% are eligible for radical surgery. Gemcitabine is an important chemotherapeutic agent for patients with PDAC; however, the efficacy of gemcitabine is limited due to resistance. Therefore, reducing gemcitabine resistance is essential for improving survival of patients with PDAC. Identifying the key target that determines gemcitabine resistance in PDAC and reversing gemcitabine resistance using target inhibitors in combination with gemcitabine are crucial steps in the quest to improve survival prognosis in patients with PDAC.<br><br>METHODS: We constructed a human genome-wide CRISPRa/dCas 9 overexpression library in PDAC cell lines to screen key targets of drug resistance based on sgRNA abundance and enrichment. Then, co-IP, ChIP, ChIP-seq, transcriptome sequencing, and qPCR were used to determine the specific mechanism by which phospholipase D1 (PLD1) confers resistance to gemcitabine.<br><br>RESULTS: PLD1 combines with nucleophosmin 1 (NPM1) and triggers NPM1 nuclear translocation, where NPM1 acts as a transcription factor to upregulate interleukin 7 receptor (IL7R) expression. Upon interleukin 7 (IL-7) binding, IL7R activates the JAK1/STAT5 signaling pathway to increase the expression of the anti-apoptotic protein, BCL-2, and induce gemcitabine resistance. The PLD1 inhibitor, Vu0155069, targets PLD1 to induce apoptosis in gemcitabine-resistant PDAC cells.<br><br>CONCLUSIONS: PLD1 is an enzyme that has a critical role in PDAC-associated gemcitabine resistance through a non-enzymatic interaction with NPM1, further promoting the downstream JAK1/STAT5/Bcl-2 pathway. Inhibiting any of the participants of this pathway can increase gemcitabine sensitivity.},
}
@article {pmid37656446,
year = {2023},
author = {Li, Y and Zhao, Z and Liu, Y and Wang, N and Man, S and Ma, L and Wang, S},
title = {CRISPR/Cas System: The Accelerator for the Development of Non-nucleic Acid Target Detection in Food Safety.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.3c03619},
pmid = {37656446},
issn = {1520-5118},
abstract = {Non-nucleic acid targets have posed a serious challenge to food safety. The detection of non-nucleic acid targets can enable us to monitor food contamination in a timely manner. In recent years, the CRISPR/Cas system has been extensively explored in biosensing. However, there is a lack of a summary of CRISPR/Cas-powered detection tailored to non-nucleic acid targets involved in food safety. This review comprehensively summarizes the recent advances on the construction of CRISPR/Cas-powered detection and the promising applications in the field of food safety related non-nucleic acid targets. The current challenges and futuristic perspectives are also proposed accordingly. The rapidly evolving CRISPR/Cas system has provided a powerful propellant for non-nucleic acid target detection via integration with aptamer and/or DNAzyme. Compared with traditional analytical methods, CRISPR/Cas-powered detection is conceptually novel, essentially eliminates the dependence on large instruments, and also demonstrates the capability for rapid, accurate, sensitive, and on-site testing.},
}
@article {pmid37654098,
year = {2023},
author = {Gawlitt, S and Liao, C and Achmedov, T and Beisel, CL},
title = {Shortened CRISPR-Cas9 arrays enable multiplexed gene targeting in bacteria from a smaller DNA footprint.},
journal = {RNA biology},
volume = {20},
number = {1},
pages = {666-680},
doi = {10.1080/15476286.2023.2247247},
pmid = {37654098},
issn = {1555-8584},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Footprinting ; Escherichia coli/genetics ; Gene Targeting ; Bacteria/genetics ; Endonucleases ; },
abstract = {CRISPR technologies comprising a Cas nuclease and a guide RNA (gRNA) can utilize multiple gRNAs to enact multi-site editing or regulation in the same cell. Nature devised a highly compact means of encoding gRNAs in the form of CRISPR arrays composed of conserved repeats separated by targeting spacers. However, the capacity to acquire new spacers keeps the arrays longer than necessary for CRISPR technologies. Here, we show that CRISPR arrays utilized by the Cas9 nuclease can be shortened without compromising and sometimes even enhancing targeting activity. Using multiplexed gene repression in E. coli, we found that each region could be systematically shortened to varying degrees before severely compromising targeting activity. Surprisingly, shortening some spacers yielded enhanced targeting activity, which was linked to folding of the transcribed array prior to processing. Overall, shortened CRISPR-Cas9 arrays can facilitate multiplexed editing and gene regulation from a smaller DNA footprint across many bacterial applications of CRISPR technologies.},
}
@article {pmid37647403,
year = {2023},
author = {Song, N and Chu, Y and Li, S and Dong, Y and Fan, X and Tang, J and Guo, Y and Teng, G and Yao, C and Yang, D},
title = {Cascade dynamic assembly/disassembly of DNA nanoframework enabling the controlled delivery of CRISPR-Cas9 system.},
journal = {Science advances},
volume = {9},
number = {35},
pages = {eadi3602},
pmid = {37647403},
issn = {2375-2548},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *DNA/genetics ; Acrylamide ; CRISPR-Associated Protein 9/genetics ; Cystamine ; Ribonucleoproteins ; },
abstract = {CRISPR-Cas9 has been explored as a therapeutic agent for down-regulating target genes; the controlled delivery of Cas9 ribonucleoprotein (RNP) is essential for therapeutic efficacy and remains a challenge. Here, we report cascade dynamic assembly/disassembly of DNA nanoframework (NF) that enables the controlled delivery of Cas9 RNP. NF was prepared with acrylamide-modified DNA that initiated cascade hybridization chain reaction (HCR). Through an HCR, single-guide RNA was incorporated to NF; simultaneously, the internal space of NF was expanded, facilitating the loading of Cas9 protein. NF was designed with hydrophilic acylamino and hydrophobic isopropyl, allowing dynamic swelling and aggregation. The responsive release of Cas9 RNP was realized by introducing disulfide bond-containing N,N-bis(acryloyl)cystamine that was specifically in response to glutathione of cancer cells, triggering the complete disassembly of NF. In vitro and in vivo investigations demonstrated the high gene editing efficiency in cancer cells, the hypotoxicity in normal cells, and notable antitumor efficacy in a breast cancer mouse model.},
}
@article {pmid37646679,
year = {2023},
author = {Sharma, A and Boelens, JJ and Cancio, M and Hankins, JS and Bhad, P and Azizy, M and Lewandowski, A and Zhao, X and Chitnis, S and Peddinti, R and Zheng, Y and Kapoor, N and Ciceri, F and Maclachlan, T and Yang, Y and Liu, Y and Yuan, J and Naumann, U and Yu, VWC and Stevenson, SC and De Vita, S and LaBelle, JL},
title = {CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease.},
journal = {The New England journal of medicine},
volume = {389},
number = {9},
pages = {820-832},
doi = {10.1056/NEJMoa2215643},
pmid = {37646679},
issn = {1533-4406},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Fetal Hemoglobin/genetics ; *Anemia, Sickle Cell/genetics/therapy ; Erythrocytes ; Hemoglobin, Sickle ; Antigens, CD34 ; },
abstract = {BACKGROUND: Sickle cell disease is caused by a defect in the β-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease. OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product, has a targeted disruption of the HBG1 and HBG2 (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny.<br><br>METHODS: We performed a tiling CRISPR-Cas9 screen of the HBG1 and HBG2 promoters by electroporating CD34+ cells obtained from healthy donors with Cas9 complexed with one of 72 guide RNAs, and we assessed the fraction of fetal hemoglobin-immunostaining erythroblasts (F cells) in erythroid-differentiated progeny. The gRNA resulting in the highest level of F cells (gRNA-68) was selected for clinical development. We enrolled participants with severe sickle cell disease in a multicenter, phase 1-2 clinical study to assess the safety and adverse-effect profile of OTQ923.<br><br>RESULTS: In preclinical experiments, CD34+ HSPCs (obtained from healthy donors and persons with sickle cell disease) edited with CRISPR-Cas9 and gRNA-68 had sustained on-target editing with no off-target mutations and produced high levels of fetal hemoglobin after in vitro differentiation or xenotransplantation into immunodeficient mice. In the study, three participants received autologous OTQ923 after myeloablative conditioning and were followed for 6 to 18 months. At the end of the follow-up period, all the participants had engraftment and stable induction of fetal hemoglobin (fetal hemoglobin as a percentage of total hemoglobin, 19.0 to 26.8%), with fetal hemoglobin broadly distributed in red cells (F cells as a percentage of red cells, 69.7 to 87.8%). Manifestations of sickle cell disease decreased during the follow-up period.<br><br>CONCLUSIONS: CRISPR-Cas9 disruption of the HBG1 and HBG2 gene promoters was an effective strategy for induction of fetal hemoglobin. Infusion of autologous OTQ923 into three participants with severe sickle cell disease resulted in sustained induction of red-cell fetal hemoglobin and clinical improvement in disease severity. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT04443907.).},
}
@article {pmid37646520,
year = {2023},
author = {Chen, S and Cao, H and Xu, Z and Huang, J and Liu, Z and Li, T and Duan, C and Wu, W and Wen, Y and Zhang, LH and Xu, Z},
title = {A type I-F CRISPRi system unveils the novel role of CzcR in modulating multidrug resistance of Pseudomonas aeruginosa.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0112323},
doi = {10.1128/spectrum.01123-23},
pmid = {37646520},
issn = {2165-0497},
abstract = {Pseudomonas aeruginosa has abundant signaling systems that exquisitely control its antibiotic resistance in response to different environmental cues. Understanding the regulation of antibiotic resistance will provide important implications for precise antimicrobial interventions. However, efficient genetic tools for functional gene characterizations are sometimes not available, particularly, in clinically isolated strains. Here, we established a type I-F CRISPRi (CSYi) system for programmable gene silencing. By incorporating anti-CRISPR proteins, this system was even applicable to bacterial hosts encoding a native type I-F CRISPR-Cas system. With the newly developed gene-silencing system, we revealed that the response regulator CzcR from the zinc (Zn[2+])-responsive two-component system CzcS/CzcR is a repressor of efflux pumps MexAB-OprM and MexGHI-OpmD, which inhibits the expression of both operons by directly interacting with their promoters. Repression of MexAB-OprM consequently increases the susceptibility of P. aeruginosa to multiple antibiotics such as levofloxacin and amikacin. Together, this study provided a simple approach to study gene functions, which enabled us to unveil the novel role of CzcR in modulating efflux pump genes and multidrug resistance in P. aeruginosa. IMPORTANCE P. aeruginosa is a ubiquitous opportunistic pathogen frequently causing chronic infections. In addition to being an important model organism for antibiotic-resistant research, this species is also important for understanding and exploiting CRISPR-Cas systems. In this study, we established a gene-silencing system based on the most abundant type I-F CRISPR-Cas system in this species, which can be readily employed to achieve targeted gene repression in multiple bacterial species. Using this gene-silencing system, the physiological role of Zn[2+] and its responsive regulator CzcR in modulating multidrug resistance was unveiled with great convenience. This study not only displayed a new framework to expand the abundant CRISPR-Cas and anti-CRISPR systems for functional gene characterizations but also provided new insights into the regulation of multidrug resistance in P. aeruginosa and important clues for precise anti-pseudomonal therapies.},
}
@article {pmid37645763,
year = {2023},
author = {Borrajo, J and Javanmardi, K and Griffin, J and Martin, SJS and Yao, D and Hill, K and Blainey, PC and Al-Shayeb, B},
title = {Programmable multi-kilobase RNA editing using CRISPR-mediated trans-splicing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37645763},
support = {DP2 HL141005/HL/NHLBI NIH HHS/United States ; },
abstract = {UNLABELLED: Current gene editing approaches in eukaryotic cells are limited to single base edits or small DNA insertions and deletions, and remain encumbered by unintended permanent effects and significant challenges in the delivery of large DNA cargo. Here we describe Splice Editing, a generalizable platform to correct gene transcripts in situ by programmable insertion or replacement of large RNA segments. By combining CRISPR-mediated RNA targeting with endogenous cellular RNA-splicing machinery, Splice Editing enables efficient, precise, and programmable large-scale editing of gene targets without DNA cleavage or mutagenesis. RNA sequencing and measurement of spliced protein products confirm that Splice Editing achieves efficient and specific targeted RNA and protein correction. We show that Splice Editors based on novel miniature RNA-targeting CRISPR-Cas systems discovered and characterized in this work can be packaged for effective delivery to human cells and affect different types of edits across multiple targets and cell lines. By editing thousands of bases simultaneously in a single reversible step, Splice Editing could expand the treatable disease population for monogenic diseases with large allelic diversity without the permanent unintended effects of DNA editing.<br><br>ONE-SENTENCE SUMMARY: CRISPR-guided trans-splicing enables efficient and specific recombination of large RNA molecules in mammalian cells, with broad applications in therapeutic development for genetic diseases and as a research tool for the study of basic RNA biology.},
}
@article {pmid37645720,
year = {2023},
author = {Peng, R and Chen, X and Xu, F and Hailstone, R and Men, Y and Du, K},
title = {Pneumatic Nano-Sieve for CRISPR-based Detection of Drug-resistant Bacteria.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37645720},
abstract = {The increasing prevalence of antibiotic-resistant bacterial infections, particularly methicillin-resistant Staphylococcus aureus (MRSA), presents a significant public health concern. Timely detection of MRSA is crucial to enable prompt medical intervention, limit its spread, and reduce antimicrobial resistance. Here, we introduce a miniaturized nano-sieve device featuring a pneumatically-regulated chamber for highly efficient MRSA purification from human plasma samples. By using packed magnetic beads as a filter and leveraging the deformability of the nano-sieve channel, we achieve an on-chip concentration factor of 15 for MRSA. We integrated this device with recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas detection system, resulting in an on-chip limit of detection (LOD) of approximately 100 CFU/mL. This developed approach provides a rapid, precise, and centrifuge-free solution suitable for point-of-care diagnostics, with the potential to significantly improve patient outcomes in resource-limited medical conditions.},
}
@article {pmid37642743,
year = {2023},
author = {Curtin, K and Wang, J and Fike, BJ and Binkley, B and Li, P},
title = {A 3D printed microfluidic device for scalable multiplexed CRISPR-cas12a biosensing.},
journal = {Biomedical microdevices},
volume = {25},
number = {3},
pages = {34},
pmid = {37642743},
issn = {1572-8781},
support = {R01GM135432/GM/NIGMS NIH HHS/United States ; R01GM135432/GM/NIGMS NIH HHS/United States ; R01GM135432/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Lab-On-A-Chip Devices ; Microfluidics ; Printing, Three-Dimensional ; },
abstract = {Accurate, rapid, and multiplexed nucleic acid detection is critical for environmental and biomedical monitoring. In recent years, CRISPR-Cas12a has shown great potential in improving the performance of DNA biosensing. However, the nonspecific trans-cleavage activity of Cas12a complicates the multiplexing capability of Cas12a biosensing. We report a 3D-printed composable microfluidic plate (cPlate) device that utilizes miniaturized wells and microfluidic loading for a multiplexed CRISPR-Cas12a assay. The device easily combines loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12a readout in a simple and high-throughput workflow with low reagent consumption. To ensure the maximum performance of the device, the concentration of Cas12a and detection probe was optimized, which yielded a four-fold sensitivity improvement. Our device demonstrates sensitive detection to the fg mL[- 1] level for four waterborne pathogens including shigella, campylobacter, cholera, and legionella within 1 h, making it suitable for low-resource settings.},
}
@article {pmid37573509,
year = {2023},
author = {Li, S and Mereby, SA and Rothstein, M and Johnson, MR and Brack, BJ and Mallarino, R},
title = {TIGER: Single-step in vivo genome editing in a non-traditional rodent.},
journal = {Cell reports},
volume = {42},
number = {8},
pages = {112980},
doi = {10.1016/j.celrep.2023.112980},
pmid = {37573509},
issn = {2211-1247},
mesh = {Female ; Pregnancy ; Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Rodentia/genetics ; },
abstract = {Rodents are taxonomically diverse and have evolved a variety of traits. A mechanistic understanding of such traits has remained elusive, however, largely because genome editing in non-traditional model species remains challenging. Here, using the African striped mouse (Rhabdomys pumilio), we describe TIGER (targeted in vivo genome editing in rodents), a method that relies on a simple intraoviductal injecting technique and uses recombinant adeno-associated viruses (rAAVs) as the sole vehicle to deliver reagents into pregnant females. We demonstrate that TIGER generates knockout and knockin (up to 3 kb) lines with high efficiency. Moreover, we engineer a double-cleaving repair rAAV template and find that it significantly increases knockin frequency and germline transmission rates. Lastly, we show that an oversized double-cleaving rAAV template leads to an insertion of 3.8 kb. Thus, TIGER constitutes an attractive alternative to traditional ex vivo genome-editing methods and has the potential to be extended to a broad range of species.},
}
@article {pmid37561631,
year = {2023},
author = {Liu, Z and Zheng, X and Chen, J and Zheng, L and Ma, Z and Chen, L and Deng, M and Tang, H and Zhou, L and Kang, T and Wu, Y and Liu, Z},
title = {NFYC-37 promotes tumor growth by activating the mevalonate pathway in bladder cancer.},
journal = {Cell reports},
volume = {42},
number = {8},
pages = {112963},
doi = {10.1016/j.celrep.2023.112963},
pmid = {37561631},
issn = {2211-1247},
mesh = {Humans ; Mevalonic Acid/metabolism ; *Hydroxymethylglutaryl-CoA Reductase Inhibitors ; RNA, Guide, CRISPR-Cas Systems ; *Urinary Bladder Neoplasms/genetics/pathology ; Transcription Factors/metabolism ; },
abstract = {Dysregulation of transcription is a hallmark of cancer, including bladder cancer (BLCA). CRISPR-Cas9 screening using a lentivirus library with single guide RNAs (sgRNAs) targeting human transcription factors and chromatin modifiers is used to reveal genes critical for the proliferation and survival of BLCA cells. As a result, the nuclear transcription factor Y subunit gamma (NFYC)-37, but not NFYC-50, is observed to promote cell proliferation and tumor growth in BLCA. Mechanistically, NFYC-37 interacts with CBP and SREBP2 to activate mevalonate pathway transcription, promoting cholesterol biosynthesis. However, NFYC-50 recruits more of the arginine methyltransferase CARM1 than NFYC-37 to methylate CBP, which prevents the CBP-SREBP2 interaction and subsequently inhibits the mevalonate pathway. Importantly, statins targeting the mevalonate pathway can suppress NFYC-37-induced cell proliferation and tumor growth, indicating the need for conducting a clinical trial with statins for treating patients with BLCA and high NFYC-37 levels, as most patients with BLCA have high NFYC-37 levels.},
}
@article {pmid37544379,
year = {2023},
author = {Agrawal, P and Harish, V and Mohd, S and Singh, SK and Tewari, D and Tatiparthi, R and Harshita, and Vishwas, S and Sutrapu, S and Dua, K and Gulati, M},
title = {Role of CRISPR/Cas9 in the treatment of Duchenne muscular dystrophy and its delivery strategies.},
journal = {Life sciences},
volume = {330},
number = {},
pages = {122003},
doi = {10.1016/j.lfs.2023.122003},
pmid = {37544379},
issn = {1879-0631},
mesh = {Humans ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Dystrophin/genetics ; CRISPR-Cas Systems/genetics ; Mutation ; Exons ; },
abstract = {Duchenne muscular dystrophy (DMD) is a neuromuscular disorder brought on by mutations in the DMD gene, which prevent muscle cells from expressing the dystrophin protein. CRISPR/Cas9 technology has evolved as potential option to treat DMD due to its ability to permanently skip exons, restoring the disrupted DMD reading frame and leading to dystrophin restoration. Even though, having potential to treat DMD, the delivery, safety and efficacy of this technology is still challenging. Several delivery methods, including viral vectors, nanoparticles, and electroporation, have been explored to deliver CRISPR/Cas9 to the targeted cells. Despite the potential of CRISPR/Cas9 technology in the treatment of DMD, several limitations need to be addressed. The off-target effects of CRISPR/Cas9 are a major concern that needs to be addressed to avoid unintended mutations. The delivery of CRISPR/Cas9 to the target cells and the immune response due to the viral vectors used for delivery are a few other limitations. The clinical trials of CRISPR/Cas9 for DMD provide valuable insights into the safety and efficacy of this technology in humans and the limitations that need to be known. Therefore, in this review we insightfully discussed the challenges and limitations of CRISPR/Cas9 in the treatment of DMD and delivery strategies used, and the ongoing efforts to overcome these challenges and restore dystrophin expression in DMD patients in the ongoing trials.},
}
@article {pmid37486124,
year = {2023},
author = {Garrigues, S and Peng, M and Kun, RS and de Vries, RP},
title = {Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9.},
journal = {mBio},
volume = {14},
number = {4},
pages = {e0066823},
pmid = {37486124},
issn = {2150-7511},
support = {15807//Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Mutation ; Genetic Engineering ; Aspergillus niger/genetics ; },
abstract = {CRISPR/Cas9 genome editing technology has been implemented in almost all living organisms. Its editing precision appears to be very high and therefore could represent a big change from conventional genetic engineering approaches. However, guide RNA binding to nucleotides similar to the target site could result in undesired off-target mutations. Despite this, evaluating whether mutations occur is rarely performed in genome editing studies. In this study, we generated CRISPR/Cas9-derived filamentous fungal strains and analyzed them for the occurrence of mutations, and to which extent genome stability affects their occurrence. As a test case, we deleted the (hemi-)cellulolytic regulator-encoding gene xlnR in two Aspergillus niger strains: a wild type (WT) and a non-homologous end-joining (NHEJ)-deficient strain ΔkusA. Initial phenotypic analysis suggested a much higher prevalence of mutations in the WT compared to NHEJ-deficient strains, which was confirmed and quantified by whole-genome sequencing analysis. Our results clearly demonstrate that CRISPR/Cas9 applied to an NHEJ-deficient strain is an efficient strategy to avoid unwanted mutations. IMPORTANCE Filamentous fungi are commonly used biofactories for the production of industrially relevant proteins and metabolites. Often, fungal biofactories undergo genetic development (genetic engineering, genome editing, etc.) aimed at improving production yields. In this context, CRISPR/Cas9 has gained much attention as a genome editing strategy due to its simplicity, versatility, and precision. However, despite the high level of accuracy reported for CRISPR/Cas9, in some cases unintentional cleavages in non-targeted loci-known as off-target mutations-could arise. While biosafety should be a central feature of emerging biotechnologies to minimize unintended consequences, few studies quantitatively evaluate the risk of off-target mutations. This study demonstrates that the use of non-homologous end-joining-deficient fungal strains drastically reduces the number of unintended genomic mutations, ensuring that CRISPR/Cas9 can be safely applied for strain development.},
}
@article {pmid36931732,
year = {2023},
author = {Sun, R and Raban, R and Akbari, OS},
title = {CRISPR-Cas9 Methods and Key Considerations in the Production of Aedes aegypti Mutant Strains.},
journal = {Cold Spring Harbor protocols},
volume = {2023},
number = {9},
pages = {pdb.top107693},
doi = {10.1101/pdb.top107693},
pmid = {36931732},
issn = {1559-6095},
mesh = {Animals ; Humans ; CRISPR-Cas Systems ; *Aedes/genetics ; *Yellow Fever/genetics/prevention &amp; control ; Gene Editing/methods ; Animals, Genetically Modified ; },
abstract = {Since the characterization of the CRISPR-Cas9 system in prokaryotes, it has become the prime choice in gene editing because of its exceptional flexibility, ease of use, high efficiency, and superior specificity. As a result, CRISPR-Cas9-mediated gene-editing technologies have enabled researchers not only to engineer transgenic animal strains with site-directed insertions more efficiently but also to generate desired mutants for previously intractable species. One such species is the invasive yellow fever mosquito, Aedes aegypti, which is notorious for its ability to transmit many blood-borne human pathogens. Methods for developing new transgenic strains of the yellow fever mosquito may aid in the effort to control its populations and provide significant benefits for the public. Here, we provide an overview of injection and noninjection methods for generating transgenic mosquitoes and also highlight important experimental design features.},
}
@article {pmid35736243,
year = {2023},
author = {Geisert, RD and Johns, DN and Pfeiffer, CA and Sullivan, RM and Lucas, CG and Simintiras, CA and Redel, BK and Wells, KD and Spencer, TE and Prather, RS},
title = {Gene editing provides a tool to investigate genes involved in reproduction of pigs.},
journal = {Molecular reproduction and development},
volume = {90},
number = {7},
pages = {459-468},
doi = {10.1002/mrd.23620},
pmid = {35736243},
issn = {1098-2795},
support = {2017-67015-26451//National Institute of Food and Agriculture/ ; },
mesh = {Pregnancy ; Female ; Animals ; Swine/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Reproduction/genetics ; Endometrium/metabolism ; },
abstract = {CRISPR-Cas9 gene editing technology provides a method to generate loss-of-function studies to investigate, in vivo, the specific role of specific genes in regulation of reproduction. With proper design and selection of guide RNAs (gRNA) designed to specifically target genes, CRISPR-Cas9 gene editing allows investigation of factors proposed to regulate biological pathways involved with establishment and maintenance of pregnancy. The advantages and disadvantages of using the current gene editing technology in a large farm species is discussed. CRISPR-Cas9 gene editing of porcine conceptuses has generated new perspectives for the regulation of endometrial function during the establishment of pregnancy. The delicate orchestration of conceptus factors facilitates an endometrial proinflammatory response while regulating maternal immune cell migration and expansion at the implantation site is essential for establishment and maintenance of pregnancy. Recent developments and use of endometrial epithelial "organoids" to study endometrial function in vitro provides a future method to screen and target specific endometrial genes as an alternative to generating a gene edited animal model. With continuing improvements in gene editing technology, future researchers will be able to design studies to enhance our knowledge of mechanisms essential for early development and survival of the conceptus.},
}
@article {pmid37642232,
year = {2023},
author = {Li, J and Tang, C and Liang, G and Tian, H and Lai, G and Wu, Y and Liu, S and Zhang, W and Liu, S and Shao, H},
title = {Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 System: Factors Affecting Precision Gene Editing Efficiency and Optimization Strategies.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2023.115},
pmid = {37642232},
issn = {1557-7422},
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)- CRISPR-associated (Cas) system is a powerful genomic DNA editing tool. The increased applications of gene editing tools, including the CRISPR-Cas system, have contributed to recent advances in biological fields, such as genetic disease therapy, disease-associated gene screening and detection, and cancer therapy. However, the major limiting factor for the wide application of gene editing tools is gene editing efficiency. This review summarizes the recent advances in factors affecting the gene editing efficiency of the CRISPR-Cas9 system and the CRISPR-Cas9 system optimization strategies. The homology-directed repair efficiency-related signal pathways and the form and delivery method of the CRISPR-Cas9 system are the major factors that influence the repair efficiency of gene editing tools. Based on these influencing factors, several strategies have been developed to improve the repair efficiency of gene editing tools. This review provided novel insights for improving the repair efficiency of the CRISPR-Cas9 gene editing system, which may enable the development and improvement of gene editing tools.},
}
@article {pmid37641539,
year = {2023},
author = {Li, Y and Huang, B and Chen, J and Huang, L and Xu, J and Wang, Y and Cui, G and Zhao, H and Xin, B and Song, W and Zhu, JK and Lai, J},
title = {Targeted large fragment deletion in plants using paired crRNAs with type I CRISPR system.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14122},
pmid = {37641539},
issn = {1467-7652},
support = {B21HJ0509//the Hainan Yazhou Bay Seed Lab/ ; SYND-2022-03//the Yazhou Bay Science and Technology City Administration/ ; SYND-2022-06//the Yazhou Bay Science and Technology City Administration/ ; },
abstract = {The CRISPR-Cas systems have been widely used as genome editing tools, with type II and V systems typically introducing small indels, and type I system mediating long-range deletions. However, the precision of type I systems for large fragment deletion is still remained to be optimized. Here, we developed a compact Cascade-Cas3 Dvu I-C system with Cas11c for plant genome editing. The Dvu I-C system was efficient to introduce controllable large fragment deletion up to at least 20 kb using paired crRNAs. The paired-crRNAs design also improved the controllability of deletions for the type I-E system. Dvu I-C system was sensitive to spacer length and mismatch, which was benefit for target specificity. In addition, we showed that the Dvu I-C system was efficient for generating stable transgenic lines in maize and rice with the editing efficiency up to 86.67%. Overall, Dvu I-C system we developed here is powerful for achieving controllable large fragment deletions.},
}
@article {pmid37641170,
year = {2023},
author = {Chen, K and Wang, Y},
title = {CRISPR/Cas systems for in situ imaging of intracellular nucleic acids: Concepts and applications.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.28543},
pmid = {37641170},
issn = {1097-0290},
support = {//National Natural Science Foundation of China/ ; //Beijing Hospitals Authority Youth Programme/ ; 82002198//National Science Foundation of China/ ; },
abstract = {Accurate and precise localization of intracellular nucleic acids is crucial for regulating genetic information transcription and diagnosing diseases. Although intracellular nucleic acid imaging methods are available for various cell types, their widespread utilization is impeded by the intricate nature of the process and its exorbitant cost. Recently, numerous intracellular nucleic acid labeling techniques based on clustered regularly interspaced short palindromic repeats (CRISPR) have been established due to their modularity, flexibility, and specificity. In this work, we present various CRISPR methods that are currently employed for visualizing intracellular genomic sequences and RNA, based on their detection principles and application scenarios. Furthermore, we discuss the advantages and drawbacks of the existing CRISPR imaging methods, as well as future research directions. We anticipate that with continued refinement, more advanced CRISPR-based imaging techniques can be developed to better elucidate the localization and dynamics of intracellular nucleic acids, thereby providing a powerful tool for molecular biology research and clinical molecular pathology diagnosis.},
}
@article {pmid37637458,
year = {2023},
author = {Liu, S and Huang, S and Li, F and Sun, Y and Fu, J and Xiao, F and Jia, N and Huang, X and Sun, C and Zhou, J and Wang, Y and Qu, D},
title = {Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system.},
journal = {Frontiers in cellular and infection microbiology},
volume = {13},
number = {},
pages = {1239269},
pmid = {37637458},
issn = {2235-2988},
mesh = {*Recombinases ; *Pseudomonas aeruginosa/genetics ; CRISPR-Cas Systems ; Nucleotidyltransferases ; Hospitals ; },
abstract = {Pseudomonas aeruginosa (P. aeruginosa) is an important bacterial pathogen involved in a wide range of infections and antimicrobial resistance. Rapid and reliable diagnostic methods are of vital important for early identification, treatment, and stop of P. aeruginosa infections. In this study, we developed a simple, rapid, sensitive, and specific detection platform for P. aeruginosa infection diagnosis. The method integrated recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 12a (Cas12a) biosensing system and was termed P. aeruginosa-CRISPR-RPA assay. The P. aeruginosa-CRISPR-RPA assay was subject to optimization of reaction conditions and evaluation of sensitivity, specificity, and clinical feasibility with the serial dilutions of P. aeruginosa genomic DNA, the non-P. aeruginosa strains, and the clinical samples. As a result, the P. aeruginosa-CRISPR-RPA assay was able to complete P. aeruginosa detection within half an hour, including RPA reaction at 42°C for 20 min and CRISPR-Cas12a detection at 37°C for 10 min. The diagnostic method exhibited high sensitivity (60 fg per reaction, ~8 copies) and specificity (100%). The results of the clinical samples by P. aeruginosa-CRISPR-RPA assay were consistent to that of the initial result by microfluidic chip method. These data demonstrated that the newly developed P. aeruginosa-CRISPR-RPA assay was reliable for P. aeruginosa detection. In summary, the P. aeruginosa-CRISPR-RPA assay is a promising tool to early and rapid diagnose P. aeruginosa infection and stop its wide spread especially in the hospital settings.},
}
@article {pmid37637117,
year = {2023},
author = {Dong, Y and Huang, Y and Fan, H and Song, L and An, X and Xu, S and Li, M and Tong, Y},
title = {Characterization, complete genome sequencing, and CRISPR/Cas9 system-based decontamination of a novel Escherichia coli phage TR1 from fermentation substrates.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1230775},
pmid = {37637117},
issn = {1664-302X},
abstract = {Phage contamination has become a major concern for industrial bacteria, such as Escherichia coli BL21(DE3), used in fermentation processes. Herein, we report a CRISPR/Cas9 defense system-based strategy to precisely prey and degrade phage DNA to decontaminate target phages. First, we isolated a novel phage from fermentation substrates with BL21(DE3) as the host, named TR1. It showed a typical podovirus morphology with a head diameter of 51.46 ± 2.04 nm and a tail length of 9.31 ± 2.77 nm. The burst size of phage TR1 was 151 PFU/cell, suggesting its strong fecundity in the fermentation system. Additionally, whole-genome sequencing revealed that phage TR1 has a DNA genome of 44,099 bp in length with a 43.8% GC content, encoding a total of 68 open reading frames. Comparative genomics and phylogenetic analysis designated this phage to be a new species of the genus Christensenvirus. To counteract phage TR1, we employed the CRISPR/Cas9 system-based strategy and constructed two phage-resistant E. coli strains, BL21-C and BL21-T, based on conserved genes. Both EOP assays and growth curves indicated strong phage resistance of the recombinant strains, without affecting cell growth. Therefore, this study aimed to provide a resilient strategy to respond to ever-changing phages and ongoing phage-host arm race in industrial fermentation environments by the personalized design of spacers in the recombinant CRISPR/Cas system-containing plasmid. More importantly, our research sparks the use of phage defense mechanism to prevent phage contamination in extensive biotechnological applications.},
}
@article {pmid37315207,
year = {2023},
author = {Sretenovic, S and Green, Y and Wu, Y and Cheng, Y and Zhang, T and Van Eck, J and Qi, Y},
title = {Genome- and transcriptome-wide off-target analyses of a high-efficiency adenine base editor in tomato.},
journal = {Plant physiology},
volume = {193},
number = {1},
pages = {291-303},
doi = {10.1093/plphys/kiad347},
pmid = {37315207},
issn = {1532-2548},
support = {2018-33522-28789//U.S. Department of Agriculture Biotechnology Risk Assessment Grant Program/ ; IOS-2029889//National Science Foundation Plant Genome Research Program/ ; //Foundation for Food and Agriculture Research Fellow/ ; },
mesh = {*Solanum lycopersicum/genetics ; Transcriptome/genetics ; Adenine/metabolism ; Mutation/genetics ; Gene Editing ; RNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)-expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)-dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA-level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato.},
}
@article {pmid37294917,
year = {2023},
author = {Vavassori, V and Ferrari, S and Beretta, S and Asperti, C and Albano, L and Annoni, A and Gaddoni, C and Varesi, A and Soldi, M and Cuomo, A and Bonaldi, T and Radrizzani, M and Merelli, I and Naldini, L},
title = {Lipid nanoparticles allow efficient and harmless ex vivo gene editing of human hematopoietic cells.},
journal = {Blood},
volume = {142},
number = {9},
pages = {812-826},
doi = {10.1182/blood.2022019333},
pmid = {37294917},
issn = {1528-0020},
mesh = {Humans ; *Gene Editing/methods ; *Tumor Suppressor Protein p53/genetics/metabolism ; Hematopoietic Stem Cells/metabolism ; RNA/metabolism ; CRISPR-Cas Systems ; },
abstract = {Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses the delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo via electroporation, and when aiming for homology-driven correction of a DNA template, often provided by viral vectors together with a nuclease editor. Although HSPCs activate a robust p53-dependent DNA damage response upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multiomics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism, and inducing an inflammatory response. Nuclease RNA delivery using lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding a higher number of edited cells compared with using electroporation. Transient transcriptomic changes upon LNP treatment were mostly caused by cellular loading with exogenous cholesterol, whose potentially detrimental impact could be overcome by limiting exposure. Notably, LNP-based HSPC editing dampened p53 pathway induction and supported higher clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared with electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for the treatment of human diseases.},
}
@article {pmid37636103,
year = {2023},
author = {Ashraf, S and Ahmad, A and Khan, SH and Jamil, A and Sadia, B and Brown, JK},
title = {LbCas12a mediated suppression of Cotton leaf curl Multan virus.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1233295},
pmid = {37636103},
issn = {1664-462X},
abstract = {Begomoviruses are contagious and severely affect commercially important fiber and food crops. Cotton leaf curl Multan virus (CLCuMuV) is one of the most dominant specie of Begomovirus and a major constraint on cotton yield in Pakistan. Currently, the field of plant genome editing is being revolutionized by the CRISPR/Cas system applications such as base editing, prime editing and CRISPR based gene drives. CRISPR/Cas9 system has successfully been used against biotic and abiotic plant stresses with proof-of-concept studies in both model and crop plants. CRISPR/Cas12 and CRISPR/Cas13 have recently been applied in plant sciences for basic and applied research. In this study, we used a novel approach, multiplexed crRNA-based Cas12a toolbox to target the different ORFs of the CLCuMuV genome at multiple sites simultaneously. This method successfully eliminated the symptoms of CLCuMuV in Nicotiana benthamiana and Nicotiana tabacum. Three individual crRNAs were designed from the CLCuMuV genome, targeting the specific sites of four different ORFs (C1, V1 and overlapping region of C2 and C3). The Cas12a-based construct Cas12a-MV was designed through Golden Gate three-way cloning for precise editing of CLCuMuV genome. Cas12a-MV construct was confirmed through whole genome sequencing using the primers Ubi-intron-F1 and M13-R1. Transient assays were performed in 4 weeks old Nicotiana benthamiana plants, through the agroinfiltration method. Sanger sequencing indicated that the Cas12a-MV constructs made a considerable mutations at the target sites of the viral genome. In addition, TIDE analysis of Sanger sequencing results showed the editing efficiency of crRNA1 (21.7%), crRNA2 (24.9%) and crRNA3 (55.6%). Furthermore, the Cas12a-MV construct was stably transformed into Nicotiana tabacum through the leaf disc method to evaluate the potential of transgenic plants against CLCuMuV. For transgene analysis, the DNA of transgenic plants of Nicotiana tabacum was subjected to PCR to amplify Cas12a genes with specific primers. Infectious clones were agro-inoculated in transgenic and non-transgenic plants (control) for the infectivity assay. The transgenic plants containing Cas12a-MV showed rare symptoms and remained healthy compared to control plants with severe symptoms. The transgenic plants containing Cas12a-MV showed a significant reduction in virus accumulation (0.05) as compared to control plants (1.0). The results demonstrated the potential use of the multiplex LbCas12a system to develop virus resistance in model and crop plants against begomoviruses.},
}
@article {pmid37636063,
year = {2023},
author = {Wei, XY and Liu, L and Hu, H and Jia, HJ and Bu, LK and Pei, DS},
title = {Ultra-sensitive detection of ecologically rare fish from eDNA samples based on the RPA-CRISPR/Cas12a technology.},
journal = {iScience},
volume = {26},
number = {9},
pages = {107519},
pmid = {37636063},
issn = {2589-0042},
abstract = {Environmental DNA (eDNA) research holds great promise for improving biodiversity science and conservation efforts by enabling worldwide species censuses in near real-time. Current eDNA methods face challenges in detecting low-abundance ecologically important species. In this study, we used isothermal recombinase polymerase amplification (RPA)-CRISPR/Cas detection to test Ctenopharyngodon idella. RPA-CRISPR-Cas12a detected 6.0 eDNA copies/μL within 35 min. Ecologically rare species were identified in the Three Gorges Reservoir Area (TGRA) using functional distinctiveness and geographical restrictiveness, with seven fish species (9%) classified as potentially ecologically rare including three species in this investigation. RPA-CRISPR/Cas12a-FQ outperformed high-throughput sequencing (HTS) and qPCR in detecting low-abundance eDNA (AUC = 0.883∗∗). A significant linear correlation (R[2] = 0.682∗∗) between RPA-CRISPR/Cas12a-FQ and HTS quantification suggests its potential for predicting species abundance and enhancing eDNA-based fish biodiversity monitoring. This study highlights the value of RPA-CRISPR/Cas12a-FQ as a tool for advancing eDNA research and conservation efforts.},
}
@article {pmid37580593,
year = {2023},
author = {Reysenbach, AL and Terns, MP},
title = {CRISPR-influenced symbiosis.},
journal = {Nature microbiology},
volume = {8},
number = {9},
pages = {1611-1612},
pmid = {37580593},
issn = {2058-5276},
mesh = {*Symbiosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
}
@article {pmid37491642,
year = {2023},
author = {Kiani, L},
title = {New CRISPR-based strategies for Alzheimer disease.},
journal = {Nature reviews. Neurology},
volume = {19},
number = {9},
pages = {507},
doi = {10.1038/s41582-023-00856-5},
pmid = {37491642},
issn = {1759-4766},
mesh = {Humans ; *Alzheimer Disease/genetics/therapy ; Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37343700,
year = {2023},
author = {Song, Z and Zhang, G and Huang, S and Liu, Y and Li, G and Zhou, X and Sun, J and Gao, P and Chen, Y and Huang, X and Liu, J and Wang, X},
title = {PE-STOP: A versatile tool for installing nonsense substitutions amenable for precise reversion.},
journal = {The Journal of biological chemistry},
volume = {299},
number = {8},
pages = {104942},
pmid = {37343700},
issn = {1083-351X},
mesh = {Animals ; Humans ; *Gene Editing/methods ; *Codon, Nonsense ; Gene Silencing ; Codon, Terminator/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {The rapid advances in genome editing technologies have revolutionized the study of gene functions in cell or animal models. The recent generation of double-stranded DNA cleavage-independent base editors has been suitably adapted for interrogation of protein-coding genes on the basis of introducing premature stop codons or disabling the start codons. However, such versions of stop/start codon-oriented genetic tools still present limitations on their versatility, base-level precision, and target specificity. Here, we exploit a newly developed prime editor (PE) that differs from base editors by its adoption of a reverse transcriptase activity, which enables incorporation of various types of precise edits templated by a specialized prime editing guide RNA. Based on such a versatile platform, we established a prime editing-empowered method (PE-STOP) for installation of nonsense substitutions, providing a complementary approach to the present gene-targeting tools. PE-STOP is bioinformatically predicted to feature substantially expanded coverage in the genome space. In practice, PE-STOP introduces stop codons with good efficiencies in human embryonic kidney 293T and N2a cells (with medians of 29% [ten sites] and 25% [four sites] editing efficiencies, respectively), while exhibiting minimal off-target effects and high on-target precision. Furthermore, given the fact that PE installs prime editing guide RNA-templated mutations, we introduce a unique strategy for precise genetic rescue of PE-STOP-dependent nonsense mutation via the same PE platform. Altogether, the present work demonstrates a versatile and specific tool for gene inactivation and for functional interrogation of nonsense mutations.},
}
@article {pmid37186122,
year = {2023},
author = {Guo, Z and Hu, YH and Feng, GS and Valenzuela Ripoll, C and Li, ZZ and Cai, SD and Wang, QQ and Luo, WW and Li, Q and Liang, LY and Wu, ZK and Zhang, JG and Javaheri, A and Wang, L and Lu, J and Liu, PQ},
title = {JMJD6 protects against isoproterenol-induced cardiac hypertrophy via inhibition of NF-κB activation by demethylating R149 of the p65 subunit.},
journal = {Acta pharmacologica Sinica},
volume = {44},
number = {9},
pages = {1777-1789},
pmid = {37186122},
issn = {1745-7254},
mesh = {Rats ; Animals ; *NF-kappa B/metabolism ; Isoproterenol/toxicity ; *Heart Failure/metabolism ; Histones/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Rats, Sprague-Dawley ; Stroke Volume ; Cardiomegaly/chemically induced/prevention &amp; control/drug therapy ; Myocytes, Cardiac/metabolism ; },
abstract = {Histone modification plays an important role in pathological cardiac hypertrophy and heart failure. In this study we investigated the role of a histone arginine demethylase, Jumonji C domain-containing protein 6 (JMJD6) in pathological cardiac hypertrophy. Cardiac hypertrophy was induced in rats by subcutaneous injection of isoproterenol (ISO, 1.2 mg·kg[-1]·d[-1]) for a week. At the end of the experiment, the rats underwent echocardiography, followed by euthanasia and heart collection. We found that JMJD6 levels were compensatorily increased in ISO-induced hypertrophic cardiac tissues, but reduced in patients with heart failure with reduced ejection fraction (HFrEF). Furthermore, we demonstrated that JMJD6 overexpression significantly attenuated ISO-induced hypertrophy in neonatal rat cardiomyocytes (NRCMs) evidenced by the decreased cardiomyocyte surface area and hypertrophic genes expression. Cardiac-specific JMJD6 overexpression in rats protected the hearts against ISO-induced cardiac hypertrophy and fibrosis, and rescued cardiac function. Conversely, depletion of JMJD6 by single-guide RNA (sgRNA) exacerbated ISO-induced hypertrophic responses in NRCMs. We revealed that JMJD6 interacted with NF-κB p65 in cytoplasm and reduced nuclear levels of p65 under hypertrophic stimulation in vivo and in vitro. Mechanistically, JMJD6 bound to p65 and demethylated p65 at the R149 residue to inhibit the nuclear translocation of p65, thus inactivating NF-κB signaling and protecting against pathological cardiac hypertrophy. In addition, we found that JMJD6 demethylated histone H3R8, which might be a new histone substrate of JMJD6. These results suggest that JMJD6 may be a potential target for therapeutic interventions in cardiac hypertrophy and heart failure.},
}
@article {pmid37636024,
year = {2023},
author = {Nouri Nojadeh, J and Bildiren Eryilmaz, NS and Ergüder, BI},
title = {CRISPR/Cas9 genome editing for neurodegenerative diseases.},
journal = {EXCLI journal},
volume = {22},
number = {},
pages = {567-582},
pmid = {37636024},
issn = {1611-2156},
abstract = {Gene therapy has emerged as a promising therapeutic strategy for various conditions, including blood disorders, ocular disease, cancer, and nervous system disorders. The advent of gene editing techniques has facilitated the ability of researchers to specifically target and modify the eukaryotic cell genome, making it a valuable tool for gene therapy. This can be performed through either in vivo or ex vivo approaches. Gene editing tools, such as zinc finger nucleases, transcription activator-like effector nucleases, and CRISPR-Cas-associated nucleases, can be employed for gene therapy purposes. Among these tools, CRISPR-Cas-based gene editing stands out because of its ability to introduce heritable genome changes by designing short guide RNAs. This review aims to provide an overview of CRISPR-Cas technology and summarizes the latest research on the application of CRISPR/Cas9 genome editing technology for the treatment of the most prevalent neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, and Spinocerebellar ataxia.},
}
@article {pmid37633620,
year = {2023},
author = {Liu, Z and Liu, J and Yang, Z and Zhu, L and Zhu, Z and Huang, H and Jiang, L},
title = {Endogenous CRISPR-Cas mediated in situ genome editing: State-of-the-art and the road ahead for engineering prokaryotes.},
journal = {Biotechnology advances},
volume = {68},
number = {},
pages = {108241},
doi = {10.1016/j.biotechadv.2023.108241},
pmid = {37633620},
issn = {1873-1899},
abstract = {The CRISPR-Cas systems have shown tremendous promise as heterologous tools for genome editing in various prokaryotes. However, the perturbation of DNA homeostasis and the inherent toxicity of Cas9/12a proteins could easily lead to cell death, which led to the development of endogenous CRISPR-Cas systems. Programming the widespread endogenous CRISPR-Cas systems for in situ genome editing represents a promising tool in prokaryotes, especially in genetically intractable species. Here, this review briefly summarizes the advances of endogenous CRISPR-Cas-mediated genome editing, covering aspects of establishing and optimizing the genetic tools. In particular, this review presents the application of different types of endogenous CRISPR-Cas tools for strain engineering, including genome editing and genetic regulation. Notably, this review also provides a detailed discussion of the transposon-associated CRISPR-Cas systems, and the programmable RNA-guided transposition using endogenous CRISPR-Cas systems to enable editing of microbial communities for understanding and control. Therefore, they will be a powerful tool for targeted genetic manipulation. Overall, this review will not only facilitate the development of standard genetic manipulation tools for non-model prokaryotes but will also enable more non-model prokaryotes to be genetically tractable.},
}
@article {pmid37633433,
year = {2023},
author = {Zhao, F and Ding, X and Liu, Z and Yan, X and Chen, Y and Jiang, Y and Chen, S and Wang, Y and Kang, T and Xie, C and He, M and Zheng, J},
title = {Application of CRISPR/Cas9-based genome editing in ecotoxicology.},
journal = {Environmental pollution (Barking, Essex : 1987)},
volume = {336},
number = {},
pages = {122458},
doi = {10.1016/j.envpol.2023.122458},
pmid = {37633433},
issn = {1873-6424},
abstract = {Chemicals are widely used and released into the environment, and their degradation, accumulation, migration, and transformation processes in the environment can pose a threat to the ecosystem. The advancement in analytical methods with high-throughput screening of biomolecules has revolutionized the way toxicologists used to explore the effects of chemicals on organisms. CRISPR/Cas is a newly developed tool, widely used in the exploration of basic science and biologically engineered products given its high efficiency and low cost. For example, it can edit target genes efficiently, and save loss of the crop yield caused by environmental pollution as well as gain a better understanding of the toxicity mechanisms from various chemicals. This review briefly introduces the development history of CRISPR/Cas and summarizes the current application of CRISPR/Cas in ecotoxicology, including its application on improving crop yield and drug resistance towards agricultural pollution, antibiotic pollution and other threats. The benefits by applying the CRISPR/Cas9 system in conventional toxicity mechanism studies are fully demonstrated here together with its foreseeable expansions in other area of ecotoxicology. Finally, the prospects and disadvantages of CRISPR/Cas system in the field of ecotoxicology are also discussed.},
}
@article {pmid37631249,
year = {2023},
author = {Titze-de-Almeida, SS and Titze-de-Almeida, R},
title = {Progress in circRNA-Targeted Therapy in Experimental Parkinson's Disease.},
journal = {Pharmaceutics},
volume = {15},
number = {8},
pages = {},
pmid = {37631249},
issn = {1999-4923},
abstract = {Circular RNAs (circRNAs) are single-stranded RNA molecules often circularized by backsplicing. Growing evidence implicates circRNAs in the underlying mechanisms of various diseases, such as Alzheimer's and Parkinson's disease (PD)-the first and second most prevalent neurodegenerative disorders. In this sense, circSNCA, circHIPK2, circHIPK3, and circSLC8A1 are circRNAs that have been related to the neurodegenerative process of PD. Gain-of-function and loss-of-function studies on circRNAs have shed light on their roles in the pathobiology of various diseases. Gain-of-function approaches typically employ viral or non-viral vectors that hyperexpress RNA sequences capable of circularizing to form the specific circRNA under investigation. In contrast, loss-of-function studies utilize CRISPR/Cas systems, antisense oligonucleotides (ASOs), or RNAi techniques to knock down the target circRNA. The role of aberrantly expressed circRNAs in brain pathology has raised a critical question: could circRNAs serve as viable targets for neuroprotective treatments? Translating any oligonucleotide-based therapy, including those targeting circRNAs, involves developing adequate brain delivery systems, minimizing off-target effects, and addressing the high costs of treatment. Nonetheless, RNAi-based FDA-approved drugs have entered the market, and circRNAs have attracted significant attention and investment from major pharmaceutical companies. Spanning from bench to bedside, circRNAs present a vast opportunity in biotechnology for oligonucleotide-based therapies designed to slow or even halt the progression of neurodegenerative diseases.},
}
@article {pmid37630620,
year = {2023},
author = {Yadalam, PK and Arumuganainar, D and Anegundi, RV and Shrivastava, D and Alftaikhah, SAA and Almutairi, HA and Alobaida, MA and Alkaberi, AA and Srivastava, KC},
title = {CRISPR-Cas-Based Adaptive Immunity Mediates Phage Resistance in Periodontal Red Complex Pathogens.},
journal = {Microorganisms},
volume = {11},
number = {8},
pages = {},
pmid = {37630620},
issn = {2076-2607},
abstract = {Periodontal diseases are polymicrobial immune-inflammatory diseases that can severely destroy tooth-supporting structures. The critical bacteria responsible for this destruction include red complex bacteria such as Porphoromonas gingivalis, Tanerella forsythia and Treponema denticola. These organisms have developed adaptive immune mechanisms against bacteriophages/viruses, plasmids and transposons through clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas). The CRISPR-Cas system contributes to adaptive immunity, and this acquired genetic immune system of bacteria may contribute to moderating the microbiome of chronic periodontitis. The current research examined the role of the CRISPR-Cas system of red complex bacteria in the dysbiosis of oral bacteriophages in periodontitis. Whole-genome sequences of red complex bacteria were obtained and investigated for CRISPR using the CRISPR identification tool. Repeated spacer sequences were analyzed for homologous sequences in the bacteriophage genome and viromes using BLAST algorithms. The results of the BLAST spacer analysis for T. denticola spacers had a 100% score (e value with a bacillus phage), and the results for T. forsthyia and P. gingivalis had a 56% score with a pectophage and cellulophage (e value: 0.21), respectively. The machine learning model of the identified red complex CRISPR sequences predicts with area an under the curve (AUC) accuracy of 100 percent, indicating phage inhibition. These results infer that red complex bacteria could significantly inhibit viruses and phages with CRISPR immune sequences. Therefore, the role of viruses and bacteriophages in modulating sub-gingival bacterial growth in periodontitis is limited or questionable.},
}
@article {pmid37630508,
year = {2023},
author = {Alkompoz, AK and Hamed, SM and Zaid, ASA and Almangour, TA and Al-Agamy, MH and Aboshanab, KM},
title = {Correlation of CRISPR/Cas and Antimicrobial Resistance in Klebsiella pneumoniae Clinical Isolates Recovered from Patients in Egypt Compared to Global Strains.},
journal = {Microorganisms},
volume = {11},
number = {8},
pages = {},
pmid = {37630508},
issn = {2076-2607},
support = {RGP-038//Deanship of Scientific Research at King Saud University/ ; },
abstract = {The CRISPR/Cas system has been long known to interfere with the acquisition of foreign genetic elements and was recommended as a tool for fighting antimicrobial resistance. The current study aimed to explore the prevalence of the CRISPR/Cas system in Klebsiella pneumoniae isolates recovered from patients in Egypt in comparison to global strains and correlate the CRISPR/Cas to susceptibility to antimicrobial agents. A total of 181 clinical isolates were PCR-screened for cas and selected antimicrobial resistance genes (ARGs). In parallel, 888 complete genome sequences were retrieved from the NCBI database for in silico analysis. CRISPR/Cas was found in 46 (25.4%) isolates, comprising 18.8% type I-E and 6.6% type I-E*. Multidrug resistance (MDR) and extensive drug resistance (XDR) were found in 73.5% and 25.4% of the isolates, respectively. More than 95% of the CRISPR/Cas-bearing isolates were MDR (65.2%) or XDR (32.6%). No significant difference was found in the susceptibility to the tested antimicrobial agents among the CRISPR/Cas-positive and -negative isolates. The same finding was obtained for the majority of the screened ARGs. Among the published genomes, 23.2% carried CRISPR/Cas, with a higher share of I-E* (12.8%). They were confined to specific sequence types (STs), most commonly ST147, ST23, ST15, and ST14. More plasmids and ARGs were carried by the CRISPR/Cas-negative group than others, but their distribution in the two groups was not significantly different. The prevalence of some ARGs, such as blaKPC, blaTEM, and rmtB, was significantly higher among the genomes of the CRISPR/Cas-negative strains. A weak, nonsignificant positive correlation was found between the number of spacers and the number of resistance plasmids and ARGs. In conclusion, the correlation between CRISPR/Cas and susceptibility to antimicrobial agents or bearing resistance plasmids and ARGs was found to be nonsignificant. Plasmid-targeting spacers might not be naturally captured by CRISPR/Cas. Spacer match analysis is recommended to provide a clearer image of the exact behavior of CRISPR/Cas towards resistance plasmids.},
}
@article {pmid37629024,
year = {2023},
author = {Yin, X and Harmancey, R and McPherson, DD and Kim, H and Huang, SL},
title = {Liposome-Based Carriers for CRISPR Genome Editing.},
journal = {International journal of molecular sciences},
volume = {24},
number = {16},
pages = {},
pmid = {37629024},
issn = {1422-0067},
support = {HL135092, McPherson/NH/NIH HHS/United States ; NS098918, Booker and Huang/NH/NIH HHS/United States ; },
mesh = {*Gene Editing ; *Liposomes ; RNA, Guide, CRISPR-Cas Systems ; Biotechnology ; Cues ; },
abstract = {The CRISPR-based genome editing technology, known as clustered regularly interspaced short palindromic repeats (CRISPR), has sparked renewed interest in gene therapy. This interest is accompanied by the development of single-guide RNAs (sgRNAs), which enable the introduction of desired genetic modifications at the targeted site when used alongside the CRISPR components. However, the efficient delivery of CRISPR/Cas remains a challenge. Successful gene editing relies on the development of a delivery strategy that can effectively deliver the CRISPR cargo to the target site. To overcome this obstacle, researchers have extensively explored non-viral, viral, and physical methods for targeted delivery of CRISPR/Cas9 and a guide RNA (gRNA) into cells and tissues. Among those methods, liposomes offer a promising approach to enhance the delivery of CRISPR/Cas and gRNA. Liposomes facilitate endosomal escape and leverage various stimuli such as light, pH, ultrasound, and environmental cues to provide both spatial and temporal control of cargo release. Thus, the combination of the CRISPR-based system with liposome delivery technology enables precise and efficient genetic modifications in cells and tissues. This approach has numerous applications in basic research, biotechnology, and therapeutic interventions. For instance, it can be employed to correct genetic mutations associated with inherited diseases and other disorders or to modify immune cells to enhance their disease-fighting capabilities. In summary, liposome-based CRISPR genome editing provides a valuable tool for achieving precise and efficient genetic modifications. This review discusses future directions and opportunities to further advance this rapidly evolving field.},
}
@article {pmid37432544,
year = {2023},
author = {Banerjee, S and Mukherjee, A and Kundu, A},
title = {The current scenario and future perspectives of transgenic oilseed mustard by CRISPR-Cas9.},
journal = {Molecular biology reports},
volume = {50},
number = {9},
pages = {7705-7728},
pmid = {37432544},
issn = {1573-4978},
support = {TAR/2021/000233//Department of Science and Technology, Government of West Bengal/ ; },
mesh = {*Mustard Plant/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Genome, Plant ; *Brassica napus/genetics ; },
abstract = {PURPOSE: Production of a designer crop having added attributes is the primary goal of all plant biotechnologists. Specifically, development of a crop with a simple biotechnological approach and at a rapid pace is most desirable. Genetic engineering enables us to displace genes among species. The newly incorporated foreign gene(s) in the host genome can create a new trait(s) by regulating the genotypes and/or phenotypes. The advent of the CRISPR-Cas9 tools has enabled the modification of a plant genome easily by introducing mutation or replacing genomic fragment. Oilseed mustard varieties (e.g., Brassica juncea, Brassica nigra, Brassica napus, and Brassica carinata) are one such plants, which have been transformed with different genes isolated from the wide range of species. Current reports proved that the yield and value of oilseed mustard has been tremendously improved by the introduction of stably inherited new traits such as insect and herbicide resistance. However, the genetic transformation of oilseed mustard remains incompetent due to lack of potential plant transformation systems. To solve numerous complications involved in genetically modified oilseed mustard crop varieties regeneration procedures, scientific research is being conducted to rectify the unwanted complications. Thus, this study provides a broader overview of the present status of new traits introduced in each mentioned varieties of oilseed mustard plant by different genetical engineering tools, especially CRISPR-Cas9, which will be useful to improve the transformation system of oilseed mustard crop plants.<br><br>METHODS: This review presents recent improvements made in oilseed mustard genetic engineering methodologies by using CRISPR-Cas9 tools, present status of new traits introduced in oilseed mustard plant varieties.<br><br>RESULTS: The review highlighted that the transgenic oilseed mustard production is a challenging process and the transgenic varieties of oilseed mustard provide a powerful tool for enhanced mustard yield. Over expression studies and silencing of desired genes provide functional importance of genes involved in mustard growth and development under different biotic and abiotic stress conditions. Thus, it can be expected that in near future CRISPR can contribute enormously in improving the mustard plant's architecture and develop stress resilient oilseed mustard plant species.},
}
@article {pmid37403571,
year = {2023},
author = {Donà, M and Bradamante, G and Bogojevic, Z and Gutzat, R and Streubel, S and Mosiolek, M and Dolan, L and Mittelsten Scheid, O},
title = {A versatile CRISPR-based system for lineage tracing in living plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {115},
number = {5},
pages = {1169-1184},
doi = {10.1111/tpj.16378},
pmid = {37403571},
issn = {1365-313X},
support = {WWTF WWTF LS13-057//Vienna Science and Technology Fund/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Mutation ; *Frameshift Mutation ; Phenotype ; Cell Lineage/genetics ; },
abstract = {Individual cells give rise to diverse cell lineages during the development of multicellular organisms. Understanding the contribution of these lineages to mature organisms is a central question of developmental biology. Several techniques to document cell lineages have been used, from marking single cells with mutations that express a visible marker to generating molecular bar codes by CRISPR-induced mutations and subsequent single-cell analysis. Here, we exploit the mutagenic activity of CRISPR to allow lineage tracing within living plants with a single reporter. Cas9-induced mutations are directed to correct a frameshift mutation that restores expression of a nuclear fluorescent protein, labelling the initial cell and all progenitor cells with a strong signal without modifying other phenotypes of the plants. Spatial and temporal control of Cas9 activity can be achieved using tissue-specific and/or inducible promoters. We provide proof of principle for the function of lineage tracing in two model plants. The conserved features of the components and the versatile cloning system, allowing for easy exchange of promoters, are expected to make the system widely applicable.},
}
@article {pmid36694315,
year = {2023},
author = {Liu, J and Shang, X and Yao, B and Zhang, Y and Huang, S and Guo, Y and Wang, X},
title = {Design and Construction of Carboxylesterase 2c Gene Knockout Rats by CRISPR/Cas9.},
journal = {Current drug metabolism},
volume = {24},
number = {3},
pages = {190-199},
doi = {10.2174/1389200224666230123140919},
pmid = {36694315},
issn = {1875-5453},
support = {011//East China Normal University/ ; 18430760400//Science and Technology Commission of Shanghai Municipality/ ; 2019CNECNUPI02//Shanghai Science and Technology Development Foundation/ ; 81773808//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {Rats ; Humans ; Animals ; Gene Knockout Techniques ; *CRISPR-Cas Systems ; *Diltiazem/metabolism ; Liver/metabolism ; Aspirin/metabolism ; },
abstract = {BACKGROUND: Carboxylesterase 2 (CES2) is mainly distributed in the human liver and gut, and plays an active role in the metabolic activation of many prodrugs and lipid metabolism. Although CES2 is of great significance, there are still few animal models related to CES2.<br><br>OBJECTIVES: This research aims to construct Ces2c gene knockout (KO) rats and further study the function of CES2.<br><br>METHODS: CRISPR/Cas9 gene editing technology was used to target and cleave the rat Ces2c gene. Compensatory effects of major CES subtypes both in the liver and small intestine of KO rats were detected at mRNA levels. Meanwhile, diltiazem and aspirin were used as substrates to test the metabolic capacity of Ces2c in KO rats.<br><br>RESULTS: This Ces2c KO rat model showed normal growth and breeding without off-target effects. The metabolic function of Ces2c KO rats was verified by the metabolic study of CES2 substrates in vitro. The results showed that the metabolic capacity of diltiazem in KO rats was weakened, while the metabolic ability of aspirin did not change significantly. In addition, the serum physiological indexes showed that the Ces2c deletion did not affect the liver function of rats..<br><br>CONCLUSION: The Ces2c KO rat model was successfully constructed by CRISPR/Cas9 system. This rat model can not only be used as an important tool to study the drug metabolism mediated by CES2, but also as an important animal model to study the physiological function of CES2.},
}
@article {pmid37628816,
year = {2023},
author = {Liesenhoff, C and Paulus, SM and Havertz, C and Geerlof, A and Priglinger, S and Priglinger, CS and Ohlmann, A},
title = {Endogenous Galectin-1 Modulates Cell Biological Properties of Immortalized Retinal Pigment Epithelial Cells In Vitro.},
journal = {International journal of molecular sciences},
volume = {24},
number = {16},
pages = {},
pmid = {37628816},
issn = {1422-0067},
support = {OH 214/4-3; PR 1248/2-3//Deutsche Forschungsgemeinschaft/ ; },
mesh = {Humans ; *Galectin 1/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Actins ; Epithelial Cells ; Retinal Pigments ; },
abstract = {In the eye, an increase in galectin-1 is associated with various chorioretinal diseases, in which retinal pigment epithelium (RPE) cells play a crucial role in disease development and progression. Since little is known about the function of endogenous galectin-1 in these cells, we developed a galectin-1-deficient immortalized RPE cell line (ARPE-19-LGALS1[-/-]) using a sgRNA/Cas9 all-in-one expression vector and investigated its cell biological properties. Galectin-1 deficiency was confirmed by Western blot analysis and immunocytochemistry. Cell viability and proliferation were significantly decreased in ARPE-19-LGALS1[-/-] cells when compared to wild-type controls. Further on, an increased attachment of galectin-1-deficient RPE cells was observed by cell adhesion assay when compared to control cells. The diminished viability and proliferation, as well as the enhanced adhesion of galectin-1-deficient ARPE-19 cells, could be blocked, at least in part, by the additional treatment with human recombinant galectin-1. In addition, a significantly reduced migration was detected in ARPE-19-LGALS1[-/-] cells. In comparison to control cells, galectin-1-deficient RPE cells had enhanced expression of sm-α-actin and N-cadherin, whereas expression of E-cadherin showed no significant alteration. Finally, a compensatory expression of galectin-8 mRNA was observed in ARPE-19-LGALS1[-/-] cells. In conclusion, in RPE cells, endogenous galectin-1 has crucial functions for various cell biological processes, including viability, proliferation, migration, adherence, and retaining the epithelial phenotype.},
}
@article {pmid37628594,
year = {2023},
author = {Sauvagère, S and Siatka, C},
title = {CRISPR-Cas: 'The Multipurpose Molecular Tool' for Gene Therapy and Diagnosis.},
journal = {Genes},
volume = {14},
number = {8},
pages = {},
pmid = {37628594},
issn = {2073-4425},
mesh = {*CRISPR-Cas Systems/genetics ; *Genetic Therapy ; DNA Damage ; Technology ; },
abstract = {Since the discovery of the CRISPR-Cas engineering system in 2012, several approaches for using this innovative molecular tool in therapeutic strategies and even diagnosis have been investigated. The use of this tool requires a global approach to DNA damage processes and repair systems in cells. The diversity in the functions of various Cas proteins allows for the use of this technology in clinical applications and trials. Wide variants of Cas12 and Cas13 are exploited using the collateral effect in many diagnostic applications. Even though this tool is well known, its use still raises real-world ethical and regulatory questions.},
}
@article {pmid37626297,
year = {2023},
author = {Jia, T and Yuan, F and Tao, J and Wang, G and Zhang, X and Zhang, B and Li, H},
title = {CRISPR/Cas13d targeting GZMA in PARs pathway regulates the function of osteoclasts in chronic apical periodontitis.},
journal = {Cellular &amp; molecular biology letters},
volume = {28},
number = {1},
pages = {70},
pmid = {37626297},
issn = {1689-1392},
support = {2022M723863//Project funded by China Postdoctoral Science Foundation/ ; 2017YFA0106200//National Key Research and Development Program of China/ ; },
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Osteoclasts ; Apoptosis/genetics ; RNA, Guide, CRISPR-Cas Systems ; Transforming Growth Factor beta ; *Periapical Periodontitis/genetics ; Granzymes ; },
abstract = {Chronic apical periodontitis is a prevalent oral disease characterized by bone loss, and its underlying mechanisms remain unclear. This study aimed to investigate the role and mechanism of the serine protease GZMA in osteoclasts during chronic apical periodontitis. To address this, we employed crRNA/Cas13d to inhibit GZMA expression and examined its impact on osteoclast behavior. Our findings revealed that GZMA plays a significant role in promoting osteoclast cell proliferation while inhibiting cell apoptosis. Additionally, the inhibition of GZMA led to a notable increase in miR-25-3p expression, which, in turn, downregulated the expression of TGF-β. Consequently, the reduction in TGF-β expression led to a decrease in PAR1 expression within the PARs pathway. These results suggest that GZMA might serve as a promising therapeutic target for the treatment of chronic apical periodontitis. Furthermore, our study highlights the potential of targeting GZMA using crRNA/Cas13d as a valuable approach for future therapeutic interventions.},
}
@article {pmid37626063,
year = {2023},
author = {Tsai, HH and Kao, HJ and Kuo, MW and Lin, CH and Chang, CM and Chen, YY and Chen, HH and Kwok, PY and Yu, AL and Yu, J},
title = {Whole genomic analysis reveals atypical non-homologous off-target large structural variants induced by CRISPR-Cas9-mediated genome editing.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {5183},
pmid = {37626063},
issn = {2041-1723},
support = {OMRPG3C0048//Chang Gung Medical Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; Genomics ; Cell Line ; },
abstract = {CRISPR-Cas9 genome editing has promising therapeutic potential for genetic diseases and cancers, but safety could be a concern. Here we use whole genomic analysis by 10x linked-read sequencing and optical genome mapping to interrogate the genome integrity after editing and in comparison to four parental cell lines. In addition to the previously reported large structural variants at on-target sites, we identify heretofore unexpected large chromosomal deletions (91.2 and 136 Kb) at atypical non-homologous off-target sites without sequence similarity to the sgRNA in two edited lines. The observed large structural variants induced by CRISPR-Cas9 editing in dividing cells may result in pathogenic consequences and thus limit the usefulness of the CRISPR-Cas9 editing system for disease modeling and gene therapy. In this work, our whole genomic analysis may provide a valuable strategy to ensure genome integrity after genomic editing to minimize the risk of unintended effects in research and clinical applications.},
}
@article {pmid37624748,
year = {2023},
author = {Lira, C and Cunha, DP and Vasconcelos, ZFM},
title = {Biotechnological advances in gene therapy of hematopoietic stem cells: Systematic review and meta-analysis.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2022.237},
pmid = {37624748},
issn = {1557-7422},
abstract = {Gene therapy (GT) has emerged as a promising treatment option for disorders in the hematopoietic system, particularly primary immunodeficiencies (PID). Hematopoietic stem cells (HSC) have gained attention due to their ability to support long-term hematopoiesis. In this study, we present a summary of research evaluating the most effective method of gene editing in HSC for translational medicine. We conducted a systematic literature search in various databases, including Cochrane, LILACs, Scielo, and Pubmed (MEDLINE), covering the period from January 1989 to June 10th, 2023. Our aim was to identify articles that assessed the efficiency of gene editing in HSC and clinical trials focusing on PID. Our research protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO; registration number CRD42022349850). Out of the 470 studies identified in our search, 77 met the inclusion criteria. Among these, 61 studies were included in strategy 1 (gene therapy using HSC - GT-HSC) of the systematic review (SR). We performed a meta-analysis on 17 of these studies. Additionally, 16 studies were categorized under strategy 2 (clinical trials for PID). While clinical trials have demonstrated the potential benefits of GT-HSC, the safety and efficacy of gene editing still pose significant challenges. Various viral and non-viral approaches for gene delivery have been explored in basic and clinical research, with viral vectors being the most commonly used method in HSC therapeutics. Although promising, recent technologies like CRISPR/Cas are not yet ready for efficient long-term restoration of the immune system as a whole.},
}
@article {pmid37623635,
year = {2023},
author = {Wu, Y and Li, X and Dong, L and Liu, T and Tang, Z and Lin, R and Norvienyeku, J and Xing, M},
title = {A New Insight into 6-Pentyl-2H-pyran-2-one against Peronophythora litchii via TOR Pathway.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {9},
number = {8},
pages = {},
doi = {10.3390/jof9080863},
pmid = {37623635},
issn = {2309-608X},
support = {No. 32260649//the National Natural Science Foundation of China/ ; 320RC482//the Natural Science Foundation of Hainan Province/ ; },
abstract = {The litchi downy blight disease of litchi caused by Peronophythora litchii accounts for severe losses in the field and during storage. While ample quantitative studies have shown that 6-pentyl-2H-pyran-2-one (6PP) possesses antifungal activities against multiple plant pathogenic fungi, the regulatory mechanisms of 6PP-mediated inhibition of fungal pathogenesis and growth are still unknown. Here, we investigated the potential molecular targets of 6PP in the phytopathogenic oomycetes P. litchii through integrated deployment of RNA-sequencing, functional genetics, and biochemical techniques to investigate the regulatory effects of 6PP against P. litchii. Previously we demonstrated that 6PP exerted significant oomyticidal activities. Also, comparative transcriptomic evaluation of P. litchii strains treated with 6PP Revealed significant up-regulations in the expression profile of TOR pathway-related genes, including PlCytochrome C and the transcription factors PlYY1. We also noticed that 6PP treatment down-regulated putative negative regulatory genes of the TOR pathway, including PlSpm1 and PlrhoH12 in P. litchii. Protein-ligand binding analyses revealed stable affinities between PlYY1, PlCytochrome C, PlSpm1, PlrhoH12 proteins, and the 6PP ligand. Phenotypic characterization of PlYY1 targeted gene deletion strains generated in this study using CRISPR/Cas9 and homologous recombination strategies significantly reduced the vegetative growth, sporangium, encystment, zoospore release, and pathogenicity of P. litchii. These findings suggest that 6PP-mediated activation of PlYY1 expression positively regulates TOR-related responses and significantly influences vegetative growth and the virulence of P. litchii. The current investigations revealed novel targets for 6PP and underscored the potential of deploying 6PP in developing management strategies for controlling the litchi downy blight pathogen.},
}
@article {pmid37622910,
year = {2023},
author = {Burkin, KM and Ivanov, AV and Zherdev, AV and Dzantiev, BB and Safenkova, IV},
title = {A Critical Study on DNA Probes Attached to Microplate for CRISPR/Cas12 Trans-Cleavage Activity.},
journal = {Biosensors},
volume = {13},
number = {8},
pages = {},
pmid = {37622910},
issn = {2079-6374},
support = {//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {Humans ; *COVID-19 ; CRISPR-Cas Systems ; SARS-CoV-2/genetics ; DNA Probes ; Antibodies ; DNA, Single-Stranded ; },
abstract = {CRISPR/Cas12-based biosensors are emerging tools for diagnostics. However, their application of heterogeneous formats needs the efficient detection of Cas12 activity. We investigated DNA probes attached to the microplate surface and cleaved by Cas12a. Single-stranded (ss) DNA probes (19 variants) and combined probes with double-stranded (ds) and ssDNA parts (eight variants) were compared. The cleavage efficiency of dsDNA-probes demonstrated a bell-shaped dependence on their length, with a cleavage maximum of 50%. On the other hand, the cleavage efficiency of ssDNA probes increased monotonously, reaching 70%. The most effective ssDNA probes were integrated with fluorescein, antibodies, and peroxidase conjugates as reporters for fluorescent, lateral flow, and chemiluminescent detection. Long ssDNA probes (120-145 nt) proved the best for detecting Cas12a trans-activity for all of the tested variants. We proposed a test system for the detection of the nucleocapsid (N) gene of SARS-CoV-2 based on Cas12 and the ssDNA-probe attached to the microplate surface; its fluorescent limit of detection was 0.86 nM. Being united with pre-amplification using recombinase polymerase, the system reached a detection limit of 0.01 fM, thus confirming the effectiveness of the chosen ssDNA probe for Cas12-based biosensors.},
}
@article {pmid37622855,
year = {2023},
author = {Kwak, N and Park, BJ and Song, YJ},
title = {A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V.},
journal = {Biosensors},
volume = {13},
number = {8},
pages = {},
pmid = {37622855},
issn = {2079-6374},
support = {HI22C0286//Korea Health Industry Development Institute/Republic of Korea ; 202110510001//Gachon University/ ; },
mesh = {Humans ; Animals ; *Encephalitis Virus, Japanese/genetics ; CRISPR-Cas Systems ; Antiviral Agents ; China ; Genotype ; },
abstract = {The Japanese encephalitis virus (JEV) is prevalent in Asian countries, including Korea, Japan, China, Vietnam, and India. JEV is transmitted to humans by Culex mosquitoes. Despite extensive research efforts, no approved antiviral agents are currently available, although JE can be prevented by vaccination. DNA endonuclease-targeted CRISPR trans reporter (DETECTR) is a newly emerging CRISPR-Cas12a-based molecular diagnostic method combined with isothermal nucleic acid amplification. In this study, DETECTR with reverse transcription-recombinase polymerase amplification (RT-RPA) was effectively utilized for JEV diagnosis and detected down to 10 RNA copies for JEV genotype I (GI) and 1 × 10[2] copies for both GIII and GV, achieving similar sensitivity to RT-PCR while displaying no cross-reaction with other viruses. A one-tube, one-temperature format of DETECTR was further developed, and its efficiency compared with that of conventional DETECTR.},
}
@article {pmid37622352,
year = {2023},
author = {Zhou, Y and Ruan, Z and Fang, C and Chen, X and Xu, H and Wang, Z and Yuan, Z},
title = {[Bioconversion of C1 gases and genetic engineering modification of gas-utilizing microorganisms].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {8},
pages = {3125-3142},
doi = {10.13345/j.cjb.220738},
pmid = {37622352},
issn = {1872-2075},
mesh = {*Gene Editing ; *Gases ; Carbon Dioxide ; Genetic Engineering ; Cloning, Molecular ; },
abstract = {C1 gases including CO, CO2 and CH4, are mainly derived from terrestrial biological activities, industrial waste gas and gasification syngas. Particularly, CO2 and CH4 are two of the most important greenhouse gases contributing to climate change. Bioconversion of C1 gases is not only a promising solution to addressing the problem of waste gases emission, but also a novel route to produce fuels or chemicals. In the past few years, C1-gas-utilizing microorganisms have drawn much attention and a variety of gene-editing technologies have been applied to improve their product yields or to expand product portfolios. This article reviewed the biological characteristics, aerobic or anaerobic metabolic pathways as well as the metabolic products of methanotrophs, autotrophic acetogens, and carboxydotrophic bacteria. In addition, gene-editing technologies (e.g. gene interruption technology using homologous recombination, group Ⅱ intron ClosTron technology, CRISPR/Cas gene editing and phage recombinase-mediated efficient integration of large DNA fragments) and their application in these C1-gas-utilizing microorganisms were also summarized.},
}
@article {pmid37620620,
year = {2023},
author = {Zhan, X and Tu, Z and Song, W and Nie, B and Li, S and Zhang, J and Zhang, F},
title = {Cas13a-based multiplex RNA targeting for potato virus Y.},
journal = {Planta},
volume = {258},
number = {4},
pages = {70},
pmid = {37620620},
issn = {1432-2048},
support = {32271912//National Natural Science Foundation of China/ ; 32271546//National Natural Science Foundation of China/ ; },
mesh = {*Potyvirus/genetics ; RNA ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; RNA Processing, Post-Transcriptional ; },
abstract = {The Cas13a-based multiplex RNA targeting system can be engineered to confer resistance to RNA viruses, whereas the number and expression levels of gRNAs have no significant effect on viral interference. The CRISPR-Cas systems provide adaptive immunity to bacterial and archaeal species against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However, whether the number and expression levels of guide RNAs (gRNAs) have effects on the efficiency of RNA virus inhibition is unknown. Here, we repurpose CRISPR/Cas13a in combination with an endogenous tRNA-processing system (polycistronic tRNA-gRNA) to target four genes of potato virus Y (PVY) with varying expression levels. We expressed Cas13a and four different gRNAs in potato lines, and the transgenic plants expressing multiple gRNAs displayed similar suppression of PVY accumulation and reduced disease symptoms as those expressing a single gRNA. Moreover, PTG/Cas13a-transformed plants with different expression levels of multiple gRNAs displayed similar resistance to PVY strains. Collectively, this study suggests that the Cas13a-based multiplex RNA targeting system can be utilized to engineer resistance to RNA viruses in plants, whereas the number and expression levels of gRNAs have no significant effect on CRISPR/Cas13a-mediated viral interference in plants.},
}
@article {pmid37620295,
year = {2023},
author = {Deng, C and Li, S and Liu, Y and Bao, W and Xu, C and Zheng, W and Wang, M and Ma, X},
title = {Split-Cas9-based targeted gene editing and nanobody-mediated proteolysis-targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells.},
journal = {Clinical and translational medicine},
volume = {13},
number = {8},
pages = {e1382},
pmid = {37620295},
issn = {2001-1326},
support = {2018YFA0902804//National Key Research and Development Project of China/ ; 31670944//National Natural Science Foundation/ ; 81673345//National Natural Science Foundation/ ; 31870861//National Natural Science Foundation/ ; 17431904600//Science and Technology Innovation Action Plan of Shanghai/ ; },
mesh = {Humans ; Animals ; Survivin/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Proteolysis ; Apoptosis/genetics ; Disease Models, Animal ; *Neoplasms/genetics/therapy ; },
abstract = {BACKGROUND: Precise regulation of partial critical proteins in cancer cells, such as anti-apoptotic proteins, is one of the crucial strategies for treating cancer and discovering related molecular mechanisms. Still, it is also challenging in actual research and practice. The widely used CRISPR/Cas9-based gene editing technology and proteolysis-targeting chimeras (PROTACs) have played an essential role in regulating gene expression and protein function in cells. However, the accuracy and controllability of their targeting remain necessary.<br><br>METHODS: Construction of UMUC-3-EGFP stable transgenic cell lines using the Sleeping Beauty system, Flow cytometry, quantitative real-time PCR, western blot, fluorescence microplate reader and fluorescence inverted microscope analysis of EGFP intensity. Characterization of Survivin inhibition was done by using Annexin V-FITC/PI apoptosis, calcein/PI/DAPI cell viability/cytotoxicity assay, cloning formation assay and scratch assay. The cell-derived xenograft (CDX) model was constructed to assess the in vivo effects of reducing Survivin expression.<br><br>RESULTS: Herein, we established a synergistic control platform that coordinated photoactivatable split-Cas9 targeted gene editing and light-induced protein degradation, on which the Survivin gene in the nucleus was controllably edited by blue light irradiation (named paCas9-Survivin) and simultaneously the Survivin protein in the cytoplasm was degraded precisely by a nanobody-mediated target (named paProtacL-Survivin). Meanwhile, in vitro experiments demonstrated that reducing Survivin expression could effectively promote apoptosis and decrease the proliferation and migration of bladder cancerous cells. Furthermore, the CDX model was constructed using UMUC-3 cell lines, results from animal studies indicated that both the paCas9-Survivin system and paProtacL-Survivin significantly inhibited tumour growth, with higher inhibition rates when combined.<br><br>CONCLUSIONS: In short, the coordinated regulatory strategies and combinable technology platforms offer clear advantages in controllability and targeting, as well as an excellent reference value and universal applicability in controlling the fate of cancer cells through multi-level regulation of key intracellular factors.},
}
@article {pmid37488781,
year = {2023},
author = {Miranda, JA and Fenner, K and McKinzie, PB and Dobrovolsky, VN and Revollo, JR},
title = {Unbiased whole genome detection of ultrarare off-target mutations in genome-edited cell populations by HiFi sequencing.},
journal = {Environmental and molecular mutagenesis},
volume = {64},
number = {7},
pages = {374-381},
doi = {10.1002/em.22566},
pmid = {37488781},
issn = {1098-2280},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Escherichia coli/genetics ; Mutation ; DNA/genetics ; },
abstract = {DNA base editors (BEs) composed of a nuclease-deficient Cas9 fused to a DNA-modifying enzyme can achieve on-target mutagenesis without creating double-strand DNA breaks (DSBs). As a result, BEs generate far less DNA damage than traditional nuclease-proficient Cas9 systems, which do rely on the creation of DSBs to achieve on-target mutagenesis. The inability of BEs to create DSBs makes the detection of their undesired off-target effects very difficult. PacBio HiFi sequencing can efficiently detect ultrarare mutations resulting from chemical mutagenesis in whole genomes with a sensitivity ~1 × 10[-8] mutations per base pair. In this proof-of-principle study, we evaluated whether this technique could also detect the on- and off-target mutations generated by a cytosine-to-thymine (C>T) BE targeting the LacZ gene in Escherichia coli (E. coli). HiFi sequencing detected on-target mutant allele fractions ranging from ~7% to ~63%, depending on the single-guide RNA (sgRNA) used, while no on-target mutations were detected in controls lacking the BE. The presence of the BE resulted in a ~3-fold increase in mutation frequencies compared to controls lacking the BE, irrespective of the sgRNA used. These increases were mostly composed of C:G>T:A substitutions distributed throughout the genome. Our results demonstrate that HiFi sequencing can efficiently identify on- and off-target mutations in cell populations that have undergone genome editing.},
}
@article {pmid37475654,
year = {2023},
author = {Choi, H and Park, SW and Oh, J and Kim, CS and Sung, GH and Sang, H},
title = {Efficient disruption of CmHk1 using CRISPR/Cas9 ribonucleoprotein delivery in Cordyceps militaris.},
journal = {FEMS microbiology letters},
volume = {370},
number = {},
pages = {},
doi = {10.1093/femsle/fnad072},
pmid = {37475654},
issn = {1574-6968},
support = {PJ01653303//Rural Development Administration/ ; 2020R1C1C1010108//National Research Foundation of Korea/ ; },
mesh = {*CRISPR-Cas Systems ; *Cordyceps/genetics ; Ribonucleoproteins/genetics ; Mutation ; },
abstract = {Cordyceps militaris, an entomopathogenic ascomycete, produces edible medicinal mushrooms known to have medicinal and therapeutic functions. To develop the genetic transformation system in C. militaris, green fluorescent protein (GFP) mutants of C. militaris were generated by PEG-mediated protoplast transformation. The CRISPR/Cas9 ribonucleoprotein (RNP) targeting the class III histidine kinase of C. militaris (CmHk1) was then delivered into protoplasts of C. militaris through the transformation system. Mutations induced by the RNP in selected mutants were detected: 1 nt deletion (6 mutants), 3 nt deletion with substitution of 1 nt (1 mutant), insertion of 85 nts (1 mutant), 41 nts (2 mutants), and 35 nts (5 mutants). An in vitro sensitivity assay of the mutants indicated that knockout of CmHk1 reduced sensitivity to two fungicides, iprodione and fludioxonil, but increased sensitivity to osmotic stresses compared to the wild type. Summing up, the CRISPR/Cas9 RNP delivery system was successfully developed, and our results revealed that CmHk1 was involved in the fungicide resistance and osmotic stress in C. militaris.},
}
@article {pmid37471041,
year = {2023},
author = {Liu, W and Zuo, S and Shao, Y and Bi, K and Zhao, J and Huang, L and Xu, Z and Lian, J},
title = {Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli.},
journal = {Nucleic acids research},
volume = {51},
number = {15},
pages = {8293-8307},
pmid = {37471041},
issn = {1362-4962},
support = {2021YFC2103200//National Key Research and Development Program of China/ ; 22278361//National Natural Science Foundation of China/ ; LR20B060003//Natural Science Foundation of Zhejiang Province/ ; 226-2023-00015//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics/metabolism ; Metabolic Engineering/methods ; Mutagenesis ; CRISPR-Cas Systems/genetics ; },
abstract = {While there are several genome editing techniques available, few are suitable for dynamic and simultaneous mutagenesis of arbitrary targeted sequences in prokaryotes. Here, to address these limitations, we present a versatile and multiplex retron-mediated genome editing system (REGES). First, through systematic optimization of REGES, we achieve efficiency of ∼100%, 85 ± 3%, 69 ± 14% and 25 ± 14% for single-, double-, triple- and quadruple-locus genome editing, respectively. In addition, we employ REGES to generate pooled and barcoded variant libraries with degenerate RBS sequences to fine-tune the expression level of endogenous and exogenous genes, such as transcriptional factors to improve ethanol tolerance and biotin biosynthesis. Finally, we demonstrate REGES-mediated continuous in vivo protein evolution, by combining retron, polymerase-mediated base editing and error-prone transcription. By these case studies, we demonstrate REGES as a powerful multiplex genome editing and continuous evolution tool with broad applications in synthetic biology and metabolic engineering.},
}
@article {pmid37468054,
year = {2023},
author = {Wang, L and Fu, D and Weng, S and Xu, H and Liu, L and Guo, C and Ren, Y and Liu, Z and Han, X},
title = {Genome-scale CRISPR-Cas9 screening stratifies pancreatic cancer with distinct outcomes and immunotherapeutic efficacy.},
journal = {Cellular signalling},
volume = {110},
number = {},
pages = {110811},
doi = {10.1016/j.cellsig.2023.110811},
pmid = {37468054},
issn = {1873-3913},
mesh = {Humans ; *Early Detection of Cancer ; CRISPR-Cas Systems/genetics ; *Pancreatic Neoplasms/diagnosis/genetics/therapy ; Immunotherapy ; },
abstract = {Pancreatic cancer (PC) was featured by dramatic heterogeneity and dismal outcomes. An ideal classification strategy capable of achieving risk stratification and individualized treatment is urgently needed to significantly improve prognosis. In this study, using the 105 prognostic cancer essential genes identified by genome-scale CRISPR-Cas9 screening and univariate Cox analysis, we established and verified three heterogeneous subtypes via non-negative matrix factorization (NMF) and nearest template prediction (NTP) algorithms in the TCGA-PAAD cohort (176 samples) and four multi-center cohorts (233 samples), respectively. Among them, C1 with the worst prognosis was enriched in immune-related pathways, possessed superior infiltration abundance of immune cells and immune checkpoint molecules expression, and might be most sensitive to immunotherapy. C3, owing a moderate prognosis, might be featured by proliferative biological function, and despite its highest immunogenicity, the defects in antigen processing and presentation ability coupled with barren immune environment render it ineffective for immunotherapy, while it had potential sensitivity to paclitaxel and methotrexate. Besides, C2 harbored the best prognosis and was characterized by metabolism-related functions. These results could deepen our understanding of PC molecular heterogeneity and provide a trustworthy reference for prognostic stratification management and precision medicine in clinical practice.},
}
@article {pmid37464068,
year = {2023},
author = {Neldeborg, S and Soerensen, JF and Møller, CT and Bill, M and Gao, Z and Bak, RO and Holm, K and Sorensen, B and Nyegaard, M and Luo, Y and Hokland, P and Stougaard, M and Ludvigsen, M and Holm, CK},
title = {Dual intron-targeted CRISPR-Cas9-mediated disruption of the AML RUNX1-RUNX1T1 fusion gene effectively inhibits proliferation and decreases tumor volume in vitro and in vivo.},
journal = {Leukemia},
volume = {37},
number = {9},
pages = {1792-1801},
pmid = {37464068},
issn = {1476-5551},
support = {NA//Karen Elise Jensens Fond (Karen Elise Jensen Foundation)/ ; },
mesh = {Animals ; Mice ; RUNX1 Translocation Partner 1 Protein/genetics ; *Translocation, Genetic ; Introns/genetics ; Core Binding Factor Alpha 2 Subunit/genetics/metabolism ; Tumor Burden ; CRISPR-Cas Systems ; *Leukemia, Myeloid, Acute/genetics/therapy ; Cell Proliferation ; Oncogene Proteins, Fusion/genetics/metabolism ; },
abstract = {Oncogenic fusion drivers are common in hematological cancers and are thus relevant targets of future CRISPR-Cas9-based treatment strategies. However, breakpoint-location variation in patients pose a challenge to traditional breakpoint-targeting CRISPR-Cas9-mediated disruption strategies. Here we present a new dual intron-targeting CRISPR-Cas9 treatment strategy, for targeting t(8;21) found in 5-10% of de novo acute myeloid leukemia (AML), which efficiently disrupts fusion genes without prior identification of breakpoint location. We show in vitro growth rate and proliferation reduction by 69 and 94% in AML t(8;21) Kasumi-1 cells, following dual intron-targeted disruption of RUNX1-RUNX1T1 compared to a non t(8;21) AML control. Furthermore, mice injected with RUNX1-RUNX1T1-disrupted Kasumi-1 cells had in vivo tumor growth reduction by 69 and 91% compared to controls. Demonstrating the feasibility of RUNX1-RUNX1T1 disruption, these findings were substantiated in isolated primary cells from a patient diagnosed with AML t(8;21). In conclusion, we demonstrate proof-of-principle of a dual intron-targeting CRISPR-Cas9 treatment strategy in AML t(8;21) without need for precise knowledge of the breakpoint location.},
}
@article {pmid37369005,
year = {2023},
author = {Dickson, KA and Field, N and Blackman, T and Ma, Y and Xie, T and Kurangil, E and Idrees, S and Rathnayake, SNH and Mahbub, RM and Faiz, A and Marsh, DJ},
title = {CRISPR single base-editing: in silico predictions to variant clonal cell lines.},
journal = {Human molecular genetics},
volume = {32},
number = {17},
pages = {2704-2716},
doi = {10.1093/hmg/ddad105},
pmid = {37369005},
issn = {1460-2083},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Tumor Suppressor Protein p53/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line ; Adenine/metabolism ; Cytosine/metabolism ; },
abstract = {Engineering single base edits using CRISPR technology including specific deaminases and single-guide RNA (sgRNA) is a rapidly evolving field. Different types of base edits can be constructed, with cytidine base editors (CBEs) facilitating transition of C-to-T variants, adenine base editors (ABEs) enabling transition of A-to-G variants, C-to-G transversion base editors (CGBEs) and recently adenine transversion editors (AYBE) that create A-to-C and A-to-T variants. The base-editing machine learning algorithm BE-Hive predicts which sgRNA and base editor combinations have the strongest likelihood of achieving desired base edits. We have used BE-Hive and TP53 mutation data from The Cancer Genome Atlas (TCGA) ovarian cancer cohort to predict which mutations can be engineered, or reverted to wild-type (WT) sequence, using CBEs, ABEs or CGBEs. We have developed and automated a ranking system to assist in selecting optimally designed sgRNA that considers the presence of a suitable protospacer adjacent motif (PAM), the frequency of predicted bystander edits, editing efficiency and target base change. We have generated single constructs containing ABE or CBE editing machinery, an sgRNA cloning backbone and an enhanced green fluorescent protein tag (EGFP), removing the need for co-transfection of multiple plasmids. We have tested our ranking system and new plasmid constructs to engineer the p53 mutants Y220C, R282W and R248Q into WT p53 cells and shown that these mutants cannot activate four p53 target genes, mimicking the behaviour of endogenous p53 mutations. This field will continue to rapidly progress, requiring new strategies such as we propose to ensure desired base-editing outcomes.},
}
@article {pmid37345937,
year = {2023},
author = {Reichard, WD and Smith, SE and Robertson, JB},
title = {BLINCAR: a reusable bioluminescent and Cas9-based genetic toolset for repeatedly modifying wild-type Scheffersomyces stipitis.},
journal = {mSphere},
volume = {8},
number = {4},
pages = {e0022423},
pmid = {37345937},
issn = {2379-5042},
mesh = {*Xylose ; *Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; Cellulose ; Ethanol ; Anti-Bacterial Agents ; },
abstract = {Scheffersomyces stipitis is a yeast that robustly ferments the 5-carbon sugar xylose, making the yeast a valuable candidate for lignocellulosic ethanol fermentation. However, the non-canonical codon usage of S. stipitis is an obstacle for implementing molecular tools that were developed for other yeast species, thereby limiting the molecular toolset available for S. stipitis. Here, we developed a series of molecular tools for S. stipitis including BLINCAR, a Bio-Luminescent Indicator that is Nullified by Cas9-Actuated Recombination, which can be used repeatedly to add different exogenous DNA payloads to the wild-type S. stipitis genome or used repeatedly to remove multiple native S. stipitis genes from the wild-type genome. Through the use of BLINCAR tools, one first produces antibiotic-resistant, bioluminescent colonies of S. stipitis whose bioluminescence highlights those clones that have been genetically modified; then second, once candidate clones have been confirmed, one uses a transient Cas9-producing plasmid to nullify the antibiotic resistance and bioluminescent markers from the prior introduction, thereby producing non-bioluminescent colonies that highlight those clones which have been re-sensitized to the antibiotic and are therefore susceptible to another round of BLINCAR implementation. IMPORTANCE Cellulose and hemicellulose that comprise a large portion of sawdust, leaves, and grass can be valuable sources of fermentable sugars for ethanol production. However, some of the sugars liberated from hemicellulose (like xylose) are not easily fermented using conventional glucose-fermenting yeast like Saccharomyces cerevisiae, so engineering robust xylose-fermenting yeast that is not inhibited by other components liberated from cellulose/hemicellulose will be important for maximizing yield and making lignocellulosic ethanol fermentation cost efficient. The yeast Scheffersomyces stipitis is one such yeast that can ferment xylose; however, it possesses several barriers to genetic manipulation. It is difficult to transform, has only a few antibiotic resistance markers, and uses an alternative genetic code from most other organisms. We developed a genetic toolset for S. stipitis that lowers these barriers and allows a user to deliver and/or delete multiple genetic elements to/from the wild-type genome, thereby expanding S. stipitis's potential.},
}
@article {pmid36958601,
year = {2023},
author = {Liang, Z and Wu, Y and Guo, Y and Wei, S},
title = {Addition of the T5 exonuclease increases the prime editing efficiency in plants.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {50},
number = {8},
pages = {582-588},
doi = {10.1016/j.jgg.2023.03.008},
pmid = {36958601},
issn = {1673-8527},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Genome ; Mutation ; Plants, Genetically Modified/genetics ; },
abstract = {Prime editing (PE) is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates, but is limited by low editing efficiency. We previously fused the M-MLV reverse transcriptase to the Cas9 nickase, generating the PE2 (v1) system, but the editing efficiency of this system is still low. Here we develop different versions of PE2 by adding the 5'-to-3' exonuclease at different positions of the nCas9-M-MLV RT fusion protein. PE2 (v2), in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions, deletions, and insertions at several genomic sites by 1.7- to 2.9-fold in plant cells compared to PE2 (v1). The improved editing efficiency of PE2 (v2) is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1, PE-P2, and PPE systems. Using PE2 (v2), we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites. The PE efficiency is further improved by combining PE2 (v2) and dual-pegRNAs. Taken together, the increased genome editing efficiency of PE2 (v2) developed in this study may enhance the applications of PE in plants.},
}
@article {pmid36878350,
year = {2023},
author = {Ryu, J and Adashi, EY and Hennebold, JD},
title = {The history, use, and challenges of therapeutic somatic cell and germline gene editing.},
journal = {Fertility and sterility},
volume = {120},
number = {3 Pt 1},
pages = {528-538},
doi = {10.1016/j.fertnstert.2023.02.040},
pmid = {36878350},
issn = {1556-5653},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; Mutation ; Germ Cells ; Precision Medicine ; },
abstract = {The advent of directed gene-editing technologies now over 10 years ago ushered in a new era of precision medicine wherein specific disease-causing mutations can be corrected. In parallel with developing new gene-editing platforms, optimizing their efficiency and delivery has been remarkable. With their development, there has been interest in using gene-editing systems for correcting disease mutations in differentiated somatic cells ex vivo or in vivo or for germline gene editing in gametes or 1-cell embryos to potentially limit genetic diseases in the offspring and in future generations. This review details the development and history of the current gene-editing systems and the advantages and challenges in their use for somatic cell and germline gene editing.},
}
@article {pmid36781946,
year = {2023},
author = {Yaméogo, P and Gérard, C and Majeau, N and Tremblay, JP},
title = {Removal of the GAA repeat in the heart of a Friedreich's ataxia mouse model using CjCas9.},
journal = {Gene therapy},
volume = {30},
number = {7-8},
pages = {612-619},
pmid = {36781946},
issn = {1476-5462},
support = {//CIHR/Canada ; },
mesh = {Mice ; Animals ; *Friedreich Ataxia/genetics/therapy/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Disease Models, Animal ; Iron-Binding Proteins/genetics/metabolism ; Trinucleotide Repeat Expansion/genetics ; },
abstract = {Most Friedreich ataxia (FRDA) cases are caused by the elongation of the GAA repeat (GAAr) sequence in the first intron of the FXN gene, leading to a decrease of the frataxin protein expression. Deletion of this GAAr with CRISPR/Cas9 technology leads to an increase in frataxin expression in vitro. We are therefore aiming to develop FRDA treatment based on the deletion of GAAr with CRISPR/Cas9 technology using a single AAV expressing a small Cas9 (CjCas9) and two single guide RNAs (sgRNAs) targeting the FXN gene. This AAV was intraperitoneally administrated to YG8sR (250-300 GAAr) and to YG8-800 (800 GAAr) mice. DNA and RNA were extracted from different organs a month later. PCR amplification of part of intron 1 of the FXN gene detected some GAAr deletion in some cells in heart and liver of both mouse models, but the editing rate was not sufficient to cause an increase in frataxin mRNA in the heart. However, the correlation observed between the editing rate and the distribution of AAV suggests a possible therapy based on the removal of the GAAr with a better delivery tool of the CRISPR/Cas9 system.},
}
@article {pmid34744169,
year = {2023},
author = {Simhadri, VL and McGill, JR and Sauna, ZE},
title = {Endotoxin contamination in commercially available Cas9 proteins potentially induces T-cell mediated responses.},
journal = {Gene therapy},
volume = {30},
number = {7-8},
pages = {575-580},
pmid = {34744169},
issn = {1476-5462},
support = {/FD/FDA HHS/United States ; },
mesh = {*T-Lymphocytes ; *CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Interferon-gamma/genetics ; Endotoxins/genetics ; },
abstract = {Immune responses to Cas proteins have been demonstrated recently and these may prove to be an impediment to their clinical use in gene editing. To make meaningful assessments of Cas9 immunogenicity during drug development and licensure it is imperative the reagents are free of impurities that could affect in vitro assessments of immunogenicity. Here we address the issue of endotoxin levels in laboratory grade Cas9 proteins used to measure T-cell memory responses. Many of these reagents have not been developed for immunogenicity assays, are or microbial origin and carry varying levels of endotoxin. The use of these reagents, off the shelf, without measuring endotoxin levels is likely to introduce incorrect estimates of the prevalence of memory T-cell responses in research studies. We demonstrate wide variation in endotoxin levels in Cas9 proteins from seven suppliers. Different lots from the same supplier also contained varying levels of endotoxin. ELISPOT assays showed similar large variations in the interferon-γ signals. Finally, when we carried out endotoxin depletion in four Cas9 proteins with strong signals in the ELISPOT assay, we found dampening of the interferon-γ signals.},
}
@article {pmid37619525,
year = {2023},
author = {Jose, B and Punetha, M and Tripathi, MK and Khanna, S and Yadav, V and Singh, AK and Kumar, B and Singh, K and Chouhan, VS and Sarkar, M},
title = {CRISPR/Cas mediated disruption of BMPR-1B gene and introduction of FecB mutation into the Caprine embryos using Easi-CRISPR strategy.},
journal = {Theriogenology},
volume = {211},
number = {},
pages = {125-133},
doi = {10.1016/j.theriogenology.2023.08.008},
pmid = {37619525},
issn = {1879-3231},
abstract = {Bone Morphogenetic Proteins play a significant role in ovarian physiology and contribute to the reproductive fitness of mammals. The BMPR-1B/FecB mutation, a loss of function mutation increases litter size by 1-2 with each number of mutated alleles in sheep. Considering demand-supply gap of the meat industry, and low replacement rate of indigenous caprine species, the conservative BMPR-1B locus can be explored, and FecB mutated goats can be produced. The experiment one produced CRISPR/Cas mediated KO transferable caprine embryos, and experiment two generated caprine embryos with desired FecB mutation using Easi-CRISPR strategy. In the KO experiment, Cas9 and BMPR-1B guide RNA (100:100ng/ul) were electroporated into single stage caprine zygotes at 750V, 10 ms and 1pulse using Neon transfection system. In the second experiment, phosphorothioate (PS) modified single-stranded oligodeoxynucleotide (ssODN) was used as an HDR template along with CRISPR components (100:100ng/ul, ssODN 100ng/ul). The precise time and method of electroporation, RNP format of CRISPR components and PS modified asymmetric ssODN were the factors that affected the production of mosaicism free BMPR-1B edited caprine embryos. The editing efficiency of KO and KI experiments was 68.52 and 63.16% respectively, and successful production of goats with higher mean ovulation rate can be realized with addition of embryo transfer technology to these experiments.},
}
@article {pmid37615340,
year = {2023},
author = {Luo, W and Zhang, Z and Zhou, D and Jiang, Y and Yang, J and He, B and Yu, H and Song, Y},
title = {Deep Tumor Penetration of CRISPR-Cas System for Photothermal-Sensitized Immunotherapy via Probiotics.},
journal = {Nano letters},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.nanolett.3c02061},
pmid = {37615340},
issn = {1530-6992},
abstract = {Since central cells are more malignant and aggressive in solid tumors, improving penetration of therapeutic agents and activating immunity in tumor centers exhibit great potential in cancer therapies. Here, polydopamine-coated Escherichia coli Nissle 1917 (EcN) bearing CRISPR-Cas9 plasmid-loaded liposomes (Lipo-P) are applied for enhanced immunotherapy in deep tumors through activation of innate and adaptive immunity simultaneously. After accumulation in the tumor center through hypoxia targeting, Lipo-P could be detached under the reduction of reactive oxygen species (ROS)-responsive linkers, lowering the thermal resistance of cancer cells via Hsp90α depletion. Owing to that, heating induced by polydopamine upon near-infrared irradiation could achieve effective tumor ablation. Furthermore, mild photothermal therapy induces immunogenic cell death, as bacterial infections in tumor tissues trigger innate immunity. This bacteria-assisted approach provides a promising photothermal-sensitized immunotherapy in deep tumors.},
}
@article {pmid37612816,
year = {2023},
author = {Yin, W and Zhuang, J and Li, J and Xia, L and Hu, K and Yin, J and Mu, Y},
title = {Digital Recombinase Polymerase Amplification, Digital Loop-Mediated Isothermal Amplification, and Digital CRISPR-Cas Assisted Assay: Current Status, Challenges, and Perspectives.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e2303398},
doi = {10.1002/smll.202303398},
pmid = {37612816},
issn = {1613-6829},
support = {62201497//National Natural Science Foundation of China/ ; LQ22F010005//Zhejiang Provincial Natural Science Foundation of China/ ; 2023RC053//Zhejiang Provincial Medical and Health Technology/ ; },
abstract = {Digital nucleic acid detection based on microfluidics technology can quantify the initial amount of nucleic acid in the sample with low equipment requirements and simple operations, which can be widely used in clinical and in vitro diagnosis. Recently, isothermal amplification technologies such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats-CRISPR associated proteins (CRISPR-Cas) assisted technologies have become a hot spot of attention and state-of-the-art digital nucleic acid chips have provided a powerful tool for these technologies. Herein, isothermal amplification technologies including RPA, LAMP, and CRISPR-Cas assisted methods, based on digital nucleic acid microfluidics chips recently, have been reviewed. Moreover, the challenges of digital isothermal amplification and possible strategies to address them are discussed. Finally, future directions of digital isothermal amplification technology, such as microfluidic chip and device manufacturing, multiplex detection, and one-pot detection, are outlined.},
}
@article {pmid37612728,
year = {2023},
author = {Krill-Burger, JM and Dempster, JM and Borah, AA and Paolella, BR and Root, DE and Golub, TR and Boehm, JS and Hahn, WC and McFarland, JM and Vazquez, F and Tsherniak, A},
title = {Partial gene suppression improves identification of cancer vulnerabilities when CRISPR-Cas9 knockout is pan-lethal.},
journal = {Genome biology},
volume = {24},
number = {1},
pages = {192},
pmid = {37612728},
issn = {1474-760X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Neoplasms/genetics ; Genetic Techniques ; RNA Interference ; Cell Line ; },
abstract = {BACKGROUND: Hundreds of functional genomic screens have been performed across a diverse set of cancer contexts, as part of efforts such as the Cancer Dependency Map, to identify gene dependencies-genes whose loss of function reduces cell viability or fitness. Recently, large-scale screening efforts have shifted from RNAi to CRISPR-Cas9, due to superior efficacy and specificity. However, many effective oncology drugs only partially inhibit their protein targets, leading us to question whether partial suppression of genes using RNAi could reveal cancer vulnerabilities that are missed by complete knockout using CRISPR-Cas9. Here, we compare CRISPR-Cas9 and RNAi dependency profiles of genes across approximately 400 matched cancer cell lines.<br><br>RESULTS: We find that CRISPR screens accurately identify more gene dependencies per cell line, but the majority of each cell line's dependencies are part of a set of 1867 genes that are shared dependencies across the entire collection (pan-lethals). While RNAi knockdown of about 30% of these genes is also pan-lethal, approximately 50% have selective dependency patterns across cell lines, suggesting they could still be cancer vulnerabilities. The accuracy of the unique RNAi selectivity is supported by associations to multi-omics profiles, drug sensitivity, and other expected co-dependencies.<br><br>CONCLUSIONS: Incorporating RNAi data for genes that are pan-lethal knockouts facilitates the discovery of a wider range of gene targets than could be detected using the CRISPR dataset alone. This can aid in the interpretation of contrasting results obtained from CRISPR and RNAi screens and reinforce the importance of partial gene suppression methods in building a cancer dependency map.},
}
@article {pmid37610064,
year = {2023},
author = {Li, Y and Huang, C and Liu, Y and Zeng, J and Yu, H and Tong, Z and Yuan, X and Sui, X and Fang, D and Xiao, B and Zhao, S and Yuan, C},
title = {CRISPR/Cas9-mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV-U1 strain in regenerated Nicotiana tabacum.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14159},
pmid = {37610064},
issn = {1467-7652},
support = {31860499//National Natural Science Foundation of China/ ; 2022530000241014//YNTC/ ; 2019530000241002//YNTC/ ; },
abstract = {CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.},
}
@article {pmid37609704,
year = {2023},
author = {Ma, X and Zhou, F and Yang, D and Chen, Y and Li, M and Wang, P},
title = {miRNA Detection for Prostate Cancer Diagnosis by miRoll-Cas: miRNA Rolling Circle Transcription for CRISPR-Cas Assay.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c02231},
pmid = {37609704},
issn = {1520-6882},
abstract = {Micro-RNA (miRNA) emerges as a promising type of biomarker for cancer diagnosis. There is an urgent need for developing rapid, convenient, and precise miRNA detection methods that may be conducted with limited laboratory facilities, especially in underdeveloped areas. Herein, we developed a miRNA detection method termed miRoll-Cas, where miRNA is first amplified by rolling circle transcription and then subject to CRISPR-Cas13a assay. Using miRoll-Cas, we realized the sensitive detection of multiple cancer-relevant miRNA markers (miR21, miR141, and Let7b) and specifically identified other variants of the Let7 family, which can accurately discriminate prostate cancer patients from healthy people. We envision that miRoll-Cas may be readily translated to clinical applications in the diagnosis of a variety of diseases beyond cancer.},
}
@article {pmid37608945,
year = {2023},
author = {Wang, L and Chen, X and Pan, F and Yao, G and Chen, J},
title = {Development of a rapid detection method for Karenia mikimotoi by using CRISPR-Cas12a.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1205765},
pmid = {37608945},
issn = {1664-302X},
abstract = {Harmful algal blooms (HABs), mainly formed by dinoflagellates, have detrimental effects on marine ecosystems and public health. Therefore, detecting HABs is crucial for early warning and prevention of HABs as well as the mitigation of their adverse effects. Although various methods, such as light microscopy, electron microscopy, real-time PCR, and microarrays, have already been established for the detection of HABs, they are still cumbersome to be exploited in the field. Therefore, rapid nucleic detection methods such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)-lateral flow dipstick (LFD) have been developed for monitoring bloom-forming algae. However, the CRISPR/Cas-based detection of HABs has yet to be applied to this field. In this study, we developed a method for detecting Karenia mikimotoi (K. mikimotoi), a typical ichthyotoxic dinoflagellate responsible for global blooms. Our method utilized Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) to target and cleave the internal transcribed spacer (ITS) of K. mikimotoi, guided by RNA. We leveraged the target-activated non-specific single-stranded deoxyribonuclease cleavage activity of LbCas12a to generate signals that can be detected using fluorescence-read machines or LFDs. By combining RPA and LbCas12a with reporters, we significantly enhanced the sensitivity, enabling the detection of ITS-harboring plasmids at concentrations as low as 9.8 aM and genomic DNA of K. mikimotoi at levels as low as 3.6 × 10[-5] ng/μl. Moreover, we simplified the genomic DNA extraction method using cellulose filter paper (CFP) by directly eluting the DNA into RPA reactions, reducing the extraction time to < 30 s. The entire process, from genomic DNA extraction to result reporting, takes less than an hour, enabling the identification of nearly a single cell. In conclusion, our method provided an easy, specific, and sensitive approach for detecting K. mikimotoi, offering the potential for efficient monitoring and management of K. mikimotoi blooms.},
}
@article {pmid37608792,
year = {2023},
author = {Liu, P and Huang, L and Song, CQ},
title = {Editorial: Genome editing applications of CRISPR/Cas9 in metabolic diseases, hormonal system and cancer research.},
journal = {Frontiers in endocrinology},
volume = {14},
number = {},
pages = {1256966},
pmid = {37608792},
issn = {1664-2392},
mesh = {Humans ; CRISPR-Cas Systems ; Gene Editing ; *Neoplasms/genetics/therapy ; *Metabolic Diseases/genetics/therapy ; Research ; },
}
@article {pmid37607619,
year = {2023},
author = {Dhingra, Y and Sashital, DG},
title = {Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {105178},
doi = {10.1016/j.jbc.2023.105178},
pmid = {37607619},
issn = {1083-351X},
abstract = {CRISPR-Cas adaptive immune systems uptake short 'spacer' sequences from foreign DNA and incorporate them into the host genome to serve as templates for crRNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence-specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn[2+] dependent, Cas1 preferentially uses Mg[2+] over Mn[2+]. The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing, making the adaptation module self-reliant for prespacer maturation and directional integration.},
}
@article {pmid37606708,
year = {2023},
author = {Gautam, R and Shukla, P and Kirti, PB},
title = {Male sterility in plants: an overview of advancements from natural CMS to genetically manipulated systems for hybrid seed production.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {136},
number = {9},
pages = {195},
pmid = {37606708},
issn = {1432-2242},
mesh = {Male ; Humans ; Cytoplasm ; *Seeds/genetics ; Fertility ; Pollen ; *Infertility, Male ; },
abstract = {The male sterility system in plants has traditionally been utilized for hybrid seed production. In last three decades, genetic manipulation for male sterility has revolutionized this area of research related to hybrid seed production technology. Here, we have surveyed some of the natural cytoplasmic male sterility (CMS) systems that existed/ were developed in different crop plants for developing male sterility-fertility restoration systems used in hybrid seed production and highlighted some of the recent biotechnological advancements in the development of genetically engineered systems that occurred in this area. We have indicated the possible future directions toward the development of engineered male sterility systems. Cytoplasmic male sterility (CMS) is an important trait that is naturally prevalent in many plant species, which has been used in the development of hybrid varieties. This is associated with the use of appropriate genes for fertility restoration provided by the restorer line that restores fertility on the corresponding CMS line. The development of hybrids based on a CMS system has been demonstrated in several different crops. However, there are examples of species, which do not have usable cytoplasmic male sterility and fertility restoration systems (Cytoplasmic Genetic Male Sterility Systems-CGMS) for hybrid variety development. In such plants, it is necessary to develop usable male sterile lines through genetic engineering with the use of heterologous expression of suitable genes that control the development of male gametophyte and fertile male gamete formation. They can also be developed through gene editing using the recently developed CRISPR-Cas technology to knock out suitable genes that are responsible for the development of male gametes. The present review aims at providing an insight into the development of various technologies for successful production of hybrid varieties and is intended to provide only essential information on male sterility systems starting from naturally occurring ones to the genetically engineered systems obtained through different means.},
}
@article {pmid37486064,
year = {2023},
author = {Bielczyk-Maczynska, E and Sharma, D and Blencowe, M and Saliba Gustafsson, P and Gloudemans, MJ and Yang, X and Carcamo-Orive, I and Wabitsch, M and Svensson, KJ and Park, CY and Quertermous, T and Knowles, JW and Li, J},
title = {A single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes.},
journal = {American journal of physiology. Cell physiology},
volume = {325},
number = {3},
pages = {C648-C660},
doi = {10.1152/ajpcell.00148.2023},
pmid = {37486064},
issn = {1522-1563},
support = {R01 DK125260/DK/NIDDK NIH HHS/United States ; UM1 HG011972/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; Adipogenesis/genetics ; Adipocytes/metabolism ; Cell Line ; *Metabolic Diseases/metabolism ; Cell Differentiation/genetics ; },
abstract = {CROP-Seq combines gene silencing using CRISPR interference with single-cell RNA sequencing. Here, we applied CROP-Seq to study adipogenesis and adipocyte biology. Human preadipocyte SGBS cell line expressing KRAB-dCas9 was transduced with a sgRNA library. Following selection, individual cells were captured using microfluidics at different timepoints during adipogenesis. Bioinformatic analysis of transcriptomic data was used to determine the knockdown effects, the dysregulated pathways, and to predict cellular phenotypes. Single-cell transcriptomes recapitulated adipogenesis states. For all targets, over 400 differentially expressed genes were identified at least at one timepoint. As a validation of our approach, the knockdown of PPARG and CEBPB (which encode key proadipogenic transcription factors) resulted in the inhibition of adipogenesis. Gene set enrichment analysis generated hypotheses regarding the molecular function of novel genes. MAFF knockdown led to downregulation of transcriptional response to proinflammatory cytokine TNF-α in preadipocytes and to decreased CXCL-16 and IL-6 secretion. TIPARP knockdown resulted in increased expression of adipogenesis markers. In summary, this powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte, and adipocyte function associated with metabolic disease.NEW &amp; NOTEWORTHY Genomics efforts led to the identification of many genomic loci that are associated with metabolic traits, many of which are tied to adipose tissue function. However, determination of the causal genes, and their mechanism of action in metabolism, is a time-consuming process. Here, we use an approach to determine the transcriptional outcome of candidate gene knockdown for multiple genes at the same time in a human cell model of adipogenesis.},
}
@article {pmid36877372,
year = {2023},
author = {Dawes, P and Murray, LF and Olson, MN and Barton, NJ and Smullen, M and Suresh, M and Yan, G and Zhang, Y and Fernandez-Fontaine, A and English, J and Uddin, M and Pak, C and Church, GM and Chan, Y and Lim, ET},
title = {oFlowSeq: a quantitative approach to identify protein coding mutations affecting cell type enrichment using mosaic CRISPR-Cas9 edited cerebral organoids.},
journal = {Human genetics},
volume = {142},
number = {8},
pages = {1281-1291},
pmid = {36877372},
issn = {1432-1203},
support = {R01 MH113279/MH/NIMH NIH HHS/United States ; RM1 HG008525/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mutation ; Organoids ; Genotype ; },
abstract = {Cerebral organoids are comprised of diverse cell types found in the developing human brain, and can be leveraged in the identification of critical cell types perturbed by genetic risk variants in common, neuropsychiatric disorders. There is great interest in developing high-throughput technologies to associate genetic variants with cell types. Here, we describe a high-throughput, quantitative approach (oFlowSeq) by utilizing CRISPR-Cas9, FACS sorting, and next-generation sequencing. Using oFlowSeq, we found that deleterious mutations in autism-associated gene KCTD13 resulted in increased proportions of Nestin[+] cells and decreased proportions of TRA-1-60[+] cells within mosaic cerebral organoids. We further identified that a locus-wide CRISPR-Cas9 survey of another 18 genes in the 16p11.2 locus resulted in most genes with > 2% maximum editing efficiencies for short and long indels, suggesting a high feasibility for an unbiased, locus-wide experiment using oFlowSeq. Our approach presents a novel method to identify genotype-to-cell type imbalances in an unbiased, high-throughput, quantitative manner.},
}
@article {pmid37605748,
year = {2023},
author = {Tong, C and Liang, Y and Zhang, Z and Wang, S and Zheng, X and Liu, Q and Song, B},
title = {Review of knockout technology approaches in bacterial drug resistance research.},
journal = {PeerJ},
volume = {11},
number = {},
pages = {e15790},
pmid = {37605748},
issn = {2167-8359},
mesh = {*Craniocerebral Trauma ; Drug Resistance, Bacterial/genetics ; Gene Knockout Techniques ; Genes, Bacterial ; Technology ; Transcription Activator-Like Effector Nucleases ; Zinc Finger Nucleases ; Animals ; },
abstract = {Gene knockout is a widely used method in biology for investigating gene function. Several technologies are available for gene knockout, including zinc-finger nuclease technology (ZFN), suicide plasmid vector systems, transcription activator-like effector protein nuclease technology (TALEN), Red homologous recombination technology, CRISPR/Cas, and others. Of these, Red homologous recombination technology, CRISPR/Cas9 technology, and suicide plasmid vector systems have been the most extensively used for knocking out bacterial drug resistance genes. These three technologies have been shown to yield significant results in researching bacterial gene functions in numerous studies. This study provides an overview of current gene knockout methods that are effective for genetic drug resistance testing in bacteria. The study aims to serve as a reference for selecting appropriate techniques.},
}
@article {pmid37605597,
year = {2023},
author = {S Teixeira, LF and Bellini, MH},
title = {NF-ĸΒ1 knockout reduces IL6 expression under hypoxia in renal cell carcinoma.},
journal = {Cellular and molecular biology (Noisy-le-Grand, France)},
volume = {69},
number = {6},
pages = {8-14},
doi = {10.14715/cmb/2023.69.6.2},
pmid = {37605597},
issn = {1165-158X},
mesh = {Adult ; Humans ; *Carcinoma, Renal Cell/genetics ; Interleukin-6/genetics ; NF-kappa B/genetics ; *Kidney Neoplasms/genetics ; *Craniocerebral Trauma ; Hypoxia ; },
abstract = {Renal cell carcinoma (RCC) is the most common adult renal epithelial cancer, accounting for more than 90% of all renal neoplasms. Clear cell RCC (ccRCC) is the most common subtype of RCC. Most patients with ccRCC have a mutation in the von Hippel-Lindau (VHL) tumor suppressor gene, which encodes a protein that downregulates various intracellular proteins, including hypoxia-inducible factor (HIF). Many molecules have been identified to be responsible for the aggressive phenotype of ccRCC, including the transcription factor nuclear factor kappa B (NF-кB). The increase in NF-кB activity observed in RCC is correlated with an increase in angiogenesis markers, such as interleukin 6 (IL-6). In recent years, several groups have demonstrated the functional role of NF-кB1 in RCC tumorigenicity. Herein, we used the CRISPR/Cas-9 technique to obtain an NF-кB1 knockout-human renal adenocarcinoma cell line. Expression of IL-6 at the mRNA and protein levels was analyzed under normoxia and hypoxia by real time-polymerase chain reaction and multiplex assay, respectively. The CRISPR/Cas9 technique was effective in producing 786-0 knockout cells for NF-κB1 (p105/p50), as confirmed by western blot analysis. Suppression of p50 expression in 786-0 single guide RNA (sg)1, 786-0 sg2 and 786-0 sg3 cells downregulated IL-6 mRNA and protein expression under normoxia and hypoxia. The observed decrease in the differential expression of IL-6 in hypoxia/normoxia is suggestive of a change in cellular responsiveness to hypoxia with respect to IL-6.},
}
@article {pmid37603175,
year = {2023},
author = {Mencarelli, G and Pieroni, B and Murphy, KM and Gargaro, M},
title = {Use of CRISPR/CAS9 Technologies to Study the Role of TLR in Dendritic Cell Subsets.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2700},
number = {},
pages = {77-92},
pmid = {37603175},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *CD40 Antigens ; Cytokines ; Genes, MHC Class II ; Pathogen-Associated Molecular Pattern Molecules ; Dendritic Cells ; },
abstract = {Dendritic cells (DCs) have a significant role in coordinating both innate and adaptive immunity by serving as sentinels that detect invaders and initiate immune responses to eliminate them, as well as presenting antigens to activate adaptive immune responses that are specific to the antigen and the context in which it was detected. The regulation of DC functions is complex and involves intracellular drivers such as transcription factors and signaling pathways, as well as intercellular interactions with adhesion molecules, chemokines, and their receptors in the microenvironment. Toll-like receptors (TLRs) are crucial for DCs to detect pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways that lead to DC maturation and education in bridging with adaptive immunity, including the upregulation of MHC class II expression, induction of CD80, CD86, and CD40, and production of innate cytokines. Understanding the TLR pathways that DCs use to respond to innate immune stimuli and convert them into adaptive responses is important for new therapeutic targets identification.We present a novel platform that offers a fast and affordable CRISPR-Cas9 screening of genes that are involved in dendritic cells' TLR-dependent activation. Using CRISPR/Cas9 screening to target individual TLR genes in different dendritic cell subsets allows the identification of TLR-dependent pathways that regulate dendritic cell activation and cytokine production. This approach offers the efficient targeting of TLR driver genes to modulate the immune response and identify novel immune response regulators, establishing a causal link between these regulators and functional phenotypes based on genotypes.},
}
@article {pmid37601766,
year = {2023},
author = {Mazloum, A and Karagyaur, M and Chernyshev, R and van Schalkwyk, A and Jun, M and Qiang, F and Sprygin, A},
title = {Post-genomic era in agriculture and veterinary science: successful and proposed application of genetic targeting technologies.},
journal = {Frontiers in veterinary science},
volume = {10},
number = {},
pages = {1180621},
pmid = {37601766},
issn = {2297-1769},
abstract = {Gene editing tools have become an indispensable part of research into the fundamental aspects of cell biology. With a vast body of literature having been generated based on next generation sequencing technologies, keeping track of this ever-growing body of information remains challenging. This necessitates the translation of genomic data into tangible applications. In order to address this objective, the generated Next Generation Sequencing (NGS) data forms the basis for targeted genome editing strategies, employing known enzymes of various cellular machinery, in generating organisms with specifically selected phenotypes. This review focuses primarily on CRISPR/Cas9 technology in the context of its advantages over Zinc finger proteins (ZNF) and Transcription activator-like effector nucleases (TALEN) and meganucleases mutagenesis strategies, for use in agricultural and veterinary applications. This review will describe the application of CRISPR/Cas9 in creating modified organisms with custom-made properties, without the undesired non-targeted effects associated with virus vector vaccines and bioactive molecules produced in bacterial systems. Examples of the successful and unsuccessful applications of this technology to plants, animals and microorganisms are provided, as well as an in-depth look into possible future trends and applications in vaccine development, disease resistance and enhanced phenotypic traits will be discussed.},
}
@article {pmid37598400,
year = {2023},
author = {Hu, S and Chen, Y and Xie, D and Xu, K and Fu, Y and Chi, W and Liu, H and Huang, J},
title = {Nme2 Cas9-mediated therapeutic editing in inhibiting angiogenesis after wet age-related macular degeneration onset.},
journal = {Clinical and translational medicine},
volume = {13},
number = {8},
pages = {e1383},
pmid = {37598400},
issn = {2001-1326},
support = {31971365//National Natural Science Foundation of China/ ; 32001063//National Natural Science Foundation of China/ ; 82271688//National Natural Science Foundation of China/ ; 2019BT02Y276//Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program/ ; 2020B1515120090//Guangdong Basic and Applied Basic Research Foundation/ ; 2023A1515010176//Guangdong Basic and Applied Basic Research Foundation/ ; 2023A04J1952//Guangzhou Science and Technology Planning Project/ ; 23ptpy59//Fundamental Research Funds for the Central Universities, Sun Yat-sen University/ ; },
mesh = {Aged ; Humans ; *Vascular Endothelial Growth Factor A/genetics ; CRISPR-Cas Systems/genetics ; *Macular Degeneration/genetics/therapy ; Immunotherapy ; Gene Editing ; },
abstract = {BACKGROUND: Age-related macular degeneration (AMD), particularly wet AMD characterised by choroidal neovascularization (CNV), is a leading cause of vision loss in the elderly. The hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) pathway contributes to CNV pathogenesis. Previous gene editing research indicated that disrupting these genes in retinal pigment epithelial cells could have a preventive effect on CNV progression. However, no studies have yet been conducted using gene editing to disrupt VEGF signalling after CNV induction for therapeutic validation, which is critical to the clinical application of wet AMD gene editing therapies.<br><br>METHOD: Here, we employed the single-adeno-associated virus-mediated Nme2 Cas9 to disrupt key molecules in VEGF signalling, Hif1&#x3b1;, Vegfa and Vegfr2 after inducing CNV and estimated their therapeutic effects.<br><br>RESULTS: We found that Nme2 Cas9 made efficient editing in target genes up to 71.8% post 11 days in vivo. And only Nme2 Cas9-Vegfa treatment during the early stage of CNV development reduced the CNV lesion area by 49.5%, compared to the negative control, while Nme2 Cas9-Hif1&#x3b1; or Nme2 Cas9-Vegfr2 treatment did not show therapeutic effect. Besides, no off-target effects were observed in Nme2 Cas9-mediated gene editing in vivo.<br><br>CONCLUSIONS: This study provides proof-of-concept possibility of employing Nme2 Cas9 for potential anti-angiogenesis therapy in wet AMD.},
}
@article {pmid37596335,
year = {2023},
author = {Yuzbashev, TV and Yuzbasheva, EY and Melkina, OE and Patel, D and Bubnov, D and Dietz, H and Ledesma-Amaro, R},
title = {A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {858},
pmid = {37596335},
issn = {2399-3642},
support = {BB/R01602X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; *Metabolic Engineering ; Engineering ; Escherichia coli/genetics ; Gene Library ; },
abstract = {CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we developed the YaliCraft toolkit for Yarrowia lipolytica, which is composed of a basic set of 147 plasmids and 7 modules with different purposes. We used the toolkit to generate and characterize a library of 137 promoters and to build a de novo strain synthetizing 373.8 mg/L homogentisic acid.},
}
@article {pmid37595758,
year = {2023},
author = {Dettmer-Monaco, V and Weißert, K and Ammann, S and Monaco, G and Lei, L and Gräßel, L and Rhiel, M and Rositzka, J and Kaufmann, MM and Geiger, K and Andrieux, G and Lao, J and Thoulass, G and Schell, C and Boerries, M and Illert, AL and Cornu, TI and Ehl, S and Aichele, P and Cathomen, T},
title = {Gene editing of hematopoietic stem cells restores T cell response in familial hemophagocytic lymphohistiocytosis.},
journal = {The Journal of allergy and clinical immunology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jaci.2023.08.003},
pmid = {37595758},
issn = {1097-6825},
abstract = {BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for ∼30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene, which result in impaired degranulation of cytotoxic vesicles and hence compromised T and NK cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality.<br><br>OBJECTIVE: We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site (cSD) in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV).<br><br>METHODS: We employed CRISPR-Cas technology to delete the disease-underlying mutation in HSCs, and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and CAST-Seq based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection.<br><br>RESULTS: Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wildtype cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH.<br><br>CONCLUSION: Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T cell response in the absence of genotoxicity-associated clonal outgrowth.},
}
@article {pmid37595547,
year = {2023},
author = {Wilbanks, B and Pearson, K and Maher, LJ},
title = {A non-rational approach to optimized targeting of CRISPR-Cas9 complexes.},
journal = {Cell chemical biology},
volume = {30},
number = {8},
pages = {855-857},
doi = {10.1016/j.chembiol.2023.07.012},
pmid = {37595547},
issn = {2451-9448},
mesh = {*CRISPR-Cas Systems/genetics ; *Genomics ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {In this issue of Cell Chemical Biology, Bush et al.[1] report an in vitro selection method for optimizing CRISPR-Cas9 single-guide RNAs. This approach may be useful in targeting previously intractable genomic sequences. The results also provide insights into which positions in single-guide RNAs are most amenable to modification.},
}
@article {pmid37594269,
year = {2023},
author = {Barrangou, R},
title = {CRISPR Milestones for Sustainable Agriculture and Forestry.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {303-304},
doi = {10.1089/crispr.2023.29163.editorial},
pmid = {37594269},
issn = {2573-1602},
mesh = {*Forestry ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid35474898,
year = {2021},
author = {Kramme, C and Plesa, AM and Wang, HH and Wolf, B and Smela, MP and Guo, X and Kohman, RE and Chatterjee, P and Church, GM},
title = {An integrated pipeline for mammalian genetic screening.},
journal = {Cell reports methods},
volume = {1},
number = {6},
pages = {100082},
pmid = {35474898},
issn = {2667-2375},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Mammals/genetics ; Gene Library ; Genetic Testing ; },
abstract = {With the recent advancements in genome editing, next-generation sequencing (NGS), and scalable cloning techniques, scientists can now conduct genetic screens at unprecedented levels of scale and precision. With such a multitude of technologies, there is a need for a simple yet comprehensive pipeline to enable systematic mammalian genetic screening. In this study, we develop unique algorithms for target identification and a toxin-less Gateway cloning tool, termed MegaGate, for library cloning which, when combined with existing genetic perturbation methods and NGS-coupled readouts, enable versatile engineering of relevant mammalian cell lines. Our integrated pipeline for sequencing-based target ascertainment and modular perturbation screening (STAMPScreen) can thus be utilized for a host of cell state engineering applications.},
}
@article {pmid34935002,
year = {2021},
author = {Xiong, K and Karottki, KJC and Hefzi, H and Li, S and Grav, LM and Li, S and Spahn, P and Lee, JS and Ventina, I and Lee, GM and Lewis, NE and Kildegaard, HF and Pedersen, LE},
title = {An optimized genome-wide, virus-free CRISPR screen for mammalian cells.},
journal = {Cell reports methods},
volume = {1},
number = {4},
pages = {},
pmid = {34935002},
issn = {2667-2375},
support = {R35 GM119850/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Cricetinae ; *CRISPR-Cas Systems/genetics ; CHO Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cricetulus ; Genome ; Lentivirus/genetics ; },
abstract = {Pooled CRISPR screens have been widely applied to mammalian and other organisms to elucidate the interplay between genes and phenotypes of interest. The most popular method for delivering the CRISPR components into mammalian cells is lentivirus based. However, because lentivirus is not always an option, virus-free protocols are starting to emerge. Here, we demonstrate an improved virus-free, genome-wide CRISPR screening platform for Chinese hamster ovary cells with 75,488 gRNAs targeting 15,028 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad, resulting in a very high percentage of single gRNA insertions and minimal clonal variation. Using this platform, we perform a negative selection screen on cell proliferation that identifies 1,980 genes that affect proliferation and a positive selection screen on the toxic endoplasmic reticulum stress inducer, tunicamycin, that identifies 77 gene knockouts that improve survivability.},
}
@article {pmid37594268,
year = {2023},
author = {Karvelis, T and Siksnys, V},
title = {Fanzors: Mysterious TnpB-Like Bacterial Transposon-Related RNA-Guided DNA Nucleases of Eukaryotes.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {310-312},
doi = {10.1089/crispr.2023.29164.tka},
pmid = {37594268},
issn = {2573-1602},
mesh = {*Eukaryota ; *CRISPR-Cas Systems ; Gene Editing ; RNA ; },
}
@article {pmid37594267,
year = {2023},
author = {Willmann, MR},
title = {CRISPR and the Plant Pathologists' Holy Grail.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {308-309},
doi = {10.1089/crispr.2023.29165.mwi},
pmid = {37594267},
issn = {2573-1602},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Pathologists ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid37593347,
year = {2023},
author = {Zhang, M and Zhu, Z and Xun, G and Zhao, H},
title = {To Cut or not to Cut: Next-generation Genome Editors for Precision Genome Engineering.},
journal = {Current opinion in biomedical engineering},
volume = {28},
number = {},
pages = {},
pmid = {37593347},
issn = {2468-4511},
support = {U54 DK107965/DK/NIDDK NIH HHS/United States ; UM1 HG009402/HG/NHGRI NIH HHS/United States ; },
abstract = {Since the original report of repurposing the CRISPR/Cas9 system for genome engineering, the past decade has witnessed profound improvement in our ability to efficiently manipulate the mammalian genome. However, significant challenges lie ahead that hinder the translation of CRISPR-based gene editing technologies into safe and effective therapeutics. The CRISPR systems often have a limited target scope due to PAM restrictions, and the off-target activity also poses serious risks for therapeutic applications. Moreover, the first-generation genome editors typically achieve desired genomic modifications by inducing double-strand breaks (DSBs) at target site(s). Despite being highly efficient, this "cut and fix" strategy is less favorable in clinical settings due to drawbacks associated with the nuclease-induced DSBs. In this review, we focus on recent advances that help address these challenges, including the engineering and discovery of novel CRISPR/Cas systems with improved functionalities and the development of DSB-free genome editors.},
}
@article {pmid37591976,
year = {2023},
author = {Zhan, X and Liu, W and Nie, B and Zhang, F and Zhang, J},
title = {Cas13d-mediated multiplex RNA targeting confers a broad-spectrum resistance against RNA viruses in potato.},
journal = {Communications biology},
volume = {6},
number = {1},
pages = {855},
pmid = {37591976},
issn = {2399-3642},
support = {32271912//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31600408//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {RNA ; *Solanum tuberosum/genetics ; *RNA Viruses ; RNA Processing, Post-Transcriptional ; Archaea ; Crops, Agricultural ; },
abstract = {CRISPR-Cas systems endow the bacterial and archaeal species with adaptive immune mechanisms to fend off invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13d has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However a vast number of different viruses can potentially infect the same host plant resulting in mixed infection, thus necessitating the generation of crops with broad-spectrum resistance to multiple viruses. Here we report the repurposing of CRISPR/Cas13d coupled with an endogenous tRNA-processing system (polycistronic tRNA-gRNA, PTG) to target the multiple potato RNA viruses. Expression of Cas13d and four different gRNAs were observed in transgenic potato lines expressing the Cas13d/PTG construct. We show that the Cas13d/PTG transgenic plants exhibit resistance to either PVY, PVS, PVX or PLRV alone or two/three viruses simultaneously by reducing viral accumulation in plant cells. In sum, our findings provide an efficient strategy for engineering crops that can simultaneously resist infection by multiple RNA viruses.},
}
@article {pmid37591832,
year = {2023},
author = {Geurts, MH and Gandhi, S and Boretto, MG and Akkerman, N and Derks, LLM and van Son, G and Celotti, M and Harshuk-Shabso, S and Peci, F and Begthel, H and Hendriks, D and Schürmann, P and Andersson-Rolf, A and Chuva de Sousa Lopes, SM and van Es, JH and van Boxtel, R and Clevers, H},
title = {One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4998},
pmid = {37591832},
issn = {2041-1723},
support = {C6307/A29058//Cancer Research UK (CRUK)/ ; C6307/A29058//Cancer Research UK (CRUK)/ ; },
mesh = {Adult ; Humans ; *RNA, Guide, CRISPR-Cas Systems ; Oncogenes ; Carcinogenesis/genetics ; Cell Transformation, Neoplastic ; *Adult Stem Cells ; Organoids ; },
abstract = {Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.},
}
@article {pmid37591406,
year = {2023},
author = {Peng, K and Chen, X and Pei, K and Wang, X and Ma, X and Liang, C and Dong, Q and Liu, Z and Han, M and Liu, G and Yang, H and Zheng, M and Liu, G and Gao, M},
title = {Lipodystrophic gene Agpat2 deficiency aggravates hyperlipidemia and atherosclerosis in Ldlr[-/-] mice.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {},
number = {},
pages = {166850},
doi = {10.1016/j.bbadis.2023.166850},
pmid = {37591406},
issn = {1879-260X},
abstract = {AIMS: Dysfunction of adipose tissue increases the risk of cardiovascular disease. It was well established that obesity aggravates atherosclerosis, but the effect of adipose tissue loss on atherosclerosis has been less studied. Agpat2 is the first causative gene of congenital generalized lipodystrophy (CGL), but the role of Agpat2 on atherosclerosis has not been reported. Hypertriglyceridemia is one of the clinical manifestations of CGL patients, but it is usually absent in CGL mouse model on a normal diet. This study will investigate the effect of Agpat2 on hyperlipidemia and atherosclerosis.<br><br>METHODS AND RESULTS: In this study, Agpat2 knockout (Agpat2[-/-]) mice were generated using CRISPR/Cas system, which showed severe loss of adipose tissue and fatty liver, consistent with previous reports. Agpat2[-/-] mice were then crossed with hypercholesterolemic and atherosclerotic prone LDL receptor knockout (Ldlr[-/-]) mice to obtain double knockout mouse model (Agpat2[-/-]Ldlr[-/-]). Plasma lipid profile, insulin resistance, fatty liver, and atherosclerotic lesions were observed after 12&#x202f;weeks of the atherogenic high-fat diet (HFD) feeding. We found that compared with Ldlr[-/-] mice, Agpat2[-/-]Ldlr[-/-] mice showed significantly higher plasma total cholesterol and triglycerides after HFD feeding. Agpat2[-/-]Ldlr[-/-] mice also developed hyperglycemia and hyperinsulinemia, with increased pancreatic islet area. The liver weight of Agpat2[-/-]Ldlr[-/-] mice was about 4 times higher than that of Ldlr[-/-] mice. The liver lipid deposition was severe and Sirius red staining showed liver fibrosis. In addition, in Agpat2[-/-]Ldlr[-/-] mice, the area of atherosclerotic lesions in aortic arch and aortic root was significantly increased.<br><br>CONCLUSIONS: Our results show that Agpat2 deficiency led to more severe hyperlipidemia, liver fibrosis and aggravation of atherosclerosis in Ldlr[-/-] mice. This study provided additional insights into the role of adipose tissue in hyperlipidemia and atherosclerosis.},
}
@article {pmid37402217,
year = {2023},
author = {Tripathi, L and Ntui, VO and Tripathi, JN and Norman, D and Crawford, J},
title = {A new and novel high-fidelity genome editing tool for banana using Cas-CLOVER.},
journal = {Plant biotechnology journal},
volume = {21},
number = {9},
pages = {1731-1733},
pmid = {37402217},
issn = {1467-7652},
support = {//CGIAR Research Program for root, tubers ad banana/ ; },
mesh = {*Gene Editing ; *Musa/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; },
}
@article {pmid37378559,
year = {2023},
author = {Jiang, T and Hu, X and Shen, J},
title = {Establishment of a Novel Detection Platform for Clostridioides difficile Toxin Genes Based on Orthogonal CRISPR.},
journal = {Microbiology spectrum},
volume = {11},
number = {4},
pages = {e0188623},
pmid = {37378559},
issn = {2165-0497},
support = {Grant No. GXXT-2020-016//2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Departtment of Education/ ; Grant No. AHWJ2021a011//2021 Anhui Provincial Health and Health Commission Key Scientific Research Project/ ; },
mesh = {*Clostridioides difficile/isolation &amp; purification ; *Clostridium Infections/diagnosis ; *CRISPR-Cas Systems ; *Genetic Techniques ; Chromatography, Affinity/methods ; Sensitivity and Specificity ; Humans ; },
abstract = {Clostridioides difficile is one of the leading pathogens causing nosocomial infection. The infection can range from mild to severe, and rapid identification is pivotal for early clinical diagnosis and appropriate treatment. Here, a genetic testing platform for toxins, referred to as OC-MAB (orthogonal CRISPR system combined with multiple recombinase polymerase amplification [RPA]), was developed to detect the C. difficile toxin genes tcdA and tcdB. While recognizing the amplified products of the tcdA gene and the tcdB gene, Cas13a and Cas12a could activate their cleavage activities to cut labeled RNA and DNA probes, respectively. The cleaved products were subsequently identified by dual-channel fluorescence using a quantitative PCR (qPCR) instrument. Finally, they could also be combined with labeled antibodies on immunochromatographic test strips to achieve visual detection. The OC-MAB platform exhibited ultrahigh sensitivity in detecting the tcdA and tcdB genes at levels of as low as 10[2] to 10[1] copies/mL. When testing 72 clinical stool samples, the sensitivity (95% confidence interval [CI], 0.90, 1) and specificity (95% CI, 0.84, 1) of the single-tube method based on the fluorescence readout was 100%, with a positive predictive value (PPA) value of 100% (95% CI, 0.90, 1) and a negative predictive value (NPA) value of 100% (95% CI, 0.84, 1), compared to the results of qPCR. Likewise, the sensitivity of the 2-step method based on the test strip readout was 100% (95% CI, 0.90, 1), while the specificity was 96.3% (95% CI, 0.79, 0.99), with a PPA of 98% (95% CI, 0.87, 0.99) and an NPA of 100% (95% CI, 0.90, 1). In short, orthogonal CRISPR technology is a promising tool for the detection of C. difficile toxin genes. IMPORTANCE C. difficile is currently the primary causative agent of hospital-acquired antibiotic-induced diarrhea, and timely and accurate diagnosis is crucial for hospital-acquired infection control and epidemiological investigation. Here, a new method for the identification of C. difficile was developed based on the recently popular CRISPR technology, and an orthogonal CRISPR dual system was utilized for the simultaneous detection of toxin genes A and B. It also uses a currently rare CRISPR dual-target lateral flow strip with powerful color-changing capabilities, which is appropriate for point-of-care testing (POCT).},
}
@article {pmid37323119,
year = {2023},
author = {Liu, X and Yu, Y and Yao, W and Yin, Z and Wang, Y and Huang, Z and Zhou, JQ and Liu, J and Lu, X and Wang, F and Zhang, G and Chen, G and Xiao, Y and Deng, H and Tang, W},
title = {CRISPR/Cas9-mediated simultaneous mutation of three salicylic acid 5-hydroxylase (OsS5H) genes confers broad-spectrum disease resistance in rice.},
journal = {Plant biotechnology journal},
volume = {21},
number = {9},
pages = {1873-1886},
pmid = {37323119},
issn = {1467-7652},
support = {32172078//National Natural Science Foundation of China/ ; U22A20502//National Natural Science Foundation of China/ ; 2021JJ40238//Natural Science Foundation of Hunan Province/ ; 2022JJ30285//Natural Science Foundation of Hunan Province/ ; 2021JC0007//the Science and Technology Innovation Program of Hunan Province/ ; 2021NK1001//the Science and Technology Innovation Program of Hunan Province/ ; 2023NK1010//the Science and Technology Innovation Program of Hunan Province/ ; },
mesh = {Disease Resistance/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; Salicylic Acid/pharmacology/metabolism ; Mutation/genetics ; *Oryza/metabolism ; Plant Diseases/microbiology ; *Xanthomonas ; Gene Expression Regulation, Plant ; },
abstract = {Salicylic acid (SA) is an essential plant hormone that plays critical roles in basal defence and amplification of local immune responses and establishes resistance against various pathogens. However, the comprehensive knowledge of the salicylic acid 5-hydroxylase (S5H) in rice-pathogen interaction is still elusive. Here, we reported that three OsS5H homologues displayed salicylic acid 5-hydroxylase activity, converting SA into 2,5-dihydroxybenzoic acid (2,5-DHBA). OsS5H1, OsS5H2, and OsS5H3 were preferentially expressed in rice leaves at heading stage and responded quickly to exogenous SA treatment. We found that bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) strongly induced the expression of OsS5H1, OsS5H2, and OsS5H3. Rice plants overexpressing OsS5H1, OsS5H2, and OsS5H3 showed significantly decreased SA contents and increased 2,5-DHBA levels, and were more susceptible to bacterial blight and rice blast. A simple single guide RNA (sgRNA) was designed to create oss5h1oss5h2oss5h3 triple mutants through CRISPR/Cas9-mediated gene mutagenesis. The oss5h1oss5h2oss5h3 exhibited stronger resistance to Xoo than single oss5h mutants. And oss5h1oss5h2oss5h3 plants displayed enhanced rice blast resistance. The conferred pathogen resistance in oss5h1oss5h2oss5h3 was attributed to the significantly upregulation of OsWRKY45 and pathogenesis-related (PR) genes. Besides, flg22-induced reactive oxygen species (ROS) burst was enhanced in oss5h1oss5h2oss5h3. Collectively, our study provides a fast and effective approach to generate rice varieties with broad-spectrum disease resistance through OsS5H gene editing.},
}
@article {pmid37590503,
year = {2023},
author = {Craft, J and Truong, T and Penn, BH},
title = {High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {198},
pages = {},
doi = {10.3791/65264},
pmid = {37590503},
issn = {1940-087X},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Macrophages ; Cell Line ; Electroporation ; },
abstract = {Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the physiology of macrophages in vivo more closely than immortalized macrophage cell lines and can be derived from mice already carrying defined genetic changes. However, disrupting gene function in BMDMs remains technically challenging. Here, we provide a protocol for efficient CRISPR/Cas9 genome editing in BMDMs, which allows for the introduction of small insertions and deletions (indels) that result in frameshift mutations that disrupt gene function. The protocol describes how to synthesize single-guide RNAs (sgRNA-Cas9) and form purified sgRNA-Cas9 ribonucleoprotein complexes (RNPs) that can be delivered by electroporation. It also provides an efficient method for monitoring editing efficiency using routine Sanger sequencing and a freely available online analysis program. The protocol can be performed within 1 week and does not require plasmid construction; it typically results in 85% to 95% editing efficiency.},
}
@article {pmid37590316,
year = {2023},
author = {Dardiry, M and Eberhard, G and Witte, H and Rödelsperger, C and Lightfoot, JW and Sommer, RJ},
title = {Divergent combinations of cis-regulatory elements control the evolution of phenotypic plasticity.},
journal = {PLoS biology},
volume = {21},
number = {8},
pages = {e3002270},
doi = {10.1371/journal.pbio.3002270},
pmid = {37590316},
issn = {1545-7885},
abstract = {The widespread occurrence of phenotypic plasticity across all domains of life demonstrates its evolutionary significance. However, how plasticity itself evolves and how it contributes to evolution is poorly understood. Here, we investigate the predatory nematode Pristionchus pacificus with its feeding structure plasticity using recombinant-inbred-line and quantitative-trait-locus (QTL) analyses between natural isolates. We show that a single QTL at a core developmental gene controls the expression of the cannibalistic morph. This QTL is composed of several cis-regulatory elements. Through CRISPR/Cas-9 engineering, we identify copy number variation of potential transcription factor binding sites that interacts with a single intronic nucleotide polymorphism. Another intronic element eliminates gene expression altogether, mimicking knockouts of the locus. Comparisons of additional isolates further support the rapid evolution of these cis-regulatory elements. Finally, an independent QTL study reveals evidence for parallel evolution at the same locus. Thus, combinations of cis-regulatory elements shape plastic trait expression and control nematode cannibalism.},
}
@article {pmid37588594,
year = {2023},
author = {van der Wal, YA and Nordli, H and Akandwanaho, A and Greiner-Tollersrud, L and Kool, J and Jørgensen, JB},
title = {CRISPR-Cas- induced IRF3 and MAVS knockouts in a salmonid cell line disrupt PRR signaling and affect viral replication.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1214912},
pmid = {37588594},
issn = {1664-3224},
mesh = {Animals ; *Salmonidae/genetics ; CRISPR-Cas Systems ; Signal Transduction ; Cell Line ; Salmon/genetics ; *Infectious pancreatic necrosis virus ; Mammals ; },
abstract = {BACKGROUND: Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means.<br><br>METHODS: By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection.<br><br>RESULTS: We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication.<br><br>CONCLUSION: Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.},
}
@article {pmid37588264,
year = {2023},
author = {Huang, S and Baskin, JM},
title = {Adding a Chemical Biology Twist to CRISPR Screening.},
journal = {Israel journal of chemistry},
volume = {63},
number = {1-2},
pages = {},
pmid = {37588264},
issn = {0021-2148},
support = {R01 GM131101/GM/NIGMS NIH HHS/United States ; R01 GM143367/GM/NIGMS NIH HHS/United States ; T32 GM138826/GM/NIGMS NIH HHS/United States ; },
abstract = {In less than a decade, CRISPR screening has revolutionized forward genetics and cell and molecular biology. Advances in screening technologies, including sgRNA libraries, Cas9-expressing cell lines, and streamlined sequencing pipelines, have democratized pooled CRISPR screens at genome-wide scale. Initially, many such screens were survival-based, identifying essential genes in physiological or perturbed processes. With the application of new chemical biology tools to CRISPR screening, the phenotypic space is no longer limited to live/dead selection or screening for levels of conventional fluorescent protein reporters. Further, the resolution has been increased from cell populations to single cells or even the subcellular level. We highlight advances in pooled CRISPR screening, powered by chemical biology, that have expanded phenotypic space, resolution, scope, and scalability as well as strengthened the CRISPR/Cas enzyme toolkit to enable biological hypothesis generation and discovery.},
}
@article {pmid37587688,
year = {2023},
author = {Li, X and Liao, F and Gao, J and Song, G and Zhang, C and Ji, N and Wang, X and Wen, J and He, J and Wei, Y and Zhang, H and Li, Z and Yu, G and Yin, H},
title = {Inhibitory mechanism of CRISPR-Cas9 by AcrIIC4.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkad669},
pmid = {37587688},
issn = {1362-4962},
support = {2022YFC3400400//National Key R&amp;D Program of China/ ; 32200496//National Natural Science Foundation of China/ ; 2022KJ192//Scientific Research Program of Tianjin Municipal Education Commission/ ; ZRMS2022000096//Hubei Provincial Natural Science Foundation/ ; //Scientific Research Program of Hubei Provincial Department of Education/ ; },
abstract = {CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti-CRISPR (Acr) proteins to inactivate the CRISPR-Cas systems. Recently, AcrIIC4, identified from Haemophilus parainfluenzae phage, has been reported to inhibit the endonuclease activity of Cas9 from Neisseria meningitidis (NmeCas9), but the inhibition mechanism is not clear. Here, we biochemically and structurally investigated the anti-CRISPR activity of AcrIIC4. AcrIIC4 folds into a helix bundle composed of three helices, which associates with the REC lobe of NmeCas9 and sgRNA. The REC2 domain of NmeCas9 is locked by AcrIIC4, perturbing the conformational dynamics required for the target DNA binding and cleavage. Furthermore, mutation of the key residues in the AcrIIC4-NmeCas9 and AcrIIC4-sgRNA interfaces largely abolishes the inhibitory effects of AcrIIC4. Our study offers new insights into the mechanism of AcrIIC4-mediated suppression of NmeCas9 and provides guidelines for the design of regulatory tools for Cas9-based gene editing applications.},
}
@article {pmid37587435,
year = {2023},
author = {Liu, W and Wang, X and Liu, R and Liao, Y and Peng, Z and Jiang, H and Jing, Q and Xing, Y},
title = {Efficient delivery of a large-size Cas9-EGFP vector in porcine fetal fibroblasts using a Lonza 4D-Nucleofector system.},
journal = {BMC biotechnology},
volume = {23},
number = {1},
pages = {29},
pmid = {37587435},
issn = {1472-6750},
support = {2016ZX08006-003//National Science and Technology Major Project/ ; 31771372//National Natural Science Foundation of China/ ; },
mesh = {Swine ; Animals ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Fetus ; Electroporation ; Fibroblasts ; },
abstract = {BACKGROUND: Porcine fetal fibroblasts (PFFs) are important donor cells for generating genetically modified pigs, but the transfection efficiencies of PFFs are often unsatisfactory especially when large-size vectors are to be delivered. In this study, we aimed to optimize the transfection conditions for delivery of a large-size vector in PFFs using Lonza 4D-Nucleofector™ vessels and strips.<br><br>METHODS: We firstly delivered a 13&#xa0;kb Cas9-EGFP and a 3.5&#xa0;kb pMAX-GFP vector into PFFs via 7 programs recommended by the Lonza basic protocol. We then tested 6 customized dual-electroporation programs for delivering the 13&#xa0;kb plasmid into PFFs. In addition, we screened potential alternative electroporation buffers to the Nucleofector&#x2122; P3 solution. Finally, three CRISPR/Cas9-sgRNAs targeting Rosa26, H11, and Cep112 loci were delivered into PFFs with different single and dual-electroporation programs.<br><br>RESULTS: Notably lower transfection efficiencies were observed when delivering the 13&#xa0;kb vector than delivering the 3.5&#xa0;kb vector in PFFs via the single-electroporation programs. The customized dual-electroporation program FF-113&#x2009;+&#x2009;CA-137 exhibited higher transfection efficiencies than any of the single-electroporation programs using vessels (98.1%) or strips (89.1%) with acceptable survival rates for the 13&#xa0;kb vector. Entranster-E buffer generated similar transfection efficiencies and 24-hour survival rates to those from the P3 solution, thus can be used as an alternative electroporation buffer. In the genome-editing experiments, the FF-113&#x2009;+&#x2009;CA-137 and CA-137&#x2009;+&#x2009;CA-137 programs showed significantly superior (P&#x2009;<&#x2009;0.01) efficiencies to ones from the single-electroporation programs in vessels and strips. Entranster-E buffer produced higher indel efficiencies than the P3 buffer.<br><br>CONCLUSIONS: We markedly increased the delivery efficiencies for a large vector via customized dual-electroporation programs using Lonza 4D-Nucleofector&#x2122; system, and Entranster-E buffer can be used as an alternative electroporation buffer to Nucleofector&#x2122; P3 buffer.},
}
@article {pmid37584388,
year = {2023},
author = {Doering, L and Cornean, A and Thumberger, T and Benjaminsen, J and Wittbrodt, B and Kellner, T and Hammouda, OT and Gorenflo, M and Wittbrodt, J and Gierten, J},
title = {CRISPR-based knockout and base editing confirm the role of MYRF in heart development and congenital heart disease.},
journal = {Disease models &amp; mechanisms},
volume = {16},
number = {8},
pages = {},
doi = {10.1242/dmm.049811},
pmid = {37584388},
issn = {1754-8411},
support = {810172/ERC_/European Research Council/International ; },
mesh = {Humans ; Child ; *Gene Editing ; Mutation/genetics ; Point Mutation ; Transcription Factors/metabolism ; *Heart Defects, Congenital/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite for translating genomic data into clinical care. We used CRISPR-Cas9-mediated targeting of 12 candidate genes in the vertebrate model medaka (Oryzias latipes), five of which displayed a novel cardiovascular phenotype spectrum in F0 (crispants): mapre2, smg7, cdc42bpab, ankrd11 and myrf, encoding a transcription factor recently linked to cardiac-urogenital syndrome. Our myrf mutant line showed particularly prominent embryonic cardiac defects recapitulating phenotypes of pediatric patients, including hypoplastic ventricle. Mimicking human mutations, we edited three sites to generate specific myrf single-nucleotide variants via cytosine and adenine base editors. The Glu749Lys missense mutation in the conserved intramolecular chaperon autocleavage domain fully recapitulated the characteristic myrf mutant phenotype with high penetrance, underlining the crucial function of this protein domain. The efficiency and scalability of base editing to model specific point mutations accelerate gene validation studies and the generation of human-relevant disease models.},
}
@article {pmid37584144,
year = {2023},
author = {Chen, L and Deng, L and Sun, W and Liu, J and Zhang, T and Li, S},
title = {[Development of a tau-V337M mouse model using CRISPR/Cas9 system and enhanced ssODN-mediated recombination].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {7},
pages = {3003-3014},
doi = {10.13345/j.cjb.221052},
pmid = {37584144},
issn = {1872-2075},
mesh = {Animals ; Male ; Female ; Mice ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Rad51 Recombinase/genetics ; Mice, Inbred C57BL ; Disease Models, Animal ; Recombination, Genetic ; },
abstract = {The generation of a tau-V337M point mutation mouse model using gene editing technology can provide an animal model with fast disease progression and more severe symptoms, which facilitate the study of pathogenesis and treatment of Alzheimer's disease (AD). In this study, single guide RNAs (sgRNA) and single-stranded oligonucleotides (ssODN) were designed and synthesized in vitro. The mixture of sgRNA, Cas9 protein and ssODN was microinjected into the zygotes of C57BL/6J mice. After DNA cutting and recombination, the site homologous to human 337 valine (GTG) in exon 11 was mutated into methionine (ATG). In order to improve the efficiency of recombination, a Rad51 protein was added. The female mice mated with the nonvasectomy male mice were used as the surrogates. Subsequently, the 2-cell stage gene edited embryos were transferred into the unilateral oviduct, and the F0 tau-V337M mutation mice were obtained. Higher mutation efficiency could be obtained by adding Rad51 protein. The F0 tau-V337M point mutation mice can pass the mutation on to the F1 generation mice. In conclusion, this study successfully established the first tau-V337M mutation mouse by using Cas9, ssODN and Rad51. These results provide a new method for developing AD mice model which can be used in further research on the pathogenesis and treatment of AD.},
}
@article {pmid37552581,
year = {2023},
author = {Torello Pianale, L and Olsson, L},
title = {ScEnSor Kit for Saccharomyces cerevisiae Engineering and Biosensor-Driven Investigation of the Intracellular Environment.},
journal = {ACS synthetic biology},
volume = {12},
number = {8},
pages = {2493-2497},
doi = {10.1021/acssynbio.3c00124},
pmid = {37552581},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems ; Plasmids/genetics ; Genome, Fungal ; },
abstract = {In this study, the three-step build-transform-assess toolbox for real-time monitoring of the yeast intracellular environment has been expanded and upgraded to the two-module ScEnSor (S. cerevisiae Engineering + Biosensor) Kit. The Biosensor Module includes eight fluorescent reporters for the intracellular environment; three of them (unfolded protein response, pyruvate metabolism, and ethanol consumption) were newly implemented to complement the original five. The Genome-Integration Module comprises a set of backbone plasmids for the assembly of 1-6 transcriptional units (each consisting of promoter, coding sequence, and terminator) for efficient marker-free single-locus genome integration (in HO and/or X2 loci). Altogether, the ScEnSor Kit enables rapid and easy construction of strains with new transcriptional units as well as high-throughput investigation of the yeast intracellular environment.},
}
@article {pmid37531174,
year = {2023},
author = {Yan, H and Han, S and Hughes, S and Zeng, Y},
title = {Extraction-free, one-pot CRISPR/Cas12a detection of microRNAs directly from extracellular vesicles.},
journal = {Chemical communications (Cambridge, England)},
volume = {59},
number = {67},
pages = {10165-10168},
doi = {10.1039/d3cc02982d},
pmid = {37531174},
issn = {1364-548X},
mesh = {*MicroRNAs/genetics ; CRISPR-Cas Systems/genetics ; *Extracellular Vesicles ; },
abstract = {Current methods for extracellular vesicle (EV) miRNA analysis mostly require RNA extraction, which results in a multi-step, time-consuming workflow. This study reports an extraction-free method that combines thermolysis treatment of EVs with a one-pot EXTRA-CRISPR assay, enabling the vastly simplified analysis of EV miRNAs with a comparable performance to that of the extraction-based assays.},
}
@article {pmid37523223,
year = {2023},
author = {Li, G and Qi, Y},
title = {CRISPR Empowers Tree Bioengineering for a Sustainable Future.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {305-307},
doi = {10.1089/crispr.2023.29161.gli},
pmid = {37523223},
issn = {2573-1602},
mesh = {*Trees ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Bioengineering ; },
}
@article {pmid37505910,
year = {2023},
author = {Ji, H and Xiong, W and Guo, S and Wang, S and Xing, X and Tian, T and Zhou, X},
title = {Isonitrile-Tetrazine Click-and-Release Chemistry for Controlling RNA-Guided Nucleic Acid Cleavage.},
journal = {ACS chemical biology},
volume = {18},
number = {8},
pages = {1829-1837},
doi = {10.1021/acschembio.3c00255},
pmid = {37505910},
issn = {1554-8937},
mesh = {Humans ; *RNA ; RNA, Guide, CRISPR-Cas Systems ; *Heterocyclic Compounds ; Gene Editing ; Click Chemistry ; },
abstract = {With the increasing demand for the regulation of CRISPR systems, a considerable number of studies have been conducted to control their excessive activity levels. In this context, we propose a method that involves a bioorthogonal cleavage reaction between isonitrile and tetrazine to modulate the cleavage activity of the CRISPR system. Importantly, isonitrile demonstrates significant potential for modifying sgRNAs, making it a promising candidate for bioorthogonal reactions, a phenomenon that has not been previously reported. Our approach utilizes the 3-isocyanopropyl-carbonate group as a caging group to deactivate the CRISPR systems, while tetrazine acts as an activator to restore their activities. Through the implementation of post-synthetic modifications and click-and-release chemistry, we have successfully achieved the regulation of RNA-guided nucleic acid cleavage, which holds great promise for controlling gene editing in human cells.},
}
@article {pmid37486342,
year = {2023},
author = {Gossing, M and Limeta, A and Skrekas, C and Wigglesworth, M and Davis, A and Siewers, V and David, F},
title = {Multiplexed Guide RNA Expression Leads to Increased Mutation Frequency in Targeted Window Using a CRISPR-Guided Error-Prone DNA Polymerase in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {12},
number = {8},
pages = {2271-2277},
doi = {10.1021/acssynbio.2c00689},
pmid = {37486342},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics ; *Mutation Rate ; CRISPR-Cas Systems/genetics ; DNA ; DNA-Directed DNA Polymerase/genetics ; RNA ; Mutation ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, with its ability to target a specific DNA locus using guide RNAs (gRNAs), is particularly suited for targeted mutagenesis. The targeted diversification of nucleotides in Saccharomyces cerevisiae using a CRISPR-guided error-prone DNA polymerase─called yEvolvR─was recently reported. Here, we investigate the effect of multiplexed expression of gRNAs flanking a short stretch of DNA on reversion and mutation frequencies using yEvolvR. Phenotypic assays demonstrate that higher reversion frequencies are observed when expressing multiple gRNAs simultaneously. Next generation sequencing reveals a synergistic effect of multiple gRNAs on mutation frequencies, which is more pronounced in a mutant with a partially defective DNA mismatch repair system. Additionally, we characterize a galactose-inducible yEvolvR, which enables temporal control of mutagenesis. This study demonstrates that multiplex expression of gRNAs and induction of mutagenesis greatly improves the capabilities of yEvolvR for generation of genetic libraries in vivo.},
}
@article {pmid37486333,
year = {2023},
author = {Skrekas, C and Limeta, A and Siewers, V and David, F},
title = {Targeted In Vivo Mutagenesis in Yeast Using CRISPR/Cas9 and Hyperactive Cytidine and Adenine Deaminases.},
journal = {ACS synthetic biology},
volume = {12},
number = {8},
pages = {2278-2289},
doi = {10.1021/acssynbio.2c00690},
pmid = {37486333},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Mutagenesis/genetics ; Mutagenesis, Site-Directed ; Gene Editing ; },
abstract = {Directed evolution is a preferred strategy to improve the function of proteins such as enzymes that act as bottlenecks in metabolic pathways. Common directed evolution approaches rely on error-prone PCR-based libraries where the number of possible variants is usually limited by cellular transformation efficiencies. Targeted in vivo mutagenesis can advance directed evolution approaches and help to overcome limitations in library generation. In the current study, we aimed to develop a high-efficiency time-controllable targeted mutagenesis toolkit in the yeast Saccharomyces cerevisiae by employing the CRISPR/Cas9 technology. To that end, we fused the dCas9 protein with hyperactive variants of adenine and cytidine deaminases aiming to create an inducible CRISPR-based mutagenesis tool targeting a specific DNA sequence in vivo with extended editing windows and high mutagenesis efficiency. We also investigated the effect of guide RNA multiplexing on the mutagenesis efficiency both phenotypically and on the DNA level.},
}
@article {pmid37429825,
year = {2023},
author = {Abdelhady, AM and Phillips, JA and Xu, Y and Stroh, M},
title = {Clinical Pharmacology and Translational Considerations in the Development of CRISPR-Based Therapies.},
journal = {Clinical pharmacology and therapeutics},
volume = {114},
number = {3},
pages = {591-603},
doi = {10.1002/cpt.3000},
pmid = {37429825},
issn = {1532-6535},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Pharmacology, Clinical ; Gene Editing/methods ; },
abstract = {Genome editing holds the potential for curative treatments of human disease, however, clinical realization has proven to be a challenging journey with incremental progress made up until recently. Over the last decade, advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have provided the necessary breakthrough for genome editing in the clinic. The progress of investigational CRISPR therapies from bench to bedside reflects the culmination of multiple advances occurring in parallel, several of which intersect with clinical pharmacology and translation. Directing the CRISPR therapy to the intended site of action has necessitated novel delivery platforms, and this has resulted in special considerations for the complete characterization of distribution, metabolism, and excretion, as well as immunogenicity. Once at the site of action, CRISPR therapies aim to make permanent alterations to the genome and achieve therapeutically relevant effects with a single dose. This fundamental aspect of the mechanism of action for CRISPR therapies results in new considerations for clinical translation and dose selection. Early advances in model-informed development of CRISPR therapies have incorporated key facets of the mechanism of action and have captured hallmark features of clinical pharmacokinetics and pharmacodynamics from phase I investigations. Given the recent emergence of CRISPR therapies in clinical development, the landscape continues to evolve rapidly with ample opportunity for continued innovation. Here, we provide a snapshot of selected topics in clinical pharmacology and translation that has supported the advance of systemically administered in vivo and ex vivo CRISPR-based investigational therapies in the clinic.},
}
@article {pmid37406248,
year = {2023},
author = {Monte, DFM},
title = {My CRISPR Story: Back to Brazil.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {313-315},
doi = {10.1089/crispr.2023.0032},
pmid = {37406248},
issn = {2573-1602},
mesh = {Brazil ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; },
}
@article {pmid37402223,
year = {2023},
author = {Whitford, CM and Gren, T and Palazzotto, E and Lee, SY and Tong, Y and Weber, T},
title = {Systems Analysis of Highly Multiplexed CRISPR-Base Editing in Streptomycetes.},
journal = {ACS synthetic biology},
volume = {12},
number = {8},
pages = {2353-2366},
doi = {10.1021/acssynbio.3c00188},
pmid = {37402223},
issn = {2161-5063},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Actinomycetales/genetics ; Systems Analysis ; },
abstract = {CRISPR tools, especially Cas9n-sgRNA guided cytidine deaminase base editors such as CRISPR-BEST, have dramatically simplified genetic manipulation of streptomycetes. One major advantage of CRISPR base editing technology is the possibility to multiplex experiments in genomically instable species. Here, we demonstrate scaled up Csy4 based multiplexed genome editing using CRISPR-mcBEST in Streptomyces coelicolor. We evaluated the system by simultaneously targeting 9, 18, and finally all 28 predicted specialized metabolite biosynthetic gene clusters in a single experiment. We present important insights into the performance of Csy4 based multiplexed genome editing at different scales. Using multiomics analysis, we investigated the systems wide effects of such extensive editing experiments and revealed great potentials and important bottlenecks of CRISPR-mcBEST. The presented analysis provides crucial data and insights toward the development of multiplexed base editing as a novel paradigm for high throughput engineering of Streptomyces chassis and beyond.},
}
@article {pmid37347931,
year = {2023},
author = {Lamothe, G and Carbonneau, J and Joly Beauparlant, C and Vincent, T and Quessy, P and Guedon, A and Kobinger, G and Lemay, JF and Boivin, G and Droit, A and Turgeon, N and Tremblay, JP},
title = {Rapid and Technically Simple Detection of SARS-CoV-2 Variants Using CRISPR Cas12 and Cas13.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {369-385},
doi = {10.1089/crispr.2023.0007},
pmid = {37347931},
issn = {2573-1602},
mesh = {Humans ; *SARS-CoV-2/genetics ; *COVID-19/diagnosis/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {The worldwide proliferation of the SARS-CoV-2 virus in the past 3 years has allowed the virus to accumulate numerous mutations. Dangerous lineages have emerged one after another, each leading to a new wave of the pandemic. In this study, we have developed the THRASOS pipeline to rapidly discover lineage-specific mutation signatures and thus advise the development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostic tests. We also optimized a strategy to modify loop-mediated isothermal amplification amplicons for downstream use with Cas12 and Cas13 for future multiplexing. The close ancestry of the BA.1 and BA.2 variants of SARS-CoV-2 (Omicron) made these excellent candidates for the development of a first test using this workflow. With a quick turnaround time and low requirements for laboratory equipment, the test we have created is ideally suited for settings such as mobile clinics lacking equipment such as Next-Generation Sequencers or Sanger Sequencers and the personnel to run these devices.},
}
@article {pmid37335046,
year = {2023},
author = {Luo, M and Ma, J and Cheng, X and Wu, S and Bartels, DJ and Guay, D and Engelhardt, JF and Liu, X},
title = {Genome Editing in Ferret Airway Epithelia Mediated by CRISPR/Nucleases Delivered with Amphiphilic Shuttle Peptides.},
journal = {Human gene therapy},
volume = {34},
number = {15-16},
pages = {705-718},
doi = {10.1089/hum.2023.016},
pmid = {37335046},
issn = {1557-7422},
support = {R01 HL165404/HL/NHLBI NIH HHS/United States ; P01 HL152960/HL/NHLBI NIH HHS/United States ; 75N92019C00010/HL/NHLBI NIH HHS/United States ; P30 DK054759/DK/NIDDK NIH HHS/United States ; P01 HL158506/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Gene Editing/methods ; Ferrets/genetics ; Epithelium ; Peptides/genetics ; *Lung Diseases/genetics ; CRISPR-Cas Systems ; },
abstract = {Gene editing strategies are attractive for treating genetic pulmonary diseases such as cystic fibrosis (CF). However, challenges have included the development of safe and effective vector systems for gene editing of airway epithelia and model systems to report their efficiency and durability. The domestic ferret (Mustela putorius furo) has a high degree of conservation in lung cellular anatomy with humans, and has served as an excellent model for many types of lung diseases, including CF. In this study, we evaluated the efficiency of amphiphilic shuttle peptide S10 for protein delivery and gene editing using SpCas9, and AsCas12a (Cpf1) ribonucleoproteins (RNPs). These approaches were evaluated in proliferating ferret airway basal cells, polarized airway epithelia in vitro, and lungs in vivo, by accessing the editing efficiency using reporter ferrets and measuring indels at the ferret CFTR locus. Our results demonstrate that shuttle peptides efficiently enable delivery of reporter proteins/peptides and gene editing SpCas9 or Cpf1 RNP complexes to ferret airway epithelial cells in vitro and in vivo. We measured S10 delivery efficiency of green fluorescent protein (GFP)-nuclear localization signal (NLS) protein or SpCas9 RNP into ferret airway basal cells and fully differentiated ciliated and nonciliated epithelial cells in vitro. In vitro and in vivo gene editing efficiencies were determined by Cas/LoxP-gRNA RNP-mediated conversion of a ROSA-TG Cre recombinase reporter using transgenic primary cells and ferrets. S10/Cas9 RNP was more effective, relative to S10/Cpf1 RNP at gene editing of the ROSA-TG locus. Intratracheal lung delivery of the S10 shuttle combined with GFP-NLS protein or D-Retro-Inverso (DRI)-NLS peptide demonstrated efficiencies of protein delivery that were ∼3-fold or 14-fold greater, respectively, than the efficiency of gene editing at the ROSA-TG locus using S10/Cas9/LoxP-gRNA. Cpf1 RNPs was less effective than SpCas9 at gene editing of LoxP locus. These data demonstrate the feasibility of shuttle peptide delivery of Cas RNPs to the ferret airways and the potential utility for developing ex vivo stem cell-based and in vivo gene editing therapies for genetic pulmonary diseases such as CF.},
}
@article {pmid37307061,
year = {2023},
author = {Chen, YH and Sharma, S and Bewg, WP and Xue, LJ and Gizelbach, CR and Tsai, CJ},
title = {Multiplex Editing of the Nucleoredoxin1 Tandem Array in Poplar: From Small Indels to Translocations and Complex Inversions.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {339-349},
doi = {10.1089/crispr.2022.0096},
pmid = {37307061},
issn = {2573-1602},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Populus/genetics ; DNA Copy Number Variations ; Mutagenesis ; Translocation, Genetic/genetics ; },
abstract = {The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. In this study, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ∼100 kb in Populus tremula × Populus alba. We demonstrated efficient multiplex editing with one single guide RNA in 42 transgenic lines. The mutation profiles ranged from small insertions and deletions and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid future functional characterization.},
}
@article {pmid37276175,
year = {2023},
author = {Huang, L and Yang, C and Chen, Y and Deng, H and Liao, Z and Xiao, H},
title = {CRISPR-Mediated Base Editing: Promises and Challenges for a Viable Oncotherapy Strategy.},
journal = {Human gene therapy},
volume = {34},
number = {15-16},
pages = {669-681},
doi = {10.1089/hum.2023.045},
pmid = {37276175},
issn = {1557-7422},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; DNA/genetics ; },
abstract = {Base editing technology, developed from the CRISPR/Cas9 system, is able to efficiently implement single-base substitutions at specific DNA or RNA sites without generating double-strand breaks with precision and efficiency. Point mutations account for 58% of disease-causing genetic mutations in humans, and single nucleotide variants are an important cause of tumorigenesis, and the advent of base editors offers new hope for the study or treatment of such diseases. Although it has some limitations, base editors have been continuously improved in terms of editing efficiency, specificity, and product purity since their development. In this review, we examine the main base editing technologies and discuss their applications and prospects in tumor research and therapy, as well as elaborate on their mode of delivery.},
}
@article {pmid36912815,
year = {2023},
author = {Mayes, CM and Santarpia, JL},
title = {Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2.},
journal = {The CRISPR journal},
volume = {6},
number = {4},
pages = {359-368},
doi = {10.1089/crispr.2022.0095},
pmid = {36912815},
issn = {2573-1602},
mesh = {Humans ; SARS-CoV-2/genetics ; *Coronavirus 229E, Human/genetics ; *Coronavirus OC43, Human/genetics ; *COVID-19/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined that several CRISPR targets significantly reduce viral titer, despite the presence of single nucleotide polymorphisms in the gRNA when compared with a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% and >99% in HCoV-OC43, between 78% and >99% in HCoV-229E, and between 70% and 94% in SARS-CoV-2 when compared with an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system that is capable of reducing viable virus in both Risk Group 2 and Risk Group 3 HCoV pathogens.},
}
@article {pmid37584115,
year = {2023},
author = {Xue, S and Wang, S and Liu, L and Zhong, Q and Cheng, Z and Xiao, S},
title = {[Precision gene editing technologies based on CRISPR/Cas9: a review].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {39},
number = {7},
pages = {2566-2578},
doi = {10.13345/j.cjb.220879},
pmid = {37584115},
issn = {1872-2075},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Mutation ; Genome ; },
abstract = {Gene editing technology is a genetic operation technology that can modify the DNA sequence at the genomic level. The precision gene editing technology based on CRISPR/Cas9 system is a gene editing technology that is easy to operate and widely used. Unlike the traditional CRISPR/Cas9 system, the precision gene editing technology can perform site-directed mutation of genes without DNA template. This review summarizes the recent development of precision gene editing technology based on CRISPR/Cas9, and prospects the challenges and opportunities of this technology.},
}
@article {pmid37583711,
year = {2023},
author = {Wang, Z and Wang, Z and Zhang, F and Wu, L},
title = {Thermus thermophilus Argonaute-based signal amplifier for highly sensitive and specific microRNA detection.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1221943},
pmid = {37583711},
issn = {2296-4185},
abstract = {The prokaryote-derived gene defense system as a new generation of nucleic acid detection tool exhibits impressive performance in the field of molecular diagnosis. Prokaryotic Argonaute (Ago) is a CRISPR-associated protein that is guided by a short DNA (gDNA) and then efficiently cleaves gDNA-complementary nucleic acids and presents unique characteristics that are different from the CRISPR/Cas system. However, the application of Ago in biosensing is still relatively scarce, and many properties of Ago need to be further clarified. In this study, we aim to systematically explore the properties of Thermus thermophilus Argonaute (TtAgo), including the dependence of TtAgo activity on guide DNA (gDNA) length, substrates' length, and the position of gDNA complementary region on the substrate. Based on these properties, we constructed an exonuclease III-assisted target-recycled amplification system (exoAgo) for sensitive miRNA detection. The result showed that exoAgo can be used for miRNA profiling with a detection limit of 12.2 pM and single-base-resolution and keep good performance for the detection of complex samples, which indicates that Ago has great application potential in the detection of nucleic acids. In conclusion, this study will provide guidance for further development and utilization of Ago in the field of biosensing.},
}
@article {pmid37582836,
year = {2023},
author = {Tao, Y and Lamas, V and Du, W and Zhu, W and Li, Y and Whittaker, MN and Zuris, JA and Thompson, DB and Rameshbabu, AP and Shu, Y and Gao, X and Hu, JH and Pei, C and Kong, WJ and Liu, X and Wu, H and Kleinstiver, BP and Liu, DR and Chen, ZY},
title = {Treatment of monogenic and digenic dominant genetic hearing loss by CRISPR-Cas9 ribonucleoprotein delivery in vivo.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4928},
pmid = {37582836},
issn = {2041-1723},
support = {UG3 AI150551/AI/NIAID NIH HHS/United States ; R01 DC012115/DC/NIDCD NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 DC019404/DC/NIDCD NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; U24 HG010423/HG/NHGRI NIH HHS/United States ; UH3 TR002636/TR/NCATS NIH HHS/United States ; R01 DC016875/DC/NIDCD NIH HHS/United States ; R01 DC005575/DC/NIDCD NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Ribonucleoproteins/genetics ; Liposomes ; RNA, Guide, CRISPR-Cas Systems ; *Hearing Loss/genetics/therapy ; *Deafness/genetics ; },
abstract = {Mutations in Atp2b2, an outer hair cell gene, cause dominant hearing loss in humans. Using a mouse model Atp2b2[Obl/+], with a dominant hearing loss mutation (Oblivion), we show that liposome-mediated in vivo delivery of CRISPR-Cas9 ribonucleoprotein complexes leads to specific editing of the Obl allele. Large deletions encompassing the Obl locus and indels were identified as the result of editing. In vivo genome editing promotes outer hair cell survival and restores their function, leading to hearing recovery. We further show that in a double-dominant mutant mouse model, in which the Tmc1 Beethoven mutation and the Atp2b2 Oblivion mutation cause digenic genetic hearing loss, Cas9/sgRNA delivery targeting both mutations leads to partial hearing recovery. These findings suggest that liposome-RNP delivery can be used as a strategy to recover hearing with dominant mutations in OHC genes and with digenic mutations in the auditory hair cells, potentially expanding therapeutics of gene editing to treat hearing loss.},
}
@article {pmid37137337,
year = {2023},
author = {Nizan, S and Amitzur, A and Dahan-Meir, T and Benichou, JIC and Bar-Ziv, A and Perl-Treves, R},
title = {Mutagenesis of the melon Prv gene by CRISPR/Cas9 breaks papaya ringspot virus resistance and generates an autoimmune allele with constitutive defense responses.},
journal = {Journal of experimental botany},
volume = {74},
number = {15},
pages = {4579-4596},
pmid = {37137337},
issn = {1460-2431},
mesh = {*Disease Resistance/genetics ; Alleles ; *Cucurbitaceae/genetics ; CRISPR-Cas Systems ; Mutagenesis ; Plant Diseases/genetics ; },
abstract = {The majority of plant disease resistance (R) genes encode nucleotide binding-leucine-rich repeat (NLR) proteins. In melon, two closely linked NLR genes, Fom-1 and Prv, were mapped and identified as candidate genes that control resistance to Fusarium oxysporum f.sp. melonis races 0 and 2, and to papaya ringspot virus (PRSV), respectively. In this study, we validated the function of Prv and showed that it is essential for providing resistance against PRSV infection. We generated CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] mutants using Agrobacterium-mediated transformation of a PRSV-resistant melon genotype, and the T1 progeny proved susceptible to PRSV, showing strong disease symptoms and viral spread upon infection. Three alleles having 144, 154, and ~3 kb deletions, respectively, were obtained, all of which caused loss of resistance. Interestingly, one of the Prv mutant alleles, prvΔ154, encoding a truncated product, caused an extreme dwarf phenotype, accompanied by leaf lesions, high salicylic acid levels, and defense gene expression. The autoimmune phenotype observed at 25 °C proved to be temperature dependent, being suppressed at 32 °C. This is a first report on the successful application of CRISPR/Cas9 to confirm R gene function in melon. Such validation opens up new opportunities for molecular breeding of disease resistance in this important vegetable crop.},
}
@article {pmid37580710,
year = {2023},
author = {Zhao, N and Weng, S and Liu, Z and Xu, H and Ren, Y and Guo, C and Liu, L and Zhang, Z and Ji, Y and Han, X},
title = {CRISPR-Cas9 identifies growth-related subtypes of glioblastoma with therapeutical significance through cell line knockdown.},
journal = {BMC cancer},
volume = {23},
number = {1},
pages = {749},
pmid = {37580710},
issn = {1471-2407},
support = {81702465//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Glioblastoma/genetics/therapy/metabolism ; DNA Copy Number Variations ; CRISPR-Cas Systems ; *Brain Neoplasms/genetics/therapy/metabolism ; Cell Line ; ErbB Receptors/metabolism ; Prognosis ; },
abstract = {BACKGROUND: Glioblastoma (GBM) is a type of highly malignant brain tumor that is known for its significant intratumoral heterogeneity, meaning that there can be a high degree of variability within the tumor tissue. Despite the identification of several subtypes of GBM in recent years, there remains to explore a classification based on genes related to proliferation and growth.<br><br>METHODS: The growth-related genes of GBM were identified by CRISPR-Cas9 and univariate Cox regression analysis. The expression of these genes in the Cancer Genome Atlas cohort (TCGA) was used to construct growth-related genes subtypes (GGSs) via consensus clustering. Validation of this subtyping was performed using the nearest template prediction (NTP) algorithm in two independent Gene Expression Omnibus (GEO) cohorts and the ZZ cohort. Additionally, copy number variations, biological functions, and potential drugs were analyzed for each of the different subtypes separately.<br><br>RESULTS: Our research established multicenter-validated GGSs. GGS1 exhibits the poorest prognosis, with the highest frequency of chr 7 gain &amp; chr 10 loss, and the lowest frequency of chr 19 &amp; 20 co-gain. Additionally, GGS1 displays the highest expression of EGFR. Furthermore, it is significantly enriched in metabolic, stemness, proliferation, and signaling pathways. Besides we showed that Foretinib may be a potential therapeutic agent for GGS1, the worst prognostic subtype, through data screening and in vitro experiments. GGS2 has a moderate prognosis, with a slightly higher proportion of chr 7 gain &amp; chr 10 loss, and the highest proportion of chr 19 &amp; 20 co-gain. The prognosis of GGS3 is the best, with the least chr 7 gain &amp; 10 loss and EGFR expression.<br><br>CONCLUSIONS: These results enhance our understanding of the heterogeneity of GBM and offer insights for stratified management and precise treatment of GBM patients.},
}
@article {pmid37580703,
year = {2023},
author = {Qureshi, A and Connolly, JB},
title = {Bioinformatic and literature assessment of toxicity and allergenicity of a CRISPR-Cas9 engineered gene drive to control Anopheles gambiae the mosquito vector of human malaria.},
journal = {Malaria journal},
volume = {22},
number = {1},
pages = {234},
pmid = {37580703},
issn = {1475-2875},
mesh = {Animals ; Humans ; Mosquito Vectors/genetics ; *Anopheles/genetics ; CRISPR-Cas Systems ; *Gene Drive Technology/methods ; Allergens/genetics ; *Malaria ; *MicroRNAs ; },
abstract = {BACKGROUND: Population suppression gene drive is currently being evaluated, including via environmental risk assessment (ERA), for malaria vector control. One such gene drive involves the dsxF[CRISPRh] transgene encoding (i) hCas9 endonuclease, (ii) T1 guide RNA (gRNA) targeting the doublesex locus, and (iii) DsRed fluorescent marker protein, in genetically-modified mosquitoes (GMMs). Problem formulation, the first stage of ERA, for environmental releases of dsxF[CRISPRh] previously identified nine potential harms to the environment or health that could occur, should expressed products of the transgene cause allergenicity or toxicity.<br><br>METHODS: Amino acid sequences of hCas9 and DsRed were interrogated against those of toxins or allergens from NCBI, UniProt, COMPARE and AllergenOnline bioinformatic databases and the gRNA was compared with microRNAs from the miRBase database for potential impacts on gene expression associated with toxicity or allergenicity. PubMed was also searched for any evidence of toxicity or allergenicity of Cas9 or DsRed, or of the donor organisms from which these products were originally derived.<br><br>RESULTS: While Cas9 nuclease activity can be toxic to some cell types in vitro and hCas9 was found to share homology with the prokaryotic toxin VapC, there was no evidence from previous studies of a risk of toxicity to humans and other animals from hCas9. Although hCas9 did contain an 8-mer epitope found in the latex allergen Hev b 9, the full amino acid sequence of hCas9 was not homologous to any known allergens. Combined with a lack of evidence in the literature of Cas9 allergenicity, this indicated negligible risk to humans of allergenicity from hCas9. No matches were found between the gRNA and microRNAs from either Anopheles or humans. Moreover, potential exposure to dsxF[CRISPRh] transgenic proteins from environmental releases was assessed as negligible.<br><br>CONCLUSIONS: Bioinformatic and literature assessments found no convincing evidence to suggest that transgenic products expressed from dsxF[CRISPRh] were allergens or toxins, indicating that environmental releases of this population suppression gene drive for malaria vector control should not result in any increased allergenicity or toxicity in humans or animals. These results should also inform evaluations of other GMMs being developed for vector control and in vivo clinical applications of CRISPR-Cas9.},
}
@article {pmid37580318,
year = {2023},
author = {Wimberger, S and Akrap, N and Firth, M and Brengdahl, J and Engberg, S and Schwinn, MK and Slater, MR and Lundin, A and Hsieh, PP and Li, S and Cerboni, S and Sumner, J and Bestas, B and Schiffthaler, B and Magnusson, B and Di Castro, S and Iyer, P and Bohlooly-Y, M and Machleidt, T and Rees, S and Engkvist, O and Norris, T and Cadogan, EB and Forment, JV and Šviković, S and Akcakaya, P and Taheri-Ghahfarokhi, A and Maresca, M},
title = {Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4761},
pmid = {37580318},
issn = {2041-1723},
support = {765269//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Protein Kinases/genetics ; DNA Repair/genetics ; DNA/genetics ; },
abstract = {Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (Polϴ). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.},
}
@article {pmid37577534,
year = {2023},
author = {Hu, C and Myers, MT and Zhou, X and Hou, Z and Lozen, ML and Zhang, Y and Ke, A},
title = {Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.08.05.552134},
pmid = {37577534},
abstract = {Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets, and the nuclease-helicase Cas3 to degrade them. Among seven subtypes, Type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here we use four cryo-electron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling efficient NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9, that strongly inhibit N. lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through direct competition, whereas AcrIC9 achieves so through allosteric inhibition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for controllable Type I CRISPR genome engineering.},
}
@article {pmid37573099,
year = {2023},
author = {Zeng, Q and Zhou, M and Hu, Z and Deng, W and Li, Z and Wu, L and Liang, D},
title = {Rapid and sensitive Cas12a-based one-step nucleic acid detection with ssDNA-modified crRNA.},
journal = {Analytica chimica acta},
volume = {1276},
number = {},
pages = {341622},
doi = {10.1016/j.aca.2023.341622},
pmid = {37573099},
issn = {1873-4324},
mesh = {Humans ; Female ; *RNA ; DNA, Single-Stranded/genetics ; CRISPR-Cas Systems ; *Nucleic Acids ; Biological Assay ; Nucleic Acid Amplification Techniques ; },
abstract = {CRISPR-Cas12a RNA-guided complexes have been developed to facilitate the rapid and sensitive detection of nucleic acids. However, they are limited by the complexity of the operation, risk of carry-over contamination, and degradation of CRISPR RNA (crRNA). In this study, a Cas12a-based single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA)-mediated one-step diagnostic method (CasDOS) was established to overcome these drawbacks. mD-crRNA consisted of wild-type crRNA (Wt-crRNA) with ssDNA extensions at the 3' and 5' ends. Compared to Wt-crRNA, mD-crRNA exhibited a 100-1000-fold increase in sensitivity in the one-step assay, reducing the cis-cleavage activity of Cas12a to avoid excessive cleavage of the target DNA in the early stages of the reaction, leading to increased amplification and accumulation of the target amplicons, and improved the speed and intensity of the generated fluorescence signal. The detectability of CasDOS was 16.6 aM for the constructed plasmids of Streptococcus agalactiae (GBS), human papillomavirus type 16 (HPV16), and type 18 (HPV18). In clinical trials, CasDOS achieved 100% accuracy in identifying the known genotypes of the five HPV DNA samples. Moreover, CasDOS showed complete concordance with the qPCR results for GBS detection in ten vaginal or cervical swab samples, with a turnaround time from sampling to results within 30 min. In addition, mD-crRNA remained stable after Ribonuclease R treatment, suggesting that it might be more suitable as a raw material for the CRISPR detection kit. In conclusion, we have developed a universal, rapid, and highly sensitive one-step CRISPR detection assay.},
}
@article {pmid37569693,
year = {2023},
author = {Wei, W and Chen, ZN and Wang, K},
title = {CRISPR/Cas9: A Powerful Strategy to Improve CAR-T Cell Persistence.},
journal = {International journal of molecular sciences},
volume = {24},
number = {15},
pages = {},
pmid = {37569693},
issn = {1422-0067},
support = {20220309//the Young Talent fund of the University Association for Science and Technology in Shaanxi, China/ ; },
mesh = {Humans ; *Receptors, Chimeric Antigen ; T-Lymphocytes ; CRISPR-Cas Systems/genetics ; Gene Editing ; Neoplasm Recurrence, Local/genetics ; Immunotherapy, Adoptive ; *Neoplasms/genetics/therapy ; },
abstract = {As an emerging treatment strategy for malignant tumors, chimeric antigen receptor T (CAR-T) cell therapy has been widely used in clinical practice, and its efficacy has been markedly improved in the past decade. However, the clinical effect of CAR-T therapy is not so satisfying, especially in solid tumors. Even in hematologic malignancies, a proportion of patients eventually relapse after receiving CAR-T cell infusions, owing to the poor expansion and persistence of CAR-T cells. Recently, CRISPR/Cas9 technology has provided an effective approach to promoting the proliferation and persistence of CAR-T cells in the body. This technology has been utilized in CAR-T cells to generate a memory phenotype, reduce exhaustion, and screen new targets to improve the anti-tumor potential. In this review, we aim to describe the major causes limiting the persistence of CAR-T cells in patients and discuss the application of CRISPR/Cas9 in promoting CAR-T cell persistence and its anti-tumor function. Finally, we investigate clinical trials for CRISPR/Cas9-engineered CAR-T cells for the treatment of cancer.},
}
@article {pmid37569333,
year = {2023},
author = {Stepchenkova, EI and Zadorsky, SP and Shumega, AR and Aksenova, AY},
title = {Practical Approaches for the Yeast Saccharomyces cerevisiae Genome Modification.},
journal = {International journal of molecular sciences},
volume = {24},
number = {15},
pages = {},
pmid = {37569333},
issn = {1422-0067},
support = {20-15-00081//Russian Science Foundation/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; },
abstract = {The yeast S. cerevisiae is a unique genetic object for which a wide range of relatively simple, inexpensive, and non-time-consuming methods have been developed that allow the performing of a wide variety of genome modifications. Among the latter, one can mention point mutations, disruptions and deletions of particular genes and regions of chromosomes, insertion of cassettes for the expression of heterologous genes, targeted chromosomal rearrangements such as translocations and inversions, directed changes in the karyotype (loss or duplication of particular chromosomes, changes in the level of ploidy), mating-type changes, etc. Classical yeast genome manipulations have been advanced with CRISPR/Cas9 technology in recent years that allow for the generation of multiple simultaneous changes in the yeast genome. In this review we discuss practical applications of both the classical yeast genome modification methods as well as CRISPR/Cas9 technology. In addition, we review methods for ploidy changes, including aneuploid generation, methods for mating type switching and directed DSB. Combined with a description of useful selective markers and transformation techniques, this work represents a nearly complete guide to yeast genome modification.},
}
@article {pmid37569295,
year = {2023},
author = {Bekalu, ZE and Panting, M and Bæksted Holme, I and Brinch-Pedersen, H},
title = {Opportunities and Challenges of In Vitro Tissue Culture Systems in the Era of Crop Genome Editing.},
journal = {International journal of molecular sciences},
volume = {24},
number = {15},
pages = {},
pmid = {37569295},
issn = {1422-0067},
support = {8055-00038B, ReTraQue project//Innovation Fund Denmark/ ; },
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; Edible Grain/genetics ; Genotype ; Phenotype ; Genome, Plant ; CRISPR-Cas Systems ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Currently, the development of genome editing (GE) tools has provided a wide platform for targeted modification of plant genomes. However, the lack of versatile DNA delivery systems for a large variety of crop species has been the main bottleneck for improving crops with beneficial traits. Currently, the generation of plants with heritable mutations induced by GE tools mostly goes through tissue culture. Unfortunately, current tissue culture systems restrict successful results to only a limited number of plant species and genotypes. In order to release the full potential of the GE tools, procedures need to be species and genotype independent. This review provides an in-depth summary and insights into the various in vitro tissue culture systems used for GE in the economically important crops barley, wheat, rice, sorghum, soybean, maize, potatoes, cassava, and millet and uncovers new opportunities and challenges of already-established tissue culture platforms for GE in the crops.},
}
@article {pmid37563692,
year = {2023},
author = {Nakazawa, T and Morimoto, T and Maeoka, R and Matsuda, R and Nakamura, M and Nishimura, F and Ouji, N and Yamada, S and Nakagawa, I and Park, YS and Ito, T and Nakase, H and Tsujimura, T},
title = {CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {42},
number = {1},
pages = {205},
pmid = {37563692},
issn = {1756-9966},
support = {22K09213//Japan Society for the Promotion of Science/ ; 22K16667//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; Humans ; Mice ; *Brain Neoplasms/genetics/therapy ; *Carcinoma, Intraductal, Noninfiltrating ; CRISPR-Cas Systems ; *Glioblastoma/genetics/therapy ; *Hematopoietic Stem Cell Transplantation ; Killer Cells, Natural ; *Suppressor of Cytokine Signaling Proteins/genetics ; },
abstract = {BACKGROUND: Glioblastoma (GBM) is the most common malignant brain tumor and has "immunologically cold" features. Changing GBM to an "immunologically hot" tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH, or CIS) as a critical negative regulator in NKCs.<br><br>METHODS: The GBM tumor environment featured with immunological aspect was analyzed with Cancer immunogram and GlioVis. We generated human primary CIS-deleted NKCs (NK dCIS) using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting genome sites on CIS coding exons. The genome-edited NKCs underwent microarray with differential expression analysis and gene set enrichment analysis (GSEA). The anti-GBM activity of the genome-edited NKCs was evaluated by apoptosis induction effects against allogeneic GBM cells and spheroids. We further&#xa0;detected in vivo antitumor effects using xenograft brain tumor mice.<br><br>RESULTS: We successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)&#x264; and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice.<br><br>CONCLUSION: The findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM.},
}
@article {pmid37440203,
year = {2023},
author = {Giacca, M},
title = {Fulfilling the Promise of RNA Therapies for Cardiac Repair and Regeneration.},
journal = {Stem cells translational medicine},
volume = {12},
number = {8},
pages = {527-535},
pmid = {37440203},
issn = {2157-6580},
support = {787971/ERC_/European Research Council/International ; },
mesh = {*Heart/physiology ; Regeneration ; *RNA, Antisense/therapeutic use ; *Aptamers, Nucleotide/therapeutic use ; RNA Interference/drug effects ; *RNA, Guide, CRISPR-Cas Systems/therapeutic use ; Gene Editing ; Humans ; Animals ; },
abstract = {The progressive appreciation that multiple types of RNAs regulate virtually all aspects of tissue function and the availability of effective tools to deliver RNAs in vivo now offers unprecedented possibilities for obtaining RNA-based therapeutics. For the heart, RNA therapies can be developed that stimulate endogenous repair after cardiac damage. Applications in this area include acute cardioprotection after ischemia or cancer chemotherapy, therapeutic angiogenesis to promote new blood vessel formation, regeneration to form new cardiac mass, and editing of mutations to cure inherited cardiac disease. While the potential of RNA therapeutics for all these conditions is exciting, the field is still in its infancy. A number of roadblocks need to be overcome for RNA therapies to become effective, in particular, related to the problem of delivering RNA medicines into the cells and targeting them specifically to the heart.},
}
@article {pmid37429772,
year = {2023},
author = {Islam, T and Kasfy, SH},
title = {CRISPR enables heritable genome editing in planta.},
journal = {Trends in genetics : TIG},
volume = {39},
number = {9},
pages = {646-648},
doi = {10.1016/j.tig.2023.06.009},
pmid = {37429772},
issn = {0168-9525},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Plant/genetics ; Plant Breeding ; },
abstract = {Recent exciting developments in clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing showcase its potential to rapidly and efficiently edit genomes in planta, eliminating long processes of tissue culture and extensive breeding for crop improvement. These new methods offer heritable transgene-free edits in one generation, making them an attractive option for improving commercially important crops.},
}
@article {pmid37379644,
year = {2023},
author = {Fan, M and Bao, Y and Berkhout, B and Herrera-Carrillo, E},
title = {CRISPR-Cas12b enables a highly efficient attack on HIV proviral DNA in T cell cultures.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {165},
number = {},
pages = {115046},
doi = {10.1016/j.biopha.2023.115046},
pmid = {37379644},
issn = {1950-6007},
mesh = {Animals ; Humans ; *Proviruses/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *HIV Infections/genetics ; T-Lymphocytes/metabolism ; DNA, Viral/genetics ; Endonucleases/genetics/metabolism ; Cell Culture Techniques ; Mammals ; },
abstract = {BACKGROUND: The novel endonuclease Cas12b was engineered for targeted genome editing in mammalian cells and is a promising tool for certain applications because of its small size, high sequence specificity and ability to generate relatively large deletions. We previously reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections upon attack of the integrated viral DNA genome by spCas9 and Cas12a.<br><br>METHODS: We now tested the ability of the Cas12b endonuclease to suppress a spreading HIV infection in cell culture with anti-HIV gRNAs. Virus inhibition was tested in long-term HIV replication studies, which allowed us to test for viral escape and the potential for reaching a CURE of the infected T cells.<br><br>FINDINGS: We demonstrate that Cas12b can achieve complete HIV inactivation with only a single gRNA, a result for which Cas9 required two gRNAs. When the Cas12b system is programmed with two antiviral gRNAs, the overall anti-HIV potency is improved and more grossly mutated HIV proviruses are generated as a result of multiple cut-repair actions. Such "hypermutated" HIV proviruses are more likely to be defective due to mutation of multiple essential parts of the HIV genome. We report that the mutational profiles of the Cas9, Cas12a and Cas12b endonucleases differ significantly, which may have an impact on the level of virus inactivation. These combined results make Cas12b the preferred editing system for HIV-inactivation.<br><br>INTERPRETATION: These results provide in vitro "proof of concept' for CRISPR-Cas12b mediated HIV-1 inactivation.},
}
@article {pmid37183575,
year = {2023},
author = {Song, N and Chu, Y and Tang, J and Yang, D},
title = {Lipid-, Inorganic-, Polymer-, and DNA-Based Nanocarriers for Delivery of the CRISPR/Cas9 system.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {24},
number = {16},
pages = {e202300180},
doi = {10.1002/cbic.202300180},
pmid = {37183575},
issn = {1439-7633},
mesh = {*CRISPR-Cas Systems/genetics ; *Polymers/metabolism ; Gene Editing ; CRISPR-Associated Protein 9/genetics/metabolism ; Lipids ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) system has been widely explored for the precise manipulation of target DNA and has enabled efficient genomic editing in cells. Recently, CRISPR/Cas9 has shown promising potential in biomedical applications, including disease treatment, transcriptional regulation and genome-wide screening. Despite these exciting achievements, efficient and controlled delivery of the CRISPR/Cas9 system has remained a critical obstacle to its further application. Herein, we elaborate on the three delivery forms of the CRISPR/Cas9 system, and discuss the composition, advantages and limitations of these forms. Then we provide a comprehensive overview of the carriers of the system, and focus on the nonviral nanocarriers in chemical methods that facilitate efficient and controlled delivery of the CRISPR/Cas9 system. Finally, we discuss the challenges and prospects of the delivery methods of the CRISPR/Cas9 system in depth, and propose strategies to address the intracellular and extracellular barriers to delivery in clinical applications.},
}
@article {pmid37039773,
year = {2023},
author = {Hamaker, NK and Lee, KH},
title = {High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors.},
journal = {Biotechnology and bioengineering},
volume = {120},
number = {9},
pages = {2419-2440},
doi = {10.1002/bit.28393},
pmid = {37039773},
issn = {1097-0290},
support = {T32 GM008550/GM/NIGMS NIH HHS/United States ; },
mesh = {Cricetinae ; Animals ; CHO Cells ; Cricetulus ; *DNA Repair/genetics ; *CRISPR-Associated Protein 9/genetics ; Recombinant Proteins/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3-8.3-fold and 19-22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%-55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.},
}
@article {pmid36947856,
year = {2023},
author = {Ranzau, BL and Rallapalli, KL and Evanoff, M and Paesani, F and Komor, AC},
title = {The Wild-Type tRNA Adenosine Deaminase Enzyme TadA Is Capable of Sequence-Specific DNA Base Editing.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {24},
number = {16},
pages = {e202200788},
doi = {10.1002/cbic.202200788},
pmid = {36947856},
issn = {1439-7633},
support = {R35 GM138317/GM/NIGMS NIH HHS/United States ; T32 GM007240/GM/NIGMS NIH HHS/United States ; T32 GM112584/GM/NIGMS NIH HHS/United States ; },
mesh = {*Adenosine Deaminase/metabolism ; *Gene Editing ; RNA, Transfer/chemistry ; DNA/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Base editors are genome editing tools that enable site-specific base conversions through the chemical modification of nucleobases in DNA. Adenine base editors (ABEs) convert A ⋅ T to G ⋅ C base pairs in DNA by using an adenosine deaminase enzyme to modify target adenosines to inosine intermediates. Due to the lack of a naturally occurring adenosine deaminase that can modify DNA, ABEs were evolved from a tRNA-deaminating enzyme, TadA. Previous experiments with an ABE comprising a wild-type (wt) TadA showed no detectable activity on DNA, and directed evolution was therefore required to enable this enzyme to accept DNA as a substrate. Here we show that wtTadA can perform base editing in DNA in both bacterial and mammalian cells, with a strict sequence motif requirement of TAC. We leveraged this discovery to optimize a reporter assay to detect base editing levels as low as 0.01 %. Finally, we used this assay along with molecular dynamics simulations of full ABE:DNA complexes to better understand how the sequence recognition of mutant TadA variants change as they accumulate mutations to better edit DNA substrates.},
}
@article {pmid36938677,
year = {2023},
author = {Leitner, K and Motheramgari, K and Borth, N and Marx, N},
title = {Nanopore Cas9-targeted sequencing enables accurate and simultaneous identification of transgene integration sites, their structure and epigenetic status in recombinant Chinese hamster ovary cells.},
journal = {Biotechnology and bioengineering},
volume = {120},
number = {9},
pages = {2403-2418},
doi = {10.1002/bit.28382},
pmid = {36938677},
issn = {1097-0290},
mesh = {Cricetinae ; Animals ; Cricetulus ; CHO Cells ; CRISPR-Cas Systems ; *Nanopore Sequencing ; *Nanopores ; Transgenes/genetics ; },
abstract = {The integration of a transgene expression construct into the host genome is the initial step for the generation of recombinant cell lines used for biopharmaceutical production. The stability and level of recombinant gene expression in Chinese hamster ovary (CHO) can be correlated to the copy number, its integration site as well as the epigenetic context of the transgene vector. Also, undesired integration events, such as concatemers, truncated, and inverted vector repeats, are impacting the stability of recombinant cell lines. Thus, to characterize cell clones and to isolate the most promising candidates, it is crucial to obtain information on the site of integration, the structure of integrated sequence and the epigenetic status. Current sequencing techniques allow to gather this information separately but do not offer a comprehensive and simultaneous resolution. In this study, we present a fast and robust nanopore Cas9-targeted sequencing (nCats) pipeline to identify integration sites, the composition of the integrated sequence as well as its DNA methylation status in CHO cells that can be obtained simultaneously from the same sequencing run. A Cas9-enrichment step during library preparation enables targeted and directional nanopore sequencing with up to 724× median on-target coverage and up to 153 kb long reads. The data generated by nCats provides sensitive, detailed, and correct information on the transgene integration sites and the expression vector structure, which could only be partly produced by traditional Targeted Locus Amplification-seq data. Moreover, with nCats the DNA methylation status can be analyzed from the same raw data without prior DNA amplification.},
}
@article {pmid37582546,
year = {2022},
author = {Feeney, O},
title = {Genetics and Justice, Non-Ideal Theory and the Role of Patents: The Case of CRISPR-Cas9.},
journal = {European journal of health law},
volume = {29},
number = {3-5},
pages = {543-561},
doi = {10.1163/15718093-bja10075},
pmid = {37582546},
issn = {1571-8093},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing ; Social Justice ; Biotechnology ; },
abstract = {There are ongoing concerns of social justice regarding inequalities in the distribution of access to potential genome editing technologies. Working within non-ideal theory, Colin Farrelly advances a justification for the use of patents to speed up the arrival of safe and effective interventions for all, including the socially disadvantaged. This paper argues that such success is less assured when one considers the actual functioning of patents and the practical implications of the patent system in the context of biotechnological innovations. I suggest that non-ideal theoretical approaches risk reverting back to a form of ideal theory if they simply refer to such real-world constraints - e.g. patents - but do not critically assess and fully examine how such constraints manifest themselves in practice. I highlight some considerations that would be important in order to develop and foster a more robust non-ideal approach to justice in biotechnological developments.},
}
@article {pmid37582540,
year = {2022},
author = {Slokenberga, S},
title = {What Would It Take to Enable Germline Editing in Europe for Medical Purposes?.},
journal = {European journal of health law},
volume = {29},
number = {3-5},
pages = {521-542},
doi = {10.1163/15718093-bja10074},
pmid = {37582540},
issn = {1571-8093},
mesh = {Humans ; Europe ; *Gene Editing ; Human Rights ; *Medicine ; Germ Cells ; },
abstract = {Commonly, the regulation on germline editing in Europe is described through the two prohibitions: the prohibition set out in Article 13 of the Convention for the protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine and the prohibition that is set out in the EU Clinical Trials Regulation. These prohibitions reflect the European regional position regarding the ethical and legal questions raised by the technology, and an unwillingness to enable such interventions in Europe. Simultaneously, these prohibitions have been shaped prior to the recent breakthroughs in the field, such as the discovery of the CRISPR-Cas technology, which has initiated a new era in the field. This contribution examines what it would take to enable human germline gene editing in Europe for medical purposes. It scrutinises in detail the content and context of the existing bans, as well as mechanisms to lift them. It argues that the bans that are prescribed by each of the European regional legal orders are embedded in strong structures, composed of values and principles. For the human germline gene editing to be enabled in Europe for health-related purposes, the approach to these values and principles needs to change. Only then can the machinery to lift the bans lead to a change.},
}
@article {pmid37582538,
year = {2022},
author = {Slokenberga, S and Minssen, T and Nordberg, A},
title = {Governing, Protecting, and Regulating the Future of Genome Editing: The Significance of ELSPI Perspectives.},
journal = {European journal of health law},
volume = {29},
number = {3-5},
pages = {327-340},
doi = {10.1163/15718093-bja10076},
pmid = {37582538},
issn = {1571-8093},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; Genome, Plant ; },
}
@article {pmid37563544,
year = {2023},
author = {Dutta, TK and Vashisth, N and Ray, S and Phani, V and Chinnusamy, V and Sirohi, A},
title = {Functional analysis of a susceptibility gene (HIPP27) in the Arabidopsis thaliana-Meloidogyne incognita pathosystem by using a genome editing strategy.},
journal = {BMC plant biology},
volume = {23},
number = {1},
pages = {390},
pmid = {37563544},
issn = {1471-2229},
mesh = {Animals ; Gene Editing/methods ; *Arabidopsis/genetics/parasitology ; CRISPR-Cas Systems ; *Tylenchoidea ; Plant Breeding ; Plants, Genetically Modified/genetics ; },
abstract = {BACKGROUND: Plant-parasitic root-knot nematodes cause immense yield declines in crop plants that ultimately obviate global food security. They maintain an intimate relationship with their host plants and hijack the host metabolic machinery to their own advantage. The existing resistance breeding strategies utilizing RNAi and resistance (R) genes might not be particularly effective. Alternatively, knocking out the susceptibility (S) genes in crop plants appears to be a feasible approach, as the induced mutations in S genes are likely to be long-lasting and may confer broad-spectrum resistance. This could be facilitated by the use of CRISPR/Cas9-based genome editing technology that precisely edits the gene of interest using customizable guide RNAs (gRNAs) and Cas9 endonuclease.<br><br>RESULTS: Initially, we characterized the nematode-responsive S gene HIPP27 from Arabidopsis thaliana by generating HIPP27 overexpression lines, which were inoculated with Meloidogyne incognita. Next, two gRNAs (corresponding to the HIPP27 gene) were artificially synthesized using laboratory protocols, sequentially cloned into a Cas9 editor plasmid, mobilized into Agrobacterium tumefaciens strain GV3101, and transformed into Arabidopsis plants using the floral dip method. Apart from 1-3&#xa0;bp deletions and 1&#xa0;bp insertions adjacent to the PAM site, a long deletion of approximately 161&#xa0;bp was documented in the T0 generation. Phenotypic analysis of homozygous, 'transgene-free' T2 plants revealed reduced nematode infection compared to wild-type plants. Additionally, no growth impairment was observed in gene-edited plants.<br><br>CONCLUSION: Our results suggest that the loss of function of HIPP27 in A. thaliana by CRISPR/Cas9-induced mutagenesis can improve host resistance to M. incognita.},
}
@article {pmid37562410,
year = {2023},
author = {Schraivogel, D and Steinmetz, LM and Parts, L},
title = {Pooled Genome-Scale CRISPR Screens in Single Cells.},
journal = {Annual review of genetics},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-genet-072920-013842},
pmid = {37562410},
issn = {1545-2948},
abstract = {Assigning functions to genes and learning how to control their expression are part of the foundation of cell biology and therapeutic development. An efficient and unbiased method to accomplish this is genetic screening, which historically required laborious clone generation and phenotyping and is still limited by scale today. The rapid technological progress on modulating gene function with CRISPR-Cas and measuring it in individual cells has now relaxed the major experimental constraints and enabled pooled screening with complex readouts from single cells. Here, we review the principles and practical considerations for pooled single-cell CRISPR screening. We discuss perturbation strategies, experimental model systems, matching the perturbation to the individual cells, reading out cell phenotypes, and data analysis. Our focus is on single-cell RNA sequencing and cell sorting-based readouts, including image-enabled cell sorting. We expect this transformative approach to fuel biomedical research for the next several decades. Expected final online publication date for the Annual Review of Genetics, Volume 57 is November 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.},
}
@article {pmid37561739,
year = {2023},
author = {Kay, GA and Patterson, EI and Hughes, GL and Lord, JS and Reimer, LJ},
title = {Knockdown resistance allele L1014F introduced by CRISPR/Cas9 is not associated with altered vector competence of Anopheles gambiae for o'nyong nyong virus.},
journal = {PloS one},
volume = {18},
number = {8},
pages = {e0288994},
pmid = {37561739},
issn = {1932-6203},
support = {N013514//MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; O'nyong-nyong Virus ; *Anopheles/genetics ; Alleles ; CRISPR-Cas Systems/genetics ; Mosquito Vectors/genetics ; Insecticide Resistance/genetics ; *Insecticides ; },
abstract = {Knockdown resistance (kdr) alleles conferring resistance to pyrethroid insecticides are widespread amongst vector populations. Previous research has suggested that these alleles are associated with changes in the vector competence of mosquitoes for arboviruses and Plasmodium, however non-target genetic differences between mosquito strains may have had a confounding effect. Here, to minimise genetic differences, the laboratory Anopheles gambiae Kisumu strain was compared to a CRISPR/Cas9 homozygous kdr L1014F mutant Kisumu-kdr line in order to examine associations with vector competence for o'nyong nyong virus (ONNV). Mosquitoes were infected using either blood feeds or intrathoracic microinjections. There were no significant differences in the prevalence of virus in mosquito body parts between kdr mutant and wildtype lines from either oral or intrathoracic injection routes. The ONNV titre was significantly higher in the legs of the wildtype strain at 7dpi following intrathoracic microinjection, but no other significant differences in viral titre were detected. ONNV was not detected in the saliva of mosquitoes from either strain. Our findings from per os infections suggest that the kdr L1014F allele is not associated with altered infection prevalence for ONNV, a key component of vector competence.},
}
@article {pmid37558681,
year = {2023},
author = {Yasumoto, S and Muranaka, T},
title = {Foreign DNA detection in genome-edited potatoes by high-throughput sequencing.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {12246},
pmid = {37558681},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems/genetics ; *Solanum tuberosum/genetics ; Computer Simulation ; Plant Breeding ; Gene Editing/methods ; DNA ; High-Throughput Nucleotide Sequencing/methods ; Nucleotides ; },
abstract = {Genome editing is a powerful breeding technique that introduces mutations into specific gene sequences in genomes. For genome editing in higher plants, nucleotides for artificial nuclease (e.g. TALEN or CRISPR-Cas9) are transiently or stably introduced into the plant cells. After the introduction of mutations by artificial nucleases, it is necessary to select lines that do not contain the foreign nucleotides to overcome GMO regulation; however, there is still no widely legally authorized and approved method for detecting foreign genes in genome-edited crops. Recently, k-mer analysis based on next-generation sequencing (NGS) was proposed as a new method for detecting foreign DNA in genome-edited agricultural products. Compared to conventional methods, such as PCR and Southern hybridization, in principle, this method can detect short DNA fragments with high accuracy. However, this method has not yet been applied to genome-edited potatoes. In this study, we evaluated the feasibility of k-mer analysis in tetraploid potatoes by computer simulation, and also evaluated whether the k-mer method can detect foreign genes with high accuracy by analyzing samples of genome-edited potatoes. We show that when NGS data (at a depth of × 30 the genome size) are used, the k-mer method can correctly detect foreign genes in the potato genome even with the insertion of DNA fragments of 20 nt in length. Based on these findings, we expect that k-mer analysis will be one of the main methods for detecting foreign genes in genome-edited potatoes.},
}
@article {pmid37531567,
year = {2023},
author = {Fei, X and Lei, C and Ren, W and Liu, X and Liu, C},
title = {Regulating the trans-Cleavage Activity of CRISPR/Cas12a by Using an Elongation-Caged Single-Stranded DNA Activator and the Biosensing Applications.},
journal = {Analytical chemistry},
volume = {95},
number = {32},
pages = {12169-12176},
doi = {10.1021/acs.analchem.3c02471},
pmid = {37531567},
issn = {1520-6882},
mesh = {*DNA, Single-Stranded/genetics ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Endodeoxyribonucleases/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; *Biosensing Techniques ; },
abstract = {The CRISPR/Cas12a system exhibits extraordinary capability in the field of biosensing and molecular diagnosis due to its trans-cleavage ability. However, it is still desirable for precise control and programmable regulation of Cas12a trans-cleavage activity to promote the in-depth studies and application expansion of Cas12a-based sensing platforms. In this work, we have developed a new and robust CRISPR/Cas12a regulation mechanism by endowing the activator with the function of caging crRNA ingeniously. Specifically, we constructed an integrated elongation-caged activator (EL-activator) by extending the ssDNA activator on the 3'-end. We found that appending only about 8 nt that is complementary to the crRNA repeat region is enough to cage the crRNA spacer/repeat region, thus effectively inhibiting Cas12a trans-cleavage activity. The inner inhibition mechanism was further uncovered after a thorough investigation, demonstrating that the EL-activator works by impeding the conformation of crRNA required for Cas12a recognition and destroying its affinity with Cas12a. By further switching on the elongated moiety on the EL-activator using target biomarkers, the blocked trans-cleavage activity of Cas12a can be rapidly recovered. Finally, a versatile sensing platform was established based on the EL-activator regulation mechanism, expanding the conventional Cas12a system that only directly recognizes DNA to the direct detection of enzymes and RNA biomarkers. This work has enriched the CRISPR/Cas12a regulation toolbox and expanded its sensing applications.},
}
@article {pmid37527175,
year = {2023},
author = {Dong, J and Li, X and Zhou, S and Liu, Y and Deng, L and Chen, J and Hou, J and Hou, C and Huo, D},
title = {CRISPR/Cas12a-Powered EC/FL Dual-Mode Controlled-Release Homogeneous Biosensor for Ultrasensitive and Cross-Validated Detection of Messenger Ribonucleic Acid.},
journal = {Analytical chemistry},
volume = {95},
number = {32},
pages = {12122-12130},
doi = {10.1021/acs.analchem.3c02335},
pmid = {37527175},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Delayed-Action Preparations ; RNA, Messenger/genetics ; RNA ; Coloring Agents ; DNA, Single-Stranded/genetics ; Endonucleases ; Serine Proteinase Inhibitors ; *Biosensing Techniques ; },
abstract = {Accurate detection of cancer-associated mRNAs is beneficial to early diagnosis and potential treatment of cancer. Herein, for the first time, we developed a novel CRISPR/Cas12a-powered electrochemical/fluorescent (EC/FL) dual-mode controlled-release homogeneous biosensor for mRNA detection. A functionalized ssDNA P2-capped Fe3O4-NH2 loaded with methylene blue (P2@MB-Fe3O4-NH2) was synthesized as the signal probe, while survivin mRNA was chosen as the target RNA. In the presence of the target mRNA, the nicking endonuclease-mediated rolling circle amplification (NEM-RCA) was triggered to produce significant amounts of ssDNA, activating the collateral activity of Cas12a toward the surrounding single-stranded DNA. Thus, the ssDNA P1 completely complementary to ssDNA P2 was cleaved, resulting in that the ssDNA P2 bio-gate on Fe3O4-NH2 could not be opened due to electrostatic interactions. As a result, there was no or only a little MB in the supernatant after magnetic separation, and the measured EC/FL signal was exceedingly weak. On the contrary, the ssDNA P2 bio-gate was opened, enabling MB to be released into the supernatant, and generating an obvious EC/FL signal. Benefiting from the accuracy of EC/FL dual-mode cross-verification, high amplification efficiency, high specificity of NEM-RCA and CRISPR/Cas12a, and high loading of mesoporous Fe3O4-NH2 on signal molecules, the strategy shows aM-level sensitivity and single-base mismatch specificity. More importantly, the practical applicability of this dual-mode strategy was confirmed by mRNA quantification in complex serum environments and tumor cell lysates, providing a new way for developing a powerful disease diagnosis tool.},
}
@article {pmid37516291,
year = {2023},
author = {Minet, C and Chantal, I and Berthier, D},
title = {Recent advances in genome editing of bloodstream forms of Trypanosoma congolense using CRISPR-Cas9 ribonucleoproteins: Proof of concept.},
journal = {Experimental parasitology},
volume = {252},
number = {},
pages = {108589},
doi = {10.1016/j.exppara.2023.108589},
pmid = {37516291},
issn = {1090-2449},
mesh = {Animals ; Cattle ; *Trypanosoma congolense/genetics ; CRISPR-Cas Systems ; Gene Editing ; Ribonucleoproteins/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Trypanosomiasis, African/prevention &amp; control/veterinary ; *Trypanosoma/genetics ; *Trypanosoma brucei brucei/genetics ; },
abstract = {African Animal Trypanosomosis (AAT or Nagana) is a vector-borne disease caused by Trypanosomatidae, genus Trypanosoma. The disease is transmitted by the bite of infected hematophagous insects, mainly tsetse flies but also other blood-sucking insects including stomoxes and tabanids. Although many trypanosome species infect animals, the main agents responsible for this disease with a strong socio-economic and veterinary health impact are Trypanosoma congolense (T. congolense or Tc), Trypanosoma vivax (T.vivax), and to a lesser extent, Trypanosoma brucei brucei (T.brucei brucei or Tbb). These parasites mainly infect livestock, including cattle, in sub-Saharan Africa, with major repercussions in terms of animal productivity and poverty for populations which are often already very poor. As there is currently no vaccine, the fight against the disease is primarily based on diagnosis, treatment and vector control. To develop new tools (particularly therapeutic tools) to fight against the disease, we need to know both the biology and the genes involved in the pathogenicity and virulence of the parasites. To date, unlike for Trypanosoma brucei (T.brucei) or Trypanosoma cruzi (T.cruzi), genome editing tools has been relatively little used to study T. congolense. We present an efficient, reproducible and stable CRISPR-Cas9 genome editing system for use in Tc bloodstream forms (Tc-BSF). This plasmid-free system is based on transient expression of Cas9 protein and the use of a ribonucleoprotein formed by the Cas9 and sgRNA complex. This is the first proof of concept of genome editing using CRISPR-Cas9 ribonucleoproteins on Tc-BSF. This adapted protocol enriches the "toolbox" for the functional study of genes of interest in blood forms of the Trypanosoma congolense. This proof of concept is an important step for the scientific community working on the study of trypanosomes and opens up new perspectives for the control of and fight against animal trypanosomosis.},
}
@article {pmid37470475,
year = {2023},
author = {Schubert, L and Le, AT and Hinz, TK and Navarro, AC and Nelson-Taylor, SK and Nemenoff, RA and Heasley, LE and Doebele, RC},
title = {A functional sgRNA-CRISPR screening method for generating murine RET and NTRK1 rearranged oncogenes.},
journal = {Biology open},
volume = {12},
number = {8},
pages = {},
doi = {10.1242/bio.059994},
pmid = {37470475},
issn = {2046-6390},
support = {P50CA058187/NH/NIH HHS/United States ; /CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Mice ; *RNA, Guide, CRISPR-Cas Systems ; *Oncogenes ; Protein Kinase Inhibitors/pharmacology ; },
abstract = {CRISPR/Cas9 gene editing represents a powerful tool for investigating fusion oncogenes in cancer biology. Successful experiments require that sgRNAs correctly associate with their target sequence and initiate double stranded breaks which are subsequently repaired by endogenous DNA repair systems yielding fusion chromosomes. Simple tests to ensure sgRNAs are functional are not generally available and often require single cell cloning to identify successful CRISPR-editing events. Here, we describe a novel method relying on acquisition of IL3-independence in Ba/F3 cells to identify sgRNA pairs that generate oncogenic gene rearrangements of the Ret and Ntrk1 tyrosine kinases. The rearrangements were confirmed with PCR, RT-PCR and sequencing and Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNA pairs inducing the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) established from a Trim24-Ret positive tumor exhibited high in vitro sensitivity to the RET inhibitors LOXO-292 and BLU-667 and orthotopic TR.1 cell-derived tumors underwent marked shrinkage upon LOXO-292 treatment. Thus, the method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel, syngeneic murine oncogene-driven tumor models.},
}
@article {pmid37029320,
year = {2023},
author = {Alyateem, G and Wade, HM and Bickert, AA and Lipsey, CC and Mondal, P and Smith, MD and Labib, RM and Mock, BA and Robey, RW and Gottesman, MM},
title = {Use of CRISPR-based screens to identify mechanisms of chemotherapy resistance.},
journal = {Cancer gene therapy},
volume = {30},
number = {8},
pages = {1043-1050},
pmid = {37029320},
issn = {1476-5500},
support = {n/a//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; RNA, Guide, CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology/therapeutic use ; *Neoplasms/drug therapy/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Despite the development of new classes of targeted anti-cancer drugs, the curative treatment of metastatic solid tumors remains out of reach owing to the development of resistance to current chemotherapeutics. Although many mechanisms of drug resistance have been described, there is still a general lack of understanding of the many means by which cancer cells elude otherwise effective chemotherapy. The traditional strategy of isolating resistant clones in vitro, defining their mechanism of resistance, and testing to see whether these mechanisms play a role in clinical drug resistance is time-consuming and in many cases falls short of providing clinically relevant information. In this review, we summarize the use of CRISPR technology, including the promise and pitfalls, to generate libraries of cancer cells carrying sgRNAs that define novel mechanisms of resistance. The existing strategies using CRISPR knockout, activation, and inhibition screens, and combinations of these approaches are described. In addition, specialized approaches to identify more than one gene that may be contributing to resistance, as occurs in synthetic lethality, are described. Although these CRISPR-based approaches to cataloguing drug resistance genes in cancer cells are just beginning to be utilized, appropriately used they promise to accelerate understanding of drug resistance in cancer.},
}
@article {pmid37557170,
year = {2023},
author = {Wang, S and Gabel, C and Siddique, R and Klose, T and Chang, L},
title = {Molecular mechanism for Tn7-like transposon recruitment by a type I-B CRISPR effector.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2023.07.010},
pmid = {37557170},
issn = {1097-4172},
abstract = {Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key feature of CASTs is their ability to recruit Tn7-like transposons to nuclease-deficient CRISPR effectors. However, how Tn7-like transposons are recruited by diverse CRISPR effectors remains poorly understood. Here, we present the cryo-EM structure of a recruitment complex comprising the Cascade complex, TniQ, TnsC, and the target DNA in the type I-B CAST from Peltigera membranacea cyanobiont 210A. Target DNA recognition by Cascade induces conformational changes in Cas6 and primes TniQ recruitment through its C-terminal domain. The N-terminal domain of TniQ is bound to the seam region of the TnsC spiral heptamer. Our findings provide insights into the diverse mechanisms for the recruitment of Tn7-like transposons to CRISPR effectors and will aid in the development of CASTs as gene knockin tools.},
}
@article {pmid37554703,
year = {2023},
author = {Anuradha, and Kumari, M and Zinta, G and Chauhan, R and Kumar, A and Singh, S and Singh, S},
title = {Genetic resources and breeding approaches for improvement of amaranth (Amaranthus spp.) and quinoa (Chenopodium quinoa).},
journal = {Frontiers in nutrition},
volume = {10},
number = {},
pages = {1129723},
pmid = {37554703},
issn = {2296-861X},
abstract = {Nowadays, the human population is more concerned about their diet and very specific in choosing their food sources to ensure a healthy lifestyle and avoid diseases. So people are shifting to more smart nutritious food choices other than regular cereals and staple foods they have been eating for a long time. Pseudocereals, especially, amaranth and quinoa, are important alternatives to traditional cereals due to comparatively higher nutrition, essential minerals, amino acids, and zero gluten. Both Amaranchaceae crops are low-input demanding and hardy plants tolerant to stress, drought, and salinity conditions. Thus, these crops may benefit developing countries that follow subsistence agriculture and have limited farming resources. However, these are underutilized orphan crops, and the efforts to improve them by reducing their saponin content remain ignored for a long time. Furthermore, these crops have very rich variability, but the progress of their genetic gain for getting high-yielding genotypes is slow. Realizing problems in traditional cereals and opting for crop diversification to tackle climate change, research should be focused on the genetic improvement for low saponin, nutritionally rich, tolerant to biotic and abiotic stresses, location-specific photoperiod, and high yielding varietal development of amaranth and quinoa to expand their commercial cultivation. The latest technologies that can accelerate the breeding to improve yield and quality in these crops are much behind and slower than the already established major crops of the world. We could learn from past mistakes and utilize the latest trends such as CRISPR/Cas, TILLING, and RNA interference (RNAi) technology to improve these pseudocereals genetically. Hence, the study reviewed important nutrition quality traits, morphological descriptors, their breeding behavior, available genetic resources, and breeding approaches for these crops to shed light on future breeding strategies to develop superior genotypes.},
}
@article {pmid37551560,
year = {2023},
author = {Wu, F and Lu, C and Hu, W and Guo, X and Chen, J and Luo, Z},
title = {Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination.},
journal = {Journal of Zhejiang University. Science. B},
volume = {24},
number = {8},
pages = {749-754},
doi = {10.1631/jzus.B2200705},
pmid = {37551560},
issn = {1862-1783},
support = {KYCX2021-038//the Priority Academic Program Development of Jiangsu Higher Education Institutions of China, the Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; CG2232//the Lianyungang Science and Technology Program/ ; },
mesh = {*Vibrio parahaemolyticus/genetics ; Uracil-DNA Glycosidase/genetics ; Hot Temperature ; CRISPR-Cas Systems ; Food Safety ; },
abstract = {Vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.},
}
@article {pmid37550505,
year = {2023},
author = {Watts, EA and Garrett, SC and Catchpole, RJ and Clark, LM and Sanders, TJ and Marshall, CJ and Wenck, BR and Vickerman, RL and Santangelo, TJ and Fuchs, R and Robb, B and Olson, S and Graveley, BR and Terns, MP},
title = {Histones direct site-specific CRISPR spacer acquisition in model archaeon.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {37550505},
issn = {2058-5276},
support = {R35GM118160//U.S. Department of Health and Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35GM143963//U.S. Department of Health and Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; RO1GM100329//U.S. Department of Health and Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35GM118140//U.S. Department of Health and Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
abstract = {CRISPR-Cas systems provide heritable immunity against viruses and other mobile genetic elements by incorporating fragments of invader DNA into the host CRISPR array as spacers. Integration of new spacers is localized to the 5' end of the array, and in certain Gram-negative Bacteria this polarized localization is accomplished by the integration host factor. For most other Bacteria and Archaea, the mechanism for 5' end localization is unknown. Here we show that archaeal histones play a key role in directing integration of CRISPR spacers. In Pyrococcus furiosus, deletion of either histone A or B impairs integration. In vitro, purified histones are sufficient to direct integration to the 5' end of the CRISPR array. Archaeal histone tetramers and bacterial integration host factor induce similar U-turn bends in bound DNA. These findings indicate a co-evolution of CRISPR arrays with chromosomal DNA binding proteins and a widespread role for binding and bending of DNA to facilitate accurate spacer integration.},
}
@article {pmid37550295,
year = {2023},
author = {Henn, D and Zhao, D and Sivaraj, D and Trotsyuk, A and Bonham, CA and Fischer, KS and Kehl, T and Fehlmann, T and Greco, AH and Kussie, HC and Moortgat Illouz, SE and Padmanabhan, J and Barrera, JA and Kneser, U and Lenhof, HP and Januszyk, M and Levi, B and Keller, A and Longaker, MT and Chen, K and Qi, LS and Gurtner, GC},
title = {Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4729},
pmid = {37550295},
issn = {2041-1723},
support = {S10 OD025212/OD/NIH HHS/United States ; S10 OD021763/OD/NIH HHS/United States ; R21 AG077193/AG/NIA NIH HHS/United States ; P30 DK116074/DK/NIDDK NIH HHS/United States ; S10 OD018220/OD/NIH HHS/United States ; },
mesh = {Humans ; Mice ; Animals ; *CRISPR-Cas Systems ; Wound Healing/genetics ; Genes, myc ; Gene Editing ; *Craniocerebral Trauma ; Dendritic Cells ; },
abstract = {Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.},
}
@article {pmid37549968,
year = {2023},
author = {Chen, YH and Lu, J and Yang, X and Huang, LC and Zhang, CQ and Liu, QQ and Li, QF},
title = {Gene editing of non-coding regulatory DNA and its application in crop improvement.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/erad313},
pmid = {37549968},
issn = {1460-2431},
abstract = {The development of the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Protein (CRISPR/Cas) system has provided precise and efficient strategies to edit target genes and generate transgene-free crops. Significant progress has been made in the editing of protein coding genes; however, studies on the editing of non-coding DNA with regulatory roles lags far behind. Non-coding regulatory DNAs, including long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and cis-regulatory elements (CREs), play crucial roles in regulating plant growth and development. Therefore, the combination of CRISPR/Cas technology and non-coding regulatory DNA has great potential to generate novel alleles that affect various agronomic traits of crops, thus providing valuable genetic resources for crop breeding. Herein, we review recent advances in the roles of non-coding regulatory DNA, attempts to edit non-coding regulatory DNA for crop improvement, and potential application of novel editing tools in modulating non-coding regulatory DNA. Finally, the existing problems, possible solutions, and future applications of gene editing of non-coding regulatory DNA in modern crop breeding practice are also discussed.},
}
@article {pmid37547711,
year = {2023},
author = {Ye, M and Yao, M and Li, C and Gong, M},
title = {Salt and osmotic stress can improve the editing efficiency of CRISPR/Cas9-mediated genome editing system in potato.},
journal = {PeerJ},
volume = {11},
number = {},
pages = {e15771},
pmid = {37547711},
issn = {2167-8359},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Solanum tuberosum/genetics ; Sodium Chloride/pharmacology ; Osmotic Pressure ; },
abstract = {CRISPR/Cas9-mediated genome editing technology has been widely used for the study of gene function in crops, but the differences between species have led to widely varying genome editing efficiencies. The present study utilized a potato hairy root genetic transformation system and incorporated a rapid assay with GFP as a screening marker. The results clearly demonstrated that salt and osmotic stress induced by NaCl (10 to 50 mM) and mannitol (50 to 200 mM) treatments significantly increased the positive rates of genetic transformation mediated by A. rhizogenes and the editing efficiency of the CRISPR/Cas9-mediated genome editing system in potato. However, it was observed that the regeneration of potato roots was partially inhibited as a result. The analysis of CRISPR/Cas9-mediated mutation types revealed that chimeras accounted for the largest proportion, ranging from 62.50% to 100%. Moreover, the application of salt and osmotic stress resulted in an increased probability of null mutations in potato. Notably, the highest rate of null mutations, reaching 37.5%, was observed at a NaCl concentration of 10 mM. Three potential off-target sites were sequenced and no off-targeting was found. In conclusion, the application of appropriate salt and osmotic stress significantly improved the editing efficiency of the CRISPR/Cas9-mediated genome editing system in potato, with no observed off-target effects. However, there was a trade-off as the regeneration of potato roots was partially inhibited. Overall, these findings present a new and convenient approach to enhance the genome editing efficiency of the CRISPR/Cas9-mediated gene editing system in potato.},
}
@article {pmid37547075,
year = {2023},
author = {Wang, J and Tomar, D and Martin, TG and Dubey, S and Dubey, PK and Song, J and Landesberg, G and McCormick, MG and Myers, VD and Merali, S and Merali, C and Lemster, B and McTiernan, CF and Khalili, K and Madesh, M and Cheung, JY and Kirk, JA and Feldman, AM},
title = {Bag3 Regulates Mitochondrial Function and the Inflammasome Through Canonical and Noncanonical Pathways in the Heart.},
journal = {JACC. Basic to translational science},
volume = {8},
number = {7},
pages = {820-839},
pmid = {37547075},
issn = {2452-302X},
support = {K99 DK120876/DK/NIDDK NIH HHS/United States ; R00 DK120876/DK/NIDDK NIH HHS/United States ; },
abstract = {B-cell lymphoma 2-associated athanogene-3 (Bag3) is expressed in all animal species, with Bag3 levels being most prominent in the heart, the skeletal muscle, the central nervous system, and in many cancers. Preclinical studies of Bag3 biology have focused on animals that have developed compromised cardiac function; however, the present studies were performed to identify the pathways perturbed in the heart even before the occurrence of clinical signs of dilatation and failure of the heart. These studies show that hearts carrying variants that knockout one allele of BAG3 have significant alterations in multiple cellular pathways including apoptosis, autophagy, mitochondrial homeostasis, and the inflammasome.},
}
@article {pmid37547003,
year = {2023},
author = {McLean, ZL and Gao, D and Correia, K and Roy, JCL and Shibata, S and Farnum, IN and Valdepenas-Mellor, Z and Rapuru, M and Morini, E and Ruliera, J and Gillis, T and Lucente, D and Kleinstiver, BP and Lee, JM and MacDonald, ME and Wheeler, VC and Pinto, RM and Gusella, JF},
title = {PMS1 as a target for splice modulation to prevent somatic CAG repeat expansion in Huntington's disease.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37547003},
support = {R01 NS091161/NS/NINDS NIH HHS/United States ; R01 NS126420/NS/NINDS NIH HHS/United States ; R01 NS119471/NS/NINDS NIH HHS/United States ; R01 NS105709/NS/NINDS NIH HHS/United States ; DP2 CA281401/CA/NCI NIH HHS/United States ; R01 NS049206/NS/NINDS NIH HHS/United States ; },
abstract = {Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder whose motor, cognitive, and behavioral manifestations are caused by an expanded, somatically unstable CAG repeat in the first exon of HTT that lengthens a polyglutamine tract in huntingtin. Genome-wide association studies (GWAS) have revealed DNA repair genes that influence the age-at-onset of HD and implicate somatic CAG repeat expansion as the primary driver of disease timing. To prevent the consequent neuronal damage, small molecule splice modulators (e.g., branaplam) that target HTT to reduce the levels of huntingtin are being investigated as potential HD therapeutics. We found that the effectiveness of the splice modulators can be influenced by genetic variants, both at HTT and other genes where they promote pseudoexon inclusion. Surprisingly, in a novel hTERT-immortalized retinal pigment epithelial cell (RPE1) model for assessing CAG repeat instability, these drugs also reduced the rate of HTT CAG expansion. We determined that the splice modulators also affect the expression of the mismatch repair gene PMS1 , a known modifier of HD age-at-onset. Genome editing at specific HTT and PMS1 sequences using CRISPR-Cas9 nuclease confirmed that branaplam suppresses CAG expansion by promoting the inclusion of a pseudoexon in PMS1 , making splice modulation of PMS1 a potential strategy for delaying HD onset. Comparison with another splice modulator, risdiplam, suggests that other genes affected by these splice modulators also influence CAG instability and might provide additional therapeutic targets.},
}
@article {pmid37523811,
year = {2023},
author = {Jiang, C and Zheng, X and Lin, L and Li, X and Li, X and Liao, Y and Jia, W and Shu, B},
title = {CRISPR Cas12a-mediated amplification-free digital DNA assay improves the diagnosis and surveillance of Nasopharyngeal carcinoma.},
journal = {Biosensors &amp; bioelectronics},
volume = {237},
number = {},
pages = {115546},
doi = {10.1016/j.bios.2023.115546},
pmid = {37523811},
issn = {1873-4235},
mesh = {Humans ; Nasopharyngeal Carcinoma/diagnosis/genetics ; CRISPR-Cas Systems/genetics ; *Epstein-Barr Virus Infections ; *Nasopharyngeal Neoplasms/diagnosis/genetics/pathology ; *Carcinoma ; Case-Control Studies ; Herpesvirus 4, Human/genetics ; DNA, Viral/genetics ; *Biosensing Techniques ; Real-Time Polymerase Chain Reaction ; },
abstract = {Sensitive and accurate cell-free plasma Epstein-Barr virus (EBV) DNA measurement is essential in the routine diagnosis, monitoring and treatment of Nasopharyngeal Carcinoma (NPC). This measurement in commercial and in-house assay are commonly based on real-time quantitative PCR (qPCR) method, which requires reference materials for standardization and lack quantitative precision due to amplification bias or cross-contamination. To address these issues, we developed a CRISPR/Cas12a-mediated amplification-free digital DNA assay, which targets the repetitive sequences of EBV DNA and utilizes the cis-cleavage activity of CRISPR-Cas12a prior to droplet generation. By this mean, more activated Cas12a-crRNA duplexes could be produced for subsequent target detection and counting, thus improving the performance in detecting low EBV DNA load. We demonstrated that it was more robust than conventional qPCR for detecting plasma EBV DNA in a case-control study of 208 participants, especially when the target concentrations were around the diagnostic cut-off value for NPC. More importantly, this assay allowed a more accurate diagnosis of early-stage NPC, with an area under the curve (AUC) of 0.9883 (versus 0.7682 for qPCR). Furthermore, its absolute quantification capability enabled dynamic monitoring of EBV load in NPC patients during initial diagnosis, treatment, and recurrence, thereby potentially improving disease management and prognosis. Taken together, our results demonstrate that this amplification-free digital assay has the potential to be a robust tool to improve the diagnosis and surveillance of NPC.},
}
@article {pmid37459685,
year = {2023},
author = {Alon, DM and Mittelman, K and Stibbe, E and Countryman, S and Stodieck, L and Doraisingam, S and Leal Martin, DM and Hamo, ER and Pines, G and Burstein, D},
title = {CRISPR-based genetic diagnostics in microgravity.},
journal = {Biosensors &amp; bioelectronics},
volume = {237},
number = {},
pages = {115479},
doi = {10.1016/j.bios.2023.115479},
pmid = {37459685},
issn = {1873-4235},
mesh = {Humans ; *Weightlessness ; *Biosensing Techniques ; Astronauts ; Genomics ; Recombinases ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques ; },
abstract = {Monitoring astronauts' health during space missions poses many challenges, including rapid assessment of crew health conditions. Sensitive genetic diagnostics are crucial for examining crew members and the spacecraft environment. CRISPR-Cas12a, coupled with isothermal amplification, has proven to be a promising biosensing system for rapid, on-site detection of genomic targets. However, the efficiency and sensitivity of CRISPR-based diagnostics have never been tested in microgravity. We tested the use of recombinase polymerase amplification (RPA) coupled with the collateral cleavage activity of Cas12a for genetic diagnostics onboard the International Space Station. We explored the detection sensitivity of amplified and unamplified target DNA. By coupling RPA with Cas12a, we identified targets in attomolar concentrations. We further assessed the reactions' stability following long-term storage. Our results demonstrate that CRISPR-based detection is a powerful tool for on-site genetic diagnostics in microgravity, and can be further utilized for long-term space endeavors to improve astronauts' health and well-being.},
}
@article {pmid37451024,
year = {2023},
author = {Shen, J and Chen, Z and Xie, R and Li, J and Liu, C and He, Y and Ma, X and Yang, H and Xie, Z},
title = {CRISPR/Cas12a-Assisted isothermal amplification for rapid and specific diagnosis of respiratory virus on an microfluidic platform.},
journal = {Biosensors &amp; bioelectronics},
volume = {237},
number = {},
pages = {115523},
doi = {10.1016/j.bios.2023.115523},
pmid = {37451024},
issn = {1873-4235},
mesh = {Humans ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; Microfluidics ; SARS-CoV-2/genetics ; *Biosensing Techniques ; *Nucleic Acids ; Nucleic Acid Amplification Techniques ; COVID-19 Testing ; },
abstract = {Respiratory viruses have long been a major cause of a global pandemic, emphasizing the urgent need for high-sensitivity diagnostic tools. Typical PCR technology can only determine the type of virus in the sample, which is unable to detect different variants of the same virus without costly and time-consuming gene sequencing. Here, we introduce a simple, fully enclosed, and highly integrated microfluidic system based on CRISPR/Cas12a and isothermal amplification techniques (LOC-CRISPR) that can specifically identify multiple common respiratory viruses and their variants. The LOC-CRISPR chip integrates viral nucleic acid extraction, recombinant polymerase amplification, and CRISPR/Cas12a cleavage reaction-based detection, contamination-free detection. In addition, the LOC-CRISPR chip was designed for multiplexed detection (two-sample input and ten-result outputs), which can not only detect the presence of SARS-CoV-2, H1N1, H3N2, IVB and HRSV but also differentiate the BA.1, BA.2, and BA.5 variants of SARS-COV-2. For clinical validation, the LOC-CRISPR chip was used to analyze 50 nasopharyngeal swab samples (44 positive and 6 negative) and achieved excellent sensitivity (97.8%) and specificity (100%). This innovative LOC-CRISPR system has the ability to quickly, sensitively, and accurately detect multiple target nucleic acid sequences with single-base mutations, which will further improve the rapid identification and traceability of respiratory viruses infectious diseases.},
}
@article {pmid37423067,
year = {2023},
author = {Yuan, Q and Mao, D and Tang, X and Liu, C and Zhang, R and Deng, J and Zhu, X and Li, W and Man, Q and Sun, F},
title = {Biological effect abundance analysis of hemolytic pathogens based on engineered biomimetic sensor.},
journal = {Biosensors &amp; bioelectronics},
volume = {237},
number = {},
pages = {115502},
doi = {10.1016/j.bios.2023.115502},
pmid = {37423067},
issn = {1873-4235},
mesh = {Humans ; *Biomimetics ; Hemolysis ; *Biosensing Techniques ; Biological Assay ; Enzyme-Linked Immunosorbent Assay ; CRISPR-Cas Systems ; },
abstract = {Conventional pathogen detection strategies based on the molecular structure or chemical characteristics of biomarkers can only provide the "physical abundance" of microorganisms, but cannot reflect the "biological effect abundance" in the true sense. To address this issue, we report an erythrocyte membrane-encapsulated biomimetic sensor cascaded with CRISPR-Cas12a (EMSCC). Taking hemolytic pathogens as the target model, we first constructed an erythrocyte membrane-encapsulated biomimetic sensor (EMS). Only hemolytic pathogens with biological effects can disrupt the erythrocyte membrane (EM), resulting in signal generation. Then the signal was amplified by cascading CRISPR-Cas12a, and more than 6.67 × 10[4]-fold improvement in detection sensitivity compared to traditional erythrocyte hemolysis assay was achieved. Notably, compared with polymerase chain reaction (PCR) or enzyme linked immunosorbent assay (ELISA)-based quantification methods, EMSCC can sensitively respond to the pathogenicity change of pathogens. For the detection of simulated clinical samples based on EMSCC, we obtained an accuracy of 95% in 40 samples, demonstrating its potential clinical value.},
}
@article {pmid37395412,
year = {2023},
author = {Liu, Z and Jillette, N and Robson, P and Cheng, AW},
title = {Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold.},
journal = {Nucleic acids research},
volume = {51},
number = {14},
pages = {e77},
pmid = {37395412},
issn = {1362-4962},
support = {R01-HG009900/HG/NHGRI NIH HHS/United States ; R01-HG009900/HG/NHGRI NIH HHS/United States ; },
mesh = {*RNA/genetics ; *CRISPR-Cas Systems/genetics ; Transcriptome ; RNA Processing, Post-Transcriptional ; RNA Splicing ; Gene Editing/methods ; },
abstract = {RNA processing and metabolism are subjected to precise regulation in the cell to ensure integrity and functions of RNA. Though targeted RNA engineering has become feasible with the discovery and engineering of the CRISPR-Cas13 system, simultaneous modulation of different RNA processing steps remains unavailable. In addition, off-target events resulting from effectors fused with dCas13 limit its application. Here we developed a novel platform, Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST), which can simultaneously execute multiple RNA modulation functions on different RNA targets. In CREST, RNA scaffolds are appended to the 3' end of Cas13 gRNA and their cognate RNA binding proteins are fused with enzymatic domains for manipulation. Taking RNA alternative splicing, A-to-G and C-to-U base editing as examples, we developed bifunctional and tri-functional CREST systems for simultaneously RNA manipulation. Furthermore, by fusing two split fragments of the deaminase domain of ADAR2 to dCas13 and/or PUFc respectively, we reconstituted its enzyme activity at target sites. This split design can reduce nearly 99% of off-target events otherwise induced by a full-length effector. The flexibility of the CREST framework will enrich the transcriptome engineering toolbox for the study of RNA biology.},
}
@article {pmid37395400,
year = {2023},
author = {Wang, WJ and Lin, J and Wu, CQ and Luo, AL and Xing, X and Xu, L},
title = {Establishing artificial gene connections through RNA displacement-assembly-controlled CRISPR/Cas9 function.},
journal = {Nucleic acids research},
volume = {51},
number = {14},
pages = {7691-7703},
pmid = {37395400},
issn = {1362-4962},
support = {21977122//National Natural Science Foundation of China/ ; 2020YFA0211200//National Key R&amp;D Program of China/ ; 2022B1515020047//Outstanding Youth Project of Guangdong Natural Science Foundation/ ; 2021A1515010253//General Project of Natural Science Foundation of Guangdong Province/ ; 202102020523//General Project of Natural Science Foundation of Guangzhou/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Genes, Synthetic ; Gene Regulatory Networks/genetics ; *MicroRNAs ; RNA, Messenger ; Gene Editing ; Mammals/genetics ; },
abstract = {Construction of synthetic circuits that can reprogram genetic networks and signal pathways is a long-term goal for manipulation of biosystems. However, it is still highly challenging to build artificial genetic communications among endogenous RNA species due to their sequence independence and structural diversities. Here we report an RNA-based synthetic circuit that can establish regulatory linkages between expression of endogenous genes in both Escherichiacoli and mammalian cells. This design employs a displacement-assembly approach to modulate the activity of guide RNA for function control of CRISPR/Cas9. Our experiments demonstrate the great effectiveness of this RNA circuit for building artificial connections between expression of originally unrelated genes. Both exogenous and naturally occurring RNAs, including small/microRNAs and long mRNAs, are capable of controlling expression of another endogenous gene through this approach. Moreover, an artificial signal pathway inside mammalian cells is also successfully established to control cell apoptosis through our designed synthetic circuit. This study provides a general strategy for constructing synthetic RNA circuits, which can introduce artificial connections into the genetic networks of mammalian cells and alter the cellular phenotypes.},
}
@article {pmid37354728,
year = {2023},
author = {Awasthi, P and Mishra, AK and Kocábek, T and Nath, VS and Mishra, S and Hazzouri, KM and Sudalaimuthuasari, N and Stajner, N and Jakše, J and Krofta, K and Hájek, T and Amiri, KM},
title = {CRISPR/Cas9-mediated mutagenesis of the mediator complex subunits MED5a and MED5b genes impaired secondary metabolite accumulation in hop (Humulus lupulus).},
journal = {Plant physiology and biochemistry : PPB},
volume = {201},
number = {},
pages = {107851},
doi = {10.1016/j.plaphy.2023.107851},
pmid = {37354728},
issn = {1873-2690},
mesh = {*Humulus/genetics ; CRISPR-Cas Systems ; Gene Expression Profiling ; Transcriptome ; Genomics ; },
abstract = {Hop (Humulus lupulus L.) is an important commercial crop known for the biosynthesis of valuable specialized secondary metabolites in glandular trichomes (lupulin glands), which are used for the brewing industry. To achieve burgeoning market demands is the essentiality of comprehensive understanding of the mechanisms of biosynthesis of secondary metabolites in hop. Over the past year, several studies using structural biology and functional genomics approaches have shown that Mediator (MED) serves as an integrative hub for RNAP II-mediated transcriptional regulation of various physiological and cellular processes, including involvement of MED5a and MED5b in hyperaccumulation of phenylpropanoid in A. thaliana. In the present work, an unprecedented attempt was made to generate Hlmed5a/med5b double loci mutant lines in hop using a CRISPR/Cas9-based genome editing system. The Hlmed5a/med5b double loci mutant lines showed reduced expression of structural genes of the flavonoid, humulone, and terpenoid biosynthetic pathways, which was more pronounced in the lupulin gland compared to leaf tissue and was consistent with their reduced accumulation. Phenotypic and anatomical observations revealed that Hlmed5a/med5b double loci mutant line exhibited robust growth, earlier flowering, earlier cone maturity, reduced cone size, variations in floral structure patterns, and distorted lupulin glands without any remarkable changes in leaf morphology, intensity of leaf color, and chlorophyll content. Comparative transcriptome analysis of leaf and lupulin gland tissues indicates that the expression of enzymatic genes related to secondary metabolite biosynthesis, phytohormone biosynthesis, floral organs, flowering time, and trichome development, including other genes related to starch and sucrose metabolism and defense mechanisms, were differentially modulated in the Hlmed5a/med5b lines. The combined results from functional and transcriptomic analyses illuminates the pivotal function of HlMED5a and HlMED5b in homeostasis of secondary meatbolites accumulation in hop.},
}
@article {pmid37328396,
year = {2023},
author = {Barka, A and Kohli, RM and Shi, J},
title = {CRISPR tiling screen reveals cancer epigenetic 'Goldilocks' state.},
journal = {Trends in pharmacological sciences},
volume = {44},
number = {9},
pages = {555-557},
doi = {10.1016/j.tips.2023.05.007},
pmid = {37328396},
issn = {1873-3735},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Neoplasms/genetics ; Epigenomics ; Epigenesis, Genetic ; CRISPR-Cas Systems ; },
abstract = {CRISPR tiling screens offer an efficient way to identify gain-of-function mutations in targets of cancer therapy. Recently, by utilizing these screens, Kwok et al. unexpectedly discovered mutations conferring drug addiction in lymphoma, revealing a requirement for a 'just right' window of histone methylation crucial for cancer survival.},
}
@article {pmid36646933,
year = {2023},
author = {Mathis, N and Allam, A and Kissling, L and Marquart, KF and Schmidheini, L and Solari, C and Balázs, Z and Krauthammer, M and Schwank, G},
title = {Predicting prime editing efficiency and product purity by deep learning.},
journal = {Nature biotechnology},
volume = {41},
number = {8},
pages = {1151-1159},
pmid = {36646933},
issn = {1546-1696},
support = {185293/SNSF_/Swiss National Science Foundation/Switzerland ; 310030_185293//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 201184//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {Humans ; *Deep Learning ; Gene Editing ; Hepatocytes ; Mutation ; Neural Networks, Computer ; CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing is a versatile genome editing tool but requires experimental optimization of the prime editing guide RNA (pegRNA) to achieve high editing efficiency. Here we conducted a high-throughput screen to analyze prime editing outcomes of 92,423 pegRNAs on a highly diverse set of 13,349 human pathogenic mutations that include base substitutions, insertions and deletions. Based on this dataset, we identified sequence context features that influence prime editing and trained PRIDICT (prime editing guide prediction), an attention-based bidirectional recurrent neural network. PRIDICT reliably predicts editing rates for all small-sized genetic changes with a Spearman's R of 0.85 and 0.78 for intended and unintended edits, respectively. We validated PRIDICT on endogenous editing sites as well as an external dataset and showed that pegRNAs with high (>70) versus low (<70) PRIDICT scores showed substantially increased prime editing efficiencies in different cell types in vitro (12-fold) and in hepatocytes in vivo (tenfold), highlighting the value of PRIDICT for basic and for translational research applications.},
}
@article {pmid36604543,
year = {2023},
author = {Jiao, C and Reckstadt, C and König, F and Homberger, C and Yu, J and Vogel, J and Westermann, AJ and Sharma, CM and Beisel, CL},
title = {RNA recording in single bacterial cells using reprogrammed tracrRNAs.},
journal = {Nature biotechnology},
volume = {41},
number = {8},
pages = {1107-1116},
pmid = {36604543},
issn = {1546-1696},
support = {INST93/1105-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; BE 6703/1-2//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 865973//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 865973/ERC_/European Research Council/International ; SH 580/9-2//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*RNA/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Bacteria/genetics/metabolism ; DNA/genetics ; Gene Editing ; RNA, Bacterial/genetics ; },
abstract = {Capturing an individual cell's transcriptional history is a challenge exacerbated by the functional heterogeneity of cellular communities. Here, we leverage reprogrammed tracrRNAs (Rptrs) to record selected cellular transcripts as stored DNA edits in single living bacterial cells. Rptrs are designed to base pair with sensed transcripts, converting them into guide RNAs. The guide RNAs then direct a Cas9 base editor to target an introduced DNA target. The extent of base editing can then be read in the future by sequencing. We use this approach, called TIGER (transcribed RNAs inferred by genetically encoded records), to record heterologous and endogenous transcripts in individual bacterial cells. TIGER can quantify relative expression, distinguish single-nucleotide differences, record multiple transcripts simultaneously and read out single-cell phenomena. We further apply TIGER to record metabolic bet hedging and antibiotic resistance mobilization in Escherichia coli as well as host cell invasion by Salmonella. Through RNA recording, TIGER connects current cellular states with past transcriptional states to decipher complex cellular responses in single cells.},
}
@article {pmid37546822,
year = {2023},
author = {Sinha, S and Molina Vargas, AM and Arantes, PR and Patel, A and O'Connell, MR and Palermo, G},
title = {RNA-mediated Allosteric Activation in CRISPR-Cas13a.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.07.27.550797},
pmid = {37546822},
support = {R35 GM133462/GM/NIGMS NIH HHS/United States ; },
abstract = {Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. The protein uses a CRISPR RNA (crRNA) to target RNA sequences, which are cleaved by a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) catalytic domains. In this system, an intriguing form of allosteric communication controls RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory and RNA cleavage assays to decipher this activation mechanism. We show that the binding of a target RNA acts as an allosteric effector of the spatially distant HEPN catalytic cleft, by amplifying the allosteric signals over the dynamical noise, that passes through the buried HEPN interface. Critical residues in this region - N378, R973, and R377 - rearrange their interactions upon target RNA binding, and alter allosteric signalling. Alanine mutation of these residues is experimentally shown to select target RNA over an extended complementary sequence beyond guide-target duplex, for RNA cleavage. Altogether, our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of more selective RNA-based cleavage and detection tools.},
}
@article {pmid37545761,
year = {2023},
author = {Kocsisova, Z and Coneva, V},
title = {Strategies for delivery of CRISPR/Cas-mediated genome editing to obtain edited plants directly without transgene integration.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1209586},
pmid = {37545761},
issn = {2673-3439},
abstract = {Increased understanding of plant genetics and the development of powerful and easier-to-use gene editing tools over the past century have revolutionized humankind's ability to deliver precise genotypes in crops. Plant transformation techniques are well developed for making transgenic varieties in certain crops and model organisms, yet reagent delivery and plant regeneration remain key bottlenecks to applying the technology of gene editing to most crops. Typical plant transformation protocols to produce transgenic, genetically modified (GM) varieties rely on transgenes, chemical selection, and tissue culture. Typical protocols to make gene edited (GE) varieties also use transgenes, even though these may be undesirable in the final crop product. In some crops, the transgenes are routinely segregated away during meiosis by performing crosses, and thus only a minor concern. In other crops, particularly those propagated vegetatively, complex hybrids, or crops with long generation times, such crosses are impractical or impossible. This review highlights diverse strategies to deliver CRISPR/Cas gene editing reagents to regenerable plant cells and to recover edited plants without unwanted integration of transgenes. Some examples include delivering DNA-free gene editing reagents such as ribonucleoproteins or mRNA, relying on reagent expression from non-integrated DNA, using novel delivery mechanisms such as viruses or nanoparticles, using unconventional selection methods to avoid integration of transgenes, and/or avoiding tissue culture altogether. These methods are advancing rapidly and already enabling crop scientists to make use of the precision of CRISPR gene editing tools.},
}
@article {pmid37433390,
year = {2023},
author = {Gibney, TV and Favichia, M and Latifi, L and Medwig-Kinney, TN and Matus, DQ and McIntyre, DC and Arrigo, AB and Branham, KR and Bubrig, LT and Ghaddar, A and Jiranek, JA and Liu, KE and Marcucci, CG and Porter, RJ and Pani, AM},
title = {A simple method to dramatically increase C. elegans germline microinjection efficiency.},
journal = {Developmental biology},
volume = {502},
number = {},
pages = {63-67},
doi = {10.1016/j.ydbio.2023.07.003},
pmid = {37433390},
issn = {1095-564X},
support = {R35 GM142880/GM/NIGMS NIH HHS/United States ; R35 GM137975/GM/NIGMS NIH HHS/United States ; R35 GM141886/GM/NIGMS NIH HHS/United States ; F31 HD100091/HD/NICHD NIH HHS/United States ; T32 GM007267/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; Microinjections/methods ; Animals, Genetically Modified ; *Germ Cells ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Genome manipulation methods in C. elegans require microinjecting DNA or ribonucleoprotein complexes into the microscopic core of the gonadal syncytium. These microinjections are technically demanding and represent a key bottleneck for all genome engineering and transgenic approaches in C. elegans. While there have been steady improvements in the ease and efficiency of genetic methods for C. elegans genome manipulation, there have not been comparable advances in the physical process of microinjection. Here, we report a simple and inexpensive method for handling worms using a paintbrush during the injection process that nearly tripled average microinjection rates compared to traditional worm handling methods. We found that the paintbrush increased injection throughput by substantially increasing both injection speeds and post-injection survival rates. In addition to dramatically and universally increasing injection efficiency for experienced personnel, the paintbrush method also significantly improved the abilities of novice investigators to perform key steps in the microinjection process. We expect that this method will benefit the C. elegans community by increasing the speed at which new strains can be generated and will also make microinjection-based approaches less challenging and more accessible to personnel and labs without extensive experience.},
}
@article {pmid37545694,
year = {2023},
author = {Khlidj, Y},
title = {What did CRISPR-Cas9 accomplish in its first 10 years?.},
journal = {Biochemia medica},
volume = {33},
number = {3},
pages = {030601},
pmid = {37545694},
issn = {1846-7482},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; },
abstract = {It's been 10 years now from the debut of clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) era in which gene engineering has never been so accessible, precise and efficient. This technology, like a refined surgical procedure, has offered the ability of removing different types of disease causing mutations and restoring key proteins activity with ease of outperforming the previous resembling methods: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Additionally, CRISPR-Cas9 systems can systematically introduce genetic sequences to the specific sites in the human genome allowing to stimulate desired functions such as anti-tumoral and anti-infectious faculties. The present brief review provides an updated resume of CRISPR-Cas9's top achievements from its first appearance to the current date focusing on the breakthrough research including in vitro, in vivo and human studies. This enables the evaluation of the previous phase 'the proof-of-concept phase' and marks the beginning of the next phase which will probably bring a spate of clinical trials.},
}
@article {pmid37490481,
year = {2023},
author = {Li, Y and Wang, X and He, Z and Johnson, M and A, S and Lara-Sáez, I and Lyu, J and Wang, W},
title = {3D Macrocyclic Structure Boosted Gene Delivery: Multi-Cyclic Poly(β-Amino Ester)s from Step Growth Polymerization.},
journal = {Journal of the American Chemical Society},
volume = {145},
number = {31},
pages = {17187-17200},
doi = {10.1021/jacs.3c04191},
pmid = {37490481},
issn = {1520-5126},
mesh = {Polymerization ; *RNA, Guide, CRISPR-Cas Systems ; *Gene Transfer Techniques ; Polymers/chemistry ; CRISPR-Cas Systems ; },
abstract = {The topological structures of polymers play a critical role in determining their gene delivery efficiency. Exploring novel polymeric structures as gene delivery vectors is thus of great interest. In this work, a new generation of multi-cyclic poly(β-amino ester)s (CPAEs) with unique topology structure was synthesized for the first time via step growth polymerization. Through controlling the occurrence stage of cyclization, three types of CPAEs with rings of different sizes and topologies were obtained. In vitro experiments demonstrated that the CPAEs with macro rings (MCPAEs) significantly boosted the transgene expression comparing to their branched counterparts. Moreover, the MCPAE vector with optimized terminal group efficiently delivered the CRISPR plasmid coding both Staphylococcus aureus Cas9 nuclease and dual guide sgRNAs for gene editing therapy.},
}
@article {pmid37490029,
year = {2023},
author = {Jia, ZQ and Zhang, SG and Wang, Y and Pan, JH and Liu, FF and Zhan, EL and Fouad, EA and Fu, YL and Pan, QR and Zhao, CQ},
title = {Physiological Function of RDL1 and RDL2 Subunits of the Ionotropic GABA Receptor in the Spodoptera litura with the CRISPR/Cas9 System In Vivo.},
journal = {Journal of agricultural and food chemistry},
volume = {71},
number = {31},
pages = {11875-11883},
pmid = {37490029},
issn = {1520-5118},
mesh = {Animals ; *Receptors, GABA/genetics/metabolism ; Spodoptera/physiology ; *CRISPR-Cas Systems ; Larva/genetics/metabolism ; Dieldrin ; gamma-Aminobutyric Acid ; },
abstract = {In insect ionotropic γ-aminobutyric acid receptor (iGABAR) subunits, only resistance to dieldrin (RDL) can be individually and functionally expressed in vitro. In lepidopteran, two to three RDL subtypes are identified; however, their physiological roles have not been distinguished in vivo. In this study, SlRdl1 and SlRdl2 of S. litura were individually knocked out using CRISPR/Cas9, respectively. The mortality and larval and pupal duration of KOSlRdl1 and KOSlRdl2 were increased. The flight time and distance were increased by 43.30%-80.66% and 58.96%-198.22%, respectively, in KOSlRdl1. The GABA-induced current was significantly decreased by 53.57%-74.28% and 46.91%-63.34% in the ventral nerve cord, and the GABA titer was significantly reduced by 17.65%-28.05% and 19.85%-42.46% in KOSlRdl1 and KOSlRdl2, respectively. In conclusion, SlRdl1 and SlRdl2 are necessary for the transmission of GABA-induced neural signals; however, only SlRdl1 could regulate the flight capability of S. litura. Our results provided a new avenue to study lepidopteran iGABARs.},
}
@article {pmid37545273,
year = {2023},
author = {Nie, X and Wang, D and Pan, Y and Lü, P and Yang, Y},
title = {Discovery, classification and application of the CPISPR-Cas13 system.},
journal = {Technology and health care : official journal of the European Society for Engineering and Medicine},
volume = {},
number = {},
pages = {},
doi = {10.3233/THC-230258},
pmid = {37545273},
issn = {1878-7401},
abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is an acquired immune system of bacteria and archaea. Continued research has resulted in the identification of other Cas13 proteins.<br><br>OBJECTIVE: This review briefly describes the discovery, classification, and application of the CRISPR-Cas13 system, including recent technological advances in addition to factors affecting system performance.<br><br>METHODS: Cas13-based molecular therapy of human, animal, and plant transcriptomes was discussed, including regulation of gene expression to combat pathogenic RNA viruses. In addition, the latest progress, potential shortcomings, and challenges of the CRISPR-Cas system for treatment of animal and plant diseases are reviewed.<br><br>RESULTS: The CRISPR-Cas system VI is characterized by two RNA-guided higher eukaryotes and prokaryotes nucleotide-binding domains. CRISPR RNA can cleave specific RNA through the interaction between the stem-loop rich chain of uracil residues and the Cas13a protein. The CRISPR-Cas13 system has been applied for gene editing in animal and plant cells, in addition to biological detection via accurate targeting of single-stranded RNA.<br><br>CONCLUSION: The CRISPR-Cas13 system offers a high-throughput and convenient technology for detection of viruses and potentially the development of anti-cancer drugs in the near future.},
}
@article {pmid37541970,
year = {2023},
author = {Elsharawy, H and Refat, M},
title = {CRISPR/Cas9 genome editing in wheat: enhancing quality and productivity for global food security-a review.},
journal = {Functional &amp; integrative genomics},
volume = {23},
number = {3},
pages = {265},
pmid = {37541970},
issn = {1438-7948},
mesh = {Humans ; *Gene Editing/methods ; *Triticum/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; Genome, Plant ; Crops, Agricultural/genetics ; Edible Grain/genetics ; },
abstract = {Wheat (Triticum aestivum L.) is an important cereal crop that is grown all over the world for food and industrial purposes. Wheat is essential to the human diet due to its rich content of necessary amino acids, minerals, vitamins, and calories. Various wheat breeding techniques have been utilized to improve its quality, productivity, and resistance to biotic and abiotic stress impairing production. However, these techniques are expensive, demanding, and time-consuming. Additionally, these techniques need multiple generations to provide the desired results, and the improved traits could be lost over time. To overcome these challenges, researchers have developed various genome editing tools to improve the quality and quantity of cereal crops, including wheat. Genome editing technologies evolve quickly. Nowadays, single or multiple mutations can be enabled and targeted at specific loci in the plant genome, allowing controlled removal of undesirable features or insertion of advantageous ones. Clustered, regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) is a powerful genome editing tool that can be effectively used for precise genome editing of wheat and other crops. This review aims to provide a comprehensive understanding of this technology's potential applications to enhance wheat's quality and productivity. It will first explore the function of CRISPR/Cas9 in preserving the adaptive immunity of prokaryotic organisms, followed by a discussion of its current applications in wheat breeding.},
}
@article {pmid37540372,
year = {2023},
author = {Xu, Y and Gan, ES and Ito, T},
title = {Misexpression Approaches for the Manipulation of Flower Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2686},
number = {},
pages = {429-451},
pmid = {37540372},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Transcriptional Activation ; Phenotype ; *Plants/genetics ; Flowers/genetics ; },
abstract = {The generation of dominant gain-of-function mutants through activation tagging is a forward genetic approach that can be applied to study the mechanisms of flower development, complementing the screening of loss-of-function mutants. In addition, the functions of genes of interest can be further analyzed through reverse genetics. A commonly used method is gene overexpression, where ectopic expression can result in an opposite phenotype to that caused by a loss-of-function mutation. When overexpression is detrimental, the misexpression of a gene using tissue-specific promoters can be useful to study spatial-specific function. As flower development is a multistep process, it can be advantageous to control gene expression, or its protein product activity, in a temporal and/or spatial manner. This has been made possible through several inducible promoter systems as well as inducible proteins by constructing chimeric fusions between the ligand-binding domain of the glucocorticoid receptor (GR) and the protein of interest. The recently introduced CRISPR-Cas9-based platform provides a new way of bioengineering transcriptional regulators in plants. By fusing a catalytically inactive dCas9 with functional activation or repression domains, the CRISPR-Cas9 module can achieve transcriptional activation or repression of endogenous genes. All these methods allow us to genetically manipulate gene expression during flower development. In this chapter, we describe methods to produce the expression constructs, method of screening, and more general applications of the techniques.},
}
@article {pmid37538843,
year = {2023},
author = {Zhu, J and Ju, Y and Zhou, X and Chen, T and Zhuge, X and Dai, J},
title = {Epidemiological characteristics of SHV, cmlv, and FosA6-producing carbapenem-resistant Klebsiella pneumoniae based on whole genome sequences in Jiangsu, China.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1219733},
pmid = {37538843},
issn = {1664-302X},
abstract = {Carbapenem-resistant Klebsiella pneumoniae (CRKP), particularly those with high virulence, cause invasive disease in clinical settings. An epidemiological investigation was conducted on the evolution, virulence, and antimicrobial resistance of CRKP isolates in two tertiary teaching hospitals in Jiangsu, China from November 2020 to December 2021. There were 31 different CRKP strains discovered. We performed whole genome sequencing (WGS) on 13 SHV, cmlv, and FosA6-producing CRKP to reveal molecular characteristics. Five ST15/ST11 isolates had CRISPR-Cas systems. By conjugation tests, KPC-2 can be transmitted horizontally to E. coil. A conjugative pHN7A8-related multi-resistance plasmid (KPC-2, blaCTX-M-65, blaTEM-1, fosA3, catII, and rmtB) was first discovered in CRKP clinical isolates. Using bacteriological testing, a serum killing assay, and an infection model with Galleria mellonella, three ST11-K64 KPC-2 generating carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) were identified. These strains harbored a virulent plasmid and an IncFII-family pKPC/pHN7A8 conjugative plasmid, which led to hypervirulence and resistance. One of these CR-hvKPs, which co-harbored KPC-2, NDM-6, SHV-182, SHV-64, and blaCTX-M-122 genes, was first discovered. Importantly, this CR-hvKP strain also produced biofilm and had non-inferior fitness. The widespread use of ceftazidime/avibactam might provide this CR-hvKP with a selective advantage; hence, immediate action is required to stop its dissemination. Another important finding is the novel ST6136 in K. pneumoniae. Finally, the sterilization efficiency rates of Fe2C nanoparticles in CRKP were more than 98%. Furthermore, our novel antibacterial Fe2C nanoparticles may also provide a therapeutic strategy for infections.},
}
@article {pmid37493946,
year = {2023},
author = {Ma, X and Zhang, Y and Qiao, X and Yuan, Y and Sheng, Q and Yue, T},
title = {Target-Induced AIE Effect Coupled with CRISPR/Cas12a System Dual-Signal Biosensing for the Ultrasensitive Detection of Gliotoxin.},
journal = {Analytical chemistry},
volume = {95},
number = {31},
pages = {11723-11731},
doi = {10.1021/acs.analchem.3c01760},
pmid = {37493946},
issn = {1520-6882},
mesh = {*Gliotoxin ; CRISPR-Cas Systems ; *Mycotoxins ; Oligonucleotides ; DNA, Single-Stranded ; *Biosensing Techniques ; },
abstract = {Here, a novel rapid and ultrasensitive aptamer biosensor was designed for target-induced activation of AIE effect and followed by the activation of Crispr Cas12a (LbCpf1)-mediated cleavage to achieve dual-signal detection. The prepared DNA building blocks contain the target aptamer, ssDNA-Fc, and Activator1. In this system, the activation mode was divided into two steps. First, when the target interacts with the aptamers, the DNA building blocks would be disintegrated rapidly, releasing a mass of Ac1, generating ETTC-dsDNA aggregated to produce a fluorescence signal by the AIE effect. Second, with the release of Ac2, LbCpf1-crRNA was activated, which greatly improves the ssDNA-Fc cleavage efficiency to render signal amplification and ultrasensitive detection of the target. Satisfactorily, using this approach to detect gliotoxin, optimal conditions for detection was achieved for reducing the detection time to 55 min, achieving a low detection limit of 2.4 fM and a satisfactory linear in the range of 50 fM to 1 nM, which addressed the shortcoming of a weak electrochemical signal in previous sensors. The water-insoluble AIE material was coupled with DNA to obtain water-soluble ETTC-dsDNA and successfully introduced into the sensor system, with a low detection limit of 5.6 fM. Subsequently, the biosensor combined with handheld electrochemical workstation was successfully applied in the detection of gliotoxin in five actual samples, with a detection range of 32.0 to 2.09 × 10[8] pM. This strategy not only provides a novel and effective detection platform for mycotoxins in complex food matrices but also opens a promising avenue for various molecules detection in imaging and disease diagnosis.},
}
@article {pmid37488217,
year = {2023},
author = {Huang, Z and Kaller, M and Hermeking, H},
title = {CRISPR/Cas9-mediated inactivation of miR-34a and miR-34b/c in HCT116 colorectal cancer cells: comprehensive characterization after exposure to 5-FU reveals EMT and autophagy as key processes regulated by miR-34.},
journal = {Cell death and differentiation},
volume = {30},
number = {8},
pages = {2017-2034},
pmid = {37488217},
issn = {1476-5403},
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; HCT116 Cells ; Tumor Suppressor Protein p53/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Colorectal Neoplasms/drug therapy/genetics/metabolism ; Fluorouracil/pharmacology ; Autophagy/genetics ; Gene Expression Regulation, Neoplastic ; },
abstract = {The miR-34a and miR-34b/c encoding genes represent direct targets of the p53 transcription factor, and presumably mediate part of the tumor suppressive effects of p53. Here, we sought to determine their functional relevance by inactivating miR-34a and/or miR-34b/c using a CRISPR/Cas9 approach in the colorectal cancer (CRC) cell line HCT116. Concomitant deletion of miR-34a and miR-34b/c resulted in significantly reduced suppression of proliferation after p53 activation, enhanced migration, invasion and EMT, as well as reduced sensitivity to chemotherapeutics, increased stress-induced autophagic flux, decreased apoptosis and upregulation of autophagy-related genes after 5-FU treatment. However, inactivation of singular miR-34a or miR-34b/c had little effects on the aforementioned processes. RNA-Seq analysis revealed that concomitant deletion of miR-34a/b/c caused EMT signature enrichment, impaired gene repression by the p53-DREAM pathway and elevated autophagy after 5-FU treatment. A gene signature comprised of mRNAs significantly upregulated after combined inactivation of miR-34a and miR-34b/c showed a significant association with the invasive colon cancer subtype CMS4 and poor overall survival in two CRC patient cohorts, and with 5-FU resistance in CRC cell lines. In miR-34a/b/c-deficient cells the upregulated miR-34 target FOXM1 directly induced p62 and ATG9A, which increased autophagy and consequently attenuated apoptosis and rendered the miR-34a/b/c-KO cells more resistant to 5-FU. Inhibition of autophagy by depletion of ATG9A or chloroquine re-sensitized miR-34a/b/c-deficient HCT116 cells to 5-FU. In summary, our findings show a complementary role of miR-34a and miR-34b/c in the regulation of EMT and autophagy which may be relevant for CRC therapy in the future.},
}
@article {pmid37468714,
year = {2023},
author = {Coogan, M and Xing, D and Su, B and Alston, V and Johnson, A and Khan, M and Khalil, K and Elaswad, A and Li, S and Wang, J and Lu, C and Wang, W and Hettiarachchi, D and Shang, M and Hasin, T and Qin, Z and Cone, R and Butts, IAE and Dunham, RA},
title = {CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome.},
journal = {Transgenic research},
volume = {32},
number = {4},
pages = {251-264},
pmid = {37468714},
issn = {1573-9368},
support = {2015-67015-23488//National Institute of Food and Agriculture/ ; },
mesh = {Animals ; *Ictaluridae/genetics ; Fatty Acid Elongases/genetics ; CRISPR-Cas Systems/genetics ; *Carps ; RNA, Guide, CRISPR-Cas Systems ; Animals, Genetically Modified/genetics ; *Oncorhynchus/genetics ; },
abstract = {Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccβA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, β-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccβA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch (p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively (p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls (p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.},
}
@article {pmid37460719,
year = {2023},
author = {},
title = {A method to map single-cell lineages in the mouse brain by CRISPR-based barcoding.},
journal = {Nature methods},
volume = {20},
number = {8},
pages = {1139-1140},
pmid = {37460719},
issn = {1548-7105},
mesh = {Animals ; Mice ; Cell Lineage ; *CRISPR-Cas Systems/genetics ; *Gene Expression Profiling ; Brain ; DNA Barcoding, Taxonomic ; },
}
@article {pmid37431292,
year = {2023},
author = {Sun, Z and Ren, M and Shan, B and Yang, Q and Zhao, Z and Liu, X and Yin, L},
title = {One-pot synthesis of dynamically cross-linked polymers for serum-resistant nucleic acid delivery.},
journal = {Biomaterials science},
volume = {11},
number = {16},
pages = {5653-5662},
doi = {10.1039/d3bm00685a},
pmid = {37431292},
issn = {2047-4849},
mesh = {*Polymers/chemistry ; *Nucleic Acids ; Gene Transfer Techniques ; RNA, Guide, CRISPR-Cas Systems ; Transfection ; Polyethyleneimine/chemistry ; DNA/genetics ; Plasmids ; Polyphenols ; },
abstract = {Cationic polymers used for nucleic acid delivery often suffer from complicated syntheses, undesired intracellular cargo release and low serum stability. Herein, a series of ternary polymers were synthesized via facile green chemistry to achieve efficient plasmid DNA and mRNA delivery in serum. During the one-pot synthesis of the ternary polymer, acetylphenylboric acid (APBA), polyphenol and low-molecular weight polyethyleneimine (PEI 1.8k) were dynamically cross-linked with each other due to formation of an imine between PEI 1.8k and APBA and formation of a boronate ester between APBA and polyphenol. Series of polyphenols, including ellagic acid (EA), epigallocatechin gallate (EGCG), nordihydroguaiaretic acid (NDGA), rutin (RT) and rosmarinic acid (RA), and APBA molecules, including 2-acetylphenylboric acid (2-APBA), 3-acetylphenylboric acid (3-APBA) and 4-acetylphenylboric acid (4-APBA), were screened and the best-performing ternary polymer, 2-PEI-RT, constructed from RT and 2-APBA, was identified. The ternary polymer featured efficient DNA condensation to favor cellular internalization, and the acidic environment in endolysosomes triggered effective degradation of the polymer to promote cargo release. Thus, 2-PEI-RT showed robust plasmid DNA transfection efficiencies in various tumor cells in serum, outperforming the commercial reagent PEI 25k by 1-3 orders of magnitude. Moreover, 2-PEI-RT mediated efficient cytosolic delivery of Cas9-mRNA/sgRNA to enable pronounced CRISPR-Cas9 genome editing in vitro. Such a facile and robust platform holds great potential for non-viral nucleic acid delivery and gene therapy.},
}
@article {pmid37381725,
year = {2023},
author = {Zheng, Q and Wang, W and Zhou, Y and Mo, J and Chang, X and Zha, Z and Zha, L},
title = {Synthetic nanoparticles for the delivery of CRISPR/Cas9 gene editing system: classification and biomedical applications.},
journal = {Biomaterials science},
volume = {11},
number = {16},
pages = {5361-5389},
doi = {10.1039/d3bm00788j},
pmid = {37381725},
issn = {2047-4849},
mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; *Nanoparticles ; *Neoplasms/genetics ; },
abstract = {Gene editing has great potential in biomedical research including disease diagnosis and treatment. Clustered regularly interspaced short palindromic repeats (CRISPR) is the most straightforward and cost-effective method. The efficient and precise delivery of CRISPR can impact the specificity and efficacy of gene editing. In recent years, synthetic nanoparticles have been discovered as effective CRISPR/Cas9 delivery vehicles. We categorized synthetic nanoparticles for CRISPR/Cas9 delivery and discribed their advantages and disadvantages. Further, the building blocks of different kinds of nanoparticles and their applications in cells/tissues, cancer and other diseases were described in detail. Finally, the challenges encountered in the clinical application of CRISPR/Cas9 delivery materials were discussed, and potential solutions were provided regarding efficiency and biosafety issues.},
}
@article {pmid37166587,
year = {2023},
author = {Drapal, M and Enfissi, EMA and Almeida, J and Rapacz, E and Nogueira, M and Fraser, PD},
title = {The potential of metabolomics in assessing global compositional changes resulting from the application of CRISPR/Cas9 technologies.},
journal = {Transgenic research},
volume = {32},
number = {4},
pages = {265-278},
pmid = {37166587},
issn = {1573-9368},
support = {760331//Horizon 2020 Framework Programme/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; Plant Breeding ; Metabolomics ; },
abstract = {Exhaustive analysis of genetically modified crops over multiple decades has increased societal confidence in the technology. New Plant Breeding Techniques are now emerging with improved precision and the ability to generate products containing no foreign DNA and mimic/replicate conventionally bred varieties. In the present study, metabolomic analysis was used to compare (i) tobacco genotypes with and without the CRISPR associated protein 9 (Cas9), (ii) tobacco lines with the edited and non-edited DE-ETIOLATED-1 gene without phenotype and (iii) leaf and fruit tissue from stable non-edited tomato progeny with and without the Cas9. In all cases, multivariate analysis based on the difference test using LC-HRMS/MS and GC-MS data indicated no significant difference in their metabolomes. The variations in metabolome composition that were evident could be associated with the processes of tissue culture regeneration and/or transformation (e.g. interaction with Agrobacterium). Metabolites responsible for the variance included quantitative changes of abundant, well characterised metabolites such as phenolics (e.g. chlorogenic acid) and several common sugars such as fructose. This study provides fundamental data on the characterisation of gene edited crops, that are important for the evaluation of the technology and its assessment. The approach also suggests that metabolomics could contribute to routine product-based analysis of crops/foods generated from New Plant Breeding approaches.},
}
@article {pmid37533651,
year = {2023},
author = {Ittiprasert, W and Moescheid, MF and Chaparro, C and Mann, VH and Quack, T and Rodpai, R and Miller, A and Wisitpongpun, P and Buakaew, W and Mentink-Kane, M and Schmid, S and Popratiloff, A and Grevelding, CG and Grunau, C and Brindley, PJ},
title = {Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, Schistosoma mansoni.},
journal = {Cell reports methods},
volume = {3},
number = {7},
pages = {100535},
pmid = {37533651},
issn = {2667-2375},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Humans ; *Schistosoma mansoni/genetics ; Transgenes/genetics ; Animals, Genetically Modified/genetics ; *Gene Editing ; },
abstract = {The identification and characterization of genomic safe harbor sites (GSHs) can facilitate consistent transgene activity with minimal disruption to the host cell genome. We combined computational genome annotation and chromatin structure analysis to predict the location of four GSHs in the human blood fluke, Schistosoma mansoni, a major infectious pathogen of the tropics. A transgene was introduced via CRISPR-Cas-assisted homology-directed repair into one of the GSHs in the egg of the parasite. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. The approach advances functional genomics for schistosomes by providing a tractable path for generating transgenics using homology-directed, repair-catalyzed transgene insertion. We also suggest that this work will serve as a roadmap for the development of similar approaches in helminths more broadly.},
}
@article {pmid37533644,
year = {2023},
author = {Vaitsiankova, A and Thakar, T and Ciccia, A},
title = {Base-editing screens illuminate variant effects in human hematopoiesis.},
journal = {Cell reports methods},
volume = {3},
number = {7},
pages = {100541},
pmid = {37533644},
issn = {2667-2375},
support = {R01 CA197774/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Hematopoiesis/genetics ; },
abstract = {In a recent issue of Cell, Martin-Rufino et al. develop a strategy for performing high-throughput base-editing CRISPR screens coupled with single-cell readouts in the context of human hematopoiesis. Through a series of proof-of-principle experiments, the authors demonstrate the potential of base-editing screens for the study and treatment of hematological disorders.},
}
@article {pmid37532731,
year = {2023},
author = {Huang, Y and Qin, G and Cui, T and Zhao, C and Ren, J and Qu, X},
title = {A bimetallic nanoplatform for STING activation and CRISPR/Cas mediated depletion of the methionine transporter in cancer cells restores anti-tumor immune responses.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4647},
pmid = {37532731},
issn = {2041-1723},
mesh = {Female ; Mice ; Animals ; *CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems ; *Neoplasms/therapy/drug therapy ; Immunotherapy ; Methionine/metabolism ; Immunity ; },
abstract = {Lack of sufficient cytotoxic T lymphocytes (CD8[+] T cells) infiltration and dysfunctional state of CD8[+] T cells are considered enormous obstacles to antitumor immunity. Herein, we construct a synergistic nanoplatform to promote CD8[+] T cell infiltration in tumors while restoring T cell function by regulating methionine metabolism and activating the STING innate immune pathway. The CRISPR/Cas9 system down-regulates the methionine transporter SLC43A2 and restricts the methionine uptake by tumor cells, thereby relieving the methionine competition pressure of T cells; simultaneously, the released nutrition metal ions activate the cGAS/STING pathway. In this work, the described nanoplatform can enhance the effect of immunotherapy in preclinical cancer models in female mice, enhancing STING pathway mediated immunity and facilitating the development of amino acid metabolic intervention-based cancer therapy.},
}
@article {pmid37531786,
year = {2023},
author = {Mehmandar-Oskuie, A and Jahankhani, K and Rostamlou, A and Arabi, S and Sadat Razavi, Z and Mardi, A},
title = {Molecular landscape of LncRNAs in bladder cancer: From drug resistance to novel LncRNA-based therapeutic strategies.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {165},
number = {},
pages = {115242},
doi = {10.1016/j.biopha.2023.115242},
pmid = {37531786},
issn = {1950-6007},
abstract = {Bladder cancer (BC) is a common and serious type of cancer that ranks among the top ten most prevalent malignancies worldwide. Due to the high occurrence rate of BC, the aggressive nature of cancer cells, and their resistance to medication, managing this disease has become a growing challenge in clinical care. Long noncoding RNAs (lncRNAs) are a group of RNA transcripts that do not code for proteins and are more than 200 nucleotides in length. They play a significant role in controlling cellular pathways and molecular interactions during the onset, development and progression of different types of cancers. Recent advancements in high-throughput gene sequencing technology have led to the identification of various differentially expressed lncRNAs in BC, which indicate abnormal expression. In this review, we summarize that these lncRNAs have been found to impact several functions related to the development of BC, including proliferation, cell growth, migration, metastasis, apoptosis, epithelial-mesenchymal transition, and chemo- and radio-resistance. Additionally, lncRNAs may improve prognosis prediction for BC patients, indicating a future use for them as prognostic and diagnostic biomarkers for BC patients. This review highlights that genetic tools and anti-tumor agents, such as CRISPR/Cas systems, siRNA, shRNA, antisense oligonucleotides, and vectors, have been created for use in preclinical cancer models. This has led to a growing interest in using lncRNAs based on positive research findings.},
}
@article {pmid37528660,
year = {2023},
author = {Zhu, Y and Zhang, M and Guo, S and Xu, H and Jie, Z and Tao, SC},
title = {CRISPR-based diagnostics of different biomolecules from nucleic acids, proteins, and small molecules to exosomes.},
journal = {Acta biochimica et biophysica Sinica},
volume = {},
number = {},
pages = {},
doi = {10.3724/abbs.2023134},
pmid = {37528660},
issn = {1745-7270},
abstract = {CRISPR-based detection technologies have been widely explored for molecular diagnostics. However, the challenge lies in converting the signal of different biomolecules, such as nucleic acids, proteins, small molecules, exosomes, and ions, into a CRISPR-based nucleic acid detection signal. Understanding the detection of different biomolecules using CRISPR technology can aid in the development of practical and promising detection approaches. Unfortunately, existing reviews rarely provide an overview of CRISPR-based molecular diagnostics from the perspective of different biomolecules. Herein, we first introduce the principles and characteristics of various CRISPR nucleases for molecular diagnostics. Then, we focus on summarizing and evaluating the latest advancements in CRISPR-based detection of different biomolecules. Through a comparison of different methods of amplification and signal readout, we discuss how general detection methods can be integrated with CRISPR. Finally, we conclude by identifying opportunities for the improvement of CRISPR in quantitative, amplification-free, multiplex, all-in-one, and point-of-care testing (POCT) purposes.},
}
@article {pmid37526970,
year = {2023},
author = {Shangguan, Q and White, MF},
title = {Repurposing the atypical type I-G CRISPR system for bacterial genome engineering.},
journal = {Microbiology (Reading, England)},
volume = {169},
number = {8},
pages = {},
doi = {10.1099/mic.0.001373},
pmid = {37526970},
issn = {1465-2080},
support = {BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Escherichia coli/genetics/metabolism ; DNA Replication ; DNA ; CRISPR-Cas Systems ; DNA Helicases/genetics/metabolism ; Genome, Bacterial/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {The CRISPR-Cas system functions as a prokaryotic immune system and is highly diverse, with six major types and numerous sub-types. The most abundant are type I CRISPR systems, which utilize a multi-subunit effector, Cascade, and a CRISPR RNA (crRNA) to detect invading DNA species. Detection leads to DNA loading of the Cas3 helicase-nuclease, leading to long-range deletions in the targeted DNA, thus providing immunity against mobile genetic elements (MGE). Here, we focus on the type I-G system, a streamlined, 4-subunit complex with an atypical Cas3 enzyme. We demonstrate that Cas3 helicase activity is not essential for immunity against MGE in vivo and explore applications of the Thioalkalivibrio sulfidiphilus Cascade effector for genome engineering in Escherichia coli. Long-range, bidirectional deletions were observed when the lacZ gene was targeted. Deactivation of the Cas3 helicase activity dramatically altered the types of deletions observed, with small deletions flanked by direct repeats that are suggestive of microhomology mediated end joining. When donor DNA templates were present, both the wild-type and helicase-deficient systems promoted homology-directed repair (HDR), with the latter system providing improvements in editing efficiency, suggesting that a single nick in the target site may promote HDR in E. coli using the type I-G system. These findings open the way for further application of the type I-G CRISPR systems in genome engineering.},
}
@article {pmid37526217,
year = {2023},
author = {Daskalakis, V and Papapetros, S},
title = {Engineering salt-tolerant Cas12f1 variants for gene-editing applications.},
journal = {Journal of biomolecular structure &amp; dynamics},
volume = {},
number = {},
pages = {1-11},
doi = {10.1080/07391102.2023.2240418},
pmid = {37526217},
issn = {1538-0254},
abstract = {CRISPR has revolutionized the field of genome editing in life sciences by serving as a versatile and state-of-the-art tool. Cas12f1 is a small nuclease of the bacterial immunity CRISPR system with an ideal size for cellular delivery, in contrast to CRISPR-associated (Cas) proteins like Cas9 or Cas12. However, Cas12f1 works best at low salt concentrations. In this study, we find that the plasticity of certain Cas12f1 regions (K196-Y202 and I452-L515) is negatively affected by increased salt concentrations. On this line, key protein domains (REC1, WED, Nuc, lid) that are involved in the DNA-target recognition and the activation of the catalytic RuvC domain are in turn also affected. We suggest that salt concentration should be taken in to consideration for activity assessments of Cas engineered variants, especially if the mutations are on the protospacer adjacent motif interacting domain. The results can be exploited for the engineering of Cas variants and the assessment of their activity at varying salt concentrations. We propose that the K198Q mutation can restore at great degree the compromised plasticity and could potentially lead to salt-tolerant Cas12f1 variants. The methodology can be also employed for the study of biomolecules in terms of their salinity tolerance.Communicated by Ramaswamy H. Sarma.},
}
@article {pmid37526140,
year = {2023},
author = {Chen, X and McAndrew, MJ and Lapinaite, A},
title = {Unlocking the secrets of ABEs: the molecular mechanism behind their specificity.},
journal = {Biochemical Society transactions},
volume = {},
number = {},
pages = {},
doi = {10.1042/BST20221508},
pmid = {37526140},
issn = {1470-8752},
abstract = {CRISPR-Cas, the bacterial immune systems, have transformed the field of genome editing by providing efficient, easily programmable, and accessible tools for targeted genome editing. DNA base editors (BE) are state-of-the-art CRISPR-based technology, allowing for targeted modifications of individual nucleobases within the genome. Among the BEs, adenine base editors (ABEs) have shown great potential due to their ability to convert A-to-G with high efficiency. However, current ABEs have limitations in terms of their specificity and targeting range. In this review, we provide an overview of the molecular mechanism of ABEs, with a focus on the mechanism of deoxyadenosine deamination by evolved tRNA-specific adenosine deaminase (TadA). We discuss how mutations and adjustments introduced via both directed evolution as well as rational design have improved ABE efficiency and specificity. This review offers insights into the molecular mechanism of ABEs, providing a roadmap for future developments in the precision genome editing field.},
}
@article {pmid37525893,
year = {2023},
author = {Chen, Y and Ping, Y},
title = {Development of CRISPR/Cas Delivery Systems for In Vivo Precision Genome Editing.},
journal = {Accounts of chemical research},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.accounts.3c00279},
pmid = {37525893},
issn = {1520-4898},
abstract = {ConspectusClustered, regularly interspaced, short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) is emerging as a powerful genome-editing tool, enabling precise and targeted modifications of virtually any genomic sequence in living cells. These technologies have potential therapeutic applications for cancers, metabolic diseases, and genetic disorders. However, several major challenges hinder the full realization of their potential. Specifically, CRISPR-Cas9 gene editors, whether delivered as plasmid DNA, mRNA/sgRNA, or ribonucleoprotein (RNP), exhibit poor membrane permeability, restricting their access to the intracellular genome, where the editing occurs. Additionally, these editors lack tissue or organ specificity, raising concerns about off-target editing at the tissue level that causes unwanted genotoxicity. Though a range of delivery carriers has been developed to deliver Cas9 editors, their effectiveness is often limited by a number of barriers at both the extracellular and intracellular levels. Moreover, the prolonged activity of Cas9 increases the risk of off-target editing at the genomic level. Therefore, it is crucial to develop efficient delivery vectors, along with molecular switches to safely regulate Cas9 activity.In this Account, we summarize our recent achievements in developing different types of materials that can efficiently deliver the plasmid DNA encoding Cas9 protein and single-guide RNA (sgRNA), or Cas9 RNP into cells to highlight the design considerations of carriers for safe and efficient delivery in vitro and in vivo. After elucidating the chemical and physical factors that are responsible for encapsulating and delivering these biomacromolecules, we further elucidate how we design the biodegradable polymeric carriers using dynamic disulfide chemistry, emphasize their safe and efficient delivery features for genome-editing biomacromolecules, and also introduce the integration of the intracellular delivery of genome-editing biomacromolecules with microneedle-based transdermal delivery to promote therapeutic genome editing for inflammatory skin disorders. Finally, we review how we exploit optical, chemical, and genetic switches to control the Cas9 activity in conjunction with targeted delivery to address the spatiotemporal specificity of gene editing in vivo and demonstrate their precision therapy against cancer and colitis treatment as proof-of-concept examples. In the final part, we will summarize the progress we have made and propose the future directions that may impact the field based on our own research outcomes.},
}
@article {pmid37478847,
year = {2023},
author = {Zhao, Y and Tabet, D and Rubio Contreras, D and Lao, L and Kousholt, AN and Weile, J and Melo, H and Hoeg, L and Feng, S and Coté, AG and Lin, ZY and Setiaputra, D and Jonkers, J and Gingras, AC and Gómez Herreros, F and Roth, FP and Durocher, D},
title = {Genome-scale mapping of DNA damage suppressors through phenotypic CRISPR-Cas9 screens.},
journal = {Molecular cell},
volume = {83},
number = {15},
pages = {2792-2809.e9},
doi = {10.1016/j.molcel.2023.06.025},
pmid = {37478847},
issn = {1097-4164},
support = {FDN159926/HG/NHGRI NIH HHS/United States ; UM1 HG011989/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Damage ; Mutation ; DNA Repair ; Phenotype ; },
abstract = {To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance.},
}
@article {pmid37414921,
year = {2023},
author = {Weidenauer, K and Schmidt, C and Rohde, C and Pauli, C and Blank, MF and Heid, D and Waclawiczek, A and Corbacioglu, A and Göllner, S and Lotze, M and Vierbaum, L and Renders, S and Krijgsveld, J and Raffel, S and Sauer, T and Trumpp, A and Pabst, C and Müller-Tidow, C and Janssen, M},
title = {The ribosomal protein S6 kinase alpha-1 (RPS6KA1) induces resistance to venetoclax/azacitidine in acute myeloid leukemia.},
journal = {Leukemia},
volume = {37},
number = {8},
pages = {1611-1625},
pmid = {37414921},
issn = {1476-5551},
support = {2021.145.1//Wilhelm Sander-Stiftung (Wilhelm Sander Foundation)/ ; DJCLS 04 PSD/2020//Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung (Deutsche Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung)/ ; },
mesh = {Humans ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; *Azacitidine/therapeutic use ; Bridged Bicyclo Compounds, Heterocyclic/therapeutic use ; *Leukemia, Myeloid, Acute/drug therapy/genetics ; Ribosomal Protein S6 Kinases ; Ribosomal Protein S6 Kinases, 90-kDa ; RNA, Guide, CRISPR-Cas Systems ; *Drug Resistance, Neoplasm ; },
abstract = {Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.},
}
@article {pmid37403358,
year = {2023},
author = {Stahl, EC and Sabo, JK and Kang, MH and Allen, R and Applegate, E and Kim, SE and Kwon, Y and Seth, A and Lemus, N and Salinas-Rios, V and Soczek, KM and Trinidad, M and Vo, LT and Jeans, C and Wozniak, A and Morris, T and Kimberlin, A and Foti, T and Savage, DF and Doudna, JA},
title = {Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {8},
pages = {2422-2438},
doi = {10.1016/j.ymthe.2023.06.019},
pmid = {37403358},
issn = {1525-0024},
support = {F32 GM140637/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems ; Ribonucleoproteins/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; Brain/metabolism ; },
abstract = {Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.},
}
@article {pmid37402371,
year = {2023},
author = {Chen, W and Ma, J and Wu, Z and Wang, Z and Zhang, H and Fu, W and Pan, D and Shi, J and Ji, Q},
title = {Cas12n nucleases, early evolutionary intermediates of type V CRISPR, comprise a distinct family of miniature genome editors.},
journal = {Molecular cell},
volume = {83},
number = {15},
pages = {2768-2780.e6},
doi = {10.1016/j.molcel.2023.06.014},
pmid = {37402371},
issn = {1097-4164},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Endonucleases/genetics ; DNA/genetics ; RNA ; },
abstract = {Type V CRISPR-associated systems (Cas)12 family nucleases are considered to have evolved from transposon-associated TnpB, and several of these nucleases have been engineered as versatile genome editors. Despite the conserved RNA-guided DNA-cleaving functionality, these Cas12 nucleases differ markedly from the currently identified ancestor TnpB in aspects such as guide RNA origination, effector complex composition, and protospacer adjacent motif (PAM) specificity, suggesting the presence of earlier evolutionary intermediates that could be mined to develop advanced genome manipulation biotechnologies. Using evolutionary and biochemical analyses, we identify that the miniature type V-U4 nuclease (referred to as Cas12n, 400-700 amino acids) is likely the earliest evolutionary intermediate between TnpB and large type V CRISPR systems. We demonstrate that with the exception of CRISPR array emergence, CRISPR-Cas12n shares several similar characteristics with TnpB-ωRNA, including a miniature and likely monomeric nuclease for DNA targeting, origination of guide RNA from nuclease coding sequence, and generation of a small sticky end following DNA cleavage. Cas12n nucleases recognize a unique 5'-AAN PAM sequence, of which the A nucleotide at the -2 position is also required for TnpB. Moreover, we demonstrate the robust genome-editing capacity of Cas12n in bacteria and engineer a highly efficient CRISPR-Cas12n (termed Cas12Pro) with up to 80% indel efficiency in human cells. The engineered Cas12Pro enables base editing in human cells. Our results further expand the understanding regarding type V CRISPR evolutionary mechanisms and enrich the miniature CRISPR toolbox for therapeutic applications.},
}
@article {pmid37376733,
year = {2023},
author = {Bekaert, B and Boel, A and De Witte, L and Vandenberghe, W and Popovic, M and Stamatiadis, P and Cosemans, G and Tordeurs, L and De Loore, AM and Chuva de Sousa Lopes, SM and De Sutter, P and Stoop, D and Coucke, P and Menten, B and Heindryckx, B},
title = {Retained chromosomal integrity following CRISPR-Cas9-based mutational correction in human embryos.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {31},
number = {8},
pages = {2326-2341},
doi = {10.1016/j.ymthe.2023.06.013},
pmid = {37376733},
issn = {1525-0024},
mesh = {Humans ; Male ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Semen ; Mutation ; Alleles ; Chromosomes ; },
abstract = {Human germline gene correction by targeted nucleases holds great promise for reducing mutation transmission. However, recent studies have reported concerning observations in CRISPR-Cas9-targeted human embryos, including mosaicism and loss of heterozygosity (LOH). The latter has been associated with either gene conversion or (partial) chromosome loss events. In this study, we aimed to correct a heterozygous basepair substitution in PLCZ1, related to infertility. In 36% of the targeted embryos that originated from mutant sperm, only wild-type alleles were observed. By performing genome-wide double-digest restriction site-associated DNA sequencing, integrity of the targeted chromosome (i.e., no deletions larger than 3 Mb or chromosome loss) was confirmed in all seven targeted GENType-analyzed embryos (mutant editing and absence of mutation), while short-range LOH events (shorter than 10 Mb) were clearly observed by single-nucleotide polymorphism assessment in two of these embryos. These results fuel the currently ongoing discussion on double-strand break repair in early human embryos, making a case for the occurrence of gene conversion events or partial template-based homology-directed repair.},
}
@article {pmid37131068,
year = {2023},
author = {Gupta, SK and Vishwakarma, NK and Malakar, P and Vanspati, P and Sharma, NK and Chattopadhyay, D},
title = {Development of an Agrobacterium-delivered codon-optimized CRISPR/Cas9 system for chickpea genome editing.},
journal = {Protoplasma},
volume = {260},
number = {5},
pages = {1437-1451},
pmid = {37131068},
issn = {1615-6102},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Cicer/genetics ; Codon ; Agrobacterium tumefaciens ; Genome, Plant/genetics ; },
abstract = {Chickpea is considered recalcitrant to in vitro tissue culture amongst all edible legumes. The clustered, regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based genome editing in chickpea can remove the bottleneck of limited genetic variation in this cash crop, which is rich in nutrients and protein. However, generating stable mutant lines using CRISPR/Cas9 requires efficient and highly reproducible transformation protocols. As an attempt to solve this problem, we developed a modified and optimized protocol for chickpea transformation. This study transformed the single cotyledon half-embryo explants using CaMV35S promoter to drive two marker genes (β-glucuronidase gene; GUS and green fluorescent protein; GFP) through binary vectors pBI101.2 and modified pGWB2, respectively. These vectors were delivered in the explants through three different strains of Agrobacterium tumefaciens, viz., GV3101, EHA105, and LBA4404. We found better efficiency with the strain GV3101 (17.56%) compared with two other strains, i.e., 8.54 and 5.43%, respectively. We recorded better regeneration frequencies in plant tissue culture for the constructs GUS and GFP, i.e., 20.54% and 18.09%, respectively. The GV3101 was further used for the transformation of the genome editing construct. For the development of genome-edited plants, we used this modified protocol. We also used a modified binary vector pPZP200 by introducing a CaMV35S-driven chickpea codon-optimized SpCas9 gene. The promoter of the Medicago truncatula U6.1 snRNA gene was used to drive the guide RNA cassettes. This cassette targeted and edited the chickpea phytoene desaturase (CaPDS) gene. A single gRNA was found sufficient to achieve high efficiency (42%) editing with the generation of PDS mutants with albino phenotypes. A simple, rapid, highly reproducible, stable transformation and CRISPR/Cas9-based genome editing system for chickpea was established. This study aimed to demonstrate this system's applicability by performing a gene knockout of the chickpea PDS gene using an improved chickpea transformation protocol for the first time.},
}
@article {pmid37529278,
year = {2022},
author = {Santiago, JG},
title = {Inconsistent treatments of the kinetics of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) impair assessment of its diagnostic potential.},
journal = {QRB discovery},
volume = {3},
number = {},
pages = {e9},
pmid = {37529278},
issn = {2633-2892},
abstract = {The scientific and technological advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is one of the most exciting developments of the past decade, particularly in the field of gene editing. The technology has two essential components, (1) a guide RNA to match a targeted gene and (2) a CRISPR-associated protein (e.g. Cas 9, Cas12 or Cas13) that acts as an endonuclease to specifically cut DNA. This specificity and reconfigurable nature of CRISPR has also spurred intense academic and commercial interest in the development of CRISPR-based molecular diagnostics. CRISPR Cas12 and Cas13 orthologs are most commonly applied to diagnostics, and these cleave and become activated by DNA and RNA targets, respectively. Despite the intense research interest, the limits of detection (LoDs) and applications of CRISP-based diagnostics remain an open question. A major reason for this is that reports of kinetic rates have been widely inconsistent, and the vast majority of these reports contain gross errors including violations of basic conservation and kinetic rate laws. It is the intent of this Perspective to bring attention to these issues and to identify potential improvements in the manner in which CRISPR kinetic rates and assay LoDs are reported and compared. The CRISPR field would benefit from verifications of self-consistency of data, providing sufficient data for reproduction of experiments, and, in the case of reports of novel assay LoDs, concurrent reporting of the associated kinetic rate constants. The early development of CRISPR-based diagnostics calls for self-reflection and urges us to proceed with caution.},
}
@article {pmid37524043,
year = {2023},
author = {Tian, B and Wang, Y and Tang, W and Chen, J and Zhang, J and Xue, S and Zheng, S and Cheng, G and Gu, B and Chen, M},
title = {Tandem CRISPR nucleases-based lateral flow assay for amplification-free miRNA detection via the designed "locked RNA/DNA" as fuels.},
journal = {Talanta},
volume = {266},
number = {Pt 1},
pages = {124995},
doi = {10.1016/j.talanta.2023.124995},
pmid = {37524043},
issn = {1873-3573},
abstract = {Currently, available biosensors based on CRISPR/Cas typically depend on coupling with nucleic acid amplification technologies to enhance their sensitivity. However, this approach often involves intricate amplification processes, which could be time-consuming and susceptible to contamination. In addition, signal readouts often require sophisticated and cumbersome equipment, obstructing the applicability of CRISPR/Cas assays in resource-limited regions. Herein, a tandem CRISPR/Cas13a/Cas12a mechanism (tanCRISPR) has been developed via the designed "Locked RNA/DNA" probe as fuels for the trans-cleavage nucleic acid of Cas13a and activated nucleic acid of Cas12a. Meanwhile, a lateral flow assay (LFA) is designed to combine with this tandem CRISPR/Cas13a/Cas12a mechanism (termed tanCRISPR-LFA), realizing the portable monitoring of miRNA-21. The tanCRISPR could realize the limit of detection at pM levels (266 folds lower than Cas13a-based miRNA testing alone) without the resort to target amplification procedures. Furthermore, the miRNA-21 levels of MDA-MB-231 cell extracts are sensed by tanCRISPR-LFA, which is comparable to qRT-PCR. With the virtues of portability, rapidity, sensitivity, and low cost, tanCRISPR-LFA is amenable for CRISPR/Cas-based biosensing and potential applications in the clinical diagnosis of miRNA-associated diseases.},
}
@article {pmid37523020,
year = {2023},
author = {Saakre, M and Jaiswal, S and Rathinam, M and Raman, KV and Tilgam, J and Paul, K and Sreevathsa, R and Pattanayak, D},
title = {Host-Delivered RNA Interference for Durable Pest Resistance in Plants: Advanced Methods, Challenges, and Applications.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {37523020},
issn = {1559-0305},
abstract = {Insect-pests infestation greatly affects global agricultural production and is projected to become more severe in upcoming years. There is concern about pesticide application being ineffective due to insect resistance and environmental toxicity. Reduced effectiveness of Bt toxins also made the scientific community shift toward alternative strategies to control devastating agricultural pests. With the advent of host-delivered RNA interference, also known as host-induced gene silencing, targeted insect genes have been suppressed through genetic engineering tools to deliver a novel insect-pest resistance strategy for combating a number of agricultural pests. This review recapitulates the possible mechanism of host-delivered RNA interference (HD-RNAi), in particular, the silencing of target genes of insect-pests. We emphasize the development of the latest strategies against evolving insect targets including designing of artificial microRNAs, vector constructs, and the benefit of using plastid transformation to transform target RNA-interfering genes. Advantages of using HD-RNAi over other small RNA delivery modes and also the supremacy of HD-RNAi over the CRISPR-Cas system particularly for insect resistance have been described. However, the broader application of this technology is restricted due to its several limitations. Using artificial miRNA designs, the host-delivered RNAi + Bt combinatorial approach and chloroplast transformation can overcome limitations of RNAi. With careful design and delivery approaches, RNAi promises to be extremely valuable and effective plant protection strategy to attain durable insect-pest resistance in crops. Development of transgenic plant using novel strategies to achieve durable resistance against the target insect.},
}
@article {pmid37478130,
year = {2023},
author = {Feng, H and Bavister, G and Gribble, KE and Mark Welch, DB},
title = {Highly efficient CRISPR-mediated gene editing in a rotifer.},
journal = {PLoS biology},
volume = {21},
number = {7},
pages = {e3001888},
pmid = {37478130},
issn = {1545-7885},
support = {R21 AG067034/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; Female ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9 ; *Rotifera/genetics ; DNA Repair ; },
abstract = {Rotifers have been studied in the laboratory and field for over 100 years in investigations of microevolution, ecological dynamics, and ecotoxicology. In recent years, rotifers have emerged as a model system for modern studies of the molecular mechanisms of genome evolution, development, DNA repair, aging, life history strategy, and desiccation tolerance. However, a lack of gene editing tools and transgenic strains has limited the ability to link genotype to phenotype and dissect molecular mechanisms. To facilitate genetic manipulation and the creation of reporter lines in rotifers, we developed a protocol for highly efficient, transgenerational, CRISPR-mediated gene editing in the monogonont rotifer Brachionus manjavacas by microinjection of Cas9 protein and synthetic single-guide RNA into the vitellaria of young amictic (asexual) females. To demonstrate the efficacy of the method, we created knockout mutants of the developmental gene vasa and the DNA mismatch repair gene mlh3. More than half of mothers survived injection and produced offspring. Genotyping these offspring and successive generations revealed that most carried at least 1 CRISPR-induced mutation, with many apparently mutated at both alleles. In addition, we achieved precise CRISPR-mediated knock-in of a stop codon cassette in the mlh3 locus, with half of injected mothers producing F2 offspring with an insertion of the cassette. Thus, this protocol produces knockout and knock-in CRISPR/Cas9 editing with high efficiency, to further advance rotifers as a model system for biological discovery.},
}
@article {pmid37295292,
year = {2023},
author = {Cheng, Y and Wang, H and Li, M},
title = {The promise of CRISPR/Cas9 technology in diabetes mellitus therapy: How gene editing is revolutionizing diabetes research and treatment.},
journal = {Journal of diabetes and its complications},
volume = {37},
number = {8},
pages = {108524},
doi = {10.1016/j.jdiacomp.2023.108524},
pmid = {37295292},
issn = {1873-460X},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems ; Genetic Therapy ; Stem Cells ; *Diabetes Mellitus/genetics/therapy ; },
abstract = {Diabetes mellitus is a metabolic disease, characterized by chronic hyperglycemia caused by an abnormality in insulin secretion or action. Millions of people across the world are affected by diabetes mellitus which has serious implications for their health. Over the past few decades, diabetes has become a major cause of mortality and morbidity across the world due to its rapid prevalence. Treatment for diabetes that focuses on insulin secretion and sensitization can lead to unwanted side effects and/or poor compliance, as well as treatment failure. A promising way to treat diabetes is through gene-editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR/Cas9). However, issues such as efficiency and off-target effects have hindered the use of these technologies. In this review, we summarize what we know today about CRISPR/Cas9 technology's therapeutic potential for treating diabetes. We discuss how different strategies are employed, including cell-based therapies (such as stem cells and brown adipocytes), targeting critical genes involved in diabetes pathogenesis, and discussing the challenges and limitations associated with this technology. A novel and powerful treatment approach to diabetes and other diseases can be found with CRISPR/Cas9 technology, and further research should be carried out in this field.},
}
@article {pmid37201284,
year = {2023},
author = {Sanchez-Muñoz, R},
title = {Efficient cut and paste: directional oligodeoxynucleotide-based targeted insertion (DOTI) as a precise genome-editing method.},
journal = {The Plant cell},
volume = {35},
number = {8},
pages = {2697-2698},
pmid = {37201284},
issn = {1532-298X},
mesh = {*Oligodeoxyribonucleotides/genetics ; *Gene Editing ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37522927,
year = {2023},
author = {Yigider, E and Taspinar, MS and Agar, G},
title = {Advances in bread wheat production through CRISPR/Cas9 technology: a comprehensive review of quality and other aspects.},
journal = {Planta},
volume = {258},
number = {3},
pages = {55},
pmid = {37522927},
issn = {1432-2048},
mesh = {Humans ; *Triticum/genetics ; *Bread ; CRISPR-Cas Systems/genetics ; Plant Breeding ; Gene Editing ; },
abstract = {This review provides a comprehensive overview of the CRISPR/Cas9 technique and the research areas of this gene editing tool in improving wheat quality. Wheat (Triticum aestivum L.), the basic nutrition for most of the human population, contributes 20% of the daily energy needed because of its, carbohydrate, essential amino acids, minerals, protein, and vitamin content. Wheat varieties that produce high yields and have enhanced nutritional quality will be required to fulfill future demands. Hexaploid wheat has A, B, and D genomes and includes three like but not identical copies of genes that influence important yield and quality. CRISPR/Cas9, which allows multiplex genome editing provides major opportunities in genome editing studies of plants, especially complicated genomes such as wheat. In this overview, we discuss the CRISPR/Cas9 technique, which is credited with bringing about a paradigm shift in genome editing studies. We also provide a summary of recent research utilizing CRISPR/Cas9 to investigate yield, quality, resistance to biotic/abiotic stress, and hybrid seed production. In addition, we provide a synopsis of the laboratory experience-based solution alternatives as well as the potential obstacles for wheat CRISPR studies. Although wheat's extensive genome and complicated polyploid structure previously slowed wheat genetic engineering and breeding progress, effective CRISPR/Cas9 systems are now successfully used to boost wheat development.},
}
@article {pmid36959722,
year = {2023},
author = {Nakazawa, T and Inoue, C and Morimoto, R and Nguyen, DX and Bao, D and Kawauchi, M and Sakamoto, M and Honda, Y},
title = {The lignin-degrading abilities of Gelatoporia subvermispora gat1 and pex1 mutants generated via CRISPR/Cas9.},
journal = {Environmental microbiology},
volume = {25},
number = {8},
pages = {1393-1408},
doi = {10.1111/1462-2920.16372},
pmid = {36959722},
issn = {1462-2920},
support = {//Institute for Fermentation, Osaka/ ; 20F20092//Japan Society for the Promotion of Science/ ; 120208402//Japan Society for the Promotion of Science/ ; 120209920//Japan Society for the Promotion of Science/ ; 18H02254//Japan Society for the Promotion of Science/ ; 18KK0178//Japan Society for the Promotion of Science/ ; 19H03017//Japan Society for the Promotion of Science/ ; 19K22332//Japan Society for the Promotion of Science/ ; 21K18224//Japan Society for the Promotion of Science/ ; 22H00380//Japan Society for the Promotion of Science/ ; //Kansai Research Foundation for Technology Promotion/ ; },
mesh = {Lignin/metabolism ; CRISPR-Cas Systems ; *Polyporales/metabolism ; *Pleurotus/genetics/metabolism ; },
abstract = {White-rot fungi efficiently degrade wood lignin; however, the mechanisms involved remain largely unknown. Recently, a forward genetics approach to identify several genes in Pleurotus ostreatus (Agaricales) in which mutations cause defects in wood lignin degradation was used. For example, pex1 encodes a peroxisome biogenesis factor and gat1 encodes a putative Agaricomycetes-specific DNA-binding transcription factor. In this study, we examined the effects of single-gene mutations in pex1 or gat1 on wood lignin degradation in another white-rot fungus, Gelatoporia (Ceriporiopsis) subvermispora (Polyporales), to investigate conserved and derived degradation mechanisms in white-rot fungi. G. subvermispora pex1 and gat1 single-gene mutant strains were generated from a monokaryotic wild-type strain, FP-90031-Sp/1, using plasmid-based CRISPR/Cas9. As in P. ostreatus, Gsgat1 mutants were nearly unable to degrade lignin sourced from beech wood sawdust medium (BWS), while Gspex1 mutants exhibited a delay in lignin degradation. We also found that the transcripts of lignin-modifying enzyme-encoding genes, mnp4, mnp5, mnp6, mnp7, and mnp11, which predominantly accumulate in FP-90031-Sp/1 cultured with BWS, were greatly downregulated in Gsgat1 mutants. Taken together, the results suggest that Gat1 may be a conserved regulator of the ligninolytic system of white-rot fungi and that the contribution of peroxisomes to the ligninolytic system may differ among species.},
}
@article {pmid36828577,
year = {2023},
author = {Krug, J and Richter, A and Englert, C},
title = {Rapid Genotyping of Nothobranchius furzeri Embryos, Larvae, and Adult Fish via High-Resolution Melt Analysis (HRMA).},
journal = {Cold Spring Harbor protocols},
volume = {2023},
number = {8},
pages = {107744},
doi = {10.1101/pdb.prot107744},
pmid = {36828577},
issn = {1559-6095},
mesh = {Animals ; *CRISPR-Cas Systems ; Larva/genetics ; Genotype ; Mutation ; *Gene Editing/methods ; },
abstract = {CRISPR-Cas9 has eased the induction of sequence-specific mutations and has therefore become a powerful tool to generate mutant lines for studying the role of specific genes. The cellular repair of Cas9-induced double-stranded DNA breaks by the error-prone nonhomologous end-joining pathway can result in various indel mutations. Having identified and chosen a specific mutation in a target gene, the establishment of a respective mutant line requires a feasible and precise method to differentiate the genotypes of the offspring. Here, we provide a protocol that allows genotyping of large numbers of Nothobranchius furzeri embryos, larvae, and adults harboring a previously identified indel or point mutation in a short time via high-resolution melt analysis (HRMA).},
}
@article {pmid37522916,
year = {2023},
author = {Bora, J and Dey, A and Lyngdoh, AR and Dhasmana, A and Ranjan, A and Kishore, S and Rustagi, S and Tuli, HS and Chauhan, A and Rath, P and Malik, S},
title = {A critical review on therapeutic approaches of CRISPR-Cas9 in diabetes mellitus.},
journal = {Naunyn-Schmiedeberg's archives of pharmacology},
volume = {},
number = {},
pages = {},
pmid = {37522916},
issn = {1432-1912},
abstract = {Diabetes mellitus (D.M.) is a common metabolic disorder caused mainly by combining two primary factors, which are (1) defects in insulin production by the pancreatic β-cells and (2) responsiveness of insulin-sensitive tissues towards insulin. Despite the rapid advancement in medicine to suppress elevated blood glucose levels (hyperglycemia) and insulin resistance associated with this hazard, a demand has undoubtedly emerged to find more effective and curative dimensions in therapeutic approaches against D.M. The administration of diabetes treatment that emphasizes insulin production and sensitivity may result in unfavorable side effects, reduced adherence, and potential treatment ineffectiveness. Recent progressions in genome editing technologies, for instance, in zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR-Cas)-associated nucleases, have greatly influenced the gene editing technology from concepts to clinical practices. Improvements in genome editing technologies have also opened up the possibility to target and modify specific genome sequences in a cell directly. CRISPR/Cas9 has proven effective in utilizing ex vivo gene editing in embryonic stem cells and stem cells derived from patients. This application has facilitated the exploration of pancreatic beta-cell development and function. Furthermore, CRISPR/Cas9 enables the creation of innovative animal models for diabetes and assesses the effectiveness of different therapeutic strategies in treating the condition. We, therefore, present a critical review of the therapeutic approaches of the genome editing tool CRISPR-Cas9 in treating D.M., discussing the challenges and limitations of implementing this technology.},
}
@article {pmid37521929,
year = {2023},
author = {May, D and Sanchez, S and Gilby, J and Altpeter, F},
title = {Multi-allelic gene editing in an apomictic, tetraploid turf and forage grass (Paspalum notatum Flüggé) using CRISPR/Cas9.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1225775},
pmid = {37521929},
issn = {1664-462X},
abstract = {Polyploidy is common among grasses (Poaceae) and poses challenges for conventional breeding. Genome editing technology circumvents crossing and selfing, enabling targeted modifications to multiple gene copies in a single generation while maintaining the heterozygous context of many polyploid genomes. Bahiagrass (Paspalum notatum Flüggé; 2n=4x=40) is an apomictic, tetraploid C4 species that is widely grown in the southeastern United States as forage in beef cattle production and utility turf. The chlorophyll biosynthesis gene magnesium chelatase (MgCh) was selected as a rapid readout target for establishing genome editing in tetraploid bahiagrass. Vectors containing sgRNAs, Cas9 and nptII were delivered to callus cultures by biolistics. Edited plants were characterized through PCR-based assays and DNA sequencing, and mutagenesis frequencies as high as 99% of Illumina reads were observed. Sequencing of wild type (WT) bahiagrass revealed a high level of sequence variation in MgCh likely due to the presence of at least two copies with possibly eight different alleles, including pseudogenes. MgCh mutants exhibited visible chlorophyll depletion with up to 82% reductions in leaf greenness. Two lines displayed progression of editing over time which was linked to somatic editing. Apomictic progeny of a chimeric MgCh editing event were obtained and allowed identification of uniformly edited progeny plants among a range of chlorophyll depletion phenotypes. Sanger sequencing of a highly edited mutant revealed elevated frequency of a WT allele, probably due to frequent homology-directed repair (HDR). To our knowledge these experiments comprise the first report of genome editing applied in perennial, warm-season turf or forage grasses. This technology will accelerate bahiagrass cultivar development.},
}
@article {pmid37521915,
year = {2023},
author = {Farinati, S and Draga, S and Betto, A and Palumbo, F and Vannozzi, A and Lucchin, M and Barcaccia, G},
title = {Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1223861},
pmid = {37521915},
issn = {1664-462X},
abstract = {Plant male sterility (MS) represents the inability of the plant to generate functional anthers, pollen, or male gametes. Developing MS lines represents one of the most important challenges in plant breeding programs, since the establishment of MS lines is a major goal in F1 hybrid production. For these reasons, MS lines have been developed in several species of economic interest, particularly in horticultural crops and ornamental plants. Over the years, MS has been accomplished through many different techniques ranging from approaches based on cross-mediated conventional breeding methods, to advanced devices based on knowledge of genetics and genomics to the most advanced molecular technologies based on genome editing (GE). GE methods, in particular gene knockout mediated by CRISPR/Cas-related tools, have resulted in flexible and successful strategic ideas used to alter the function of key genes, regulating numerous biological processes including MS. These precision breeding technologies are less time-consuming and can accelerate the creation of new genetic variability with the accumulation of favorable alleles, able to dramatically change the biological process and resulting in a potential efficiency of cultivar development bypassing sexual crosses. The main goal of this manuscript is to provide a general overview of insights and advances into plant male sterility, focusing the attention on the recent new breeding GE-based applications capable of inducing MS by targeting specific nuclear genic loci. A summary of the mechanisms underlying the recent CRISPR technology and relative success applications are described for the main crop and ornamental species. The future challenges and new potential applications of CRISPR/Cas systems in MS mutant production and other potential opportunities will be discussed, as generating CRISPR-edited DNA-free by transient transformation system and transgenerational gene editing for introducing desirable alleles and for precision breeding strategies.},
}
@article {pmid37521328,
year = {2023},
author = {Li, T and Chen, Y and Chen, Z and Hao, Y and Liang, M and Liu, Y and Ou, G and Zhang, H and Tang, Y and Hao, Y and Wageh, S and Al-Hartomy, OA and Kalam, A and Zhang, B and Shi, X and Li, X and Zhang, H},
title = {Early and Sensitive Detection of Pathogens for Public Health and Biosafety: An Example of Surveillance and Genotyping of SARS-CoV-2 in Sewage Water by Cas12a-Facilitated Portable Plasmonic Biosensor.},
journal = {Research (Washington, D.C.)},
volume = {6},
number = {},
pages = {0205},
pmid = {37521328},
issn = {2639-5274},
abstract = {Infectious diseases severely threaten public health and global biosafety. In addition to transmission through the air, pathogenic microorganisms have also been detected in environmental liquid samples, such as sewage water. Conventional biochemical detection methodologies are time-consuming and cost-ineffective, and their detection limits hinder early diagnosis. In the present study, ultrafine plasmonic fiber probes with a diameter of 125 μm are fabricated for clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-12a-mediated sensing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Single-stranded DNA exposed on the fiber surface is trans-cleaved by the Cas12a enzyme to release gold nanoparticles that are immobilized onto the fiber surface, causing a sharp reduction in the surface plasmon resonance (SPR) wavelength. The proposed fiber probe is virus-specific with the limit of detection of ~2,300 copies/ml, and genomic copy numbers can be reflected as shifts in wavelengths. A total of 21 sewage water samples have been examined, and the data obtained are consistent with those of quantitative polymerase chain reaction (qPCR). In addition, the Omicron variant and its mutation sites have been fast detected using S gene-specific Cas12a. This study provides an accurate and convenient approach for the real-time surveillance of microbial contamination in sewage water.},
}
@article {pmid37482301,
year = {2023},
author = {Ayub, N and Soto, G},
title = {Multiple challenges in the development of commercial crops using CRISPR/Cas technology.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {335},
number = {},
pages = {111809},
doi = {10.1016/j.plantsci.2023.111809},
pmid = {37482301},
issn = {1873-2259},
abstract = {The CRISPR/Cas system is a highly efficient and versatile tool for editing plant genomes, with the potential to accelerate breeding programs and improve the sustainability of food production. Nevertheless, technical limitations delay the rapid spread of the CRISPR/Cas system benefits in agriculture. The natural features of plant species, including reproductive behavior, ploidy levels, genetic diversity, and generation times, can significantly impact the introgression of edited traits into elite germplasms. The production and selection of edited events require the same level of effort as those of their transgenic equivalents. Additionally, edited alleles tend to be recessive or not fully dominant, which differs from dominant transgenic events. To accelerate the introgression of edited events into conventional and transgenic varieties, we suggest utilizing edits on single-copy genes that induce dominant mutations. In the absence of new, simple traits that provide exceptional economic benefits for large companies, like herbicide tolerance in transgenic crops, we propose the emergence of particular public grants for edited variety productions, especially when the introgression shows a high level of technical feasibility. In the context of climate change, these public actions must be taken quickly to alleviate significant reductions in crop production.},
}
@article {pmid36897256,
year = {2023},
author = {Li, LZ and Yang, K and Jing, Y and Fan, Y and Jiang, X and Wang, S and Liu, GH and Qu, J and Ma, S and Zhang, W},
title = {CRISPR-based screening identifies XPO7 as a positive regulator of senescence.},
journal = {Protein &amp; cell},
volume = {14},
number = {8},
pages = {623-628},
pmid = {36897256},
issn = {1674-8018},
mesh = {*Cell Nucleus ; *Cellular Senescence/genetics ; CRISPR-Cas Systems/genetics ; },
}
@article {pmid37520667,
year = {2023},
author = {Wang, H and Wang, R and Yang, J and Feng, Y and Xu, S and Pei, QG},
title = {Interactions of Fibroblast Subtypes Influence Osteoclastogenesis and Alveolar Bone Destruction in Periodontitis.},
journal = {Journal of inflammation research},
volume = {16},
number = {},
pages = {3143-3156},
pmid = {37520667},
issn = {1178-7031},
abstract = {BACKGROUND: To analyze the fibroblasts subtypes in the gingival tissues of healthy controls, gingivitis and periodontitis patients, as well as the effects of interaction between subtypes on alveolar bone destruction.<br><br>METHODS: Gingival tissues were divided into three groups according to clinical and radiographic examination, and the immunostaining of EDA+FN was assessed. Fibroblasts from gingiva developed colony formation units (CFUs) and induced Trap+MNCs. The expression of osteoclastogenesis-related genes was assessed by real-time PCR. Variances in the gene profiles of CFUs were identified by principal component analysis, and cluster analysis divided CFUs into subtypes. The induction of Trap+MNCs and gene expression were compared among individual or cocultured subtypes. The fibroblast subtypes exerted critical effect on Trap+MNCs formation were selected and edited by CRISPR/Cas to investigate the influence on osteoclastogenesis in the periodontitis in mice.<br><br>RESULTS: Most periodontitis samples exhibited intensive EDA+FN staining (P < 0.05), and these fibroblasts also induced most Trap+MNCs among three groups; consistently, fibroblasts from periodontitis highly expressed genes facilitating osteoclastogenesis. According to gene profiles and osteoclastogenic induction, four clusters of CFUs were identified. The proportion of clusters was significantly different (P < 0.05) among three groups, and their interaction influenced osteoclastogenic induction. Although Cluster 4 induced less osteoclasts, it enhanced the effects of Clusters 1 and 3 on Trap+MNCs formation (P < 0.05). EDA knockout in Cluster 4 abrogated this promotion (P < 0.05), and decreased osteoclasts and alveolar bone destruction in experimental periodontitis (P < 0.05).<br><br>CONCLUSION: Heterogeneous fibroblast subtypes affect the switch or development of periodontitis. A subtype (Cluster 4) played important role during alveolar bone destruction, by regulating other subtypes via EDA+FN paracrine.},
}
@article {pmid37518013,
year = {2023},
author = {Wang, X and Cheng, M and Yang, S and Xing, C and Li, Q and Zhu, Y and Ji, Y and Du, Y},
title = {CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood.},
journal = {Parasites &amp; vectors},
volume = {16},
number = {1},
pages = {256},
pmid = {37518013},
issn = {1756-3305},
support = {2021YFC2301100//National Key Research and Development Program of China/ ; },
mesh = {Animals ; Humans ; Mice ; Swine ; CRISPR-Cas Systems ; *Toxoplasmosis/parasitology ; *Toxoplasma/genetics ; Polymerase Chain Reaction ; Sensitivity and Specificity ; DNA, Protozoan/analysis ; },
abstract = {BACKGROUND: Toxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-Associated Protein) systems are widely used for gene editing and pathogen detection. CRISPR-based diagnostics are molecular assays that have been developed to detect parasites with high sensitivity and specificity.<br><br>METHODS: This study aimed to establish a combined CRISPR/Cas12a and RPA rapid detection method for T. gondii by targeting the B1 gene and 529&#xa0;bp repeat element (529 RE). The detection results could be visualized by the fluorescence or lateral flow strips (LFS). The sensitivity and specificity of the method were evaluated, and T. gondii-infected mouse blood was used for detection.<br><br>RESULTS: The results indicated that the established method for T. gondii detection was satisfactory, with a detection limit of 1.5 cp/&#x3bc;l for the two loci. Moreover, the B1 gene could detect 1 tachyzoite per reaction, and the 529 RE could detect 0.1 tachyzoite per reaction, consistently with the highly sensitive nested polymerase chain reaction (PCR) results. The method was suitable for strains, including RH, and did not cross-react with other protozoa DNA with similar habits. The T. gondii-infected mouse blood samples were all positive for T. gondii at 1, 3, and 5&#xa0;days post infection (dpi).<br><br>CONCLUSIONS: This study established a rapid, sensitive, and time-saving DNA detection method for T. gondii that has the potential to be an alternative tool for T. gondii detection in the field.},
}
@article {pmid37517824,
year = {2023},
author = {Wan, X and Chen, J and Wu, Y and Chen, Z and Liu, Y and Li, T and Sun, J and Zhang, T and Zhou, F and Huang, X and Li, Y and Wang, X and Sun, X},
title = {Rapid and Sensitive Diagnosis of Leber Hereditary Optic Neuropathy Variants Using CRISPR/Cas12a Detection.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {25},
number = {8},
pages = {540-554},
doi = {10.1016/j.jmoldx.2023.04.006},
pmid = {37517824},
issn = {1943-7811},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Optic Atrophy, Hereditary, Leber/diagnosis/genetics ; DNA, Mitochondrial/genetics ; Mitochondria/genetics ; Mutation ; },
abstract = {Leber hereditary optic neuropathy (LHON) is the most common maternally inherited mitochondrial disease, with >90% of cases harboring one of three point variants (m.3460G>A, m.11778G>A, and m.14484T>C). Rapid and sensitive diagnosis of LHON variants is urgently needed for early diagnosis and timely treatment after onset, which is currently limited. Herein, we adapted the Cas12a-based DNA detection platform for LHON mitochondrial variant diagnosis. Single-strand guide CRISPR RNAs and enzymatic recombinase amplification primers were first screened, the CRISPR/Cas12a system was then optimized with restriction enzymes, and finally compared with Sanger sequencing and next-generation sequencing (NGS) in multicenter clinical samples. This approach can be completed within 30 minutes using only one drop of blood and could reach a sensitivity of 1% of heteroplasmy. Among the 182 multicenter clinical samples, the CRISPR/Cas12a detection system showed high consistency with Sanger sequencing and NGS in both specificity and sensitivity. Notably, a sample harboring a de novo 3.78% m.11778G>A variant detected by NGS, but not by Sanger sequencing, was successfully confirmed using the CRISPR/Cas12a assay, which proved the effectiveness of our method. Overall, our CRISPR/Cas12a detection system provides an alternative for rapid, convenient, and sensitive detection of LHON variants, exhibiting great potential for clinical practice.},
}
@article {pmid37517615,
year = {2023},
author = {Nabi, N and Singh, S and Saffeullah, P},
title = {An updated review on distribution, biosynthesis and pharmacological effects of artemisinin: A wonder drug.},
journal = {Phytochemistry},
volume = {},
number = {},
pages = {113798},
doi = {10.1016/j.phytochem.2023.113798},
pmid = {37517615},
issn = {1873-3700},
abstract = {Plant-based drugs have been used for centuries for treating different ailments. Malaria, one of the prevalent threats in many parts of the world, is treated mainly by artemisinin-based drugs derived from plants of genus Artemisia. However, the distribution of artemisinin is restricted to a few species of the genus; besides, its yield depends on ontogeny and the plant's geographical location. Here, we review the studies focusing on biosynthesis and distributional pattern of artemisinin production in species of the genus Artemisia. We also discussed various agronomic and in vitro methods and molecular approaches to increase the yield of artemisinin. We have summarized different mechanisms of artemisinin involved in its anti-malarial, anti-cancer, anti-inflammatory and anti-viral activities (like against Covid-19). Overall the current review provides a synopsis of a global view of the distribution of artemisinin, its biosynthesis, and pharmacological potential in treating various diseases like malaria, cancer, and coronavirus, which may provoke future research efforts in drug development. Nevertheless, long-term trials and molecular approaches, like CRISPR-Cas, are required for in-depth research.},
}
@article {pmid37516612,
year = {2023},
author = {Talebian, S and Dehghani, F and Weiss, PS and Conde, J},
title = {Evolution of CRISPR-enabled biosensors for amplification-free nucleic acid detection.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2023.07.004},
pmid = {37516612},
issn = {1879-3096},
abstract = {CRISPR biosensors enable rapid and accurate detection of nucleic acids without resorting to target amplification. Specifically, these systems facilitate the simultaneous detection of multiple nucleic acid targets with single-base specificity. This is an invaluable asset that can ultimately facilitate accurate diagnoses of biologically complex diseases.},
}
@article {pmid37516409,
year = {2023},
author = {Popovitz, J and Sharma, R and Hoshyar, R and Soo Kim, B and Murthy, N and Lee, K},
title = {Gene editing therapeutics based on mRNA delivery.},
journal = {Advanced drug delivery reviews},
volume = {},
number = {},
pages = {115026},
doi = {10.1016/j.addr.2023.115026},
pmid = {37516409},
issn = {1872-8294},
abstract = {The field of gene editing has received much attention in recent years due to its immense therapeutic potential. In particular, gene editing therapeutics, such as the CRISPR-Cas systems, base editors, and other emerging gene editors, offer the opportunity to address previously untreatable disorders. This review aims to summarize the therapeutic applications of gene editing based on mRNA delivery. We introduce gene editing therapeutics using mRNA and focus on engineering and improvement of gene editing technology. We subsequently examine ex vivo and in vivo gene editing techniques and conclude with an exploration of the next generation of CRISPR and base editing systems.},
}
@article {pmid37515526,
year = {2023},
author = {Singh, M and Misra, CS and Bindal, G and Rangu, SS and Rath, D},
title = {CRISPR-Cas12a assisted specific detection of mpox virus.},
journal = {Journal of medical virology},
volume = {95},
number = {8},
pages = {e28974},
doi = {10.1002/jmv.28974},
pmid = {37515526},
issn = {1096-9071},
mesh = {Humans ; CRISPR-Cas Systems ; Monkeypox virus ; *Monkeypox ; *Orthopoxvirus/genetics ; Biological Assay ; },
abstract = {Mpox virus, a member of genus Orthopoxvirus, causes rash and flu-like symptoms in humans. In the recent global outbreak, it was reported from several geographical areas that have not historically reported mpox. Point of care, sensitive and specific mpox diagnostic assays are critical in checking the spread of the disease. We have developed a clustered regularly interspaced short palindromic repeats associated Cas12a nuclease-based assay for detecting mpox virus. Mpox specific conserved sequences were identified in polA (E9L) gene which differ by a single nucleotide polymorphism (SNP) from all the viruses present in the genus Orthopoxvirus. This SNP was exploited in our assay to specifically distinguish mpox virus from other related orthopox viruses with a limit of detection of 1 copy/μl in 30 min. The assay exhibits a sensitive and specific detection of mpox virus which can prove to be of practical value for its surveillance in areas infected with multiple orthopox viruses, especially in hotspots of mpox virus infections.},
}
@article {pmid37390747,
year = {2023},
author = {Hua, Z and Xu, K and Xiao, W and Shu, C and Li, N and Li, K and Gu, H and Zhu, Z and Zhang, L and Ren, H and Zeng, Q and Yin, Y and Bi, Y},
title = {Dual single guide RNAs-mediating deletion of mature myostatin peptide results in concomitant muscle fibre hyperplasia and adipocyte hypotrophy in pigs.},
journal = {Biochemical and biophysical research communications},
volume = {673},
number = {},
pages = {145-152},
doi = {10.1016/j.bbrc.2023.06.053},
pmid = {37390747},
issn = {1090-2104},
mesh = {Animals ; Swine ; *RNA, Guide, CRISPR-Cas Systems ; Gene Knockout Techniques ; *Myostatin/genetics ; Hyperplasia/genetics/pathology ; Muscle Fibers, Skeletal ; Muscle, Skeletal/pathology ; Adipocytes ; },
abstract = {Myostatin (MSTN) is a major gene target for skeletal muscle overgrowth in animals. We hypothesized that deletion of the entire mature peptide encoded by MSTN in pigs would knock out its bioactive form and accordingly stimulate skeletal muscle overgrowth. Thus, we engineered two pairs of single-guide RNAs (sgRNAs) to target exons 1 and 3 of MSTN in primary fetal fibroblasts of Taoyuan black pigs. We found that sgRNAs targeting exon 3, which encodes the mature peptide, had higher biallelic null mutation efficiency than those targeting exon 1. Somatic cell nuclear transfer was conducted using the exon 3 mutation cells as donor cells to generate five cloned MSTN null piglets (MSTN[-/-]). Growth testing revealed that both the growth rate and average daily weight gain of MST[-/-] pigs were greater than those of wild-type (MSTN[+/+]) pigs. Slaughter data demonstrated that the lean ratio of MSTN[-/-] pigs was 11.3% higher (P < 0.01) while the back-fat thickness was 17.33% lower (P < 0.01) than those of MSTN[+/+] pigs. Haematoxylin-eosin staining indicated that the increased leanness of MSTN[-/-] pigs resulted from muscle fibre hyperplasia rather than hypertrophy.HE staining showed markedly decreased adipocyte size in MSTN[-/-] pigs. We also critically examined the off-target and random integration by resequencing, which showed that the founder MSTN[-/-] pigs contained no non-target mutations or exogenous plasmid elements. This study is the first to report the successful knock out of the mature MSTN peptide using dual sgRNA-mediated deletion, leading to the most prominent alteration of meat production traits in pigs published thus far. This new strategy is expected to have a wide impact on genetic improvements in food animals.},
}
@article {pmid37513832,
year = {2023},
author = {Junaid, M and Thirapanmethee, K and Khuntayaporn, P and Chomnawang, MT},
title = {CRISPR-Based Gene Editing in Acinetobacter baumannii to Combat Antimicrobial Resistance.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {16},
number = {7},
pages = {},
pmid = {37513832},
issn = {1424-8247},
support = {Mullika Chomnawang//Mahidol University/ ; },
abstract = {Antimicrobial resistance (AMR) poses a significant threat to the health, social, environment, and economic sectors on a global scale and requires serious attention to addressing this issue. Acinetobacter baumannii was given top priority among infectious bacteria because of its extensive resistance to nearly all antibiotic classes and treatment options. Carbapenem-resistant A. baumannii is classified as one of the critical-priority pathogens on the World Health Organization (WHO) priority list of antibiotic-resistant bacteria for effective drug development. Although available genetic manipulation approaches are successful in A. baumannii laboratory strains, they are limited when employed on newly acquired clinical strains since such strains have higher levels of AMR than those used to select them for genetic manipulation. Recently, the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system has emerged as one of the most effective, efficient, and precise methods of genome editing and offers target-specific gene editing of AMR genes in a specific bacterial strain. CRISPR-based genome editing has been successfully applied in various bacterial strains to combat AMR; however, this strategy has not yet been extensively explored in A. baumannii. This review provides detailed insight into the progress, current scenario, and future potential of CRISPR-Cas usage for AMR-related gene manipulation in A. baumannii.},
}
@article {pmid37513399,
year = {2023},
author = {Li, Q and Li, S and Fang, J and Yang, C and Zhao, X and Wang, Q and Zhou, W and Zheng, W},
title = {Artemisinin Confers Neuroprotection against 6-OHDA-Induced Neuronal Injury In Vitro and In Vivo through Activation of the ERK1/2 Pathway.},
journal = {Molecules (Basel, Switzerland)},
volume = {28},
number = {14},
pages = {},
pmid = {37513399},
issn = {1420-3049},
support = {32070969//National Natural Science Foundation of China/ ; 0127/2019/A3, 0113/2018/A3 and 0038/2020/AMJ//Science and Technology Development Fund, Macau SAR/ ; 2022-Natural Science Foundation//Guangdong Provincial Funding Committee for Basic and Applied Fundamental Research/ ; LY21H280010//Natural Science Foundation of Zhejiang Province/ ; 2021KY136//Medical and health Science and Technology Project of Zhejiang Province/ ; MYRG2018-00134-FHS and MYRG2020-00158-FSH//University of Macau/ ; },
mesh = {Rats ; Humans ; Mice ; Animals ; *Parkinson Disease/drug therapy/metabolism ; MAP Kinase Signaling System ; Oxidopamine/adverse effects ; *Neuroprotective Agents/therapeutic use ; Neuroprotection ; *Neurodegenerative Diseases/drug therapy ; RNA, Guide, CRISPR-Cas Systems ; *Neuroblastoma/drug therapy ; Apoptosis ; *Artemisinins/metabolism ; Dopaminergic Neurons ; },
abstract = {Parkinson's disease (PD) is an age-related, progressive neurodegenerative disease characterized by the gradual and massive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). We have recently reported that artemisinin, an FDA-approved first-line antimalarial drug, possesses a neuroprotective effect. However, the effects and underlying mechanisms of artemisinin on Parkinson's disease remain to be elucidated. In this study, we investigated the neuroprotective effects of artemisinin on 6-OHDA and MPP[+] in neuronal cells and animal models, as well as the underlying mechanisms. Our results showed that artemisinin significantly attenuated the loss of cell viability, LDH release, elevated levels of reactive oxygen species (ROS), the collapse of the mitochondria trans-membrane potential and cell apoptosis in PC12 cells. Western blot results showed that artemisinin stimulated the phosphorylation of ERK1/2, its upstream signaling proteins c-Raf and MEK and its downstream target CREB in PC12 cells in a time- and concentration-dependent manner. In addition, the protective effect of artemisinin was significantly reduced when the ERK pathway was blocked using the ERK pathway inhibitor PD98059 or when the expression of ERK was knocked down using sgRNA. These results indicate the essential role of ERK in the protective effect of artemisinin. Similar results were obtained in SH-SY5Y cells and primary cultured neurons treated with 6-OHDA, as well as in cellular models of MPP[+] injury. More interestingly, artemisinin attenuated PD-like behavior deficit in mice injected with 6-OHDA evaluated by behavioral tests including swimming test, pole-test, open field exploration and rotarod tests. Moreover, artemisinin also stimulated the phosphorylation of ERK1/2, inhibited apoptosis, and rescued dopaminergic neurons in SNc of these animals. Application of ERK pathway inhibitor PD98059 blocked the protective effect of artemisinin in mice during testing. Taking these results together, it was indicated that artemisinin preserves neuroprotective effects against 6-OHDA and MPP[+] induced injury both in vitro and in vivo by the stimulation of the ERK1/2 signaling pathway. Our findings support the potential therapeutic effect of artemisinin in the prevention and treatment of Parkinson's disease.},
}
@article {pmid37512821,
year = {2023},
author = {Reyes-Castillo, PA and González-Vázquez, R and Torres-Maravilla, E and Bautista-Hernández, JI and Zúñiga-León, E and Leyte-Lugo, M and Mateos-Sánchez, L and Mendoza-Pérez, F and Gutiérrez-Nava, MA and Reyes-Pavón, D and Azaola-Espinosa, A and Mayorga-Reyes, L},
title = {Bifidobacterium longum LBUX23 Isolated from Feces of a Newborn; Potential Probiotic Properties and Genomic Characterization.},
journal = {Microorganisms},
volume = {11},
number = {7},
pages = {},
pmid = {37512821},
issn = {2076-2607},
abstract = {Bifidobacterium longum is considered a microorganism with probiotic potential, which has been extensively studied, but these probiotic effects are strain dependent. This work aims to characterize the probiotic potential, based on the biochemical and genomic functionality, of B. longum LBUX23, isolated from neonates' feces. B. longum LBUX23 contains one circular genome of 2,287,838 bp with a G+C content of 60.05%, no plasmids, no CRISPR-Cas operon, possesses 56 tRNAs, 9 rRNAs, 1 tmRNA and 1776 coding sequences (CDSs). It has chromosomally encoded resistance genes to ampicillin and dicloxacillin, non-hemolytic activity, and moderate inhibition of Escherichia coli ATCC 25922 and to some emergent pathogen's clinical strains. B. longum LBUX23 was able to utilize lactose, sucrose, fructooligosaccharides (FOS), and lactulose. The maximum peak of bacterial growth was observed in sucrose and FOS at 6 h; in lactose and lactulose, it was shown at 8 h. B. longum LBUX23 can survive in gastrointestinal conditions (pH 4 to 7). A decrease in survival (96.5 and 93.8%) was observed at pH 3 and 3.5 during 120 min. argC, argH, and dapA genes could be involved in this tolerance. B. longum LBUX23 can also survive under primary and secondary glyco- or tauro-conjugated bile salts, and a mixture of bile salts due to the high extracellular bile salt hydrolase (BSH) activity (67.3 %), in taurocholic acid followed by taurodeoxycholic acid (48.5%), glycocholic acid (47.1%), oxgall (44.3%), and glycodeoxycholic acid (29.7%) probably due to the presence of the cbh and gnlE genes which form an operon (start: 119573 and end: 123812). Low BSH activity was determined intracellularly (<7%), particularly in glycocholic acid; no intracellular activity was shown. B. longum LBUX23 showed antioxidant effects in DPPH radical, mainly in intact cells (27.4%). In the case of hydroxyl radical scavenging capacity, cell debris showed the highest reduction (72.5%). In the cell-free extract, superoxide anion radical scavenging capacity was higher (90.5%). The genome of B. longum LBUX23 contains PNPOx, AhpC, Bcp, trxA, and trxB genes, which could be involved in this activity. Regarding adherence, it showed adherence up to 5% to Caco-2 cells. B. longum LBUX23 showed in vitro potential probiotic properties, mainly in BSH activity and antioxidant capacity, which indicates that it could be a good candidate for antioxidant or anti-cholesterol tests using in vivo models.},
}
@article {pmid37512736,
year = {2023},
author = {Romero Deza, AA and Schaumburg, F and Berli, CLA},
title = {Valveless On-Chip Aliquoting for Molecular Diagnosis.},
journal = {Micromachines},
volume = {14},
number = {7},
pages = {},
pmid = {37512736},
issn = {2072-666X},
support = {PIP 11220200101392CO//National Scientific and Technical Research Council/ ; },
abstract = {The detection of nucleic acids as specific markers of infectious diseases is commonly implemented in molecular biology laboratories. The translation of these benchtop assays to a lab-on-a-chip format demands huge efforts of integration and automation. The present work is motivated by a strong requirement often posed by molecular assays that combine isothermal amplification and CRISPR/Cas-based detection: after amplification, a 2-8 microliter aliquot of the reaction products must be taken for the subsequent reaction. In order to fulfill this technical problem, we have designed and prototyped a microfluidic device that is able to meter and aliquot in the required range during the stepped assay. The operation is achieved by integrating a porous material that retains the desired amount of liquid after removing the excess reaction products, an innovative solution that avoids valving and external actuation. The prototypes were calibrated and experimentally tested to demonstrate the overall performance (general fluidics, metering, aliquoting, mixing and reaction). The proposed aliquoting method is fully compatible with additional functions, such as sample concentration or reagent storage, and could be further employed in alternative applications beyond molecular diagnosis.},
}
@article {pmid37511831,
year = {2023},
author = {Yadav, RK and Tripathi, MK and Tiwari, S and Tripathi, N and Asati, R and Chauhan, S and Tiwari, PN and Payasi, DK},
title = {Genome Editing and Improvement of Abiotic Stress Tolerance in Crop Plants.},
journal = {Life (Basel, Switzerland)},
volume = {13},
number = {7},
pages = {},
pmid = {37511831},
issn = {2075-1729},
abstract = {Genome editing aims to revolutionise plant breeding and could assist in safeguarding the global food supply. The inclusion of a 12-40 bp recognition site makes mega nucleases the first tools utilized for genome editing and first generation gene-editing tools. Zinc finger nucleases (ZFNs) are the second gene-editing technique, and because they create double-stranded breaks, they are more dependable and effective. ZFNs were the original designed nuclease-based approach of genome editing. The Cys2-His2 zinc finger domain's discovery made this technique possible. Clustered regularly interspaced short palindromic repeats (CRISPR) are utilized to improve genetics, boost biomass production, increase nutrient usage efficiency, and develop disease resistance. Plant genomes can be effectively modified using genome-editing technologies to enhance characteristics without introducing foreign DNA into the genome. Next-generation plant breeding will soon be defined by these exact breeding methods. There is abroad promise that genome-edited crops will be essential in the years to come for improving the sustainability and climate-change resilience of food systems. This method also has great potential for enhancing crops' resistance to various abiotic stressors. In this review paper, we summarize the most recent findings about the mechanism of abiotic stress response in crop plants and the use of the CRISPR/Cas mediated gene-editing systems to improve tolerance to stresses including drought, salinity, cold, heat, and heavy metals.},
}
@article {pmid37511604,
year = {2023},
author = {Zhang, Y and Mo, Y and Han, L and Sun, Z and Xu, W},
title = {Exploring Transcriptional Regulation of Hyperaccumulation in Sedum plumbizincicola through Integrated Transcriptome Analysis and CRISPR/Cas9 Technology.},
journal = {International journal of molecular sciences},
volume = {24},
number = {14},
pages = {},
pmid = {37511604},
issn = {1422-0067},
support = {32172666//National Natural Science Foundation of China/ ; },
mesh = {Cadmium/toxicity/metabolism ; *Sedum/metabolism ; CRISPR-Cas Systems ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Transcription Factors/genetics ; *Soil Pollutants ; Biodegradation, Environmental ; Plant Roots/genetics/metabolism ; },
abstract = {The cadmium hyperaccumulator Sedum plumbizincicola has remarkable abilities for cadmium (Cd) transport, accumulation and detoxification, but the transcriptional regulation mechanisms responsible for its Cd hyperaccumulation remain unknown. To address this knowledge gap, we conducted a comparative transcriptome study between S. plumbizincicola and the non-hyperaccumulating ecotype (NHE) of Sedum alfredii with or without Cd treatment. Our results revealed many differentially expressed genes involved in heavy metal transport and detoxification that were abundantly expressed in S. plumbizincicola. Additionally, we identified a large number of differentially expressed transcription factor genes, highlighting the complexity of transcriptional regulatory networks. We further screened four transcription factor genes that were highly expressed in the roots of S. plumbizincicola as candidate genes for creating CRISPR/Cas9 knockout mutations. Among these, the SpARR11 and SpMYB84 mutant lines exhibited decreased Cd accumulation in their aboveground parts, suggesting that these two transcription factors may play a role in the regulation of the Cd hyperaccumulation in S. plumbizincicola. Although further research will be required to determine the precise targeted genes of these transcription factors, combined transcriptome analysis and CRISPR/Cas9 technology provides unprecedented opportunities for identifying transcription factors related to Cd hyperaccumulation and contributes to the understanding of the transcriptional regulation mechanism of hyperaccumulation in S. plumbizincicola.},
}
@article {pmid37511168,
year = {2023},
author = {Wichmann, M and Maire, CL and Nuppenau, N and Habiballa, M and Uhde, A and Kolbe, K and Schröder, T and Lamszus, K and Fehse, B and Głów, D},
title = {Deep Characterization and Comparison of Different Retrovirus-like Particles Preloaded with CRISPR/Cas9 RNPs.},
journal = {International journal of molecular sciences},
volume = {24},
number = {14},
pages = {},
pmid = {37511168},
issn = {1422-0067},
support = {SFB841/SP2 (BF)//Deutsche Forschungsgemeinschaft/ ; DZG Innovation Fund "Gene and Cell Therapy": DZG 01.202 (BF)//German Center for Infection Research/ ; MD grant to MW//C3i-Hamburg Graduate School/ ; },
mesh = {Mice ; Animals ; Humans ; *CRISPR-Cas Systems/genetics ; Retroviridae/genetics ; Gene Editing/methods ; Lentivirus/genetics ; *Nanoparticles ; },
abstract = {The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced as promising vectors for the delivery of CRISPR/Cas genome editing components. Here, we characterized and directly compared three different types of retrovirus-based (R) VLPs, two derived from the γ-retrovirus murine leukemia virus (gRVLPs and "enhanced" egRVLPs) and one from the lentivirus human immunodeficiency virus, HIV (LVLPs). First, we unified and optimized the production of the different RVLPs. To ensure maximal comparability of the produced RVLPs, we adapted several assays, including nanoparticle tracking analysis (NTA), multi-parametric imaging flow cytometry (IFC), and Cas9-ELISA, to analyze their morphology, surface composition, size, and concentration. Next, we comparatively tested the three RVLPs targeting different genes in 293T model cells. Using identical gRNAs, we found egRVLPs to mediate the most efficient editing. Functional analyses indicated better cargo (i.e., Cas9) transfer and/or release as the underlying reason for their superior performance. Finally, we compared on- and off-target activities of the three RVLPs in human-induced pluripotent stem cells (hiPSC) exploiting the clinically relevant C-C motif chemokine receptor 5 (CCR5) as the target. Again, egRVLPs facilitated the highest, almost 100% knockout rates, importantly with minimal off-target activity. In conclusion, in direct comparison, egRVLPs were the most efficient RVLPs. Moreover, we established methods for in-depth characterization of VLPs, facilitating their validation and thus more predictable and safe application.},
}
@article {pmid37511044,
year = {2023},
author = {Peixoto, J and Príncipe, C and Pestana, A and Osório, H and Pinto, MT and Prazeres, H and Soares, P and Lima, RT},
title = {Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma.},
journal = {International journal of molecular sciences},
volume = {24},
number = {14},
pages = {},
pmid = {37511044},
issn = {1422-0067},
support = {PTDC/MEC-ONC/31520/2017//FEEI, FEDER through COMPETE 2020 -POCI, Portugal 2020, and by Portuguese funds through FCT/Minist&#xe9;rio da Ci&#xea;ncia, Tecnologia e Ensino Superior/ ; POCI-01-0145-FEDER-028779 (PTDC/BIA-MIC/28779/2017)//FEEI, FEDER through COMPETE 2020 -POCI, Portugal 2020, and by Portuguese funds through FCT/Minist&#xe9;rio da Ci&#xea;ncia, Tecnologia e Ensino Superior/ ; project "Institute for Research and Innovation in Health Sciences" (UID/BIM/04293/2019)//FEEI, FEDER through COMPETE 2020 -POCI, Portugal 2020, and by Portuguese funds through FCT/Minist&#xe9;rio da Ci&#xea;ncia, Tecnologia e Ensino Superior/ ; "Cancer Research on Therapy Resistance: From Basic Mechanisms to Novel Targets"-NORTE-01-0145-FEDER-000051//Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF/ ; The Porto Comprehensive Cancer Center" with the reference NORTE-01-0145-FEDER-072678 - Cons&#xf3;rcio PORTO.CCC - Porto.Comprehensive Cancer Center Raquel Seruca//European Regional Development Fund/ ; ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125//Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infra-structures of Strategic Relevance/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Glioblastoma/genetics ; Proteomics ; Gene Editing/methods ; Receptors, LDL/genetics ; },
abstract = {LRP1B remains one of the most altered genes in cancer, although its relevance in cancer biology is still unclear. Recent advances in gene editing techniques, particularly CRISPR/Cas9 systems, offer new opportunities to evaluate the function of large genes, such as LRP1B. Using a dual sgRNA CRISPR/Cas9 gene editing approach, this study aimed to assess the impact of disrupting LRP1B in glioblastoma cell biology. Four sgRNAs were designed for the dual targeting of two LRP1B exons (1 and 85). The U87 glioblastoma (GB) cell line was transfected with CRISPR/Cas9 PX459 vectors. To assess LRP1B-gene-induced alterations and expression, PCR, Sanger DNA sequencing, and qRT-PCR were carried out. Three clones (clones B9, E6, and H7) were further evaluated. All clones presented altered cellular morphology, increased cellular and nuclear size, and changes in ploidy. Two clones (E6 and H7) showed a significant decrease in cell growth, both in vitro and in the in vivo CAM assay. Proteomic analysis of the clones' secretome identified differentially expressed proteins that had not been previously associated with LRP1B alterations. This study demonstrates that the dual sgRNA CRISPR/Cas9 strategy can effectively edit LRP1B in GB cells, providing new insights into the impact of LRP1B deletions in GBM biology.},
}
@article {pmid37510242,
year = {2023},
author = {Parra-Sánchez, &#xc1; and Antequera-Zambrano, L and Martínez-Navarrete, G and Zorrilla-Muñoz, V and Paz, JL and Alvarado, YJ and González-Paz, L and Fernández, E},
title = {Comparative Analysis of CRISPR-Cas Systems in Pseudomonas Genomes.},
journal = {Genes},
volume = {14},
number = {7},
pages = {},
pmid = {37510242},
issn = {2073-4425},
mesh = {Humans ; *Genome, Bacterial ; *CRISPR-Cas Systems/genetics ; Pseudomonas/genetics ; Plasmids ; RNA ; },
abstract = {Pseudomonas is a bacterial genus with some saprophytic species from land and others associated with opportunistic infections in humans and animals. Factors such as pathogenicity or metabolic aspects have been related to CRISPR-Cas, and in silico studies into it have focused more on the clinical and non-environmental setting. This work aimed to perform an in silico analysis of the CRISPR-Cas systems present in Pseudomonas genomes. It analyzed 275 complete genomic sequences of Pseudomonas taken from the NCBI database. CRISPR loci were obtained from CRISPRdb. The genes associated with CRISPR (cas) and CAS proteins, and the origin and diversity of spacer sequences, were identified and compared by BLAST. The presence of self-targeting sequences, PAMs, and the conservation of DRs were visualized using WebLogo 3.6. The CRISPR-like RNA secondary structure prediction was analyzed using RNAFold and MFold. CRISPR structures were identified in 19.6% of Pseudomonas species. In all, 113 typical CRISPR arrays with 18 putative cas were found, as were 2050 spacers, of which 52% showed homology to bacteriophages, 26% to chromosomes, and 22% to plasmids. No potential self-targeting was detected within the CRISPR array. All the found DRs can form thermodynamically stable secondary RNA structures. The comparison of the CRISPR/Cas system can help understand the environmental adaptability of each evolutionary lineage of clinically and environmentally relevant species, providing data support for bacterial typing, traceability, analysis, and exploration of unconventional CRISPR.},
}
@article {pmid37510232,
year = {2023},
author = {Kanai, M and Hikino, K and Mano, S},
title = {Cloning and Functional Verification of Endogenous U6 Promoters for the Establishment of Efficient CRISPR/Cas9-Based Genome Editing in Castor (Ricinus communis).},
journal = {Genes},
volume = {14},
number = {7},
pages = {},
pmid = {37510232},
issn = {2073-4425},
mesh = {*Ricinus/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Seeds/genetics ; *Ricin/genetics/metabolism ; Cloning, Molecular ; },
abstract = {Castor (Ricinus communis) seeds are rich in a type of hydroxy fatty acid called ricinoleic acid, which is in high demand for the production of plant-based plastics, lubricants, and hydraulic oils. However, the high content of ricin, a toxic protein, in these seeds has restricted further expansion in the area of castor cultivation. Therefore, the development of ricin-free castor is needed. Genome editing technology, although successfully applied in several plant species, is still in the developing stages in castor and awaits the identification of an endogenous U6 promoter with robust function. Here, we searched for U6 small nuclear RNA (snRNA) genes in the castor genome. This led to the identification of six U6 snRNA genes. The promoters of these U6 snRNA genes were cloned, and their function was examined in castor cells using the particle delivery method. The results showed that a U6 promoter length of approximately 300 bp from the transcription start site was sufficient to activate gene expression. This study provides insights into the endogenous castor U6 promoter sequences and outlines a method for verifying the function of U6 promoters in plants using the particle delivery system.},
}
@article {pmid37508483,
year = {2023},
author = {Wang, JH and Wu, SJ and Li, Y and Zhao, Y and Liu, ZM and Deng, SL and Lian, ZX},
title = {Improving the Efficiency of Precise Genome Editing with CRISPR/Cas9 to Generate Goats Overexpressing Human Butyrylcholinesterase.},
journal = {Cells},
volume = {12},
number = {14},
pages = {},
pmid = {37508483},
issn = {2073-4409},
mesh = {Animals ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Butyrylcholinesterase/genetics ; Goats/genetics ; Transfection ; },
abstract = {The CRISPR/Cas9 system is widely used for genome editing in livestock production, although off-target effects can occur. It is the main method to produce genome-edited goats by somatic cell nuclear transfer (SCNT) of CRISPR/Cas9-mediated genome-edited primary goat fetal fibroblast cells (GFFs). Improving the double-strand break (DSB) efficiency of Cas9 in primary cells would improve the homologous repair (HR) efficiency. The low efficiency of HR remains a major hurdle in CRISPR/Cas9-mediated precise genome editing, increasing the work required to screen the genome-edited primary cell clones. In this study, we modified several essential parameters that affect the efficiency of the CRISPR/Cas9-mediated knock-in GFF cloning system, including establishing a high-efficiency transfection system for primary cells via nucleofection and optimizing homology arm (HA) length during HR. Here, we specifically inserted a recombinant human butyrylcholinesterase gene (rhBChE) into the goat fibroblast growth factor (FGF)-5 locus through the CRISPR/Cas9 system, thereby achieving simultaneous rhBChE insertion and FGF5 knock-out. First, this study introduced the Cas9, FGF5 knock-out small guide RNA, and rhBChE knock-in donors into GFFs by electroporation and obtained positive cell clones without off-target effects. Then, we demonstrated the expression of rhBChE in GFF clones and verified its function. Finally, we obtained a CRISPR/Cas9-mediated rhBChE-overexpression goat.},
}
@article {pmid37506757,
year = {2023},
author = {Muzyukina, P and Soutourina, O},
title = {CRISPR genotyping methods: Tracing the evolution from spoligotyping to machine learning.},
journal = {Biochimie},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.biochi.2023.07.017},
pmid = {37506757},
issn = {1638-6183},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with adaptive immunity defenses against foreign genetic invaders. The identification of CRISPR-Cas function is among the most impactful discoveries of recent decades that have shaped the development of genome editing in various organisms paving the way for a plethora of promising applications in biotechnology and health. Even before the discovery of CRISPR-Cas biological role, the particular structure of CRISPR loci has been explored for epidemiological genotyping of bacterial pathogens. CRISPR-Cas loci are arranged in CRISPR arrays of mostly identical direct repeats intercalated with invader-derived spacers and an operon of cas genes encoding the Cas protein components. Each small CRISPR RNA (crRNA) encoded within the CRISPR array constitutes a key functional unit of this RNA-based CRISPR-Cas defense system guiding the Cas effector proteins toward the foreign nucleic acids for their destruction. The information acquired from prior invader encounters and stored within CRISPR arrays turns out to be extremely valuable in tracing the microevolution and epidemiology of major bacterial pathogens. We review here the history of CRISPR-based typing strategies highlighting the first PCR-based methods that have set the stage for recent developments of high-throughput sequencing and machine learning-based approaches. A great amount of whole genome sequencing and metagenomic data accumulated in recent years opens up new avenues for combining experimental and computational approaches of high-resolution CRISPR-based typing.},
}
@article {pmid37506507,
year = {2023},
author = {Tanny, T and Sallam, M and Soda, N and Nguyen, NT and Alam, M and Shiddiky, MJA},
title = {CRISPR/Cas-Based Diagnostics in Agricultural Applications.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.3c00913},
pmid = {37506507},
issn = {1520-5118},
abstract = {Pests and disease-causing pathogens frequently impede agricultural production. An early and efficient diagnostic tool is crucial for effective disease management. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein (Cas) have recently been harnessed to develop diagnostic tools. The CRISPR/Cas system, composed of the Cas endonuclease and guide RNA, enables precise identification and cleavage of the target nucleic acids. The inherent sensitivity, high specificity, and rapid assay time of the CRISPR/Cas system make it an effective alternative for diagnosing plant pathogens and identifying genetically modified crops. Furthermore, its potential for multiplexing and suitability for point-of-care testing at the field level provide advantages over traditional diagnostic systems such as RT-PCR, LAMP, and NGS. In this review, we discuss the recent developments in CRISPR/Cas based diagnostics and their implications in various agricultural applications. We have also emphasized the major challenges with possible solutions and provided insights into future perspectives and potential applications of the CRISPR/Cas system in agriculture.},
}
@article {pmid37504099,
year = {2023},
author = {Ivanov, AV and Safenkova, IV and Zherdev, AV and Wan, Y and Dzantiev, BB},
title = {Comparison of Single-Stranded DNA Probes Conjugated with Magnetic Particles for Trans-Cleavage in Cas12a-Based Biosensors.},
journal = {Biosensors},
volume = {13},
number = {7},
pages = {},
pmid = {37504099},
issn = {2079-6374},
support = {075-15-2022-318//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {*DNA, Single-Stranded ; CRISPR-Cas Systems ; DNA ; *Biosensing Techniques ; Magnetic Phenomena ; },
abstract = {Biosensors based on endonuclease Cas12 provide high specificity in pathogen detection. Sensitive detection using Cas12-based assays can be achieved using trans-cleaved DNA probes attached to simply separated carriers, such as magnetic particles (MPs). The aim of this work was to compare polyA, polyC, and polyT single-stranded (ss) DNA with different lengths (from 10 to 145 nt) as trans-target probes were immobilized on streptavidin-covered MPs. Each ssDNA probe was labeled using fluorescein (5') and biotin (3'). To compare the probes, we used guide RNAs that were programmed for the recognition of two bacterial pathogens: Dickeya solani (causing blackleg and soft rot) and Erwinia amylovora (causing fire blight). The Cas12 was activated by targeting double-stranded DNA fragments of D. solani or E. amylovora and cleaved the MP-ssDNA conjugates. The considered probes demonstrated basically different dependencies in terms of cleavage efficiency. PolyC was the most effective probe when compared to polyA or polyT probes of the same length. The minimal acceptable length for the cleavage follows the row: polyC < polyT < polyA. The efficiencies of polyC and polyT probes with optimal length were proven for the DNA targets' detection of D. solani and E. amylovora. The regularities found can be used in Cas12a-based detection of viruses, bacteria, and other DNA/RNA-containing analytes.},
}
@article {pmid37503583,
year = {2023},
author = {Wang, BZ and Zhang, C and Zhang, JL and Sun, J},
title = {Conditional editing of the Drosophila melanogaster genome using single transcripts expressing Cas9 and sgRNA.},
journal = {Yi chuan = Hereditas},
volume = {45},
number = {7},
pages = {593-601},
doi = {10.16288/j.yczz.23-099},
pmid = {37503583},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Drosophila melanogaster/genetics ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; CRISPR-Associated Protein 9/genetics ; },
abstract = {The CRISPR/Cas9(clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR- associated protein 9) system, a highly efficient, simple, and easy genome editing technology, offers significant potential for genetic engineering and has been commonly applied in gene function studies in Drosophila melanogaster. However, when using CRISPR/Cas9 system to edit Drosophila melanogaster gene, Cas9 and sgRNA expression elements exist in different Drosophila melanogaster individuals, and Cas9 and sgRNA must be integrated into an individual through a complex genetic hybridization process, which has a long and complex operation cycle In this study, on the basis of the CRISPR/Cas9 system, we introduced the tRNA-sgRNA system and triplex elements, used triplex elements to link Cas9 and tRNA-sgRNA genes, stabilized the end of Cas9 mRNA after single transcript cutting, and made the expression of both Cas9 protein and sgRNA with a single transcript a reality. And as we obtained the corresponding phenotypic progeny in one hybridization, genetic manipulation was simplified. We found that conditional knockout of the white(w) gene in the Drosophila melanogaster eye and the broad(br) gene in the adult wing disc resulted in corresponding phenotypes that matched expectations using our new conditional gene editing system. So the significant advances in this new conditional gene editing system over the existing CRISPR/Cas9 system are that it is more efficient, extendable, and easy to use.},
}
@article {pmid37498808,
year = {2023},
author = {Hausjell, CS and Klausberger, M and Ernst, W and Grabherr, R},
title = {Evaluation of an inducible knockout system in insect cells based on co-infection and CRISPR/Cas9.},
journal = {PloS one},
volume = {18},
number = {7},
pages = {e0289178},
pmid = {37498808},
issn = {1932-6203},
mesh = {Animals ; *CRISPR-Cas Systems ; *Coinfection/genetics ; RNA, Guide, CRISPR-Cas Systems ; Baculoviridae/genetics ; Insecta/genetics ; },
abstract = {Due to comparably high product titers and low production costs, the baculovirus/insect cell expression system is considered a versatile production platform in the biopharmaceutical industry. Its excellence in producing complex multimeric protein assemblies, including virus-like particles (VLPs), which are considered promising vaccine candidates to counter emerging viral threats, made the system even more attractive. However, the co-formation of budded baculovirus during VLP production poses a severe challenge to downstream processing. In order to reduce the amount of budded baculovirus in the expression supernatant we developed an inducible knockout system based on CRISPR/Cas9 and co-infection with two baculoviral vectors: one bringing along the Cas9 nuclease and the other one having incorporated the sequence for sgRNA expression. With our set-up high titer viruses can be generated separately, as only when both viruses infect cells simultaneously a knockout can occur. When budding essential genes gp64 and vp80 were targeted for knockout, we measured a reduction in baculovirus titer by over 90%. However, as a consequence, we also determined lower overall eYFP fluorescence intensity showing reduced recombinant protein production, indicating that further improvements in engineering as well as purification are required in order to ultimately minimize costs and timeframes for vaccine production utilizing the baculovirus/insect cell expression system.},
}
@article {pmid37458780,
year = {2023},
author = {Jayaprakash, P and Barroso, L and Vajente, M and Maestroni, L and Louis, EJ and Morrissey, JP and Branduardi, P},
title = {CRISPR-Cas9 engineering in the hybrid yeast Zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.},
journal = {FEMS yeast research},
volume = {23},
number = {},
pages = {},
pmid = {37458780},
issn = {1567-1364},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Chromosomes ; Loss of Heterozygosity ; },
abstract = {The hybrid yeast Zygosaccharomyces parabailii holds potential as a cell factory mainly because of its robustness in withstanding stressors that often characterize bio-based processes. However, a complex genome and a lack of gene editing tools hinder the capacity to engineer this yeast. In this work, we developed a CRISPR-Cas9 gene editing system for Z. parabailii that allows simultaneous disruption or deletion of both alleles of a gene. We evaluated four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements and established that the most efficient systems used an RNA Pol II promoter followed by a 5'tRNA flanking the gRNA. This gRNA system was then used to construct a strain of Z. parabailii in which both alleles of DNL4 were inactivated and so relied on homologous recombination to repair double-stranded breaks. Our system can be used for gene inactivation in a wild-type strain and precise deletion with marker insertion in a dnl4 mutant. In some cases, we observed inter-chromosomal recombination around the site of the DSB that could cause loss of heterozygosity through gene conversion or deletion. Although an additional aspect that needs to be monitored during strain engineering, this phenomenon also offers opportunities to explore genome plasticity in hybrid yeasts.},
}
@article {pmid37402133,
year = {2023},
author = {Yu, L and Peng, Y and Sheng, M and Wang, Q and Huang, J and Yang, X},
title = {Sensitive and Amplification-Free Electrochemiluminescence Biosensor for HPV-16 Detection Based on CRISPR/Cas12a and DNA Tetrahedron Nanostructures.},
journal = {ACS sensors},
volume = {8},
number = {7},
pages = {2852-2858},
doi = {10.1021/acssensors.3c00806},
pmid = {37402133},
issn = {2379-3694},
mesh = {CRISPR-Cas Systems ; Human papillomavirus 16/genetics ; Gold/chemistry ; *Metal Nanoparticles/chemistry ; Luminescent Measurements/methods ; DNA/genetics/chemistry ; *Biosensing Techniques/methods ; *Nanostructures/chemistry ; },
abstract = {Rapid and accurate detection of biomarkers was very important for early screening and treatment of diseases. Herein, a sensitive and amplification-free electrochemiluminescence (ECL) biosensor based on CRISPR/Cas12a and DNA tetrahedron nanostructures (TDNs) was constructed. Briefly, 3D TDN was self-assembled on the Au nanoparticle-deposited glassy carbon electrode surface to construct the biosensing interface. The presence of the target would activate the trans-cleavage activity of Cas12a-crRNA duplex to cleave the single-stranded DNA signal probe on the vertex of TDN, causing the Ru(bpy)3[2+] to fall from the electrode surface and weakened the ECL signal. Thus, the CRISPR/Cas12a system transduced the change of target concentration into an ECL signal enabling the detection of HPV-16. The specific recognition of CRISPR/Cas12a to HPV-16 made the biosensor have good selectivity, while the TDN-modified sensing interface could reduce the cleaving steric resistance and improve the cleaving performance of CRISPR/Cas12a. In addition, the pretreated biosensor could complete sample detection within 100 min with a detection limit of 8.86 fM, indicating that the developed biosensor possesses the potential application prospect for fast and sensitive nucleic acid detection.},
}
@article {pmid37220911,
year = {2023},
author = {Dasgupta, S and LaDu, JK and Garcia, GR and Li, S and Tomono-Duval, K and Rericha, Y and Huang, L and Tanguay, RL},
title = {A CRISPR-Cas9 mutation in sox9b long intergenic noncoding RNA (slincR) affects zebrafish development, behavior, and regeneration.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {194},
number = {2},
pages = {153-166},
pmid = {37220911},
issn = {1096-0929},
support = {P30 ES030287/ES/NIEHS NIH HHS/United States ; R01 ES030017/ES/NIEHS NIH HHS/United States ; P42 ES016465/ES/NIEHS NIH HHS/United States ; R35 ES031709/ES/NIEHS NIH HHS/United States ; R01-ES030017/ES/NIEHS NIH HHS/United States ; /NH/NIH HHS/United States ; },
mesh = {Animals ; Zebrafish ; Zebrafish Proteins/metabolism ; *RNA, Long Noncoding/genetics/metabolism ; CRISPR-Cas Systems ; Receptors, Aryl Hydrocarbon/metabolism ; *Polychlorinated Dibenzodioxins ; Mutation ; RNA, Messenger/metabolism ; Regeneration ; },
abstract = {The role of long noncoding RNAs (lncRNAs) regulators of toxicological responses to environmental chemicals is gaining prominence. Previously, our laboratory discovered an lncRNA, sox9b long intergenic noncoding RNA (slincR), that is activated by multiple ligands of aryl hydrocarbon receptor (AHR). Within this study, we designed a CRISPR-Cas9-mediated slincR zebrafish mutant line to better understand its biological function in presence or absence of a model AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The slincRosu3 line contains an 18 bp insertion within the slincR sequence that changes its predicted mRNA secondary structure. Toxicological profiling showed that slincRosu3 is equally or more sensitive to TCDD for morphological and behavioral phenotypes. Embryonic mRNA-sequencing showed differential responses of 499 or 908 genes in slincRosu3 in absence or presence of TCDD Specifically, unexposed slincRosu3 embryos showed disruptions in metabolic pathways, suggesting an endogenous role for slincR. slincRosu3 embryos also had repressed mRNA levels of sox9b-a transcription factor that slincR is known to negatively regulate. Hence, we studied cartilage development and regenerative capacity-both processes partially regulated by sox9b. Cartilage development was disrupted in slincRosu3 embryos both in presence and absence of TCDD. slincRosu3 embryos also displayed a lack of regenerative capacity of amputated tail fins, accompanied by a lack of cell proliferation. In summary, using a novel slincR mutant line, we show that a mutation in slincR can have widespread impacts on gene expression and structural development endogenously and limited, but significant impacts in presence of AHR induction that further highlights its importance in the developmental process.},
}
@article {pmid36894722,
year = {2023},
author = {Kim, YH and Kim, N and Okafor, I and Choi, S and Min, S and Lee, J and Bae, SM and Choi, K and Choi, J and Harihar, V and Kim, Y and Kim, JS and Kleinstiver, BP and Lee, JK and Ha, T and Kim, HH},
title = {Sniper2L is a high-fidelity Cas9 variant with high activity.},
journal = {Nature chemical biology},
volume = {19},
number = {8},
pages = {972-980},
pmid = {36894722},
issn = {1552-4469},
support = {2022R1C1C2004229//National Research Foundation of Korea (NRF)/ ; 20012443//Ministry of Trade, Industry and Energy (Ministry of Trade, Industry and Energy, Korea)/ ; HI21C1314//Ministry of Health and Welfare (Ministry of Health, Welfare and Family Affairs)/ ; R35GM122569//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 2022R1A3B1078084//National Research Foundation of Korea (NRF)/ ; 2018R1A5A2025079//National Research Foundation of Korea (NRF)/ ; R35 GM122569/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; DNA/genetics ; },
abstract = {Although several high-fidelity SpCas9 variants have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper-Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences and developed DeepSniper, a deep learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.},
}
@article {pmid36879061,
year = {2023},
author = {Wei, CT and Popp, NA and Peleg, O and Powell, RL and Borenstein, E and Maly, DJ and Fowler, DM},
title = {A chemically controlled Cas9 switch enables temporal modulation of diverse effectors.},
journal = {Nature chemical biology},
volume = {19},
number = {8},
pages = {981-991},
pmid = {36879061},
issn = {1552-4469},
support = {F30 HL151075/HL/NHLBI NIH HHS/United States ; R01 GM145011/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Kinetics ; Nucleotides ; Adenine ; },
abstract = {CRISPR-Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies.},
}
@article {pmid37505340,
year = {2023},
author = {Ma, J and Zhang, P and Zheng, M and Wang, B and Gao, P and Qu, L and Zheng, F},
title = {A strain of Vibrio alginolyticus isolated from Azumapecten farreri and its pathogenic mechanism using CRISPR-Cas9 technology.},
journal = {Biotechnology letters},
volume = {},
number = {},
pages = {},
pmid = {37505340},
issn = {1573-6776},
support = {2019GHY112003//the Key Research and Development Program of Shandong Province/ ; No. ZR2016DM18//the National Natural Science Foundation of Shandong Province/ ; No. 2017YFC1404504//the National Key R&amp;D Program of China/ ; },
abstract = {Scallops have become an important aquaculture species in China because they contain high-quality protein, and scallops are important health food that combines multiple effects and high economic benefits. However, scallop aquaculture is perennially threatened by various pathogenic Vibrio species, leading to great economic losses. We obtained a strain of pathogenic bacteria, identified as Vibrio alginolyticus, from the diseased Azumapecten farreri in the scallop farming area of Huangdao District in 2018, and V. alginolyticus is one of the major shellfish pathogens. We showed that V. alginolyticus was isolated and identified as a pathogen in A. farreri for the first time. In this study, we evaluated its morphology and performed a phylogenetic analysis based on 16S rRNA gene sequencing. In addition, we performed a preliminary analysis of its pathogenic mechanisms. The Hfq protein in V. alginolyticus is an important RNA-binding protein in the quorum-sensing system that not only affects the sensitivity of Vibrio to environmental stress but also regulates a variety of functions, such as cell membrane formation, motility, and virulence towards the host. However, its effect on the pathogenesis of V. alginolyticus to A. farreri is unclear. To further investigate the pathogenic mechanism of the Hfq protein in V. alginolyticus to A. farreri, we used the CRISPR-Cas9 system to target and deplete the hfq gene fragment in V. alginolyticus and obtained the mutant strain V. ΔHfq[-]. We found that the peripheral flagellum of the mutant strain was lost, which reduced the motility of V. alginolyticus. Therefore, the deletion of target genes by the CRISPR/Cas9 genome editing system confirmed that the Hfq protein played a key role in reducing the ability of V. alginolyticus to infect A. farreri. In conclusion, our current findings provided valuable insights into the healthy culture of scallops.},
}
@article {pmid37504649,
year = {2023},
author = {Yu, Y and Li, T and Guo, M and Xiong, R and Yan, D and Chen, P},
title = {Possible Regulation of Larval Juvenile Hormone Titers in Bombyx mori by BmFAMeT6.},
journal = {Insects},
volume = {14},
number = {7},
pages = {},
pmid = {37504649},
issn = {2075-4450},
abstract = {Juvenile hormone (JH) plays a vital role in the growth, development, and reproduction of insects and other arthropods. Previous experiments have suggested that BmFAMeT6 could affect the duration of the silk moth's larval stage. In this study, we established the BmFAMeT6 overexpression strain and BmFAMeT6 knockout strain using the GAL4/UAS binary hybrid system and CRISPR/Cas 9 system, respectively, and found that the larval stage of the overexpression strain was shorter, while the knockout strain was longer. Our results exhibited that both the JH titers and BmKr-h1 levels in the larvae of the third instar were reduced significantly by BmFAMeT6 overexpression, but were increased obviously by BmFAMeT6 knockout. In addition, injection of farnesoic acid induced changes in the JH I and JH II levels in the hemolymphs of larvae. This study is the first to directly reveal the role of BmFAMeT6 in the regulation of insect JH titers and the relationship between farnesoic acid and JH (JH I and JH II). This provides a new perspective on regulating the growth and development of insects such as Bombyx mori.},
}
@article {pmid37504509,
year = {2023},
author = {de Dieu Habimana, J and Mukama, O and Amissah, OB and Sun, Y and Karangwa, E and Liu, Y and Mugisha, S and Cheng, N and Wang, L and Chen, J and Deng, S and Huang, R and Li, Z},
title = {A Rationally Designed CRISPR/Cas12a Assay Using a Multimodal Reporter for Various Readouts.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c01876},
pmid = {37504509},
issn = {1520-6882},
abstract = {The CRISPR/Cas systems offer a programmable platform for nucleic acid detection, and CRISPR/Cas-based diagnostics (CRISPR-Dx) have demonstrated the ability to target nucleic acids with greater accuracy and flexibility. However, due to the configuration of the reporter and the underlying labeling mechanism, almost all reported CRISPR-Dx rely on a single-option readout, resulting in limitations in end-point result readouts. This is also associated with high reagent consumption and delays in diagnostic reports due to protocol differences. Herein, we report for the first time a rationally designed Cas12a-based multimodal universal reporter (CAMURE) with improved sensitivity that harnesses a dual-mode reporting system, facilitating options in end-point readouts. Through systematic configurations and optimizations, our novel universal reporter achieved a 10-fold sensitivity enhancement compared to the DETECTR reporter. Our unique and versatile reporter could be paired with various readouts, conveying the same diagnostic results. We applied our novel reporter for the detection of staphylococcal enterotoxin A due to its high implication in staphylococcal food poisoning. Integrated with loop-mediated isothermal amplification, our multimodal reporter achieved 10 CFU/mL sensitivity and excellent specificity using a real-time fluorimeter, in-tube fluorescence, and lateral flow strip readouts. We also propose, using artificially contaminated milk samples, a fast (2-5 min) Triton X-100 DNA extraction approach with a comparable yield to the commercial extraction kit. Our CAMURE could be leveraged to detect all gene-encoding SEs by simply reprogramming the guide RNA and could also be applied to the detection of other infections and disease biomarkers.},
}
@article {pmid37504071,
year = {2023},
author = {Liu, CC and Dai, Y},
title = {Application of CRISPR Cas Systems for Biosensing.},
journal = {Biosensors},
volume = {13},
number = {7},
pages = {},
pmid = {37504071},
issn = {2079-6374},
abstract = {The essential properties of a biosensor are its sensitivity and selectivity to detect, monitor and quantify the biomarker(s) for the interests of medicine [...].},
}
@article {pmid37503092,
year = {2023},
author = {George, JT and Acree, C and Park, JU and Kong, M and Wiegand, T and Pignot, YL and Kellogg, EH and Greene, EC and Sternberg, SH},
title = {Mechanism of target site selection by type V-K CRISPR-associated transposases.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.07.14.548620},
pmid = {37503092},
abstract = {Unlike canonical CRISPR-Cas systems that rely on RNA-guided nucleases for target cleavage, CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to facilitate RNA-guided transposition of large genetic payloads. Type V-K CASTs offer several potential upsides for genome engineering, due to their compact size, easy programmability, and unidirectional integration. However, these systems are substantially less accurate than type I-F CASTs, and the molecular basis for this difference has remained elusive. Here we reveal that type V-K CASTs undergo two distinct mobilization pathways with remarkably different specificities: RNA-dependent and RNA-independent transposition. Whereas RNA-dependent transposition relies on Cas12k for accurate target selection, RNA-independent integration events are untargeted and primarily driven by the local availability of TnsC filaments. The cryo-EM structure of the untargeted complex reveals a TnsB-TnsC-TniQ transpososome that encompasses two turns of a TnsC filament and otherwise resembles major architectural aspects of the Cas12k-containing transpososome. Using single-molecule experiments and genome-wide meta-analyses, we found that AT-rich sites are preferred substrates for untargeted transposition and that the TnsB transposase also imparts local specificity, which collectively determine the precise insertion site. Knowledge of these motifs allowed us to direct untargeted transposition events to specific hotspot regions of a plasmid. Finally, by exploiting TnsB's preference for on-target integration and modulating the availability of TnsC, we suppressed RNA-independent transposition events and increased type V-K CAST specificity up to 98.1%, without compromising the efficiency of on-target integration. Collectively, our results reveal the importance of dissecting target site selection mechanisms and highlight new opportunities to leverage CAST systems for accurate, kilobase-scale genome engineering applications.},
}
@article {pmid37502928,
year = {2023},
author = {Hu, K and Chou, CW and Wilke, CO and Finkelstein, IJ},
title = {Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37502928},
abstract = {CRISPR-associated transposons (CASTs) co-opt CRISPR-Cas proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we show that CASTs instead co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that all CAST sub-types co-occur with defense-associated CRISPR-Cas systems. Using an E. coli quantitative transposition assay, we show that CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA reveals that Cas6 recognizes direct repeats via sequence-independent π - π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via a crRNA-independent unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA (tracrRNA) or a single guide RNA (sgRNA) reduces, but does not abrogate, off-target integration for type V CASTs. Exploiting new spacers in defense-associated CRISPR arrays explains how CASTs horizontally transfer to new hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.},
}
@article {pmid37502876,
year = {2023},
author = {Ahmed, NM and Joglekar, P and Deming, C and , and Lemon, KP and Kong, HH and Segre, JA and Conlan, S},
title = {Genomic characterization of the C. tuberculostearicum species complex, a ubiquitous member of the human skin microbiome.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {37502876},
abstract = {UNLABELLED: Corynebacterium is a predominant genus in the skin microbiome, yet its genetic diversity on skin is incompletely characterized and lacks a comprehensive set of reference genomes. Our work aims to investigate the distribution of Corynebacterium species on the skin, as well as to expand the existing genome reference catalog to enable more complete characterization of skin metagenomes. We used V1-V3 16S rRNA gene sequencing data from 14 body sites of 23 healthy volunteers to characterize Corynebacterium diversity and distribution across healthy human skin. Corynebacterium tuberculostearicum is the predominant species found on human skin and we identified two distinct C. tuberculostearicum ribotypes (A &amp; B) that can be distinguished by variation in the 16S rRNA V1-V3 sequence. One is distributed across all body sites and the other found primarily on the feet. We performed whole genome sequencing of 40 C. tuberculostearicum isolates cultured from the skin of five healthy individuals across seven skin sites. We generated five closed genomes of diverse C. tuberculostearicum which revealed that C. tuberculostearicum isolates are largely syntenic and carry a diversity of methylation patterns, plasmids and CRISPR/Cas systems. The pangenome of C. tuberculostearicum is open with a core genome size of 1806 genes and a pangenome size of 5451 total genes. This expanded pangenome enabled the mapping of 24% more C. tuberculostearicum reads from shotgun metagenomic datasets derived from skin body sites. Finally, while the genomes from this study all fall within a C. tuberculostearicum species complex, the ribotype B isolates may constitute a new species.<br><br>IMPORTANCE: Amplicon sequencing data combined with isolate whole genome sequencing has expanded our understanding of Corynebacterium on the skin. Human skin is characterized by a diverse collection of Corynebacterium species but C. tuberculostearicum predominates many sites. Our work supports the emerging idea that C. tuberculostearicum is a species complex encompassing several distinct species. We produced a collection of genomes that help define this complex including a potentially new species which we are calling C. hallux based on a preference for sites on the feet, whole-genome average nucleotide identity, pangenomics and growth in skin-like media. This isolate collection and high-quality genome resource sets the stage for developing engineered strains for both basic and translational clinical studies. Microbiomes are shaped by taxa that are both characteristic to those sites and functionally important to that community. The genus Corynebacterium is one such taxa for the human skin and nares. Foundational studies using 16S rRNA gene sequencing and shotgun metagenomics by our lab (1, 2) and others (3) have established Corynebacterium as common members of the skin microbiome. While Corynebacterium have been positively correlated with the resolution of dysbiosis associated with eczema flares (4), the importance of the Corynebacterium spp. is less defined for skin disease severity in primary immune deficient patients (5, 6). Corynebacterium spp. are predominant members of the human aerodigestive tract microbiome (nares, oral cavity and respiratory tract) (3) and participate in microbe-microbe interactions with members of nasal microbiome (7, 8). Corynebacterium have been shown to engage with the host immune system, specifically C. accolens -promoted IL23-dependent inflammation in mice on a high-fat diet (9). C. bovis and C. mastiditis have been shown to predominate the microbiome of a ADAM10-deficient mouse model (10) as well as an ADAM17-deficient mouse model of eczema (11). Finally, C. tuberculostearicum has been shown to induce inflammation in human epidermal keratinocyte cell cultures (12). These studies establish Corynebacterium spp. as key members of the skin microbiome capable of both microbe-microbe and microbe-host interactions. A critical resource for understanding the biology of Corynebacterium on the skin is a robust collection of complete reference genomes, including isolates collected from a variety of individuals and body sites. Previously published genome collections from skin- or nares-resident species include Staphylococcus epidermidis (13), Cutibacterium acnes (14) and the recent comparative analysis of Dolosigranulum pigrum (15). Of note, while emerging bioinformatic methods and pipelines are now being employed to extract nearly-complete genomes (MAGs) from metagenomic assemblies of skin samples (16), MAGs are not yet a substitute for genomes from cultured isolates to understand strain level or pangenomic diversity. In addition to functional prediction, comparative genomics is increasingly being used to augment conventional microbiological methods to define or redefine taxonomic boundaries (17, 18), as well as describe the full extent of diversity within these boundaries (19). A pangenome, which encompasses the complete set of genes present within a set of genome sequences, enables the characterization of gene-level heterogeneity within a taxonomic group. The pangenome is commonly subdivided into the 'core' genome, referring to genes present in all strains, and the 'accessory' or 'dispensable' genome, referring to those present in only one or some isolates. (The accessory pangenome can be further subdivided to reflect a wider range of gene uniqueness, e.g. singletons.) Thorough characterization of taxa is limited by the availability of representative and high-quality genome assemblies. Unfortunately, with the exceptions of clinically relevant Corynebacterium spp. (e.g. , C. diphtheriae , C. striatum and C. pseudotuberculosis), the genus is inadequately sequenced, with 75% of species having fewer than six genomes. This includes common skin-associated species like C. tuberculostearicum with just five unique isolate genomes, only two of which are from skin. This work seeks first to characterize the distribution of Corynebacterium across 14 skin sites from 23 healthy volunteers. The second goal of this work focuses on what we identify as the predominant skin Corynebacterium species, C. tuberculostearicum . We have sequenced 23 distinct C. tuberculostearicum strains (n=40 genomes before dereplication), a five-fold increase in the number of publicly available, unique genomes (n=5). In addition to short-read assemblies, we generated five complete genomes which, along with the type strain (DSM44922), demonstrate that C. tuberculostearicum genomes are largely syntenic and carry a number of methylation systems as well as a CRISPR/Cas system. Genes from the C. tuberculostearicum genomes in our collection fall into 5451 gene clusters comprising the species pangenome. This expanded pangenome, as compared to existing public references, improved the mapping of C. tuberculostearicum metagenomic reads from unrelated healthy volunteers. In addition, we have identified a distinct C. tuberculostearicum clade that is highly enriched on the feet that may represent a new species, tentatively designated Corynebacterium hallux .},
}
@article {pmid37501535,
year = {2023},
author = {Pavia, MJ and Finn, D and Macedo-Tafur, F and Tello-Espinoza, R and Penaccio, C and Bouskill, N and Cadillo-Quiroz, H},
title = {Genes and genome-resolved metagenomics reveal the microbial functional make up of Amazon peatlands under geochemical gradients.},
journal = {Environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1462-2920.16469},
pmid = {37501535},
issn = {1462-2920},
support = {CAREER-1749252//National Science Foundation/ ; DE-AC02-05CH11231//the US Department of Energy/ ; },
abstract = {The Pastaza-Marañón Foreland Basin (PMFB) holds the most extensive tropical peatland area in South America. PMFB peatlands store ~7.07 Gt of organic carbon interacting with multiple microbial heterotrophic, methanogenic, and other aerobic/anaerobic respirations. Little is understood about the contribution of distinct microbial community members inhabiting tropical peatlands. Here, we studied the metagenomes of three geochemically distinct peatlands spanning minerotrophic, mixed, and ombrotrophic conditions. Using gene- and genome-centric approaches, we evaluate the functional potential of the underlying microbial communities. Abundance analyses show significant differences in C, N, P, and S acquisition genes. Furthermore, community interactions mediated by toxin-antitoxin and CRISPR-Cas systems were enriched in oligotrophic soils, suggesting that non-metabolic interactions may exert additional controls in low-nutrient environments. Additionally, we reconstructed 519 metagenome-assembled genomes spanning 28 phyla. Our analyses detail key differences across the geochemical gradient in the predicted microbial populations involved in degradation of organic matter, and the cycling of N and S. Notably, we observed differences in the nitric oxide (NO) reduction strategies between sites with high and low N2 O fluxes and found phyla putatively capable of both NO and sulfate reduction. Our findings detail how gene abundances and microbial populations are influenced by geochemical differences in tropical peatlands.},
}
@article {pmid37500801,
year = {2023},
author = {Esser, SP and Rahlff, J and Zhao, W and Predl, M and Plewka, J and Sures, K and Wimmer, F and Lee, J and Adam, PS and McGonigle, J and Turzynski, V and Banas, I and Schwank, K and Krupovic, M and Bornemann, TLV and Figueroa-Gonzalez, PA and Jarett, J and Rattei, T and Amano, Y and Blaby, IK and Cheng, JF and Brazelton, WJ and Beisel, CL and Woyke, T and Zhang, Y and Probst, AJ},
title = {A predicted CRISPR-mediated symbiosis between uncultivated archaea.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {37500801},
issn = {2058-5276},
support = {PR1603/2-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; RA3432/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; BE6703/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 1553211//National Science Foundation (NSF)/ ; },
abstract = {CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages.},
}
@article {pmid37498289,
year = {2023},
author = {Sánchez-Costa, M and Gola, S and Rodríguez-Sáiz, M and Barredo, JL and Hidalgo, A and Berenguer, J},
title = {From accurate genome sequence to biotechnological application: The thermophile Mycolicibacterium hassiacum as experimental model.},
journal = {Microbial biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1751-7915.14290},
pmid = {37498289},
issn = {1751-7915},
support = {GA 685474-2//H2020 European Research Council/ ; PID2019-109073RB-100//Ministerio de Ciencia e Innovaci&#xf3;n/ ; },
abstract = {Mycobacteria constitute a large group of microorganisms belonging to the phylum Actinobacteria encompassing some of the most relevant pathogenic bacteria and many saprophytic isolates that share a unique and complex cell envelope. Also unique to this group is the extensive capability to use and synthesize sterols, a class of molecules that include active signalling compounds of pharmaceutical use. However, few mycobacterial species and strains have been established as laboratory models to date, Mycolicibacterium smegmatis mc[2] 155 being the most common one. In this work, we focus on the use of a thermophilic mycobacterium, Mycolicibacterium hassiacum, which grows optimally above 50°C, as an emerging experimental model valid to extend our general knowledge of mycobacterial biology as well as for application purposes. To that end, accurate genomic sequences are key for gene mining, the study of pathogenicity or lack thereof and the potential for gene transfer. The combination of long- and short-massive sequencing technologies is strictly necessary to remove biases caused by errors specific to long-reads technology. By doing so in M. hassiacum, we obtained from the curated genome clues regarding the genetic manipulation potential of this microorganism from the presence of insertion sequences, CRISPR-Cas, type VII ESX secretion systems, as well as lack of plasmids. Finally, as a proof of concept of the applicability of M. hassiacum as a laboratory and industrial model, we used this high-quality genome of M. hassiacum to successfully knockout a gene involved in the use of phytosterols as source of carbon and energy, using an improved gene cassette for thermostable selection and a transformation protocol at high temperature.},
}
@article {pmid37496635,
year = {2023},
author = {Jiang, C and She, Q and Wang, H},
title = {Editorial: Insights in genome editing tools and mechanisms: 2022.},
journal = {Frontiers in genome editing},
volume = {5},
number = {},
pages = {1240576},
pmid = {37496635},
issn = {2673-3439},
}
@article {pmid37495710,
year = {2023},
author = {Kim, C and Cnaani, A and Kültz, D},
title = {Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {12086},
pmid = {37495710},
issn = {2045-2322},
mesh = {Animals ; *Tilapia/metabolism ; CRISPR-Cas Systems/genetics ; Osmoregulation ; Gene Expression Regulation ; Cell Line ; },
abstract = {MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia (Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic (salinity) stress at both the whole organism and cellular levels of biological organization. Here, we utilize an O. mossambicus brain cell line and an optimized vector-based CRISPR/Cas9 system to functionally disrupt MYC in the tilapia genome and to establish causal links between MYC and cell functions, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonal myca (a gene encoding MYC) knockout (ko) cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2%). Subsequent isolation and dilution of cells from these pools produced a myca ko cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonal myca ko cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linking myca to cellular osmoregulation and other cell functions.},
}
@article {pmid37494395,
year = {2023},
author = {Sun, W and Cheng, Z and Wang, J and Yang, J and Li, X and Wang, J and Chen, M and Yang, X and Sheng, G and Lou, J and Wang, Y},
title = {AcrIIC4 inhibits type II-C Cas9 by preventing R-loop formation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {31},
pages = {e2303675120},
doi = {10.1073/pnas.2303675120},
pmid = {37494395},
issn = {1091-6490},
mesh = {*CRISPR-Cas Systems ; R-Loop Structures ; RNA, Guide, CRISPR-Cas Systems ; DNA/metabolism ; *Bacteriophages/genetics ; Gene Editing ; },
abstract = {Anti-CRISPR (Acr) proteins are encoded by phages and other mobile genetic elements and inhibit host CRISPR-Cas immunity using versatile strategies. AcrIIC4 is a broad-spectrum Acr that inhibits the type II-C CRISPR-Cas9 system in several species by an unknown mechanism. Here, we determined a series of structures of Haemophilus parainfluenzae Cas9 (HpaCas9)-sgRNA in complex with AcrIIC4 and/or target DNA, as well as the crystal structure of AcrIIC4 alone. We found that AcrIIC4 resides in the crevice between the REC1 and REC2 domains of HpaCas9, where its extensive interactions restrict the mobility of the REC2 domain and prevent the unwinding of target double-stranded (ds) DNA at the PAM-distal end. Therefore, the full-length guide RNA:target DNA heteroduplex fails to form in the presence of AcrIIC4, preventing Cas9 nuclease activation. Altogether, our structural and biochemical studies illuminate a unique Acr mechanism that allows DNA binding to the Cas9 effector complex but blocks its cleavage by preventing R-loop formation, a key step supporting DNA cleavage by Cas9.},
}
@article {pmid37493508,
year = {2023},
author = {Ricci, L and Selma-Royo, M and Golzato, D and Nabinejad, A and Servais, C and Armanini, F and Asnicar, F and Pinto, F and Tamburini, S and Segata, N},
title = {Draft genome sequence of a representative strain of the Catenibacterium genus isolated from human feces.},
journal = {Microbiology resource announcements},
volume = {},
number = {},
pages = {e0032923},
doi = {10.1128/MRA.00329-23},
pmid = {37493508},
issn = {2576-098X},
abstract = {A strain from a previously undescribed species belonging to the Catenibacterium genus was isolated from the stool of a healthy volunteer. The strain is strictly anaerobic, and the genome encodes a CRISPR-Cas system and genes related to trimethylamine production.},
}
@article {pmid37491415,
year = {2023},
author = {Matrishin, CB and Haase, EM and Dewhirst, FE and Mark Welch, JL and Miranda-Sanchez, F and Chen, T and MacFarland, DC and Kauffman, KM},
title = {Phages are unrecognized players in the ecology of the oral pathogen Porphyromonas gingivalis.},
journal = {Microbiome},
volume = {11},
number = {1},
pages = {161},
pmid = {37491415},
issn = {2049-2618},
support = {T32DE023526/DE/NIDCR NIH HHS/United States ; R01DE016937/DE/NIDCR NIH HHS/United States ; R01DE016937/DE/NIDCR NIH HHS/United States ; R01DE016937/DE/NIDCR NIH HHS/United States ; },
mesh = {Humans ; *Bacteriophages/genetics ; Porphyromonas gingivalis/genetics ; Prophages/genetics ; *Periodontal Diseases ; Base Sequence ; },
abstract = {BACKGROUND: Porphyromonas gingivalis (hereafter "Pg") is an oral pathogen that has been hypothesized to act as a keystone driver of inflammation and periodontal disease. Although Pg is most readily recovered from individuals with actively progressing periodontal disease, healthy individuals and those with stable non-progressing disease are also colonized by Pg. Insights into the factors shaping the striking strain-level variation in Pg, and its variable associations with disease, are needed to achieve a more mechanistic understanding of periodontal disease and its progression. One of the key forces often shaping strain-level diversity in microbial communities is infection of bacteria by their viral (phage) predators and symbionts. Surprisingly, although Pg has been the subject of study for over 40 years, essentially nothing is known of its phages, and the prevailing paradigm is that phages are not important in the ecology of Pg.<br><br>RESULTS: Here we systematically addressed the question of whether Pg are infected by phages-and we found that they are. We found that prophages are common in Pg, they are genomically diverse, and they encode genes that have the potential to alter Pg physiology and interactions. We found that phages represent unrecognized targets of the prevalent CRISPR-Cas defense systems in Pg, and that Pg strains encode numerous additional mechanistically diverse candidate anti-phage defense systems. We also found that phages and candidate anti-phage defense system elements together are major contributors to strain-level diversity and the species pangenome of this oral pathogen. Finally, we demonstrate that prophages harbored by a model Pg strain are active in culture, producing extracellular viral particles in broth cultures.<br><br>CONCLUSION: This work definitively establishes that phages are a major unrecognized force shaping the ecology and intra-species strain-level diversity of the well-studied oral pathogen Pg. The foundational phage sequence datasets and model systems that we establish here add to the rich context of all that is already known about Pg, and point to numerous avenues of future inquiry that promise to shed new light on fundamental features of phage impacts on human health and disease broadly. Video Abstract.},
}
@article {pmid37490214,
year = {2023},
author = {Chen, T and Chen, Z and Zhang, H and Li, Y and Yao, L and Zeng, B and Zhang, Z},
title = {Development of a CRISPR/Cpf1 system for multiplex gene editing in Aspergillus oryzae.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {37490214},
issn = {1874-9356},
abstract = {CRISPR/Cas technology is a powerful tool for genome engineering in Aspergillus oryzae as an industrially important filamentous fungus. Previous study has reported the application of the CRISPR/Cpf1 system based on the Cpf1 (LbCpf1) from Lachnospiraceae bacterium in A. oryzae. However, multiplex gene editing have not been investigated using this system. Here, we presented a new CRISPR/Cpf1 multiplex gene editing system in A. oryzae, which contains the Cpf1 nuclease (FnCpf1) from Francisella tularensis subsp. novicida U112 and CRISPR-RNA expression cassette. The crRNA cassette consisted of direct repeats and guide sequences driven by the A. oryzae U6 promoter and U6 terminator. Using the constructed FnCpf1 gene editing system, the wA and pyrG genes were mutated successfully. Furthermore, simultaneous editing of wA and pyrG genes in A. oryzae was performed using two guide sequences targeting these gene loci in a single crRNA array. This promising CRISPR/Cpf1 genome-editing system provides a powerful tool for genetically engineering A. oryzae.},
}
@article {pmid37489761,
year = {2023},
author = {Li, H and Song, W and Li, H and Cui, J and Xie, Y and Wu, B and Chen, R},
title = {Advances in isothermal nucleic acid amplification methods for hepatitis B virus detection.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3an00700f},
pmid = {37489761},
issn = {1364-5528},
abstract = {Hepatitis B virus (HBV) infection is a major global health problem of widespread concern. Clinically, serological assays are the most widely used diagnostic tests for HBV infection, with the presence of HBsAg in the serum being indicative of acute and chronic hepatitis B infection. However, increased identification of HBV DNA positive but HBsAg negative cases has greatly promoted the use of molecular assays for more accurate HBV diagnosis. Over the past few decades, especially since the outbreak of COVID-19, significant advancements have been made in the techniques and devices for nucleic acid testing (NAT). Nowadays, the mainstream NAT techniques can broadly be split into two categories: PCR-based methods and non-PCR-based isothermal amplification methods. As achieving point-of-care testing (POCT) or on-site testing is an important development tendency for the next-generation NAT, non-PCR-based isothermal amplification methods like nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA) have garnered significant attention in recent years. In this review, we provide a comprehensive overview of the nucleic acid isothermal amplification technologies currently used for HBV detection. The analytical performances of different methods are compared and their integration with microfluidics, lateral flow assays, and CRISPR/Cas systems is also discussed.},
}
@article {pmid37489056,
year = {2023},
author = {Ma, J and Li, X and Lou, C and Lin, X and Zhang, Z and Chen, D and Yang, S},
title = {Utility of CRISPR/Cas mediated electrochemical biosensors.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d3ay00903c},
pmid = {37489056},
issn = {1759-9679},
abstract = {Electrochemical biosensors represent a class of sensors that employ biological materials as sensitive elements, electrodes as conversion elements, and potential or current as detection signals. The integration of CRISPR/Cas systems into electrochemical biosensors holds immense potential, offering enhanced versatility, heightened sensitivity and specificity, reduced recovery time, and the ability to capture and identify analytes at low concentrations. In this review, we provided a succinct summary of the fundamental principles underlying electrochemical biosensors and CRISPR/Cas systems, and new progress of electrochemical biosensors based on CRISPR/Cas systems in virus, bacteria, and cancer detections. Besides, we discussed its pros and cons, present gaps, potential problem-solvers, and future prospects. To sum up, CRISPR/Cas mediated electrochemical biosensors will surely benefit us a lot in the detection of cells and microorganisms, and of course in other promising fields.},
}
@article {pmid37487103,
year = {2023},
author = {Choi, W and Wu, H and Yserentant, K and Huang, B and Cheng, Y},
title = {Efficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {31},
pages = {e2302471120},
doi = {10.1073/pnas.2302471120},
pmid = {37487103},
issn = {1091-6490},
support = {S10 OD021741/OD/NIH HHS/United States ; R35 GM140847/GM/NIGMS NIH HHS/United States ; S10 OD020054/OD/NIH HHS/United States ; R01 GM131641/GM/NIGMS NIH HHS/United States ; R01 GM124334/GM/NIGMS NIH HHS/United States ; U54 AI170792/AI/NIAID NIH HHS/United States ; S10 OD026881/OD/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; HEK293 Cells ; Transfection ; Green Fluorescent Proteins/metabolism ; *Glyceraldehyde-3-Phosphate Dehydrogenases/genetics/metabolism ; Gene Editing ; },
abstract = {CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor templates that enable efficient tagging of endogenous proteins with affinity tags by transient transfection and selection of genome-edited cells in various human cell lines. Combined with technological advancements in single-particle cryogenic electron microscopy, this strategy allows efficient structural studies of endogenous proteins captured in their native cellular environment and during different cellular processes. We demonstrated this strategy by tagging six different human proteins in both HEK293T and Jurkat cells. Moreover, analysis of endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293T cells allowed us to follow its behavior spatially and temporally in response to prolonged oxidative stress, correlating the increased number of oxidation-induced inactive catalytic sites in GAPDH with its translocation from cytosol to nucleus.},
}
@article {pmid37486522,
year = {2023},
author = {Oliveira, VC and Roballo, KCS and Mariano Junior, CG and Ambrósio, CE},
title = {Gene Editing Technologies Targeting TFAM and Its Relation to Mitochondrial Diseases.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {173-189},
pmid = {37486522},
issn = {0065-2598},
abstract = {Mitochondria are organelles present in the cytoplasm of eukaryotic cells; they play a key role in adenosine triphosphate (ATP) synthesis and oxidative phosphorylation. Mitochondria have their own DNA, mitochondrial DNA (mtDNA), keeping the function of the mitochondria. Mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters is and considered essential in mtDNA replication and transcription. More recently, TFAM has been shown to play a central role in the maintenance and regulation of mitochondrial copy number, inflammatory response, expression regulation, and mitochondrial genome activity. Gene editing tools such as the CRISPR-Cas 9 technique, TALENs, and other gene editing tools have been used to investigate the role of TFAM in mitochondrial mechanics and biogenesis as well as its correlation to mitochondrial disorders. Thus this chapter brings a summary of mitochondria function, dysfunction, the importance of TFAM in the maintenance of mitochondria, and state of the art of gene editing tools involving TFAM and mtDNA.},
}
@article {pmid37310200,
year = {2023},
author = {Chen, H and Yang, QL and Xu, JX and Deng, X and Zhang, YJ and Liu, T and Rots, MG and Xu, GL and Huang, KY},
title = {Efficient methods for multiple types of precise gene-editing in Chlamydomonas.},
journal = {The Plant journal : for cell and molecular biology},
volume = {115},
number = {3},
pages = {846-865},
doi = {10.1111/tpj.16265},
pmid = {37310200},
issn = {1365-313X},
mesh = {CRISPR-Cas Systems ; *Chlamydomonas/genetics ; Gene Editing/methods ; *Chlamydomonas reinhardtii/genetics ; },
abstract = {Precise gene-editing using CRISPR/Cas9 technology remains a long-standing challenge, especially for genes with low expression and no selectable phenotypes in Chlamydomonas reinhardtii, a classic model for photosynthesis and cilia research. Here, we developed a multi-type and precise genetic manipulation method in which a DNA break was generated by Cas9 nuclease and the repair was mediated using a homologous DNA template. The efficacy of this method was demonstrated for several types of gene editing, including inactivation of two low-expression genes (CrTET1 and CrKU80), the introduction of a FLAG-HA epitope tag into VIPP1, IFT46, CrTET1 and CrKU80 genes, and placing a YFP tag into VIPP1 and IFT46 for live-cell imaging. We also successfully performed a single amino acid substitution for the FLA3, FLA10 and FTSY genes, and documented the attainment of the anticipated phenotypes. Lastly, we demonstrated that precise fragment deletion from the 3'-UTR of MAA7 and VIPP1 resulted in a stable knock-down effect. Overall, our study has established efficient methods for multiple types of precise gene editing in Chlamydomonas, enabling substitution, insertion and deletion at the base resolution, thus improving the potential of this alga in both basic research and industrial applications.},
}
@article {pmid37486521,
year = {2023},
author = {Paiva, IM and Damasceno, S and Cunha, TM},
title = {CRISPR Libraries and Whole-Genome Screening to Identify Essential Factors for Viral Infections.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {157-172},
pmid = {37486521},
issn = {0065-2598},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Pandemics ; *COVID-19/genetics ; *Virus Diseases/diagnosis/genetics ; Gene Editing ; },
abstract = {The CRISPR-Cas9 system has revolutionized genetics and offers a simple and inexpensive way of generating perturbation that results in gene repression, activation, or editing. The advances in this technique make possible the development of CRISPR libraries which consist of a set of sgRNAs to cause perturbations in several genes in the same cell population. The use of libraries raised the CRISPR-Cas9 technique to a genomic scale and provides a powerful approach for identifying previously unknown molecular mechanisms and pathways involved in a specific phenotype or biological process. More specifically, the CRISPRko libraries (set of sgRNAs for gene knockout) and their high-throughput screenings are widely used in research with viral agents, and it was enlarged even more with the COVID-19 pandemic. With this chapter, we aim to point out how this tool helps in understanding virus-host relationships, such as the mechanisms of virus entry into the cell, the essential factors for its replication, and the cellular pathways involved in the response against the pathogen. The chapter also provided some practical considerations for each step of an experimentation using these tools that include choosing the library and screening type, the target cell, the viral strain, the library amplification and guaranteeing its coverage, the strategies for the gene screening pipeline by bioinformatics, and finally, target validation. To conclude, it was presented a table reviewing the last updates in the research for antiviral therapies using CRISPR libraries.},
}
@article {pmid37486517,
year = {2023},
author = {Fuziwara, CS and Kimura, ET},
title = {Using CRISPR/Cas9 to Edit a Thyroid Cancer Cell Line.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {73-84},
pmid = {37486517},
issn = {0065-2598},
mesh = {Humans ; CRISPR-Cas Systems ; *Thyroid Neoplasms/genetics ; *MicroRNAs/genetics ; Cell Line, Tumor ; *RNA, Long Noncoding/genetics ; Gene Editing ; },
abstract = {Thyroid cancer is the most prevalent endocrine malignancy, comprising multiple types of cancer, with distinct clinical-pathological characteristics. The oncogenesis of thyroid cancer is related to genetic alterations in MAPK signaling that induce proliferation and modulate noncoding genes, such as microRNAs and long noncoding RNAs. In this context, CRISPR/Cas9 emerges as a potential tool to modify gene sequence and modulate gene expression in thyroid cancer cell lines. In this chapter, we explore some of the current studies in which researchers have applied CRISPR/Cas9 in vitro to investigate thyroid cancer biology (Fig. 5.1).},
}
@article {pmid37486516,
year = {2023},
author = {Bonamino, MH and Correia, EM},
title = {The CRISPR/Cas System in Human Cancer.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {59-71},
pmid = {37486516},
issn = {0065-2598},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Genome ; Models, Animal ; *Neoplasms/genetics/therapy ; },
abstract = {The use of CRISPR as a genetic editing tool modified the oncology field from its basic to applied research for opening a simple, fast, and cheaper way to manipulate the genome. This chapter reviews some of the major uses of this technique for in vitro- and in vivo-based biological screenings, for cellular and animal model generation, and new derivative tools applied to cancer research. CRISPR has opened new frontiers increasing the knowledge about cancer, pointing to new solutions to overcome several challenges to better understand the disease and design better treatments.},
}
@article {pmid37486515,
year = {2023},
author = {Furtado, CLM and da Silva Santos, R and Sales, SLA and Teixeira, LPR and Pessoa, CD&#xd3;},
title = {Long Non-coding RNAs and CRISPR-Cas Edition in Tumorigenesis.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {41-58},
pmid = {37486515},
issn = {0065-2598},
abstract = {Long non-coding RNAs (lncRNAs) are one of the most abundant and heterogeneous transcripts with key roles in chromatin remodeling and gene regulation at the transcriptional and post-transcriptional levels. Due to their role in cell growth and differentiation, lncRNAs have emerged as an important biomarker in cancer diagnosis, prognosis, and targeted treatment. Recent studies have focused on elucidating lncRNA function during malignant transformation, tumor progression and drug resistance. The advent of the CRISPR system has made it possible to precisely edit complex genomic loci such as lncRNAs. Thus, we summarized the advances in CRISPR-Cas approaches for functional studies of lncRNAs including gene knockout, knockdown, overexpression and RNA targeting in tumorigenesis and drug resistance. Additionally, we highlighted the perspectives and potential applications of CRISPR approaches to treat cancer, as an emerging and promising target therapy.},
}
@article {pmid37409577,
year = {2023},
author = {Wang, Z and Xu, C and Yu, C and Si, Z and Huang, D and Shen, P and Fang, M and Xu, Z},
title = {Integration of a CRISPR Cas12a-assisted multicolor biosensor and a micropipette tip enables visible point-of-care testing of foodborne Vibrio vulnificus.},
journal = {The Analyst},
volume = {148},
number = {15},
pages = {3509-3517},
doi = {10.1039/d3an00714f},
pmid = {37409577},
issn = {1364-5528},
mesh = {*Vibrio vulnificus/genetics ; Bacterial Proteins/genetics ; CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Foodborne pathogens cause numerous food safety problems, and as a virulent bacterium falling under this category, Vibrio vulnificus (V. vulnificus) poses a huge threat to public health. The conventional methods used for the detection of V. vulnificus, including culture-based and molecular detection methods, have a variety of drawbacks, including being time-consuming and labor-intensive, the requirement of large-scale equipment, and the lack of professional operators. This paper establishes a visible detection platform for V. vulnificus based on CRISPR/Cas12a, which is integrated with nucleic acid isothermal amplification and β-galactosidase-catalyzed visible color reaction. The specific vvhA gene and a conservative segment in the 16S rDNA gene of the Vibrio genus were selected as the detection targets. By using spectrum analysis, this CRISPR detection platform achieved sensitive detection of V. vulnificus (1 CFU per reaction) with high specificity. Through the color transformation system, as low as 1 CFU per reaction of V. vulnificus in both bacterial solution and artificially contaminated seafood could be visibly observed with the naked eye. Furthermore, the consistency between our assay and the qPCR assay in the detection of V. vulnificus spiked seafood was confirmed. In general, this visible detection platform is user-friendly, accurate, portable, and equipment-free, and is expected to provide a powerful supplement in point-of-care testing of V. vulnificus and also holds good promise for future application in foodborne pathogen detection.},
}
@article {pmid37368499,
year = {2023},
author = {Trujillo Rodríguez, L and Ellington, AJ and Reisch, CR and Chevrette, MG},
title = {CRISPR-Associated Transposase for Targeted Mutagenesis in Diverse Proteobacteria.},
journal = {ACS synthetic biology},
volume = {12},
number = {7},
pages = {1989-2003},
pmid = {37368499},
issn = {2161-5063},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Transposases/genetics ; DNA Transposable Elements/genetics ; Proteobacteria/genetics ; Mutagenesis/genetics ; Gene Editing ; Bacteria/genetics ; RNA ; CRISPR-Cas Systems/genetics ; },
abstract = {Genome editing tools, through the disruption of an organism's native genetic material or the introduction of non-native DNA, facilitate functional investigations to link genotypes to phenotypes. Transposons have been instrumental genetic tools in microbiology, enabling genome-wide, randomized disruption of genes and insertions of new genetic elements. Due to this randomness, identifying and isolating particular transposon mutants (i.e., those with modifications at a genetic locus of interest) can be laborious, often requiring one to sift through hundreds or thousands of mutants. Programmable, site-specific targeting of transposons became possible with recently described CRISPR-associated transposase (CASTs) systems, allowing the streamlined recovery of desired mutants in a single step. Like other CRISPR-derived systems, CASTs can be programmed by guide-RNA that is transcribed from short DNA sequence(s). Here, we describe a CAST system and demonstrate its function in bacteria from three classes of Proteobacteria. A dual plasmid strategy is demonstrated: (i) CAST genes are expressed from a broad-host-range replicative plasmid and (ii) guide-RNA and transposon are encoded on a high-copy, suicidal pUC plasmid. Using our CAST system, single-gene disruptions were performed with on-target efficiencies approaching 100% in Beta- and Gammaproteobacteria (Burkholderia thailandensis and Pseudomonas putida, respectively). We also report a peak efficiency of 45% in the Alphaproteobacterium Agrobacterium fabrum. In B. thailandensis, we performed simultaneous co-integration of transposons at two different target sites, demonstrating CAST's utility in multilocus strategies. The CAST system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in all three bacteria tested. Lastly, the dual plasmid system allowed for iterative transposon mutagenesis in all three bacteria without loss of efficiency. Given these iterative capabilities and large payload capacity, this system will be helpful for genome engineering experiments across several fields of research.},
}
@article {pmid37344596,
year = {2023},
author = {Abdel-Hafiz, HA and Schafer, JM and Chen, X and Xiao, T and Gauntner, TD and Li, Z and Theodorescu, D},
title = {Y chromosome loss in cancer drives growth by evasion of adaptive immunity.},
journal = {Nature},
volume = {619},
number = {7970},
pages = {624-631},
pmid = {37344596},
issn = {1476-4687},
mesh = {Animals ; Humans ; Mice ; *CD8-Positive T-Lymphocytes/immunology/pathology ; *Chromosome Deletion ; *Chromosomes, Human, Y/genetics ; Proteomics ; Tumor Microenvironment/immunology ; *Urinary Bladder Neoplasms/genetics/immunology/pathology/therapy ; *Tumor Escape/genetics/immunology ; Gene Expression Profiling ; Genomics ; Prognosis ; CRISPR-Cas Systems ; Gene Editing ; In Vitro Techniques ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; Flow Cytometry ; Immunotherapy ; },
abstract = {Loss of the Y chromosome (LOY) is observed in multiple cancer types, including 10-40% of bladder cancers[1-6], but its clinical and biological significance is unknown. Here, using genomic and transcriptomic studies, we report that LOY correlates with poor prognoses in patients with bladder cancer. We performed in-depth studies of naturally occurring LOY mutant bladder cancer cells as well as those with targeted deletion of Y chromosome by CRISPR-Cas9. Y-positive (Y[+]) and Y-negative (Y[-]) tumours grew similarly in vitro, whereas Y[-] tumours were more aggressive than Y[+] tumours in immune-competent hosts in a T cell-dependent manner. High-dimensional flow cytometric analyses demonstrated that Y[-] tumours promote striking dysfunction or exhaustion of CD8[+] T cells in the tumour microenvironment. These findings were validated using single-nuclei RNA sequencing and spatial proteomic evaluation of human bladder cancers. Of note, compared with Y[+] tumours, Y[-] tumours exhibited an increased response to anti-PD-1 immune checkpoint blockade therapy in both mice and patients with cancer. Together, these results demonstrate that cancer cells with LOY mutations alter T cell function, promoting T cell exhaustion and sensitizing them to PD-1-targeted immunotherapy. This work provides insights into the basic biology of LOY mutation and potential biomarkers for improving cancer immunotherapy.},
}
@article {pmid37486514,
year = {2023},
author = {Monteiro, CJ and Heery, DM and Whitchurch, JB},
title = {Modern Approaches to Mouse Genome Editing Using the CRISPR-Cas Toolbox and Their Applications in Functional Genomics and Translational Research.},
journal = {Advances in experimental medicine and biology},
volume = {1429},
number = {},
pages = {13-40},
pmid = {37486514},
issn = {0065-2598},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; Translational Research, Biomedical ; Genomics ; Genome/genetics ; },
abstract = {Mice have been used in biological research for over a century, and their immense contribution to scientific breakthroughs can be seen across all research disciplines, with some of the main beneficiaries being the fields of medicine and life sciences. Genetically engineered mouse models (GEMMs), along with other model organisms, are fundamentally important research tools frequently utilised to enhance our understanding of pathophysiology and biological mechanisms behind disease. In the 1980s, it became possible to precisely edit the mouse genome to create gene knockout and knock-in mice, although with low efficacy. Recent advances utilising CRISPR-Cas technologies have considerably improved our ability to do this with ease and precision, while also allowing the generation of desired genetic variants from single nucleotide substitutions to large insertions/deletions. It is now quick and relatively easy to genetically edit somatic cells which were previously more recalcitrant to traditional approaches. Further refinements have created a 'CRISPR toolkit' that has expanded the use of CRISPR-Cas beyond gene knock-ins and knockouts. In this chapter, we review some of the latest applications of CRISPR-Cas technologies in GEMMs, including nuclease-dead Cas9 systems for activation or repression of gene expression, base editing and prime editing. We also discuss improvements in Cas9 specificity, targeting efficacy and delivery methods in mice. Throughout, we provide examples wherein CRISPR-Cas technologies have been applied to target clinically relevant genes in preclinical GEMMs, both to generate humanised models and for experimental gene therapy research.},
}
@article {pmid37486077,
year = {2023},
author = {Kotapati, KV and Hamid, A and Ateka, EM and Pappu, H},
title = {CRISPR/Cas, transcriptomics, and RNA interference in virus disease management.},
journal = {Phytopathology},
volume = {},
number = {},
pages = {},
doi = {10.1094/PHYTO-01-23-0002-V},
pmid = {37486077},
issn = {0031-949X},
abstract = {Plant viruses infect a wide range of commercially important crop plants and cause significant crop production losses worldwide. Numerous alterations in plant physiology related to the reprogramming of gene expression may result from viral infections. While conventional IPM-based strategies have been effective in reducing the impact of several viral diseases, continued emergence of new viruses and strains, expanding host ranges, and emergence of resistance breaking strains necessitate a sustained effort toward the development and application of new approaches for virus management that would complement existing tactics. RNA interference-based techniques, and more recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing technologies have paved the way for precise targeting of viral transcripts and manipulation of viral genomes and host factors. In-depth knowledge of the molecular mechanisms underlying the development of disease would further expand the applicability of these recent methods. Advances in next generation/high throughput sequencing have made possible more intensive studies into host-virus interactions. Utilizing the omics data and its application has the potential to expedite in fast tracking traditional plant breeding methods, as well as applying modern molecular tools for trait enhancement, including virus resistance. Here, we summarize the recent developments in CRISPR/Cas system, transcriptomics, endogenous RNA interference and exogenous application of dsRNA in virus disease management.},
}
@article {pmid37427455,
year = {2023},
author = {Jiang, L and Zhou, B and Qian, H and Wang, H and Wang, Y and Fan, W and Zheng, G and Ge, J},
title = {Cell-type-specific CRISPRization of mitochondrial DNA using bifunctional biodegradable silica nanoparticles.},
journal = {Chemical communications (Cambridge, England)},
volume = {59},
number = {60},
pages = {9251-9254},
doi = {10.1039/d3cc01778h},
pmid = {37427455},
issn = {1364-548X},
mesh = {*DNA, Mitochondrial/genetics ; CRISPR-Cas Systems ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; *Nanoparticles ; },
abstract = {We report cell-type-specific and CRISPR/Cas9-mediated mtDNA editing platform by using bifunctional biodegradable silica nanoparticles, which were capable of selective intracellular delivery to CD44-overexpressed cells and subsequent mitochondrial localization, followed by glutathione-responsive biodegradation and release of Cas9/sgRNA to realize precise mtDNA editing.},
}
@article {pmid37380765,
year = {2023},
author = {Soden, ME and Yee, JX and Zweifel, LS},
title = {Circuit coordination of opposing neuropeptide and neurotransmitter signals.},
journal = {Nature},
volume = {619},
number = {7969},
pages = {332-337},
pmid = {37380765},
issn = {1476-4687},
mesh = {*Brain/cytology/metabolism ; Calcium/metabolism ; CRISPR-Cas Systems ; Dopamine/metabolism ; Dopaminergic Neurons/metabolism ; GABAergic Neurons ; gamma-Aminobutyric Acid/metabolism ; Gene Editing ; Hypothalamic Area, Lateral/cytology/metabolism ; *Neural Pathways ; *Neurotensin/metabolism ; *Neurotransmitter Agents/metabolism ; Receptors, Neurotensin/metabolism ; *Signal Transduction ; Ventral Tegmental Area/cytology/metabolism ; },
abstract = {Fast-acting neurotransmitters and slow, modulatory neuropeptides are co-released from neurons in the central nervous system, albeit from distinct synaptic vesicles[1]. The mechanisms of how co-released neurotransmitters and neuropeptides that have opposing actions-for example, stimulatory versus inhibitory-work together to exert control of neural circuit output remain unclear. This has been difficult to resolve owing to the inability to selectively isolate these signalling pathways in a cell- and circuit-specific manner. Here we developed a genetic-based anatomical disconnect procedure that utilizes distinct DNA recombinases to independently facilitate CRISPR-Cas9 mutagenesis[2] of neurotransmitter- and neuropeptide-related genes in distinct cell types in two different brain regions simultaneously. We demonstrate that neurons within the lateral hypothalamus that produce the stimulatory neuropeptide neurotensin and the inhibitory neurotransmitter GABA (γ-aminobutyric acid) utilize these signals to coordinately activate dopamine-producing neurons of the ventral tegmental area. We show that GABA release from lateral hypothalamus neurotensin neurons inhibits GABA neurons within the ventral tegmental area, disinhibiting dopamine neurons and causing a rapid rise in calcium, whereas neurotensin directly generates a slow inactivating calcium signal in dopamine neurons that is dependent on the expression of neurotensin receptor 1 (Ntsr1). We further show that these two signals work together to regulate dopamine neuron responses to maximize behavioural responding. Thus, a neurotransmitter and a neuropeptide with opposing signals can act on distinct timescales through different cell types to enhance circuit output and optimize behaviour.},
}
@article {pmid37282836,
year = {2023},
author = {Ma, P and Wang, Q and Luo, X and Mao, L and Wang, Z and Ye, E and Loh, XJ and Li, Z and Wu, YL},
title = {Recent advances in stimuli-responsive polymeric carriers for controllable CRISPR/Cas9 gene editing system delivery.},
journal = {Biomaterials science},
volume = {11},
number = {15},
pages = {5078-5094},
doi = {10.1039/d3bm00529a},
pmid = {37282836},
issn = {2047-4849},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Genetic Therapy ; Gene Transfer Techniques ; Endonucleases/genetics/metabolism ; Polymers ; },
abstract = {Non-viral polymeric vectors with good biocompatibility have been recently explored as delivery systems for clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nucleases. In this review, based on current limitations and critical barriers, we summarize the advantages of stimulus-responsive polymeric delivery vectors (i.e., pH, redox, or enzymes) towards controllable CRISPR/Cas9 genome editing system delivery as well as the advances in using stimulus-responsive CRISPR/Cas9 polymeric carriers towards cancer treatment. Last but not least, the key challenges and promising development strategies of stimulus-responsive polymeric vector designs for CRISPR/Cas9 systems will also be discussed.},
}
@article {pmid37128708,
year = {2023},
author = {Liu, L and Xue, Y and Luo, J and Han, M and Liu, X and Jiang, T and Zhao, Y and Xu, Y and Ma, C},
title = {Developing a UV-visible reporter-assisted CRISPR/Cas9 gene editing system to alter flowering time in Chrysanthemum indicum.},
journal = {Plant biotechnology journal},
volume = {21},
number = {8},
pages = {1519-1521},
pmid = {37128708},
issn = {1467-7652},
mesh = {*Gene Editing ; CRISPR-Cas Systems ; *Chrysanthemum/genetics ; Genes, Reporter ; Flowers/genetics ; },
}
@article {pmid37485554,
year = {2023},
author = {Yang, J and Yang, K and Du, S and Luo, W and Wang, C and Liu, H and Liu, K and Zhang, Z and Gao, Y and Han, X and Song, Y},
title = {Bioorthogonal Reaction-Mediated Tumor-Selective Delivery of CRISPR/Cas9 System for Dual-Targeted Cancer Immunotherapy.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202306863},
doi = {10.1002/anie.202306863},
pmid = {37485554},
issn = {1521-3773},
abstract = {CRISPR system-assisted immunotherapy is an attractive option in cancer therapy. However, its efficacy is still less than expected due to the limitations in delivering the CRISPR system to target cancer cells. Here, we report a new CRISPR/Cas9 tumor-targeting delivery strategy based on bioorthogonal reactions for dual-targeted cancer immunotherapy. First, selective in vivo metabolic labeling of cancer and activation of the cGAS-STING pathway was achieved simultaneously through tumor microenvironment (TME)-biodegradable hollow manganese dioxide (H-MnO2) nano-platform. Subsequently, CRISPR/Cas9 system-loaded liposome was accumulated within the modified tumor tissue through in vivo click chemistry, resulting in the loss of protein tyrosine phosphatase N2 (PTPN2) and further sensitizing tumors to immunotherapy. Overall, our strategy provides a modular platform for precise gene editing in vivo and exhibits potent antitumor response by boosting innate and adaptive antitumor immunity.},
}
@article {pmid37483607,
year = {2023},
author = {Chang, JC and Wang, CY and Lin, S},
title = {Interrogation of human microglial phagocytosis by CRISPR genome editing.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1169725},
pmid = {37483607},
issn = {1664-3224},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Microglia ; *Glioblastoma/genetics ; Amyloid beta-Peptides ; Phagocytosis/genetics ; },
abstract = {BACKGROUND: Microglia are an integral part of central nervous system, but our understanding of microglial biology is limited due to the challenges in obtaining and culturing primary human microglia. HMC3 is an important cell line for studying human microglia because it is readily accessible and straightforward to maintain in standard laboratories. Although HMC3 is widely used for microglial research, a robust genetic method has not been described. Here, we report a CRISPR genome editing platform, by the electroporation of Cas9 ribonucleoproteins (Cas9 RNP) and synthetic DNA repair templates, to enable rapid and precise genetic modifications of HMC3. For proof-of-concept demonstrations, we targeted the genes implicated in the regulation of amyloid beta (Aβ) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3.<br><br>METHODS: We performed CRISPR gene knockout (KO) in HMC3 by the electroporation of pre-assembled Cas9 RNP. Co-introduction of DNA repair templates allowed site-specific knock-in (KI) of an epitope tag, a synthetic promoter and a fluorescent reporter gene. The editing efficiencies were determined genotypically by DNA sequencing and phenotypically by immunofluorescent staining and flow cytometry. The gene-edited HMC3 cells were examined in vitro by fluorescent A&#x3b2; and glioblastoma phagocytosis assays.<br><br>RESULTS: Our platform enabled robust single (>90%) and double (>70%) KO without detectable off-target editing by high throughput DNA sequencing. We also inserted a synthetic SFFV promoter to efficiently upregulate the expression of endogenous CD14 and TREM2 genes associated with microglial phagocytosis. The CRISPR-edited HMC3 showed stable phenotypes and enhanced phagocytosis of fluorescence-labeled A&#x3b2;1-42 peptides. Confocal microscopy further confirmed the localization of A&#x3b2;1-42 aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells.<br><br>CONCLUSION: CRISPR genome editing by Cas9 RNP electroporation is a robust approach to genetically modify HMC3 for functional studies such as the interrogation of A&#x3b2; and tumor phagocytosis, and is readily adoptable to investigate other aspects of microglial biology.},
}
@article {pmid37483272,
year = {2023},
author = {Xiao, Y and Zhang, Z and Yin, S and Ma, X},
title = {Nanoplasmonic biosensors for precision medicine.},
journal = {Frontiers in chemistry},
volume = {11},
number = {},
pages = {1209744},
pmid = {37483272},
issn = {2296-2646},
abstract = {Nanoplasmonic biosensors have a huge boost for precision medicine, which allows doctors to better understand diseases at the molecular level and to improve the earlier diagnosis and develop treatment programs. Unlike traditional biosensors, nanoplasmonic biosensors meet the global health industry's need for low-cost, rapid and portable aspects, while offering multiplexing, high sensitivity and real-time detection. In this review, we describe the common detection schemes used based on localized plasmon resonance (LSPR) and highlight three sensing classes based on LSPR. Then, we present the recent applications of nanoplasmonic in other sensing methods such as isothermal amplification, CRISPR/Cas systems, lab on a chip and enzyme-linked immunosorbent assay. The advantages of nanoplasmonic-based integrated sensing for multiple methods are discussed. Finally, we review the current applications of nanoplasmonic biosensors in precision medicine, such as DNA mutation, vaccine evaluation and drug delivery. The obstacles faced by nanoplasmonic biosensors and the current countermeasures are discussed.},
}
@article {pmid37481734,
year = {2023},
author = {Liang, Y and Zhang, J and Xu, C and Wang, J and Han, W and Yang, J and Wu, S and An, J and Liu, J and Zhang, Z and Shi, J and Zhang, K},
title = {Biomimetic Mineralized CRISPR/Cas RNA Nanoparticles for Efficient Tumor-Specific Multiplex Gene Editing.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.3c04116},
pmid = {37481734},
issn = {1936-086X},
abstract = {CRISPR/Cas9 systems have great potential to achieve sophisticated gene therapy and cell engineering by editing multiple genomic loci. However, to achieve efficient multiplex gene editing, the delivery system needs adequate capacity to transfect all CRISPR/Cas9 RNA species at the required stoichiometry into the cytosol of each individual cell. Herein, inspired by biomineralization in nature, we develop an all-in-one biomimetic mineralized CRISPR/Cas9 RNA delivery system. This system allows for precise control over the coencapsulation ratio between Cas9 mRNA and multiple sgRNAs, while also exhibiting a high RNA loading capacity. In addition, it enhances the storage stability of RNA at 4 °C for up to one month, and the surface of the nanoparticles can be easily functionalized for precise targeting of RNA nanoparticles in vivo at nonliver sites. Based on the above characteristics, as a proof-of-concept, our system was able to achieve significant gene-editing at each target gene (Survivin: 31.9%, PLK1: 24.41%, HPV: 23.2%) and promote apoptosis of HeLa cells in the mouse model, inhibiting tumor growth without obvious off-target effects in liver tissue. This system addresses various challenges associated with multicomponent RNA delivery in vivo, providing an innovative strategy for the RNA-based CRISPR/Cas9 gene editing.},
}
@article {pmid37481551,
year = {2023},
author = {Wu, J and Huang, Y and Ding, X and Kang, L and Wang, X and Li, D and Cheng, W and Liu, G and Xue, J and Ding, S},
title = {CPA-Cas12a-based lateral flow strip for portable assay of Methicillin-resistant Staphylococcus aureus in clinical sample.},
journal = {Journal of nanobiotechnology},
volume = {21},
number = {1},
pages = {234},
pmid = {37481551},
issn = {1477-3155},
support = {KJQN202200431//the Project of Science and Technology Research Program of Chongqing Municipal Education Commission/ ; KJQN202000444//the Project of Science and Technology Research Program of Chongqing Municipal Education Commission/ ; //the Talents Project of University-Town Hospital of Chongqing Medical University/ ; cstc2019jcyj-msxmX0294//the Natural Science Foundation of Chongqing/ ; cstc2020jcyj-msxmX0190//the Natural Science Foundation of Chongqing/ ; cstc2019jcyj-msxmX0179//the Natural Science Foundation of Chongqing/ ; 20200154//the Project of the Basic and Frontier Funds in the Yuzhong District of Chongqing/ ; },
mesh = {*Methicillin-Resistant Staphylococcus aureus ; CRISPR-Cas Systems ; Cross-Priming ; Staphylococcus aureus ; Anti-Bacterial Agents/pharmacology ; Biological Assay ; },
abstract = {The rapid and accurate identification of methicillin-resistant Staphylococcus aureus at an early antibiotic therapy stage would be benefit to disease diagnosis and antibiotic selection. Herein, we integrated cross-priming amplification (CPA) and CRISPR/Cas 12a (designated as CPA-Cas 12a) systems to establish a sensitive and efficient lateral flow assay to detect methicillin-resistant Staphylococcus aureus. This assay relies on the CPA isothermal nucleic acid amplification strategy which can amplify the DNA extracted from Staphylococcus aureus and accompanying the indiscriminately trans-cleavage process of Cas 12a/CrRNA duplex after recognizing specific sequence. Taking the advantage of reporter and high turnover Cas 12a activity, a dramatic change in response was achieved to produce a significant increase in the analytical sensitivity. The signal conversion and output were realized using a lateral flow strip to achieve field-deployable detection. Furthermore, this bioassay was accommodated with a microfluidic device to realize automatically portable detection. This proposed assay completed within 30 min with the detection limit of 5 CFU mL[-1], was verified by testing bacterial suspension and 202 clinical samples. Given the high sensitivity, specificity and efficiency, this colorimetric readout assay through strip could be further promoted to the clinical diagnosis, clinical medication of multidrug-resistant bacteria.},
}
@article {pmid37474872,
year = {2023},
author = {Wu, P and Zhang, M and Xue, X and Ding, P and Ye, L},
title = {Dual-amplification system based on CRISPR-Cas12a and horseradish peroxidase-tethered magnetic microspheres for colorimetric detection of microcystin-LR.},
journal = {Mikrochimica acta},
volume = {190},
number = {8},
pages = {314},
pmid = {37474872},
issn = {1436-5073},
mesh = {Microspheres ; *CRISPR-Cas Systems ; Horseradish Peroxidase/metabolism ; *Colorimetry/methods ; Hydrogen Peroxide/metabolism ; DNA, Complementary ; Magnetic Phenomena ; },
abstract = {A novel dual-amplification system based on CRISPR-Cas12a and horseradish peroxidase (HRP) was developed for colorimetric determination of MC-LR. This dual-amplification was accomplished by combining the nuclease activity of CRISPR-Cas12a with the redox activity of HRP. HRP linked to magnetic beads through an ssDNA (MB-ssDNA-HRP) was used to induce a color change of the 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 chromogenic substrate solution. Specific binding of MC-LR with its aptamer initiated the release of a complementary DNA (cDNA), which was designed to activate the trans-cleavage activity of CRISPR-Cas12a. Upon activation, Cas12a cut the ssDNA linker in MB-ssDNA-HRP, causing a reduction of HRP on the magnetic beads. Consequently, the UV-Vis absorbance of the HRP-catalyzed reaction was decreased. The dual-signal amplification facilitated by CRISPR-Cas12a and HRP enabled the colorimetric detection of MC-LR in the range 0.01 to 50 ng·mL[-1] with a limit of detection (LOD) of 4.53 pg·mL[-1]. The practicability of the developed colorimetric method was demonstrated by detecting different levels of MC-LR in spiked real water samples. The recoveries ranged from 86.2 to 118.5% and the relative standard deviation (RSD) was 8.4 to 17.6%. This work provides new inspiration for the construction of effective signal amplification platforms and demonstrates a simple and user-friendly colorimetric method for determination of trace MC-LR.},
}
@article {pmid37474569,
year = {2023},
author = {Sheng, Y and Wang, H and Ou, Y and Wu, Y and Ding, W and Tao, M and Lin, S and Deng, Z and Bai, L and Kang, Q},
title = {Insertion sequence transposition inactivates CRISPR-Cas immunity.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4366},
pmid = {37474569},
issn = {2041-1723},
abstract = {CRISPR-Cas immunity systems safeguard prokaryotic genomes by inhibiting the invasion of mobile genetic elements. Here, we screened prokaryotic genomic sequences and identified multiple natural transpositions of insertion sequences (ISs) into cas genes, thus inactivating CRISPR-Cas defenses. We then generated an IS-trapping system, using Escherichia coli strains with various ISs and an inducible cas nuclease, to monitor IS insertions into cas genes following the induction of double-strand DNA breakage as a physiological host stress. We identified multiple events mediated by different ISs, especially IS1 and IS10, displaying substantial relaxed target specificity. IS transposition into cas was maintained in the presence of DNA repair machinery, and transposition into other host defense systems was also detected. Our findings highlight the potential of ISs to counter CRISPR activity, thus increasing bacterial susceptibility to foreign DNA invasion.},
}
@article {pmid37254611,
year = {2023},
author = {Li, YJ and Gu, JM and Ma, S and Xu, Y and Liu, M and Zhang, C and Liu, X and Wang, GF},
title = {Genome editing of the susceptibility gene ZmNANMT confers multiple disease resistance without agronomic penalty in maize.},
journal = {Plant biotechnology journal},
volume = {21},
number = {8},
pages = {1525-1527},
pmid = {37254611},
issn = {1467-7652},
mesh = {*Gene Editing ; *Zea mays/genetics ; Disease Resistance/genetics ; CRISPR-Cas Systems ; Genome, Plant/genetics ; },
}
@article {pmid36949241,
year = {2023},
author = {Maurya, VK and Ying, Y and Lanza, DG and Heaney, JD and Lydon, JP},
title = {A CRISPR/Cas9-engineered mouse carrying a conditional knockout allele for the early growth response-1 transcription factor.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {61},
number = {3-4},
pages = {e23515},
doi = {10.1002/dvg.23515},
pmid = {36949241},
issn = {1526-968X},
support = {P30 CA125123/CA/NCI NIH HHS/United States ; R01 HD042311/HD/NICHD NIH HHS/United States ; },
mesh = {Mice ; Female ; Animals ; *Transcription Factors/genetics ; Alleles ; *CRISPR-Cas Systems ; Exons ; },
abstract = {Early growth response 1 (EGR1) mediates transcriptional programs that are indispensable for cell division, differentiation, and apoptosis in numerous physiologies and pathophysiologies. Whole-body EGR1 knockouts in mice (Egr1[KO]) have advanced our understanding of EGR1 function in an in vivo context. To extend the utility of the mouse to investigate EGR1 responses in a tissue- and/or cell-type-specific manner, we generated a mouse model in which exon 2 of the mouse Egr1 gene is floxed by CRISPR/Cas9 engineering. The floxed Egr1 alleles (Egr1[f/f]) are designed to enable spatiotemporal control of Cre-mediated EGR1 ablation in the mouse. To confirm that the Egr1[f/f] alleles can be abrogated using a Cre driver, we crossed the Egr1[f/f] mouse with a global Cre driver to generate the Egr1 conditional knockout (Egr1[d/d]) mouse in which EGR1 expression is ablated in all tissues. Genetic and protein analysis confirmed the absence of exon 2 and loss of EGR1 expression in the Egr1[d/d] mouse, respectively. Moreover, the Egr1[d/d] female exhibits overt reproductive phenotypes previously reported for the Egr1[KO] mouse. Therefore, studies described in this short technical report underscore the potential utility of the murine Egr1 floxed allele to further resolve EGR1 function at a tissue- and/or cell-type-specific level.},
}
@article {pmid37470431,
year = {2023},
author = {Mendoza, CS and Findlay, AC and Judelson, H},
title = {An LbCas12a variant and elevated incubation temperatures enhance the rate of gene editing in the oomycete Phytophthora infestans.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {},
number = {},
pages = {},
doi = {10.1094/MPMI-05-23-0072-SC},
pmid = {37470431},
issn = {0894-0282},
abstract = {CRISPR-Cas editing systems have proved to be powerful tools for functional genomics research, but their effectiveness in many non-model species remains limited. In the potato and tomato pathogen Phytophthora infestans, an editing system was previously developed that expresses the Lachnospiracae bacterium Cas12a endonuclease (LbCas12a) and guide RNA from a DNA vector. However, the method works at low efficiency. Based on a hypothesis that editing is constrained by a mismatch between the optimal temperatures for P. infestans growth and endonuclease catalysis, we tested two strategies that increased the frequency of editing of two target genes by about ten-fold. First, we found that editing was boosted by a mutation in LbCas12a (D156R), which had been reported to expand its catalytic activity over a broader temperature range. Second, we observed that editing was enhanced by transiently incubating transformed tissue at a higher temperature. These modifications should make CRISPR-Cas12a more useful for interrogating gene and protein function in P. infestans and its relatives, especially species that grow optimally at lower temperatures.},
}
@article {pmid37469443,
year = {2023},
author = {Lee, M},
title = {Deep learning in CRISPR-Cas systems: a review of recent studies.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1226182},
pmid = {37469443},
issn = {2296-4185},
abstract = {In genetic engineering, the revolutionary CRISPR-Cas system has proven to be a vital tool for precise genome editing. Simultaneously, the emergence and rapid evolution of deep learning methodologies has provided an impetus to the scientific exploration of genomic data. These concurrent advancements mandate regular investigation of the state-of-the-art, particularly given the pace of recent developments. This review focuses on the significant progress achieved during 2019-2023 in the utilization of deep learning for predicting guide RNA (gRNA) activity in the CRISPR-Cas system, a key element determining the effectiveness and specificity of genome editing procedures. In this paper, an analytical overview of contemporary research is provided, with emphasis placed on the amalgamation of artificial intelligence and genetic engineering. The importance of our review is underscored by the necessity to comprehend the rapidly evolving deep learning methodologies and their potential impact on the effectiveness of the CRISPR-Cas system. By analyzing recent literature, this review highlights the achievements and emerging trends in the integration of deep learning with the CRISPR-Cas systems, thus contributing to the future direction of this essential interdisciplinary research area.},
}
@article {pmid37468545,
year = {2023},
author = {Higashitani, Y and Horie, K},
title = {Long-read sequence analysis of MMEJ-mediated CRISPR genome editing reveals complex on-target vector insertions that may escape standard PCR-based quality control.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {11652},
pmid = {37468545},
issn = {2045-2322},
mesh = {Animals ; Mice ; *Gene Editing ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; Alleles ; Polymerase Chain Reaction ; Sequence Analysis ; },
abstract = {CRISPR genome editing is a powerful tool for elucidating biological functions. To modify the genome as intended, it is essential to understand the various modes of recombination that can occur. In this study, we report complex vector insertions that were identified during the generation of conditional alleles by CRISPR editing using microhomology-mediated end joining (MMEJ). The targeting vector contained two loxP sequences and flanking 40-bp microhomologies. The genomic regions corresponding to the loxP sequences were cleaved with Cas9 in mouse embryonic stem cells. PCR screening for targeted recombination revealed a high frequency of bands of a larger size than expected. Nanopore sequencing of these bands revealed complex vector insertions mediated not only by MMEJ but also by non-homologous end joining and homologous recombination in at least 17% of the clones. A new band appeared upon improving the PCR conditions, suggesting the presence of unintentionally modified alleles that escape standard PCR screening. This prompted us to characterize the recombination of each allele of the genome-edited clones using heterozygous single nucleotide polymorphisms, leading to confirmation of the presence of homozygous alleles. Our study indicates that careful quality control of genome-edited clones is needed to exclude complex, unintended, on-target vector insertion.},
}
@article {pmid37467473,
year = {2023},
author = {Durut, N and Kornienko, AE and Schmidt, HA and Lettner, N and Donà, M and Nordborg, M and Scheid, OM},
title = {Long non-coding RNAs contribute to DNA damage resistance in Arabidopsis thaliana.},
journal = {Genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/genetics/iyad135},
pmid = {37467473},
issn = {1943-2631},
abstract = {Efficient repair of DNA lesions is essential for faithful transmission of genetic information between somatic cells and for genome integrity across generations. Plants have multiple, partially redundant and overlapping DNA repair pathways, probably due to the less constricted germline and the inevitable exposure to light including higher energy wavelengths. Many proteins involved in DNA repair and their mode of actions are well described. In contrast, a role for DNA damage-associated RNA components, evident from many other organisms, is less well understood. Here, we have challenged young Arabidopsis thaliana plants with two different types of genotoxic stress and performed de novo assembly and transcriptome analysis. We identified three long non-coding RNAs (lncRNAs) that are lowly or not expressed under regular conditions but up-regulated or induced by DNA damage. We generated CRISPR/Cas deletion mutants and found that the absence of the lncRNAs impairs the recovery capacity of the plants from genotoxic stress. The genetic loci are highly conserved among world-wide distributed Arabidopsis accessions and within related species in the Brassicaceae group. Together, these results suggest that the lncRNAs have a conserved function in connection with DNA damage and provide a basis for a mechanistic analysis of their role.},
}
@article {pmid37466850,
year = {2023},
author = {Pandya, K and Jagani, D and Singh, N},
title = {CRISPR-Cas Systems: Programmable Nuclease Revolutionizing the Molecular Diagnosis.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {37466850},
issn = {1559-0305},
abstract = {CRISPR-Cas system has evolved as a highly preferred genetic engineering tool to perform target gene manipulation via alteration of the guide RNA (gRNA) sequence. The ability to recognize and cleave a specific target with high precision has led to its applicability in multiple frontiers pertaining to human health and medicine. From basic research focused on understanding the molecular basis of disease to translational approach leading to early and precise disease diagnosis as well as developing effective therapeutics, the CRISPR-Cas system has proved to be a quite versatile tool. The coupling of CRISPR-Cas mediated cleavage with isothermal amplification (ISA) of target DNA, followed by a read-out using fluorescent or colorimetric reporters appears quite promising in providing a solution to the urgent need for nucleic acid-based point-of-care diagnostic. Hence, it has been recognized as a highly sophisticated molecular diagnostic tool for the detection of disease-specific biomarkers not limited to nucleic acids-based detection but also of non-nucleic acid targets such as proteins, exosomes, and other small molecules. In this review, we have presented salient features and principles of class 2 type II, V, and VI CRISPR-Cas systems represented by Cas9, Cas12, and Cas13 endonucleases which are frequently used in molecular diagnosis. The article then highlights different medical diagnostic applications of CRISPR-Cas systems focusing on the diagnosis of SARS-CoV-2, Dengue, Mycobacterium tuberculosis, and Listeria monocytogenes. Lastly, we discuss existing obstacles and potential future pathways concerning this subject in a concise manner.},
}
@article {pmid37466441,
year = {2023},
author = {Ortiz-Cartagena, C and Pablo-Marcos, D and Fernández-García, L and Blasco, L and Pacios, O and Bleriot, I and Siller, M and López, M and Fernández, J and Aracil, B and Fraile-Ribot, PA and García-Fernández, S and Fernández-Cuenca, F and Hernández-García, M and Cantón, R and Calvo-Montes, J and Tomás, M},
title = {CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0132923},
doi = {10.1128/spectrum.01329-23},
pmid = {37466441},
issn = {2165-0497},
abstract = {Carbapenem-resistant pathogens have been recognized as a health concern as they are both difficult to treat and detect in clinical microbiology laboratories. Researchers are making great efforts to develop highly specific, sensitive, accurate, and rapid diagnostic techniques, required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic methods due to their high specificity; the Cas13a endonuclease can discriminate single nucleotide changes and displays collateral cleavage activity against single-stranded RNA molecules when activated. This technology is usually combined with isothermal pre-amplification reactions in order to increase its sensitivity. We have developed a new LAMP-CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for nucleic acid purification and concentration. To evaluate the assay, we used 68 OXA-48-like-producing Klebsiella pneumoniae clinical isolates as well as 64 Enterobacter cloacae complex GES-6, 14 Pseudomonas aeruginosa GES-5, 9 Serratia marcescens GES-6, 5 P. aeruginosa GES-6, and 3 P. aeruginosa (GES-15, GES-27, and GES-40) and 1 K. pneumoniae GES-2 isolates. The assay, which takes less than 2 h and costs approximately 10 € per reaction, exhibited 100% specificity and sensitivity (99% confidence interval [CI]) for both OXA-48 and all GES carbapenemases. IMPORTANCE Carbapenems are one of the last-resort antibiotics for defense against multidrug-resistant pathogens. Multiple nucleic acid amplification methods, including multiplex PCR, multiplex loop-mediated isothermal amplification (LAMP) and multiplex RPAs, can achieve rapid, accurate, and simultaneous detection of several resistance genes to carbapenems in a single reaction. However, these assays need thermal cycling steps and specialized instruments, giving them limited application in the field. In this work, we adapted with high specificity and sensitivity values, a new LAMP CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for RNA extraction.},
}
@article {pmid37464237,
year = {2023},
author = {Mercer, M and Bhargava, V and Goldstein, CD and Buszczak, M},
title = {Targeting Endogenous Loci That Function in Drosophila Germline Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2677},
number = {},
pages = {99-112},
pmid = {37464237},
issn = {1940-6029},
support = {R35 GM144043/GM/NIGMS NIH HHS/United States ; R01 GM127569/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Drosophila/genetics ; *Drosophila melanogaster/genetics ; CRISPR-Cas Systems ; Germ Cells ; Stem Cells ; },
abstract = {CRISPR-Cas9 genome editing technology can be used to manipulate the genome of Drosophila melanogaster. The ability to delete genes, make specific mutations, add tags, or make other genetic manipulations is useful for studying germline stem cell biology. In this chapter, we will describe a method to use CRISPR-Cas9 genome editing technology to make knock-out and knock-in flies. We will cover everything from guideRNA (gRNA) and donor plasmid design and cloning to screening for positive edits.},
}
@article {pmid37464161,
year = {2023},
author = {Totorikaguena, L and Olabarrieta, E},
title = {CRISPR/Cas9 as a Simple Technique for the Generation of Murine Knockout Models for Neuropsychiatric Diseases.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2687},
number = {},
pages = {45-55},
pmid = {37464161},
issn = {1940-6029},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems/genetics ; Mice, Knockout ; *Gene Editing/methods ; Mutation ; },
abstract = {The development of schizophrenia-like rodent models is still a major challenge for the study of this mental disorder. Schizophrenia and other neuropsychiatric disorders are thought to be triggered by multiple factors, and furthermore, the genetic component of schizophrenia is highly complex. The edition of one single gene for mimicking some of the symptoms of the disorder could cause unintended mutations that could influence animal's behavior making it difficult to study. Since 2013, CRISPR-Cas gene-editing technology has been a great improvement in the specificity of transgenic model generation because of its speed, efficiency, cost, and apparent ease. This protocol describes a simple method to generate a knockout mouse model using CRISPR technology, which can be applied to any gene presumably involved in the development of schizophrenia and other neuropsychiatric disorders.},
}
@article {pmid37428865,
year = {2023},
author = {Duan, Y and Tan, Y and Chen, X and Pei, X and Li, M},
title = {Modular and Flexible Molecular Device for Simultaneous Cytosine and Adenine Base Editing at Random Genomic Loci in Filamentous Fungi.},
journal = {ACS synthetic biology},
volume = {12},
number = {7},
pages = {2147-2156},
doi = {10.1021/acssynbio.3c00229},
pmid = {37428865},
issn = {2161-5063},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Cytosine/metabolism ; Adenine/metabolism ; Genomics ; Fungi/genetics ; },
abstract = {Random base editing is regarded as a fundamental method for accelerating the genomic evolution in both scientific research and industrial applications. In this study, we designed a modular interaction-based dual base editor (MIDBE) that assembled a DNA helicase and various base editors through dockerin/cohesin-mediated protein-protein interactions, resulting in a self-assembled MIDBE complex capable of editing bases at any locus in the genome. The base editing type of MIDBE can be readily controlled by the induction of cytidine or/and adenine deaminase gene expression. MIDBE exhibited the highest editing efficiency 2.3 × 10[3] times greater than the native genomic mutation rate. To evaluate the potential of MIDBE in genomic evolution, we developed a removable plasmid-based MIDBE tool, which led to a remarkable 977.1% increase of lovastatin production in Monascus purpureus HJ11. MIDBE represents the first biological tool for generating and accumulating base mutations in Monascus chromosome and also offers a bottom-up strategy for designing the base editor.},
}
@article {pmid37404005,
year = {2023},
author = {Shen, JY and Zhao, Q and He, QL},
title = {Application of CRISPR in Filamentous Fungi and Macrofungi: From Component Function to Development Potentiality.},
journal = {ACS synthetic biology},
volume = {12},
number = {7},
pages = {1908-1923},
doi = {10.1021/acssynbio.3c00099},
pmid = {37404005},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; Fungi/genetics ; Genes, Fungal ; },
abstract = {Fungi, particularly filamentous fungi and macrofungi, have a very powerful ability to produce secondary metabolites and can serve as excellent chassis cells for the production of enzymes or natural products of great value in synthetic biology. Thus, it is imperative to establish simple, reliable, and efficient techniques for their genetic modification. However, the heterokaryosis of some fungi and the dominance of nonhomologous end-joining (NHEJ) repair mechanisms in vivo have been greatly affecting the efficiency of fungal gene editing. In recent years, the CRISPR/Cas9 system has been applied as a widely used gene editing technology in life science research and has also played an important role in the genetic modification of filamentous and macrofungi. The various functional components (cas9, sgRNA, promoter, and screening marker) of the CRISPR/Cas9 system and its development, as well as the difficulties and potential of the CRISPR/Cas9 system in filamentous fungus and macrofungi, are the main topics of this paper.},
}
@article {pmid37379837,
year = {2023},
author = {Huang, J and Lin, Q and Fei, H and He, Z and Xu, H and Li, Y and Qu, K and Han, P and Gao, Q and Li, B and Liu, G and Zhang, L and Hu, J and Zhang, R and Zuo, E and Luo, Y and Ran, Y and Qiu, JL and Zhao, KT and Gao, C},
title = {Discovery of deaminase functions by structure-based protein clustering.},
journal = {Cell},
volume = {186},
number = {15},
pages = {3182-3195.e14},
doi = {10.1016/j.cell.2023.05.041},
pmid = {37379837},
issn = {1097-4172},
mesh = {*Gene Editing ; *Proteins/metabolism ; Cytidine Deaminase/genetics/metabolism ; DNA ; CRISPR-Cas Systems ; Cytosine/metabolism ; },
abstract = {The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected deaminase proteins to analyze and identified many previously unknown properties. We were surprised to find that most proteins in the DddA-like clade were not double-stranded DNA deaminases. We engineered the smallest single-strand-specific cytidine deaminase, enabling efficient cytosine base editor (CBE) to be packaged into a single adeno-associated virus (AAV). Importantly, we profiled a deaminase from this clade that edits robustly in soybean plants, which previously was inaccessible to CBEs. These discovered deaminases, based on AI-assisted structural predictions, greatly expand the utility of base editors for therapeutic and agricultural applications.},
}
@article {pmid37358911,
year = {2023},
author = {Liu, Y and Zhang, Z and Zuo, N and Jiang, W and Gu, Y},
title = {Programmable Acetylation Modification of Bacterial Proteins by a Cas12a-Guided Acetyltransferase.},
journal = {ACS synthetic biology},
volume = {12},
number = {7},
pages = {2135-2146},
doi = {10.1021/acssynbio.3c00211},
pmid = {37358911},
issn = {2161-5063},
mesh = {*Bacterial Proteins/genetics/metabolism ; Acetylation ; *Acetyltransferases/metabolism ; CRISPR-Cas Systems ; Lysine/metabolism ; Protein Processing, Post-Translational ; Polyesters/metabolism ; },
abstract = {Protein lysine acetylation (PLA) is a crucial post-translational modification in organisms that regulates a variety of metabolic and physiological activities. Many advances have been made in PLA-related research; however, the quick and accurate identification of causal relationships between specific protein acetylation events and phenotypic outcomes at the proteome level remains challenging due to the lack of efficient targeted modification techniques. In this study, based on the characteristics of transcription-translation coupling in bacteria, we designed and constructed an in situ targeted protein acetylation (TPA) system integrating the dCas12a protein, guiding element crRNA, and bacterial acetylase At2. Rapid identification of multiple independent protein acetylation and cell phenotypic analyses in Gram-negative Escherichia coli and Gram-positive Clostridium ljungdahlii demonstrated that TPA is a specific and efficient targeting tool for protein modification studies and engineering.},
}
@article {pmid37348112,
year = {2023},
author = {Yang, S and Zhu, J and Zhou, X and Zhang, J and Li, Q and Bian, F and Zhu, J and Yan, T and Wang, X and Zhang, Y and Yang, J and Jiang, Y and Yang, S},
title = {RNA-Guided DNA Transposition in Corynebacterium glutamicum and Bacillus subtilis.},
journal = {ACS synthetic biology},
volume = {12},
number = {7},
pages = {2198-2202},
doi = {10.1021/acssynbio.3c00193},
pmid = {37348112},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Bacillus subtilis/genetics ; *Corynebacterium glutamicum/genetics ; RNA ; Gene Editing ; },
abstract = {Recently discovered CRISPR-associated transposases (CASTs) have been implemented as useful multiplexed genome editing tools, albeit only in a small group of bacteria. We demonstrated that the type I-F CAST from Vibrio cholerae could induce RNA-guided transposition in Bacillus subtilis and Corynebacterium glutamicum with efficiencies of 0.00018% and 0.027%, respectively. Lowering the culturing temperature to 16 °C in rich media increased the insertion efficiency to 3.64% in B. subtilis. By adding a positive selection against spectinomycin in the cargo DNA, up to 9 kb of the DNA fragment could be integrated at the target site with a 13.4% efficiency in C. glutamicum, which was difficult using the conventional approach. The successful implementation of CAST in these two academically important and industrial-relevant Firmicutes shows its great potential in a wide variety of bacteria and could be further optimized for multiplexed genome editing.},
}
@article {pmid37326023,
year = {2023},
author = {Zhao, Y and Coelho, C and Lauer, S and Majewski, M and Laurent, JM and Brosh, R and Boeke, JD},
title = {CREEPY: CRISPR-mediated editing of synthetic episomes in yeast.},
journal = {Nucleic acids research},
volume = {51},
number = {13},
pages = {e72},
pmid = {37326023},
issn = {1362-4962},
support = {RM1 HG009491/HG/NHGRI NIH HHS/United States ; 1RM1HG009491/GF/NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Plasmids/genetics ; DNA ; },
abstract = {Use of synthetic genomics to design and build 'big' DNA has revolutionized our ability to answer fundamental biological questions by employing a bottom-up approach. Saccharomyces cerevisiae, or budding yeast, has become the major platform to assemble large synthetic constructs thanks to its powerful homologous recombination machinery and the availability of well-established molecular biology techniques. However, introducing designer variations to episomal assemblies with high efficiency and fidelity remains challenging. Here we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, a method for rapid engineering of large synthetic episomal DNA constructs. We demonstrate that CRISPR editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes. We optimize CREEPY for efficient and precise multiplex editing of >100 kb yeast episomes, providing an expanded toolkit for synthetic genomics.},
}
@article {pmid37264902,
year = {2023},
author = {Killelea, T and Dimude, JU and He, L and Stewart, AL and Kemm, FE and Radovčić, M and Ivančić-Baće, I and Rudolph, CJ and Bolt, EL},
title = {Cas1-Cas2 physically and functionally interacts with DnaK to modulate CRISPR Adaptation.},
journal = {Nucleic acids research},
volume = {51},
number = {13},
pages = {6914-6926},
pmid = {37264902},
issn = {1362-4962},
support = {BB/T006625-1//BBSRC/ ; IP-2016-06-8861//Croatian Science Foundation/ ; //University of Nottingham/ ; },
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/metabolism ; *CRISPR-Associated Proteins/metabolism ; *Escherichia coli Proteins/metabolism ; DNA/chemistry ; CRISPR-Cas Systems ; },
abstract = {Prokaryotic Cas1-Cas2 protein complexes generate adaptive immunity to mobile genetic elements (MGEs), by capture and integration of MGE DNA in to CRISPR sites. De novo immunity relies on naive adaptation-Cas1-Cas2 targeting of MGE DNA without the aid of pre-existing immunity 'interference' complexes-by mechanisms that are not clear. Using E. coli we show that the chaperone DnaK inhibits DNA binding and integration by Cas1-Cas2, and inhibits naive adaptation in cells that results from chromosomal self-targeting. Inhibition of naive adaptation was reversed by deleting DnaK from cells, by mutation of the DnaK substrate binding domain, and by expression of an MGE (phage λ) protein. We also imaged fluorescently labelled Cas1 in living cells, observing that Cas1 foci depend on active DNA replication, and are much increased in frequency in cells lacking DnaK. We discuss a model in which DnaK provides a mechanism for restraining naive adaptation from DNA self-targeting, until DnaK is triggered to release Cas1-Cas2 to target MGE DNA.},
}
@article {pmid37246708,
year = {2023},
author = {Ponnienselvan, K and Liu, P and Nyalile, T and Oikemus, S and Maitland, SA and Lawson, ND and Luban, J and Wolfe, SA},
title = {Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency.},
journal = {Nucleic acids research},
volume = {51},
number = {13},
pages = {6966-6980},
pmid = {37246708},
issn = {1362-4962},
support = {UG3 TR002668/TR/NCATS NIH HHS/United States ; R35HL140017/NH/NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Zebrafish/genetics ; Binding Sites ; *Cold Temperature ; Cold-Shock Response ; Ribonucleoproteins ; Gene Editing ; CRISPR-Cas Systems/genetics ; Mammals ; },
abstract = {Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. Previous studies defined an optimal primer binding site (PBS) length for the pegRNA of ∼13 nucleotides depending on the sequence composition. However, optimal PBS length characterization has been based on prime editing outcomes using plasmid or lentiviral expression systems. In this study, we demonstrate that for prime editor (PE) ribonucleoprotein complexes, the auto-inhibitory interaction between the PBS and the spacer sequence affects pegRNA binding efficiency and target recognition. Destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency in multiple prime editing formats. In the case of end-protected pegRNAs, a shorter PBS length with a PBS-target strand melting temperature near 37°C is optimal in mammalian cells. Additionally, a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes for pegRNAs with optimized PBS lengths. Finally, we show that prime editor ribonucleoprotein complexes programmed with pegRNAs designed using these refined parameters efficiently correct disease-related genetic mutations in patient-derived fibroblasts and efficiently install precise edits in primary human T cells and zebrafish.},
}
@article {pmid37464084,
year = {2023},
author = {Mehta, D},
title = {EU proposal on CRISPR-edited crops is welcome - but not enough.},
journal = {Nature},
volume = {619},
number = {7970},
pages = {437},
doi = {10.1038/d41586-023-02328-8},
pmid = {37464084},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems/genetics ; *Crop Production/legislation &amp; jurisprudence/methods ; *Crops, Agricultural/genetics ; *Gene Editing/legislation &amp; jurisprudence ; *Plants, Genetically Modified/genetics ; *European Union ; },
}
@article {pmid37464007,
year = {2023},
author = {Zhou, Y and Wang, L and Lu, Z and Yu, Z and Ma, L},
title = {Optimized minimal genome-wide human sgRNA library.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {11569},
pmid = {37464007},
issn = {2045-2322},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; *Genome, Human ; Gene Library ; Genomic Library ; CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screening is revolting the genetic analysis of a cellular or molecular phenotype in question but is challenged by the large size of single-guide RNA (sgRNA) library. Here we designed a minimal genome-wide human sgRNA library, H-mLib, which is composed of 21,159 sgRNA pairs assembled based on a dedicated selection strategy from all potential SpCas9/sgRNAs in the human genome. These sgRNA pairs were cloned into a dual-gRNA vector each targeting one gene, resulting in a compact library size nearly identical to the number of human protein-coding genes. The performance of the H-mLib was benchmarked to other CRISPR libraries in a proliferation screening conducted in K562 cells. We also identified groups of core essential genes and cell-type specific essential genes by comparing the screening results from the K562 and Jurkat cells. Together, the H-mLib exemplified high specificity and sensitivity in identifying essential genes while containing minimal library complexity, emphasizing its advantages and applications in CRISPR screening with limited cell numbers.},
}
@article {pmid37463886,
year = {2023},
author = {Kang, Y and Yu, Y and Liu, Y and Pan, Y and Zhang, R and Ren, D and Cai, Z and Ma, J and Xiong, X and Zhang, Q and Zhang, C and Tu, R},
title = {Identification of USP29 as a key regulator of nucleotide biosynthesis in neuroblastoma through integrative analysis of multi-omics data.},
journal = {Cancer biology &amp; therapy},
volume = {24},
number = {1},
pages = {2237200},
pmid = {37463886},
issn = {1555-8576},
mesh = {Humans ; *Multiomics ; RNA, Guide, CRISPR-Cas Systems ; *Neuroblastoma/pathology ; Glycolysis ; Glucose ; Cell Line, Tumor ; N-Myc Proto-Oncogene Protein/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Ubiquitin-Specific Proteases/metabolism ; },
abstract = {Cancer cells show enhanced nucleotide biosynthesis, which is essential for their unlimited proliferation, but the underlying mechanisms are not entirely clear. Ubiquitin specific peptidase 29 (USP29) was reported to sustain neuroblastoma progression by promoting glycolysis and glutamine catabolism; however, its potential role in regulating nucleotide biosynthesis in tumor cells remains unknown. In this study, we depleted endogenous USP29 in MYCN-amplified neuroblastoma SK-N-BE2 cells by sgRNAs and conducted metabolomic analysis in cells with or without USP29 depletion, we found that USP29 deficiency caused a disorder of intermediates involved in glycolysis and nucleotide biosynthesis. De novo nucleotide biosynthesis was analyzed using [13]C6 glucose as a tracer under normoxia and hypoxia. The results indicated that USP29-depleted cells showed inhibition of nucleotide anabolic intermediates derived from glucose, and this inhibition was more significant under hypoxic conditions. Analysis of RNA sequencing data in SK-N-BE2 cells demonstrated that USP29 promoted the gene expression of metabolic enzymes involved in nucleotide anabolism, probably by regulating MYC and E2F downstream pathways. These findings indicated that USP29 is a key regulator of nucleotide biosynthesis in tumor cells.},
}
@article {pmid37463216,
year = {2023},
author = {Alba Burbano, D and Cardiff, RAL and Tickman, BI and Kiattisewee, C and Maranas, CJ and Zalatan, JG and Carothers, JM},
title = {Engineering activatable promoters for scalable and multi-input CRISPRa/i circuits.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {120},
number = {30},
pages = {e2220358120},
doi = {10.1073/pnas.2220358120},
pmid = {37463216},
issn = {1091-6490},
mesh = {Promoter Regions, Genetic/genetics ; *Escherichia coli/genetics ; *Escherichia coli Proteins/genetics ; Gene Regulatory Networks ; CRISPR-Cas Systems/genetics ; },
abstract = {Dynamic, multi-input gene regulatory networks (GRNs) are ubiquitous in nature. Multilayer CRISPR-based genetic circuits hold great promise for building GRNs akin to those found in naturally occurring biological systems. We develop an approach for creating high-performing activatable promoters that can be assembled into deep, wide, and multi-input CRISPR-activation and -interference (CRISPRa/i) GRNs. By integrating sequence-based design and in vivo screening, we engineer activatable promoters that achieve up to 1,000-fold dynamic range in an Escherichia coli-based cell-free system. These components enable CRISPRa GRNs that are six layers deep and four branches wide. We show the generalizability of the promoter engineering workflow by improving the dynamic range of the light-dependent EL222 optogenetic system from 6-fold to 34-fold. Additionally, high dynamic range promoters enable CRISPRa systems mediated by small molecules and protein-protein interactions. We apply these tools to build input-responsive CRISPRa/i GRNs, including feedback loops, logic gates, multilayer cascades, and dynamic pulse modulators. Our work provides a generalizable approach for the design of high dynamic range activatable promoters and enables classes of gene regulatory functions in cell-free systems.},
}
@article {pmid37460924,
year = {2023},
author = {Hussen, BM and Rasul, MF and Abdullah, SR and Hidayat, HJ and Faraj, GSH and Ali, FA and Salihi, A and Baniahmad, A and Ghafouri-Fard, S and Rahman, M and Glassy, MC and Branicki, W and Taheri, M},
title = {Targeting miRNA by CRISPR/Cas in cancer: advantages and challenges.},
journal = {Military Medical Research},
volume = {10},
number = {1},
pages = {32},
pmid = {37460924},
issn = {2054-9369},
mesh = {Humans ; *MicroRNAs/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Neoplasms/genetics/therapy ; },
abstract = {Clustered regulatory interspaced short palindromic repeats (CRISPR) has changed biomedical research and provided entirely new models to analyze every aspect of biomedical sciences during the last decade. In the study of cancer, the CRISPR/CRISPR-associated protein (Cas) system opens new avenues into issues that were once unknown in our knowledge of the noncoding genome, tumor heterogeneity, and precision medicines. CRISPR/Cas-based gene-editing technology now allows for the precise and permanent targeting of mutations and provides an opportunity to target small non-coding RNAs such as microRNAs (miRNAs). However, the development of effective and safe cancer gene editing therapy is highly dependent on proper design to be innocuous to normal cells and prevent introducing other abnormalities. This study aims to highlight the cutting-edge approaches in cancer-gene editing therapy based on the CRISPR/Cas technology to target miRNAs in cancer therapy. Furthermore, we highlight the potential challenges in CRISPR/Cas-mediated miRNA gene editing and offer advanced strategies to overcome them.},
}
@article {pmid37460897,
year = {2023},
author = {Omura, SN and Nakagawa, R and Südfeld, C and Villegas Warren, R and Wu, WY and Hirano, H and Laffeber, C and Kusakizako, T and Kise, Y and Lebbink, JHG and Itoh, Y and van der Oost, J and Nureki, O},
title = {Mechanistic and evolutionary insights into a type V-M CRISPR-Cas effector enzyme.},
journal = {Nature structural &amp; molecular biology},
volume = {},
number = {},
pages = {},
pmid = {37460897},
issn = {1545-9985},
abstract = {RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.},
}
@article {pmid37460713,
year = {2023},
author = {Zhang, J and Zhang, L and Zhang, C and Yang, Y and Liu, H and Li, L and Zhang, S and Li, X and Liu, X and Liu, Y and Wang, J and Yang, G and Xia, Q and Wang, W and Yang, J},
title = {Developing an efficient and visible prime editing system to restore tobacco 8-hydroxy-copalyl diphosphate gene for labdane diterpene Z-abienol biosynthesis.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {37460713},
issn = {1869-1889},
abstract = {Prime editing (PE) is a versatile CRISPR-Cas based precise genome-editing platform widely used to introduce a range of possible base conversions in various organisms. However, no PE systems have been shown to induce heritable mutations in tobacco, nor in any other dicot. In this study, we generated an efficient PE system in tobacco that not only introduced heritable mutations, but also enabled anthocyanin-based reporter selection of transgene-free T1 plants. This system was used to confer Z-abienol biosynthesis in the allotetraploid tobacco cultivar HHDJY by restoring a G>T conversion in the NtCPS2 gene. High levels of Z-abienol were detected in the leaves of homozygous T1 plants at two weeks after topping. This study describes an advance in PE systems and expands genome-editing toolbox in tobacco, even in dicots, for use in basic research and molecular breeding. And restoring biosynthesis of Z-abienol in tobacco might provide an efficient way to obtain Z-abienol in plants.},
}
@article {pmid37460548,
year = {2023},
author = {Yue, Q and Meng, J and Qiu, Y and Yin, M and Zhang, L and Zhou, W and An, Z and Liu, Z and Yuan, Q and Sun, W and Li, C and Zhao, H and Molnár, I and Xu, Y and Shi, S},
title = {A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4267},
pmid = {37460548},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Saccharomyces cerevisiae/genetics ; Fungi/genetics ; },
abstract = {Synthetic biology requires efficient systems that support the well-coordinated co-expression of multiple genes. Here, we discover a 9-bp nucleotide sequence that enables efficient polycistronic gene expression in yeasts and filamentous fungi. Coupling polycistronic expression to multiplexed, markerless, CRISPR/Cas9-based genome editing, we develop a strategy termed HACKing (Highly efficient and Accessible system by CracKing genes into the genome) for the assembly of multigene pathways. HACKing allows the expression level of each enzyme to be precalibrated by linking their translation to those of host proteins with predetermined abundances under the desired fermentation conditions. We validate HACKing by rapidly constructing highly efficient Saccharomyces cerevisiae cell factories that express 13 biosynthetic genes, and produce model endogenous (1,090.41 ± 80.92 mg L[-1] squalene) or heterologous (1.04 ± 0.02 mg L[-1] mogrol) terpenoid products. Thus, HACKing addresses the need of synthetic biology for predictability, simplicity, scalability, and speed upon fungal pathway engineering for valuable metabolites.},
}
@article {pmid37459160,
year = {2023},
author = {Collins, AJ and Whitaker, RJ},
title = {CRISPR Comparison Toolkit: Rapid Identification, Visualization, and Analysis of CRISPR Array Diversity.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2022.0080},
pmid = {37459160},
issn = {2573-1602},
abstract = {CRISPR-Cas systems provide immunity against mobile genetic elements (MGEs) through sequence-specific targeting by spacer sequences encoded in CRISPR arrays. Spacers are highly variable between microbial strains and can be acquired rapidly, making them well suited for use in strain typing of closely related organisms. However, no tools are currently available to automate the process of reconstructing strain histories using CRISPR spacers. We therefore developed the CRISPR Comparison Toolkit (CCTK) to enable analyses of array relationships. The CCTK includes tools to identify arrays, analyze relationships between arrays using CRISPRdiff and CRISPRtree, and predict targets of spacers. CRISPRdiff visualizes arrays and highlights the similarities between them. CRISPRtree infers a phylogenetic tree from array relationships and presents a hypothesis of the evolutionary history of the arrays. The CCTK unifies several CRISPR analysis tools into a single command line application, including the first tool to infer phylogenies from array relationships.},
}
@article {pmid37395536,
year = {2023},
author = {Lin, Y and Luo, X and Burghardt, T and Dorrer, S and Höhn, M and Wagner, E and Lächelt, U},
title = {Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery.},
journal = {Journal of the American Chemical Society},
volume = {145},
number = {28},
pages = {15171-15179},
doi = {10.1021/jacs.3c01902},
pmid = {37395536},
issn = {1520-5126},
mesh = {*CRISPR-Cas Systems/genetics ; *Ribonucleoproteins ; Evolution, Chemical ; RNA, Guide, CRISPR-Cas Systems ; Gene Editing ; },
abstract = {The introduction of the CRISPR/Cas9 system in the form of Cas9/sgRNA ribonucleoproteins (RNP) is an efficient, straightforward strategy for genome editing, and potent RNP carriers are in high demand. Here, we report a series of artificial peptides based on novel ionizable amino acids that are able to deliver Cas9 RNP into cells very efficiently. Systematic variation of hydrophobic properties revealed a relationship between the xenopeptide logD7.4 and genome editing potency. By correlating the physicochemical properties with biological activity, individual optima were found for different xenopeptide sequence architectures. The optimized amphiphilic carriers enable ∼88% eGFP knockout at an RNP dose of only 1 nM and up to 40% homology-directed repair (HDR) in eGFP/BFP switchable reporter cells by co-delivery with an ssDNA template. Mechanistic studies demonstrated that hydrophobically balanced xenopeptides are more resistant to ionic stress as well as concentration-dependent dissociation and promote endocytosis by both clathrin- and macropinocytosis-mediated pathways. The systematic study develops a versatile and adjustable carrier platform and highlights impactful structure-activity relationships, providing a new chemical guide for the design and optimization of nonviral Cas9 RNP nanocarriers.},
}
@article {pmid37279283,
year = {2023},
author = {Liu, Q and Chen, Y and Hu, S and Liu, W and Xie, D and Yang, X and Huang, W and Liu, S and Chen, X and Liu, H and Huang, J},
title = {Screening an Effective Dual-Adeno-Associated Virus Split-Cytosine Base Editor System for C-to-T Conversion In Vivo.},
journal = {Human gene therapy},
volume = {34},
number = {13-14},
pages = {629-638},
doi = {10.1089/hum.2023.055},
pmid = {37279283},
issn = {1557-7422},
mesh = {Humans ; Animals ; Mice ; *Dependovirus/genetics/metabolism ; *Cytosine/metabolism ; HeLa Cells ; Gene Editing ; Transfection ; CRISPR-Cas Systems ; },
abstract = {The cytosine base editor (CBE) has shown promise as a gene editing tool for gene therapy, as it can convert cytidine to thymidine. Adeno-associated virus (AAV) has been widely used for in vivo gene therapy, but its limited 4.7 kb packing capacity presents challenges in delivering CBE by a single AAV. To address this, one feasible solution is to split CBE into two sections for dual-AAV delivery. In this study, we utilized BE3 as an example and constructed 22 potential split-BE3 pairs with the combination of 11 splitting sites and two split-inteins (Npu and Rma). These split-BE3 pairs were initially screened in the green fluorescent protein (GFP) reporter system, with six split-BE3 pairs selected for further evaluation. The subsequent screening of split-BE3 pairs was performed at two endogenous sites in 293T and HeLa cells, revealing that the split-BE3-Rma674, split-BE3-Rma713, and split-BE3-Rma1005 displayed effective C-to-T conversion after transfection. The effectiveness of dual-AAV split-BE3 was further validated in culture cells and adult mouse eyes. Of note, the split-BE3-Rma674 demonstrated the most efficient C-to-T editing after AAV infection, with a maximal editing efficiency of 23.29% ± 10.98% in the mouse retinal pigment epithelium cells in vivo. Overall, our study presents a novel split-BE3 system with effective C-to-T conversion, which could be applied to CBE-based in vivo gene therapy.},
}
@article {pmid37265143,
year = {2023},
author = {Liu, Z and Huang, L and Deng, H and Chen, Y and Xiao, H},
title = {Characteristics, Recombination Methods, and Applications Progresses of Split-Cas9 System.},
journal = {Human gene therapy},
volume = {34},
number = {13-14},
pages = {594-604},
doi = {10.1089/hum.2022.223},
pmid = {37265143},
issn = {1557-7422},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; CRISPR-Associated Protein 9/genetics ; Genetic Vectors/genetics ; Recombination, Genetic ; },
abstract = {CRISPR technology has been used to revolutionize various facets of life sciences because of its potent gene editing capabilities. In particular, CRISPR technology is anticipated to be used to cure congenital disorders, and malignant cancers brought on by gene mutation. In this article, we introduce a Split-Cas9 system, in which Cas9 protein is split into two or more parts and recombined in cells to function specific induction circumstances. Split-Cas9 system can improve the therapeutic index of CRISPR technology by splitting Cas9 proteins into small fragments, thus enhancing their compatibility with virus vectors and precise temporal and spatial control. This article examined the combination mode of Split-Cas9 system, contrasted the differences in its split sites and activity efficiency, and discussed the use and clinical transformation in vivo and in vitro.},
}
@article {pmid37211656,
year = {2023},
author = {Ashok, K and Bhargava, CN and Asokan, R and Pradeep, C and Kennedy, JS and Rai, A and Manamohan, M},
title = {First report on the utility of pupal case for early determination of CRISPR/Cas9 ribonucleoprotein mediated genomic edits in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Tephritidae: Diptera).},
journal = {Archives of insect biochemistry and physiology},
volume = {113},
number = {4},
pages = {e22024},
doi = {10.1002/arch.22024},
pmid = {37211656},
issn = {1520-6327},
support = {//Centre for Agricultural Bioinformatics/ ; },
mesh = {Female ; Male ; Animals ; *Tephritidae/genetics ; CRISPR-Cas Systems ; Pupa/genetics ; Drosophila ; Genomics ; },
abstract = {The Oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly invasive pest of quarantine importance affecting the global fruit trade. In managing B. dorsalis, methods like cultural, biological, chemical, sterile insect technique (SIT), and semiochemical-mediated attract-and-kill are in use with varying success. The SIT approach is the method of choice for a chemical-free, long-term suppression of B. dorsalis, followed in many countries across the globe. The nonspecific mutations caused by irradiation affect the overall fitness of flies, thus requiring a more precise method for a heritable, fitness-not-compromising approach. In this regard, CRISPR/Cas9-mediated genome editing enables the creation of mutations at the precise genomic location/s through RNA-guided dsDNA cleavage. Of late, DNA-free editing employing ribonucleoprotein complex (RNP) is preferred to validate the target genes at G0 stage embryos in insects. It requires characterizing genomic edits from adults after completing their life cycle, which may entail a few days to months, depending on longevity. Additionally, edit characterization is required from each individual, as edits are unique. Therefore, all RNP-microinjected individuals must be maintained until the end of their life cycle, irrespective of editing. To overcome this impediment, we predetermine the genomic edits from the shed tissues, such as pupal cases, to maintain only edited individuals. In this study, we have shown the utility of pupal cases from five males and females of B. dorsalis to predetermine the genomic edits, which corroborated the edits from the respective adults.},
}
@article {pmid37457727,
year = {2023},
author = {Martín-Adrados, B and Wculek, SK and Fernández-Bravo, S and Torres-Ruiz, R and Valle-Noguera, A and Gomez-Sánchez, MJ and Hernández-Walias, JC and Ferreira, FM and Corraliza, AM and Sancho, D and Esteban, V and Rodriguez-Perales, S and Cruz-Adalia, A and Nakaya, HI and Salas, A and Bernardo, D and Campos-Martín, Y and Martínez-Zamorano, E and Muñoz-López, D and Gómez Del Moral, M and Cubero, FJ and Blumberg, RS and Martínez-Naves, E},
title = {Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress.},
journal = {Frontiers in immunology},
volume = {14},
number = {},
pages = {1185517},
pmid = {37457727},
issn = {1664-3224},
mesh = {Humans ; *Colitis, Ulcerative/pathology ; Caco-2 Cells ; Thapsigargin ; Endoplasmic Reticulum Stress/genetics ; *Inflammatory Bowel Diseases/metabolism ; Epithelial Cells/metabolism ; Hydroxymethylglutaryl-CoA Synthase ; },
abstract = {INTRODUCTION: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease.<br><br>METHODS: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells.<br><br>RESULTS: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines.<br><br>CONCLUSION: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.},
}
@article {pmid37455748,
year = {2023},
author = {Qi, L and Shi, M and Zhu, FC and Lian, CA and He, LS},
title = {Genomic evidence for the first symbiotic Deferribacterota, a novel gut symbiont from the deep-sea hydrothermal vent shrimp Rimicaris kairei.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1179935},
pmid = {37455748},
issn = {1664-302X},
abstract = {The genus Rimicaris is the dominant organism living in hydrothermal vents. However, little research has been done on the functions of their intestinal flora. Here, we investigated the potential functions of Deferribacterota, which is dominant in the intestine of Rimicaris kairei from the Central Indian Ridge. In total, six metagenome-assembled genomes (MAGs) of Deferribacterota were obtained using the metagenomic approach. The six Deferribacterota MAGs (Def-MAGs) were clustered into a new branch in the phylogenetic tree. The six Def-MAGs were further classified into three species, including one new order and two new genera, based on the results of phylogenetic analysis, relative evolutionary divergence (RED), average nucleotide identity (ANI), average amino acid identity (AAI) and DNA-DNA hybridization (DDH) values. The results of the energy metabolism study showed that these bacteria can use a variety of carbon sources, such as glycogen, sucrose, salicin, arbutin, glucose, cellobiose, and maltose. These bacteria have a type II secretion system and effector proteins that can transport some intracellular toxins to the extracellular compartment and a type V CRISPR-Cas system that can defend against various invasions. In addition, cofactors such as biotin, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) synthesized by R. kairei gut Deferribacterota may also assist their host in surviving under extreme conditions. Taken together, the potential function of Deferribacterota in the hydrothermal R. kairei gut suggests its long-term coevolution with the host.},
}
@article {pmid37455550,
year = {2023},
author = {Zhang, J and Zhang, Y and Khanal, S and Cao, D and Zhao, J and Dang, X and Nguyen, LNT and Schank, M and Wu, XY and Jiang, Y and Ning, S and Wang, L and El Gazzar, M and Moorman, JP and Guo, H and Yao, ZQ},
title = {Synthetic gRNA/Cas9 ribonucleoprotein targeting HBV DNA inhibits viral replication.},
journal = {Journal of medical virology},
volume = {95},
number = {7},
pages = {e28952},
doi = {10.1002/jmv.28952},
pmid = {37455550},
issn = {1096-9071},
support = {R21 AI157909/AI/NIAID NIH HHS/United States ; R15 AG069544/AG/NIA NIH HHS/United States ; S10 OD021572/OD/NIH HHS/United States ; },
mesh = {Humans ; DNA, Viral/genetics/metabolism ; CRISPR-Cas Systems ; Hepatitis B virus/genetics ; *Hepatitis B ; *Hepatitis B, Chronic ; Virus Replication ; RNA/metabolism/pharmacology ; DNA, Circular/genetics ; },
abstract = {The presence of hepatitis B virus (HBV) covalently closed circular (ccc) DNA (cccDNA), which serves as a template for viral replication and integration of HBV DNA into the host cell genome, sustains liver pathogenesis and constitutes an intractable barrier to the eradication of chronic HBV infection. The current antiviral therapy for HBV infection, using nucleos(t)ide analogues (NAs), can suppress HBV replication but cannot eliminate integrated HBV DNA and episomal cccDNA. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a powerful genetic tool that can edit integrated HBV DNA and minichromosomal cccDNA for gene therapy, but its expression and delivery require a viral vector, which poses safety concerns for therapeutic applications in humans. In the present study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a nonviral formulation to develop a novel CRISPR/Cas9-mediated gene therapy for eradicating HBV infection. We designed a series of gRNAs targeting multiple specific HBV genes and tested their antiviral efficacy and cytotoxicity in different HBV cellular models. Transfection of stably HBV-infected human hepatoma cell line HepG2.2.15 with HBV-specific gRNA/Cas9 RNPs resulted in a substantial reduction in HBV transcripts. Specifically, gRNA5 and/or gRNA9 RNPs significantly reduced HBV cccDNA, total HBV DNA, pregenomic RNA, and HBV antigen (HBsAg, HBeAg) levels. T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing confirmed specific HBV gene cleavage and mutations at or around the gRNA target sites. Notably, this gene-editing system did not alter cellular viability or proliferation in the treated cells. Because of their rapid DNA cleavage capability, low off-target effects, low risk of insertional mutagenesis, and readiness for use in clinical application, these results suggest that synthetic gRNA/Cas9 RNP-based gene-editing can be utilized as a promising therapeutic drug for eradicating chronic HBV infection.},
}
@article {pmid37451948,
year = {2023},
author = {Graver, BA and Chakravarty, N and Solomon, KV},
title = {Prokaryotic Argonautes for in vivo biotechnology and molecular diagnostics.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2023.06.010},
pmid = {37451948},
issn = {1879-3096},
abstract = {Prokaryotic Argonautes (pAgos) are an emerging class of programmable endonucleases that are believed to be more flexible than existing CRISPR-Cas systems and have significant potential for biotechnology. Current applications of pAgos include a myriad of molecular diagnostics and in vitro DNA assembly tools. However, efforts have historically been centered on thermophilic pAgo variants. To enable in vivo biotechnological applications such as gene editing, focus has shifted to pAgos from mesophilic organisms. We discuss what is known of pAgos, how they are being developed for various applications, and strategies to overcome current challenges to in vivo applications in prokaryotes and eukaryotes.},
}
@article {pmid37451945,
year = {2023},
author = {Chowdhry, R and Lu, SZ and Lee, S and Godhulayyagari, S and Ebrahimi, SB and Samanta, D},
title = {Enhancing CRISPR/Cas systems with nanotechnology.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2023.06.005},
pmid = {37451945},
issn = {1879-3096},
abstract = {CRISPR/Cas systems have revolutionized biology and medicine, and have led to new paradigms in disease diagnostics and therapeutics. However, these complexes suffer from key limitations regarding barriers to cellular entry, stability in biological environments, and off-target effects. Integrating nanotechnology with CRISPR/Cas systems has emerged as a promising strategy to overcome these challenges and has further unlocked structures that accumulate preferentially in tissues of interest, have tunable pharmacological properties, and are activated in response to desired stimuli. Nanomaterials can also enhance CRISPR/Cas-mediated detection platforms by enabling faster, more sensitive, and convenient readouts. We highlight recent advances in this rapidly growing field. We also outline areas that need further development to fully realize the potential of CRISPR technologies.},
}
@article {pmid37446383,
year = {2023},
author = {Ormazabal, ME and Pavan, E and Vaena, E and Ferino, D and Biasizzo, J and Mucci, JM and Serra, F and Cifù, A and Scarpa, M and Rozenfeld, PA and Dardis, AE},
title = {Exploring the Pathophysiologic Cascade Leading to Osteoclastogenic Activation in Gaucher Disease Monocytes Generated via CRISPR/Cas9 Technology.},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37446383},
issn = {1422-0067},
mesh = {Humans ; *Gaucher Disease/pathology ; Osteogenesis ; Monocytes/pathology ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Gaucher disease (GD) is caused by biallelic pathogenic variants in the acid β-glucosidase gene (GBA1), leading to a deficiency in the β-glucocerebrosidase (GCase) enzyme activity resulting in the intracellular accumulation of sphingolipids. Skeletal alterations are one of the most disabling features in GD patients. Although both defective bone formation and increased bone resorption due to osteoblast and osteoclast dysfunction contribute to GD bone pathology, the molecular bases are not fully understood, and bone disease is not completely resolved with currently available specific therapies. For this reason, using editing technology, our group has developed a reliable, isogenic, and easy-to-handle cellular model of GD monocytes (GBAKO-THP1) to facilitate GD pathophysiology studies and high-throughput drug screenings. In this work, we further characterized the model showing an increase in proinflammatory cytokines (Interleukin-1β and Tumor Necrosis Factor-α) release and activation of osteoclastogenesis. Furthermore, our data suggest that GD monocytes would display an increased osteoclastogenic potential, independent of their interaction with the GD microenvironment or other GD cells. Both proinflammatory cytokine production and osteoclastogenesis were restored at least, in part, by treating cells with the recombinant human GCase, a substrate synthase inhibitor, a pharmacological chaperone, and an anti-inflammatory compound. Besides confirming that this model would be suitable to perform high-throughput screening of therapeutic molecules that act via different mechanisms and on different phenotypic features, our data provided insights into the pathogenic cascade, leading to osteoclastogenesis exacerbation and its contribution to bone pathology in GD.},
}
@article {pmid37446368,
year = {2023},
author = {Shirazi Parsa, H and Sabet, MS and Moieni, A and Shojaeiyan, A and Dogimont, C and Boualem, A and Bendahmane, A},
title = {CRISPR/Cas9-Mediated Cytosine Base Editing Using an Improved Transformation Procedure in Melon (Cucumis melo L.).},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37446368},
issn = {1422-0067},
support = {101095736/ERC_/European Research Council/International ; },
mesh = {Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Cucumis melo/genetics ; *Cucurbitaceae/genetics ; Plants/genetics ; },
abstract = {Melon is a recalcitrant plant for stable genetic transformation. Various protocols have been tried to improve melon transformation efficiency; however, it remains significantly low compared to other plants such as tomato. In this study, the primary focus was on the optimization of key parameters during the inoculation and co-culture steps of the genetic transformation protocol. Our results showed that immersing the explants in the inoculation medium for 20 min significantly enhanced transformation efficiency. During the co-culture step, the use of filer paper, 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), and a temperature of 24 °C significantly enhanced the melon transformation efficiency. Furthermore, the impact of different ethylene inhibitors and absorbers on the transformation efficiency of various melon varieties was explored. Our findings revealed that the use of these compounds led to a significant improvement in the transformation efficiency of the tested melon varieties. Subsequently, using our improved protocol and reporter-gene construct, diploid transgenic melons successfully generated. The efficiency of plant genetic transformation ranged from 3.73 to 4.83%. Expanding the scope of our investigation, the optimized protocol was applied to generate stable gene-edited melon lines using the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated cytosine base editor and obtained melon lines with editions (C-to-T and C-to-G) in the eukaryotic translation initiation factor 4E, CmeIF4E gene. In conclusion, the optimized melon transformation protocol, along with the utilization of the CRISPR/Cas9-mediated cytosine base editor, provides a reliable framework for functional gene engineering in melon. These advancements hold significant promise for furthering genetic research and facilitating crop improvement in this economically important plant species.},
}
@article {pmid37440632,
year = {2023},
author = {Sulis, DB and Jiang, X and Yang, C and Marques, BM and Matthews, ML and Miller, Z and Lan, K and Cofre-Vega, C and Liu, B and Sun, R and Sederoff, H and Bing, RG and Sun, X and Williams, CM and Jameel, H and Phillips, R and Chang, HM and Peszlen, I and Huang, YY and Li, W and Kelly, RM and Sederoff, RR and Chiang, VL and Barrangou, R and Wang, JP},
title = {Multiplex CRISPR editing of wood for sustainable fiber production.},
journal = {Science (New York, N.Y.)},
volume = {381},
number = {6654},
pages = {216-221},
doi = {10.1126/science.add4514},
pmid = {37440632},
issn = {1095-9203},
mesh = {Carbohydrates/analysis ; *Gene Editing ; *Lignin/genetics ; *Wood/genetics ; CRISPR-Cas Systems ; *Populus/genetics ; Paper ; Sustainable Growth ; },
abstract = {The domestication of forest trees for a more sustainable fiber bioeconomy has long been hindered by the complexity and plasticity of lignin, a biopolymer in wood that is recalcitrant to chemical and enzymatic degradation. Here, we show that multiplex CRISPR editing enables precise woody feedstock design for combinatorial improvement of lignin composition and wood properties. By assessing every possible combination of 69,123 multigenic editing strategies for 21 lignin biosynthesis genes, we deduced seven different genome editing strategies targeting the concurrent alteration of up to six genes and produced 174 edited poplar variants. CRISPR editing increased the wood carbohydrate-to-lignin ratio up to 228% that of wild type, leading to more-efficient fiber pulping. The edited wood alleviates a major fiber-production bottleneck regardless of changes in tree growth rate and could bring unprecedented operational efficiencies, bioeconomic opportunities, and environmental benefits.},
}
@article {pmid37413791,
year = {2023},
author = {Ji, Z and Zhou, B and Shang, Z and Liu, S and Li, X and Zhang, X and Li, B},
title = {Active CRISPR-Cas12a on Hydrophilic Metal-Organic Frameworks: A Nanobiocomposite with High Stability and Activity for Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {95},
number = {28},
pages = {10580-10587},
doi = {10.1021/acs.analchem.3c00400},
pmid = {37413791},
issn = {1520-6882},
mesh = {Humans ; *COVID-19/diagnosis ; CRISPR-Cas Systems/genetics ; *Metal-Organic Frameworks ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; *Biosensing Techniques ; },
abstract = {CRISPR-Cas12a is an accurate and responsive biosensing technique, but its limited stability has restricted its widespread applications. To address this, we propose a strategy using metal-organic frameworks (MOFs) to protect Cas12a from harsh environments. After screening multiple candidate MOFs, it was found that hydrophilic MAF-7 is highly compatible with Cas12a, and the as-formed Cas12a-on-MAF-7 (COM) not only retains high enzymatic activity but also possesses excellent tolerance to heat, salt, and organic solvents. Further investigation showed that COM can serve as an analytical component for nucleic acid detection, resulting in an ultrasensitive assay for SARS-CoV-2 RNA detection with a detection limit of 1 copy. This is the first successful attempt to create an active Cas12a nanobiocomposite that functions as a biosensor without the need for shell deconstruction or enzyme release.},
}
@article {pmid37392174,
year = {2023},
author = {Xu, J and Liu, Z and Zhang, Z and Wu, T},
title = {Unlocking the Full Potential of Cas12a: Exploring the Effects of Substrate and Reaction Conditions on Trans-Cleavage Activity.},
journal = {Analytical chemistry},
volume = {95},
number = {28},
pages = {10664-10669},
doi = {10.1021/acs.analchem.3c01307},
pmid = {37392174},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Fluorescent Dyes ; Nucleotides ; *Biosensing Techniques ; },
abstract = {The trans-cleavage activity of Cas12a has been widely used with various applications. Here, we report that the trans-cleavage activity of Cas12a can be significantly affected by the fluorescent probe length and reaction buffer. The optimal probe length for Cas12a is found to be 15 nucleotides, and the optimal buffer is NEBuffer 4. Compared to the popularly used reaction conditions, the activity of Cas12a is improved by about 50-fold. In addition, the detection limit of Cas12a for DNA targets has been reduced by nearly three orders of magnitude. Our method provides a powerful tool for Cas12a trans-cleavage activity applications.},
}
@article {pmid36896979,
year = {2023},
author = {Liu, T and Ji, J and Cheng, Y and Zhang, S and Wang, Z and Duan, K and Wang, Y},
title = {CRISPR/Cas9-mediated editing of GmTAP1 confers enhanced resistance to Phytophthora sojae in soybean.},
journal = {Journal of integrative plant biology},
volume = {65},
number = {7},
pages = {1609-1612},
doi = {10.1111/jipb.13476},
pmid = {36896979},
issn = {1744-7909},
support = {31721004//National Natural Science Foundation of China/ ; 31901957//National Natural Science Foundation of China/ ; 32172499//National Natural Science Foundation of China/ ; },
mesh = {*Soybeans/genetics ; *Phytophthora ; CRISPR-Cas Systems/genetics ; Disease Resistance/genetics ; Plant Diseases/genetics ; },
abstract = {Soybean root rot disease caused by Phytophthora sojae seriously constrains soybean yield. Knocking out the susceptibility gene GmTAP1 in soybean created new soybean lines resistant to several P. sojae strains and these lines showed no agronomic penalties in the field.},
}
@article {pmid36037289,
year = {2023},
author = {Katsuda, T and Cure, H and Sussman, J and Simeonov, KP and Krapp, C and Arany, Z and Grompe, M and Stanger, BZ},
title = {Rapid in vivo multiplexed editing (RIME) of the adult mouse liver.},
journal = {Hepatology (Baltimore, Md.)},
volume = {78},
number = {2},
pages = {486-502},
doi = {10.1002/hep.32759},
pmid = {36037289},
issn = {1527-3350},
support = {R01 DK107667/DK/NIDDK NIH HHS/United States ; DK083355/DK/NIDDK NIH HHS/United States ; //Biesecker Pediatric Liver Foundation/Children's Hospital of Philadelphia/ ; },
mesh = {Mice ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Liver ; Mammals ; },
abstract = {BACKGROUND AND AIMS: Assessing mammalian gene function in vivo has traditionally relied on manipulation of the mouse genome in embryonic stem cells or perizygotic embryos. These approaches are time-consuming and require extensive breeding when simultaneous mutations in multiple genes is desired. The aim of this study is to introduce a rapid in vivo multiplexed editing (RIME) method and provide proof of concept of this system.<br><br>APPROACH AND RESULTS: RIME, a system wherein CRISPR/caspase 9 technology, paired with adeno-associated viruses (AAVs), permits the inactivation of one or more genes in the adult mouse liver. The method is quick, requiring as little as 1&#x2009;month from conceptualization to knockout, and highly efficient, enabling editing in >95% of target cells. To highlight its use, we used this system to inactivate, alone or in combination, genes with functions spanning metabolism, mitosis, mitochondrial maintenance, and cell proliferation.<br><br>CONCLUSIONS: RIME enables the rapid, efficient, and inexpensive analysis of multiple genes in the mouse liver in vivo .},
}
@article {pmid37446306,
year = {2023},
author = {Shah, R and van den Berk, PCM and Pritchard, CEJ and Song, JY and Kreft, M and Pilzecker, B and Jacobs, H},
title = {A C57BL/6J Fancg-KO Mouse Model Generated by CRISPR/Cas9 Partially Captures the Human Phenotype.},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37446306},
issn = {1422-0067},
support = {KWF NKI-2017- 10032 and KWF NKI-2017-10796 to Heinz Jacobs//Dutch Cancer Society/ ; },
mesh = {Humans ; Animals ; Mice ; *Cisplatin/metabolism ; *Fanconi Anemia/genetics/metabolism ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; DNA-Binding Proteins/metabolism ; Fibroblasts/metabolism ; Mitomycin ; Phenotype ; Fanconi Anemia Complementation Group G Protein/genetics ; },
abstract = {Fanconi anemia (FA) develops due to a mutation in one of the FANC genes that are involved in the repair of interstrand crosslinks (ICLs). FANCG, a member of the FA core complex, is essential for ICL repair. Previous FANCG-deficient mouse models were generated with drug-based selection cassettes in mixed mice backgrounds, leading to a disparity in the interpretation of genotype-related phenotype. We created a Fancg-KO (KO) mouse model using CRISPR/Cas9 to exclude these confounders. The entire Fancg locus was targeted and maintained on the immunological well-characterized C57BL/6J background. The intercrossing of heterozygous mice resulted in sub-Mendelian numbers of homozygous mice, suggesting the loss of FANCG can be embryonically lethal. KO mice displayed infertility and hypogonadism, but no other developmental problems. Bone marrow analysis revealed a defect in various hematopoietic stem and progenitor subsets with a bias towards myelopoiesis. Cell lines derived from Fancg-KO mice were hypersensitive to the crosslinking agents cisplatin and Mitomycin C, and Fancg-KO mouse embryonic fibroblasts (MEFs) displayed increased γ-H2AX upon cisplatin treatment. The reconstitution of these MEFs with Fancg cDNA corrected for the ICL hypersensitivity. This project provides a new, genetically, and immunologically well-defined Fancg-KO mouse model for further in vivo and in vitro studies on FANCG and ICL repair.},
}
@article {pmid36893939,
year = {2023},
author = {Smits, JPH and van den Brink, NJM and Meesters, LD and Hamdaoui, H and Niehues, H and Jansen, PAM and van Vlijmen-Willems, IMJJ and Rodijk-Olthuis, D and Evrard, C and Poumay, Y and van Geel, M and Hendriks, WJAJ and Schalkwijk, J and Zeeuwen, PLJM and van den Bogaard, EH},
title = {Investigations into the FLG Null Phenotype: Showcasing the Methodology for CRISPR/Cas9 Editing of Human Keratinocytes.},
journal = {The Journal of investigative dermatology},
volume = {143},
number = {8},
pages = {1520-1528.e5},
doi = {10.1016/j.jid.2023.02.021},
pmid = {36893939},
issn = {1523-1747},
mesh = {Humans ; *CRISPR-Cas Systems ; *Intermediate Filament Proteins/metabolism ; Filaggrin Proteins ; Keratinocytes/metabolism ; Phenotype ; },
abstract = {Ever since the association between FLG loss-of-function variants and ichthyosis vulgaris and atopic dermatitis disease onset was identified, FLGs function has been under investigation. Intraindividual genomic predisposition, immunological confounders, and environmental interactions complicate the comparison between FLG genotypes and related causal effects. Using CRISPR/Cas9, we generated human FLG-knockout (ΔFLG) N/TERT-2G keratinocytes. FLG deficiency was shown by immunohistochemistry of human epidermal equivalent cultures. Next to (partial) loss of structural proteins (involucrin, hornerin, keratin 2, and transglutaminase 1), the stratum corneum was denser and lacked the typical basket weave appearance. In addition, electrical impedance spectroscopy and transepidermal water loss analyses highlighted a compromised epidermal barrier in ΔFLG human epidermal equivalents. Correction of FLG reinstated the presence of keratohyalin granules in the stratum granulosum, FLG protein expression, and expression of the proteins mentioned earlier. The beneficial effects on stratum corneum formation were reflected by the normalization of electrical impedance spectroscopy and transepidermal water loss. This study shows the causal phenotypical and functional consequences of FLG deficiency, indicating that FLG is not only central in epidermal barrier function but also vital for epidermal differentiation by orchestrating the expression of other important epidermal proteins. These observations pave the way to fundamental investigations into the exact role of FLG in skin biology and disease.},
}
@article {pmid35882048,
year = {2023},
author = {Vora, DS and Jaiswal, AK and Sundar, D},
title = {Implementing accelerated dynamics to unravel the effects of high-fidelity Cas9 mutants on target DNA and guide RNA hybrid stability.},
journal = {Journal of biomolecular structure &amp; dynamics},
volume = {41},
number = {13},
pages = {6178-6190},
doi = {10.1080/07391102.2022.2103032},
pmid = {35882048},
issn = {1538-0254},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics ; Mutation ; Genome ; RNA/genetics ; },
abstract = {The clustered regularly interspersed short palindromic repeats (CRISPR) and its associated nuclease (Cas9) offers a unique and easily reprogrammable system for editing eukaryotic genomes. Cas9 is guided to the target by an RNA strand, and precise edits are created by introducing double-stranded breaks. However, nuclease activity of Cas9 is also triggered at other sites other than the target sit, which is a major limitation for various applications. Cas9 variants have been designed to improve the efficacy of the tool by introducing certain mutations. However, the on-target activity of such Cas9 variants is often seen as compromised. Hence, understanding the sub-molecular differences in the variants is essential to elucidate the factors that contribute to efficiency. The study reveals distortions in the PAM-distal regions of the nucleic hybrids as well as changes in the interactions between the Cas9 variants and RNA-DNA hybrid, contributing to the explanation for differences in on-target activity.Communicated by Ramaswamy H. Sarma.},
}
@article {pmid37446265,
year = {2023},
author = {Han, Y and Yang, J and Wu, H and Liu, F and Qin, B and Li, R},
title = {Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of SRL1 and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis.},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37446265},
issn = {1422-0067},
mesh = {*Gene Editing ; *Oryza/metabolism ; CRISPR-Cas Systems/genetics ; Plant Proteins/genetics/metabolism ; Plant Breeding ; Gene Expression Profiling ; Plant Leaves/genetics/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Leaf rolling is a crucial agronomic trait to consider in rice (Oryza sativa L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The SEMI-ROLLED LEAF 1 (SRL1) gene plays a key role in regulating leaf rolling, as it encodes a glycosylphosphatidylinositol-anchored protein located on the plasma membrane. In this study, we used CRISPR/Cas9 to target the second and third exons of the SRL1 gene in the indica rice line GXU103, which resulted in the generation of 14 T0 transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T1 generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting SRL1 homozygous double-target knockout as srl1-103. This line exhibited defects in leaf development, leaf rolling in the mature upright leaves, and a compact nature of the fully grown plants. Compared with the wild type (WT), the T2 generation of srl1-103 varied in two key aspects: the width of flag leaf (12.6% reduction compared with WT) and the leaf rolling index (48.77% increase compared with WT). In order to gain a deeper understanding of the involvement of SRL1 in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T2 generation of srl1-103. A comparison of srl1-103 with WT revealed 459 differentially expressed genes (DEGs), including 388 upregulated genes and 71 downregulated genes. In terms of the function of the DEGs, there seemed to be a significant enrichment of genes associated with cell wall synthesis (LOC_Os08g01670, LOC_Os05g46510, LOC_Os04g51450, LOC_Os10g28080, LOC_Os04g39814, LOC_Os01g71474, LOC_Os01g71350, and LOC_Os11g47600) and vacuole-related genes (LOC_Os09g23300), which may partially explain the increased leaf rolling in srl1-103. Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene (LOC_Os08g44840) could be the main reason for the decreased leaf angle and the compact nature of the mutant plants. In summary, this study successfully elucidated the gene regulatory network in which SRL1 participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement.},
}
@article {pmid37445761,
year = {2023},
author = {Xiao, Y and Zhang, Y and Xie, F and Olsen, RH and Shi, L and Li, L},
title = {Inhibition of Plasmid Conjugation in Escherichia coli by Targeting rbsB Gene Using CRISPRi System.},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37445761},
issn = {1422-0067},
mesh = {Humans ; *Escherichia coli/metabolism ; *RNA, Guide, CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Plasmids/genetics ; Anti-Bacterial Agents/pharmacology ; Conjugation, Genetic ; },
abstract = {Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic-resistant genes (ARGs) among human pathogens. The spread of ARGs can be halted or diminished by interfering with the conjugation process. In this study, we explored the possibility of using an rbsB gene as a single target to inhibit plasmid-mediated horizontal gene transfer in Escherichia coli by CRISPR interference (CRISPRi) system. Three single-guide RNAs (sgRNAs) were designed to target the rbsB gene. The transcriptional levels of the rbsB gene, the conjugation-related genes, and the conjugation efficiency in the CRISPRi strain were tested. We further explored the effect of the repressed expression of the rbsB gene on the quorum sensing (QS) system and biofilm formation. The results showed that the constructed CRISPRi system was effective in repressing the transcriptional level of the rbsB gene at a rate of 66.4%. The repressed expression of the rbsB gene resulted in the reduced conjugation rate of RP4 plasmid by 88.7%, which significantly inhibited the expression of the conjugation-related genes (trbBp, trfAp, traF and traJ) and increased the global regulator genes (korA, korB and trbA). The repressed rbsB gene expression reduced the depletion of autoinducer 2 signals (AI-2) by 12.8% and biofilm formation by a rate of 68.2%. The results of this study indicated the rbsB gene could be used as a universal target for the inhibition of conjugation. The constructed conjugative CRISPRi system has the potential to be used in ARG high-risk areas.},
}
@article {pmid37445725,
year = {2023},
author = {Chohra, I and Giri, S and Malgrange, B},
title = {Generation of a Well-Characterized Homozygous Chromodomain-Helicase-DNA-Binding Protein 4[G1003D] Mutant hESC Line Using CRISPR/eCas9 (ULIEGEe001-A-1).},
journal = {International journal of molecular sciences},
volume = {24},
number = {13},
pages = {},
pmid = {37445725},
issn = {1422-0067},
support = {R.FNRS.4649//Fund for Scientific Research/ ; 22/003//Fonds leon Fredericq/ ; FSR-R.RCFRA.3775//University of Li&#xe8;ge/ ; },
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line ; Homozygote ; DNA-Binding Proteins/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {The chromatin remodeler Chromodomain-helicase-DNA-binding protein 4 (CHD4) is crucial for the development of multiple organ systems. Functional mutations of CHD4 have recently been described in a developmental disorder, namely Siffrim-Hitz-Weiss syndrome (SIHIWES). Herein, we have generated a homozygous CHD4G1003D hESC line (WAe025-A-1) using CRISPR/eCas9-based gene editing in the WA-25 hESC line. The edited hESC line maintains normal karyotype, pluripotency, and ability to differentiate into three germ layers. This cell line will be a valuable resource for studying the functional role of CHD4 during the development and disease modeling of SIHIWES in vitro.},
}
@article {pmid37443815,
year = {2023},
author = {Zhang, T and Yao, H and Wang, H and Sui, T},
title = {Development of Woolly Hair and Hairlessness in a CRISPR-Engineered Mutant Mouse Model with KRT71 Mutations.},
journal = {Cells},
volume = {12},
number = {13},
pages = {},
pmid = {37443815},
issn = {2073-4409},
mesh = {Child, Preschool ; Humans ; Animals ; Mice ; Mice, Nude ; *RNA, Guide, CRISPR-Cas Systems ; *Hair Diseases/genetics ; Hair ; Mutation/genetics ; },
abstract = {Hypotrichosis simplex (HS) and woolly hair (WH) are rare and monogenic disorders of hair loss. HS, characterized by a diffuse loss of hair, usually begins in early childhood and progresses into adulthood. WH displays strong coiled hair involving a localized area of the scalp or covering the entire side. Mutations in the keratin K71(KRT71) gene have been reported to underlie HS and WH. Here, we report the generation of a mouse model of HS and WH by the co-injection of Cas9 mRNA and sgRNA, targeting exon6 into mouse zygotes. The Krt71-knockout (KO) mice displayed the typical phenotypes, including Krt71 protein expression deletion and curly hair in their full body. Moreover, we found that mice in 3-5 weeks showed a new phenomenon of the complete shedding of hair, which was similar to nude mice. However, we discovered that the mice exhibited no immune deficiency, which was a typical feature of nude mice. To our knowledge, this novel mouse model generated by the CRISPR/Cas9 system mimicked woolly hair and could be valuable for hair disorder studies.},
}
@article {pmid37443646,
year = {2023},
author = {Chen, SJ and Rai, CI and Wang, SC and Chen, YC},
title = {Point-of-Care Testing for Infectious Diseases Based on Class 2 CRISPR/Cas Technology.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {13},
number = {13},
pages = {},
pmid = {37443646},
issn = {2075-4418},
abstract = {The early detection of infectious diseases and microorganisms is critical for effective disease treatment, control, and prevention. Currently, nucleic acid testing and antigen-antibody serum reaction are the two methods most commonly used for the detection of infectious diseases. The former is highly accurate, specific, and sensitive, but it is time-consuming, expensive, and has special technician and instrument requirements. The latter is rapid and economical, but it may not be accurate and sensitive enough. Therefore, it is necessary to develop a quick and on-site diagnostic test for point-of-care testing (POCT) to enable the clinical detection of infectious diseases that is accurate, sensitive, convenient, cheap, and portable. Here, CRISPR/Cas-based detection methods are detailed and discussed in depth. The powerful capacity of these methods will facilitate the development of diagnostic tools for POCT, though they still have some limitations. This review explores and highlights POCT based on the class 2 CRISPR/Cas assay, such as Cas12 and Cas13 proteins, for the detection of infectious diseases. We also provide an outlook on perspectives, multi-application scenarios, clinical applications, and limitations for POCT based on class 2 CRISPR/Cas technology.},
}
@article {pmid37443005,
year = {2023},
author = {Dirkx, N and Weuring, WJ and De Vriendt, E and Smal, N and van de Vondervoort, J and van 't Slot, R and Koetsier, M and Zonnekein, N and De Pooter, T and Weckhuysen, S and Koeleman, BPC},
title = {Increased prime edit rates in KCNQ2 and SCN1A via single nicking all-in-one plasmids.},
journal = {BMC biology},
volume = {21},
number = {1},
pages = {156},
pmid = {37443005},
issn = {1741-7007},
support = {1616091 (MING)//Vrienden WKZ/ ; 1861419N//FWO-FKM/ ; G041821N//FWO-FKM/ ; 1S59221N//FWO-SB/ ; TreatKCNQ//European Joint Programme on Rare Disease JTC 2020/ ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; Peptide Elongation Factor 1/genetics ; Plasmids/genetics ; *RNA, Small Untranslated ; KCNQ2 Potassium Channel/genetics ; NAV1.1 Voltage-Gated Sodium Channel/genetics ; },
abstract = {BACKGROUND: Prime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models.<br><br>RESULTS: pAIO-EF1&#x3b1;-PE2 and pAIO-EF1&#x3b1;-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1&#x3b1;-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1&#x3b1;-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors.<br><br>CONCLUSION: Taken together, our study shows an improved efficacy of EF-1&#x3b1; driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important.},
}
@article {pmid37442412,
year = {2023},
author = {Wang, F and Zhang, C and Xu, H and Zeng, W and Ma, L and Li, Z},
title = {Structural Basis for the Ribonuclease Activity of a Thermostable CRISPR-Cas13a from Thermoclostridium caenicola.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {168197},
doi = {10.1016/j.jmb.2023.168197},
pmid = {37442412},
issn = {1089-8638},
abstract = {The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from mesophilic organisms, a newly discovered Cas13a from thermophilic bacteria Thermoclostridium caenicola (TccCas13a) shows low sequence similarity, high thermostability, and lacks pre-crRNA processing activity. The thermostability of TccCas13a has been harnessed to make a sensitive and robust tool for nucleic acid detection. Here we present the structures of TccCas13a-crRNA binary complex at 2.8 Å, and TccCas13a at 3.5 Å. Although TccCas13a shares a similarly bilobed architecture with other mesophilic organism-derived Cas13a proteins, TccCas13a displayed distinct structure features. Specifically, it holds a long crRNA 5'-flank, forming extensive polar contacts with Helical-1 and HEPN2 domains. The detailed analysis of the interaction between crRNA 5'-flank and TccCas13a suggested lack of suitable nucleophile to attack the 2'-OH of crRNA 5'-flank may explain why TccCas13a failed to cleave pre-crRNA. The stem-loop segment of crRNA spacer toggles between double-stranded and single-stranded conformational states, suggesting a potential safeguard mechanism for target recognition. Superimposition of structures of TccCas13a and TccCas13a-crRNA revealed several conformational changes required for crRNA loading, including dramatic movement of Helical-2 domain. Collectively, these structural insights expand our understanding into type VI CRISPR-Cas effectors, and would facilitate the development of TccCas13a-based applications.},
}
@article {pmid37441240,
year = {2023},
author = {Guo, Y and Xia, H and Dai, T and Liu, T and Shamoun, SF and CuiPing, W},
title = {CRISPR/Cas12a-based approaches for efficient and accurate detection of Phytophthora ramorum.},
journal = {Frontiers in cellular and infection microbiology},
volume = {13},
number = {},
pages = {1218105},
pmid = {37441240},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; *Phytophthora/genetics ; DNA ; Europe ; North America ; },
abstract = {INTRODUCTION: Phytophthora ramorum is a quarantine pathogen that causes leaf blight and shoot dieback of the crown, bark cankers and death on a number of both ornamental and forest trees, especially in North America and northern Europe, where it has produced severe outbreaks. Symptoms caused by P. ramorum can be confused with those by other Phytophthora and fungal species. Early and accurate detection of the causal pathogen P. ramorum is crucial for effective prevention and control of Sudden Oak Death.<br><br>METHODS: In this study, we developed a P. ramorum detection technique based on a combination of recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology (termed RPACRISPR/ Cas12a).<br><br>RESULTS: This novel method can be utilized for the molecular identification of P. ramorum under UV light and readout coming from fluorophores, and can specifically detect P. ramorum at DNA concentrations as low as 100 pg within 25 min at 37&#xb0;C.<br><br>DISCUSSION: We have developed a simple, rapid, sensitive, unaided-eye visualization, RPA CRISPR/Cas12a-based detection system for the molecular identification of P. ramorum that does not require technical expertise or expensive ancillary equipment. And this system is sensitive for both standard laboratory samples and samples from the field.},
}
@article {pmid37440893,
year = {2023},
author = {Brenner, E and Sreevatsan, S},
title = {Cold Cas: reevaluating the occurrence of CRISPR/Cas systems in Mycobacteriaceae.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1204838},
pmid = {37440893},
issn = {1664-302X},
abstract = {Bacterial CRISPR/Cas systems target foreign genetic elements such as phages and regulate gene expression by some pathogens, even in the host. The system is a marker for evolutionary history and has been used for inferences in Mycobacterium tuberculosis for 30 years. However, knowledge about mycobacterial CRISPR/Cas systems remains limited. It is believed that Type III-A Cas systems are exclusive to Mycobacterium canettii and the M. tuberculosis complex (MTBC) of organisms and that very few of the >200 diverse species of non-tuberculous mycobacteria (NTM) possess any CRISPR/Cas system. This study sought unreported CRISPR/Cas loci across NTM to better understand mycobacterial evolution, particularly in species phylogenetically near the MTBC. An analysis of available mycobacterial genomes revealed that Cas systems are widespread across Mycobacteriaceae and that some species contain multiple types. The phylogeny of Cas loci shows scattered presence in many NTM, with variation even within species, suggesting gains/losses of these loci occur frequently. Cas Type III-A systems were identified in pathogenic Mycobacterium heckeshornense and the geological environmental isolate Mycobacterium SM1. In summary, mycobacterial CRISPR/Cas systems are numerous, Type III-A systems are unreliable as markers for MTBC evolution, and mycobacterial horizontal gene transfer appears to be a frequent source of genetic variation.},
}
@article {pmid37440088,
year = {2023},
author = {da Costa, BL and Li, Y and Levi, SR and Tsang, SH and Quinn, PMJ},
title = {Generation of CRB1 RP Patient-Derived iPSCs and a CRISPR/Cas9-Mediated Homology-Directed Repair Strategy for the CRB1 c.2480G&gt;T Mutation.},
journal = {Advances in experimental medicine and biology},
volume = {1415},
number = {},
pages = {571-576},
pmid = {37440088},
issn = {0065-2598},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; CRISPR-Cas Systems/genetics ; DNA Copy Number Variations ; *Retinitis Pigmentosa/genetics/therapy ; Mutation ; Eye Proteins/genetics ; Membrane Proteins/genetics ; Nerve Tissue Proteins/genetics ; },
abstract = {Mutations in the Crumbs-homologue-1 (CRB1) gene lead to a spectrum of severe inherited retinal diseases, including retinitis pigmentosa (RP). The establishment of a genotype-phenotype correlation in CRB1 patients has been difficult due to the substantial variability and phenotypic overlap between CRB1-associated diseases. This phenotypic modulation may be due to several factors, including genetic modifiers, deep intronic mutations, isoform diversity, and copy number variations. Induced pluripotent stem cell (iPSC)-derived patient retinal organoids are novel tools that can provide sensitive, quantitative, and scalable phenotypic assays. CRB1 RP patient iPSC-derived retinal organoids have shown reproducible phenotypes compared to healthy retinal organoids. However, having genetically defined iPSC isogenic controls that take into account potential phenotypic modulation is crucial. In this study, we generated iPSC from an early-onset CRB1 patient and developed a correction strategy for the c.2480G>T, p.(Gly827Val) CRB1 mutation using CRISPR/Cas9-mediated homology-directed repair.},
}
@article {pmid37440022,
year = {2023},
author = {Tsai, YT and da Costa, BL and Caruso, SM and Nolan, ND and Levi, SR and Tsang, SH and Quinn, PMJ},
title = {Generation of an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase Prime Editor.},
journal = {Advances in experimental medicine and biology},
volume = {1415},
number = {},
pages = {109-114},
pmid = {37440022},
issn = {0065-2598},
mesh = {Humans ; Animals ; Mice ; *RNA-Directed DNA Polymerase/genetics/metabolism ; *Avian Myeloblastosis Virus/genetics/metabolism ; HEK293 Cells ; Gene Editing ; Moloney murine leukemia virus/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Prime editing (PE) is a novel, double-strand break (DSB)-independent gene editing technology that represents an exciting avenue for the treatment of inherited retinal diseases (IRDs). Given the extensive and heterogenous nature of the 280 genes associated with IRDs, genome editing has presented countless complications. However, recent advances in genome editing technologies have identified PE to have tremendous potential, with the capability to ameliorate small deletions and insertions in addition to all twelve possible transition and transversion mutations. The current PE system is based on the fusion of the Streptococcus pyogenes Cas9 (SpCas9) nickase H840A mutant and an optimized Moloney murine leukemia virus (MMLV) reverse-transcriptase (RT) in conjunction with a PE guide RNA (pegRNA). In this study, we developed a prime editor based on the avian myeloblastosis virus (AMV)-RT and showed its applicability for the installation of the PRPH2 c.828+1G>A mutation in HEK293 cells.},
}
@article {pmid37439822,
year = {2023},
author = {Durán-Vinet, B and Araya-Castro, K and Zaiko, A and Pochon, X and Wood, SA and Stanton, JL and Jeunen, GJ and Scriver, M and Kardailsky, A and Chao, TC and Ban, DK and Moarefian, M and Aran, K and Gemmell, NJ},
title = {CRISPR-Cas-Based Biomonitoring for Marine Environments: Toward CRISPR RNA Design Optimization Via Deep Learning.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2023.0019},
pmid = {37439822},
issn = {2573-1602},
abstract = {Almost all of Earth's oceans are now impacted by multiple anthropogenic stressors, including the spread of nonindigenous species, harmful algal blooms, and pathogens. Early detection is critical to manage these stressors effectively and to protect marine systems and the ecosystem services they provide. Molecular tools have emerged as a promising solution for marine biomonitoring. One of the latest advancements involves utilizing CRISPR-Cas technology to build programmable, rapid, ultrasensitive, and specific diagnostics. CRISPR-based diagnostics (CRISPR-Dx) has the potential to allow robust, reliable, and cost-effective biomonitoring in near real time. However, several challenges must be overcome before CRISPR-Dx can be established as a mainstream tool for marine biomonitoring. A critical unmet challenge is the need to design, optimize, and experimentally validate CRISPR-Dx assays. Artificial intelligence has recently been presented as a potential approach to tackle this challenge. This perspective synthesizes recent advances in CRISPR-Dx and machine learning modeling approaches, showcasing CRISPR-Dx potential to progress as a rising molecular tool candidate for marine biomonitoring applications.},
}
@article {pmid37438488,
year = {2023},
author = {Sowbhagya, R and Muktha, H and Ramakrishnaiah, TN and Surendra, AS and Tanvi, Y and Nivitha, K and Rajashekara, S},
title = {CRISPR/Cas-mediated genome editing in mice for the development of drug delivery mechanism.},
journal = {Molecular biology reports},
volume = {},
number = {},
pages = {},
pmid = {37438488},
issn = {1573-4978},
abstract = {BACKGROUND: To manipulate particular locations in the bacterial genome, researchers have recently resorted to a group of unique sequences in bacterial genomes that are responsible for safeguarding bacteria against bacteriophages. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) are two such systems, each of which consists of an RNA component and an enzyme component.<br><br>METHODS AND RESULTS: This review focuses primarily on how CRISPR/Cas9 technology can be used to make models to study human diseases in mice. Creating RNA molecules that direct endonucleases to a specific position in the genome are crucial for achieving a specific genetic modification. CRISPR/Cas9 technology has allowed scientists to edit the genome with greater precision than ever before. Researchers can use knock-in and knock-out methods to model human diseases such as Neurological, cardiovascular disease, and cancer.<br><br>CONCLUSIONS: In terms of developing innovative methods to discover ailments for diseases/disorders, improved CRISPR/Cas9 technology will provide easier access to valuable novel animal models.},
}
@article {pmid37437865,
year = {2023},
author = {Nayak, V and Patra, S and Singh, KR and Ganguly, B and Kumar, DN and Panda, D and Maurya, GK and Singh, J and Majhi, S and Sharma, R and Pandey, SS and Singh, RP and Kerry, RG},
title = {Advancement in precision diagnosis and therapeutic for triple-negative breast cancer: Harnessing diagnostic potential of CRISPR-cas &amp; engineered CAR T-cells mediated therapeutics.},
journal = {Environmental research},
volume = {235},
number = {},
pages = {116573},
doi = {10.1016/j.envres.2023.116573},
pmid = {37437865},
issn = {1096-0953},
abstract = {Cancer is characterized by uncontrolled cell growth, disrupted regulatory pathways, and the accumulation of genetic mutations. These mutations across different types of cancer lead to disruptions in signaling pathways and alterations in protein expression related to cellular growth and proliferation. This review highlights the AKT signaling cascade and the retinoblastoma protein (pRb) regulating cascade as promising for novel nanotheranostic interventions. Through synergizing state-of-the-art gene editing tools like the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system with nanomaterials and targeting AKT, there is potential to enhance cancer diagnostics significantly. Furthermore, the integration of modified CAR-T cells into multifunctional nanodelivery systems offers a promising approach for targeted cancer inhibition, including the eradication of cancer stem cells (CSCs). Within the context of highly aggressive and metastatic Triple-negative Breast Cancer (TNBC), this review specifically focuses on devising innovative nanotheranostics. For both pre-clinical and post-clinical TNBC detection, the utilization of the CRISPR-Cas system, guided by RNA (gRNA) and coupled with a fluorescent reporter specifically designed to detect TNBC's mutated sequence, could be promising. Additionally, a cutting-edge approach involving the engineering of TNBC-specific iCAR and syn-Notch CAR T-cells, combined with the co-delivery of a hybrid polymeric nano-liposome encapsulating a conditionally replicative adenoviral vector (CRAdV) against CSCs, could present an intriguing intervention strategy. This review thus paves the way for exciting advancements in the field of nanotheranostics for the treatment of TNBC and beyond.},
}
@article {pmid37436979,
year = {2023},
author = {Hansen, SL and Larsen, HL and Pikkupeura, LM and Maciag, G and Guiu, J and Müller, I and Clement, DL and Mueller, C and Johansen, JV and Helin, K and Lerdrup, M and Jensen, KB},
title = {An organoid-based CRISPR-Cas9 screen for regulators of intestinal epithelial maturation and cell fate.},
journal = {Science advances},
volume = {9},
number = {28},
pages = {eadg4055},
pmid = {37436979},
issn = {2375-2548},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Fetus ; *Adult Stem Cells ; Organoids ; },
abstract = {Generation of functionally mature organs requires exquisite control of transcriptional programs governing cell state transitions during development. Despite advances in understanding the behavior of adult intestinal stem cells and their progeny, the transcriptional regulators that control the emergence of the mature intestinal phenotype remain largely unknown. Using mouse fetal and adult small intestinal organoids, we uncover transcriptional differences between the fetal and adult state and identify rare adult-like cells present in fetal organoids. This suggests that fetal organoids have an inherent potential to mature, which is locked by a regulatory program. By implementing a CRISPR-Cas9 screen targeting transcriptional regulators expressed in fetal organoids, we establish Smarca4 and Smarcc1 as important factors safeguarding the immature progenitor state. Our approach demonstrates the utility of organoid models in the identification of factors regulating cell fate and state transitions during tissue maturation and reveals that SMARCA4 and SMARCC1 prevent precocious differentiation during intestinal development.},
}
@article {pmid37436915,
year = {2023},
author = {Wei, Y and Feng, LJ and Yuan, XZ and Wang, SG and Xia, PF},
title = {Developing a Base Editing System for Marine Roseobacter Clade Bacteria.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.3c00259},
pmid = {37436915},
issn = {2161-5063},
abstract = {The Roseobacter clade bacteria are of great significance in marine ecology and biogeochemical cycles, and they are potential microbial chassis for marine synthetic biology due to their versatile metabolic capabilities. Here, we adapted a CRISPR-Cas-based system, base editing, with the combination of nuclease-deactivated Cas9 and deaminase for Roseobacter clade bacteria. Taking the model roseobacter Roseovarius nubinhibens as an example, we achieved precise and efficient genome editing at single-nucleotide resolution without generating double-strand breaks or requesting donor DNAs. Since R. nubinhibens can metabolize aromatic compounds, we interrogated the key genes in the β-ketoadipate pathway with our base editing system via the introduction of premature STOP codons. The essentiality of these genes was demonstrated, and for the first time, we determined PcaQ as a transcription activator experimentally. This is the first report of CRISPR-Cas-based genome editing in the entire clade of Roseobacter bacteria. We believe that our work provides a paradigm for interrogating marine ecology and biogeochemistry with direct genotype-and-phenotype linkages and potentially opens a new avenue for the synthetic biology of marine Roseobacter bacteria.},
}
@article {pmid37435997,
year = {2023},
author = {Calorenni, P and Leonardi, AA and Sciuto, EL and Rizzo, MG and Faro, MJL and Fazio, B and Irrera, A and Conoci, S},
title = {PCR-Free Innovative Strategies for SARS-CoV-2 Detection.},
journal = {Advanced healthcare materials},
volume = {},
number = {},
pages = {e2300512},
doi = {10.1002/adhm.202300512},
pmid = {37435997},
issn = {2192-2659},
abstract = {The pandemic outbreak caused by SARS-CoV-2 coronavirus brought a crucial issue in Public Health causing up to now more than 600 million of infected people and 6.5 million of deaths. Conventional diagnostic methods are based on quantitative reverse transcription polymerase chain reaction (RT-qPCR assay) and immuno-detection (ELISA assay). However, despite these techniques have the advantages of being standardized and consolidated, they keep some main limitations in terms of accuracy (immunoassays), time/cost consumption of analysis, the need for qualified personnel and lab constrain (molecular assays). There is crucial the need to develop new diagnostic approaches for accurate, fast and portable viral detection and quantification. Among these, PCR-free biosensors represent the most appealing solution since they can allow molecular detection without the complexity of the PCR. This will enable the possibility to be integrated in portable and low-cost systems for massive and decentralized screening of SARS-CoV-2 in a point-of-care (PoC) format, pointing to achieve a performant identification and control of infection. In this review, the most recent approaches for the SARS-CoV-2 PCR-free detection are reported, describing both the instrumental and methodological features, and highlighting their suitability for a PoC application. This article is protected by copyright. All rights reserved.},
}
@article {pmid37390772,
year = {2023},
author = {Keng, CT and Yogarajah, T and Lee, RCH and Muhammad, IBH and Chia, BS and Vasandani, SR and Lim, DS and Guo, K and Wong, YH and Mok, CK and Chu, JJH and Chew, WL},
title = {AAV-CRISPR-Cas13 eliminates human enterovirus and prevents death of infected mice.},
journal = {EBioMedicine},
volume = {93},
number = {},
pages = {104682},
doi = {10.1016/j.ebiom.2023.104682},
pmid = {37390772},
issn = {2352-3964},
mesh = {Humans ; Mice ; Animals ; CRISPR-Cas Systems ; Dependovirus/genetics ; *COVID-19/genetics ; *Enterovirus/genetics ; *Enterovirus A, Human/genetics ; },
abstract = {BACKGROUND: RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that adeno-associated virus (AAV) -delivered CRISPR-Cas13 directly targets and eliminates the positive-strand EV-A71 RNA virus in cells and infected mice.<br><br>METHODS: We developed a Cas13gRNAtor bioinformatics pipeline to design CRISPR guide RNAs (gRNAs) that cleave conserved viral sequences across the virus phylogeny and developed an AAV-CRISPR-Cas13 therapeutics using in&#xa0;vitro viral plaque assay and in&#xa0;vivo EV-A71 lethally-infected mouse model.<br><br>FINDINGS: We show that treatment with a pool of AAV-CRISPR-Cas13-gRNAs designed using the bioinformatics pipeline effectively blocks viral replication and reduces viral titers in cells by >99.99%. We further demonstrate that AAV-CRISPR-Cas13-gRNAs prophylactically and therapeutically inhibited viral replication in infected mouse tissues and prevented death in a lethally challenged EV-A71-infected mouse model.<br><br>INTERPRETATION: Our results show that the bioinformatics pipeline designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting to reduce viral loads. Additionally, this new antiviral AAV-CRISPR-Cas13 modality represents an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections.<br><br>FUNDING: Agency for Science, Technology and Research (A&#x2217;STAR) Assured Research Budget, A&#x2217;STAR Central Research Fund UIBR SC18/21-1089UI, A&#x2217;STAR Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012, MOE Tier 2 2017 (MOE2017-T2-1-078; MOE-T2EP30221-0005), and NUHSRO/2020/050/RO5+5/NUHS-COVID/4.},
}
@article {pmid37353926,
year = {2023},
author = {Philippe, C and Cornuault, JK and de Melo, AG and Morin-Pelchat, R and Jolicoeur, AP and Moineau, S},
title = {The never-ending battle between lactic acid bacteria and their phages.},
journal = {FEMS microbiology reviews},
volume = {47},
number = {4},
pages = {},
doi = {10.1093/femsre/fuad035},
pmid = {37353926},
issn = {1574-6976},
support = {//Natural Sciences and Engineering Research Council of Canada/ ; },
abstract = {Over the past few decades, the interest in lactic acid bacteria (LAB) has been steadily growing. This is mainly due to their industrial use, their health benefits as probiotic bacteria and their ecological importance in host-related microbiota. Phage infection represents a significant risk for the production and industrial use of LAB. This created the need to study the various means of defense put in place by LAB to resist their viral enemies, as well as the countermeasures evolved by phages to overcome these defenses. In this review, we discuss defense systems that LAB employ to resist phage infections. We also describe how phages counter these mechanisms through diverse and sophisticated strategies. Furthermore, we discuss the way phage-host interactions shape each other's evolution. The recent discovery of numerous novel defense systems in other bacteria promises a new dawn for phage research in LAB.},
}
@article {pmid37343658,
year = {2023},
author = {Fatma, Z and Tan, SI and Boob, AG and Zhao, H},
title = {A landing pad system for multicopy gene integration in Issatchenkia orientalis.},
journal = {Metabolic engineering},
volume = {78},
number = {},
pages = {200-208},
doi = {10.1016/j.ymben.2023.06.010},
pmid = {37343658},
issn = {1096-7184},
mesh = {*CRISPR-Cas Systems ; *Saccharomyces cerevisiae/genetics ; Gene Editing/methods ; Succinates ; },
abstract = {The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user's choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.},
}
@article {pmid37279759,
year = {2023},
author = {Tian, R and Cao, C and He, D and Dong, D and Sun, L and Liu, J and Chen, Y and Wang, Y and Huang, Z and Li, L and Jin, Z and Huang, Z and Xie, H and Zhao, T and Zhong, C and Hong, Y and Hu, Z},
title = {Massively parallel CRISPR off-target detection enables rapid off-target prediction model building.},
journal = {Med (New York, N.Y.)},
volume = {4},
number = {7},
pages = {478-492.e6},
doi = {10.1016/j.medj.2023.05.005},
pmid = {37279759},
issn = {2666-6340},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Papillomavirus Infections/genetics ; Gene Editing/methods ; Genome ; },
abstract = {BACKGROUND: CRISPR (clustered regularly interspaced short palindromic repeats) genome editing holds tremendous potential in clinical translation. However, the off-target effect has always been a major concern.<br><br>METHODS: Here, we have developed a novel sensitive and specific off-target detection method, AID-seq (adaptor-mediated off-target identification by sequencing), that can comprehensively and faithfully detect the low-frequency off targets generated by different CRISPR nucleases (including Cas9 and Cas12a).<br><br>FINDINGS: Based on AID-seq, we developed a pooled strategy to simultaneously identify the on/off targets of multiple gRNAs, as well as using mixed human and human papillomavirus (HPV) genomes to screen the most efficient and safe targets from 416 HPV gRNA candidates for antiviral therapy. Moreover, we used the pooled strategy with 2,069 single-guide RNAs (sgRNAs) at a pool size of about 500 to profile the properties of our newly discovered CRISPR, FrCas9. Importantly, we successfully built an off-target detection model using these off-target data via the CRISPR-Net deep learning method (area under the receiver operating characteristic curve [AUROC]&#xa0;= 0.97, area under the precision recall curve [AUPRC]&#xa0;= 0.29).<br><br>CONCLUSIONS: To our knowledge, AID-seq is the most sensitive and specific in&#xa0;vitro off-target detection method to date. And the pooled AID-seq strategy can be used as a rapid and high-throughput platform to select the best sgRNAs and characterize the properties of new CRISPRs.<br><br>FUNDING: This work was supported by The National Natural Science Foundation of China (grant nos. 32171465 and 82102392), the General Program of Natural Science Foundation of Guangdong Province of China (grant no. 2021A1515012438), Guangdong Basic and Applied Basic Research Foundation (grant no. 2020A1515110170), and the National Ten Thousand Plan-Young Top Talents of China (grant no. 80000-41180002).},
}
@article {pmid37263857,
year = {2023},
author = {Qin, H and Xu, W and Yao, K},
title = {CRISPR-based genome editing in disease treatment.},
journal = {Trends in molecular medicine},
volume = {29},
number = {8},
pages = {673-674},
doi = {10.1016/j.molmed.2023.05.003},
pmid = {37263857},
issn = {1471-499X},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems ; },
}
@article {pmid37244368,
year = {2023},
author = {Chen, JP and Gong, JS and Su, C and Li, H and Xu, ZH and Shi, JS},
title = {Improving the soluble expression of difficult-to-express proteins in prokaryotic expression system via protein engineering and synthetic biology strategies.},
journal = {Metabolic engineering},
volume = {78},
number = {},
pages = {99-114},
doi = {10.1016/j.ymben.2023.05.007},
pmid = {37244368},
issn = {1096-7184},
mesh = {*CRISPR-Cas Systems ; *Synthetic Biology ; Gene Editing ; Protein Engineering ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Solubility and folding stability are key concerns for difficult-to-express proteins (DEPs) restricted by amino acid sequences and superarchitecture, resolved by the precise distribution of amino acids and molecular interactions as well as the assistance of the expression system. Therefore, an increasing number of tools are available to achieve efficient expression of DEPs, including directed evolution, solubilization partners, chaperones, and affluent expression hosts, among others. Furthermore, genome editing tools, such as transposons and CRISPR Cas9/dCas9, have been developed and expanded to construct engineered expression hosts capable of efficient expression ability of soluble proteins. Accounting for the accumulated knowledge of the pivotal factors in the solubility and folding stability of proteins, this review focuses on advanced technologies and tools of protein engineering, protein quality control systems, and the redesign of expression platforms in prokaryotic expression systems, as well as advances of the cell-free expression technologies for membrane proteins production.},
}
@article {pmid37236858,
year = {2023},
author = {Pan, C and Qi, Y},
title = {PrimeRoot for targeted large DNA insertion in plants.},
journal = {Trends in plant science},
volume = {28},
number = {8},
pages = {870-872},
doi = {10.1016/j.tplants.2023.05.002},
pmid = {37236858},
issn = {1878-4372},
mesh = {*Plant Breeding ; *Plants/genetics ; Gene Editing ; Genome, Plant/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems/genetics ; },
abstract = {Genome editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized plant breeding through targeted genome and transcriptome modifications. However, accurate insertion of large DNA cargoes remains challenging. Recently, Sun and colleagues introduced PrimeRoot, a groundbreaking technology that enables precise and targeted integration of large DNA cargoes into plant genomes with remarkable efficiency and accuracy.},
}
@article {pmid36967299,
year = {2023},
author = {Tanaka, M and Nakamura, T},
title = {Targeting epigenetic aberrations of sarcoma in CRISPR era.},
journal = {Genes, chromosomes &amp; cancer},
volume = {62},
number = {9},
pages = {510-525},
doi = {10.1002/gcc.23142},
pmid = {36967299},
issn = {1098-2264},
support = {17cmA106609//Japan Agency for Medical Research and Development/ ; 20cm0106277//Japan Agency for Medical Research and Development/ ; 22ama221206//Japan Agency for Medical Research and Development/ ; 21ak0101170//Japan Agency for Medical Research and Development/ ; 26250029//Japan Society for the Promotion of Science/ ; 16KM07131//Japan Society for the Promotion of Science/ ; 19K07702//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; Mice ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Methylation ; *Sarcoma/genetics ; Epigenesis, Genetic ; Tumor Microenvironment ; },
abstract = {Sarcomas are rare malignancies that exhibit diverse biological, genetic, morphological, and clinical characteristics. Genetic alterations, such as gene fusions, mutations in transcriptional machinery components, histones, and DNA methylation regulatory molecules, play an essential role in sarcomagenesis. These mutations induce and/or cooperate with specific epigenetic aberrations required for the growth and maintenance of sarcomas. Appropriate mouse models have been developed to clarify the significance of genetic and epigenetic interactions in sarcomas. Studies using the mouse models for human sarcomas have demonstrated major advances in our understanding the developmental processes as well as tumor microenvironment of sarcomas. Recent technological progresses in epigenome editing will not only improve the studies using animal models but also provide a direct clue for epigenetic therapies. In this manuscript, we review important epigenetic aberrations in sarcomas and their representative mouse models, current methods of epigenetic editing using CRISPR/dCas9 systems, and potential applications in sarcoma studies and therapeutics.},
}
@article {pmid36965130,
year = {2023},
author = {Vanoli, F and Antonescu, CR},
title = {Modeling sarcoma relevant translocations using CRISPR-Cas9 in human embryonic stem derived mesenchymal precursors.},
journal = {Genes, chromosomes &amp; cancer},
volume = {62},
number = {9},
pages = {501-509},
doi = {10.1002/gcc.23141},
pmid = {36965130},
issn = {1098-2264},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; P50 CA217694/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; P50 CA217694/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Translocation, Genetic ; CRISPR-Cas Systems ; *Sarcoma/genetics ; Gene Editing ; Gene Rearrangement ; },
abstract = {The role of cancer relevant translocations in tumorigenesis has been historically hampered by the lack of faithful in vitro and in vivo models. The development of the latest genome editing tools (e.g., CRISPR-Cas9) allowed modeling of various chromosomal translocations with different effects on proliferation and transformation capacity depending on the cell line used and secondary genetic alterations. The cellular context is particularly relevant in the case of oncogenic fusions expressed in sarcomas whose histogenesis remain uncertain. Moreover, recent studies have emphasized the increased frequency of gene fusion promiscuity across different mesenchymal tumor entities, which are clinicopathologically unrelated. This review provides a summary of different strategies utilized to generate cancer models with a focus on fusion-driven mesenchymal neoplasia.},
}
@article {pmid36959711,
year = {2023},
author = {Draper, GM and Panken, DJ and Largaespada, DA},
title = {Modeling human cancer predisposition syndromes using CRISPR/Cas9 in human cell line models.},
journal = {Genes, chromosomes &amp; cancer},
volume = {62},
number = {9},
pages = {493-500},
doi = {10.1002/gcc.23140},
pmid = {36959711},
issn = {1098-2264},
support = {R01 NS115438/NS/NINDS NIH HHS/United States ; UH3 CA244687/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Syndrome ; Gene Editing ; Disease Susceptibility ; Cell Line ; *Neoplasms/genetics ; },
abstract = {The advancement of CRISPR mediated gene engineering provides an opportunity to improve upon preclinical human cell line models of cancer predisposing syndromes. This review focuses on using CRISPR/Cas9 genome editing tools to model various human cancer predisposition syndromes. We examine the genetic mutations associated with neurofibromatosis type 1, Li-Fraumeni syndrome, Gorlin syndrome, BRCA mutant breast and ovarian cancers, and APC mutant cancers. Furthermore, we discuss the possibilities of using next-generation CRISPR-derived precision gene editing tools to introduce a variety of genetic lesions into human cell lines. The goal is to improve the quality of preclinical models surrounding these cancer predisposition syndromes through dissecting the effects of these mutations on the development of cancer and to provide new insights into the underlying mechanisms of these cancer predisposition syndromes. These studies demonstrate the continued utility and improvement of CRISPR/Cas9-induced human cell line models in studying the genetic basis of cancer.},
}
@article {pmid36593416,
year = {2023},
author = {Hu, J and Gao, C},
title = {CRISPR-edited plants by grafting.},
journal = {Nature biotechnology},
volume = {41},
number = {7},
pages = {909-910},
pmid = {36593416},
issn = {1546-1696},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Plants ; CRISPR-Cas Systems/genetics ; Gene Editing ; Plants, Genetically Modified/genetics ; },
}
@article {pmid36593415,
year = {2023},
author = {Yang, L and Machin, F and Wang, S and Saplaoura, E and Kragler, F},
title = {Heritable transgene-free genome editing in plants by grafting of wild-type shoots to transgenic donor rootstocks.},
journal = {Nature biotechnology},
volume = {41},
number = {7},
pages = {958-967},
pmid = {36593415},
issn = {1546-1696},
support = {810131//EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; 810131//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Plant Breeding ; Plants, Genetically Modified/genetics ; Transgenes/genetics ; },
abstract = {Generation of stable gene-edited plant lines using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) requires a lengthy process of outcrossing to eliminate CRISPR-Cas9-associated sequences and produce transgene-free lines. We have addressed this issue by designing fusions of Cas9 and guide RNA transcripts to tRNA-like sequence motifs that move RNAs from transgenic rootstocks to grafted wild-type shoots (scions) and achieve heritable gene editing, as demonstrated in wild-type Arabidopsis thaliana and Brassica rapa. The graft-mobile gene editing system enables the production of transgene-free offspring in one generation without the need for transgene elimination, culture recovery and selection, or use of viral editing vectors. We anticipate that using graft-mobile editing systems for transgene-free plant production may be applied to a wide range of breeding programs and crop plants.},
}
@article {pmid36593413,
year = {2023},
author = {Tou, CJ and Orr, B and Kleinstiver, BP},
title = {Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases.},
journal = {Nature biotechnology},
volume = {41},
number = {7},
pages = {968-979},
pmid = {36593413},
issn = {1546-1696},
support = {2020295403//National Science Foundation (NSF)/ ; Howard M. Goodman Fellowship//Massachusetts General Hospital (MGH)/ ; },
mesh = {Humans ; *DNA Transposable Elements ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transposases/genetics/metabolism ; Plasmids ; Endonucleases/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. However, the utility of type V-K CASTs is hindered by high off-target integration and a transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid cointegrate products. Here we overcome both limitations by engineering new CASTs with improved integration product purity and genome-wide specificity. To do so, we engineered a nicking homing endonuclease fusion to TnsB (named HELIX) to restore the 5' nicking capability needed for cargo excision on the DNA donor. HELIX enables cut-and-paste DNA insertion with up to 99.4% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. HELIX has substantially higher on-target specificity than canonical CASTs, and we identify several novel factors that further regulate targeted and genome-wide integration. Finally, we extend HELIX to other type V-K orthologs and demonstrate the feasibility of HELIX-mediated integration in human cell contexts.},
}
@article {pmid35932337,
year = {2023},
author = {de Mélo, AHF and Nunes, AL and Carvalho, PH and da Silva, MF and Teixeira, GS and Goldbeck, R},
title = {Evaluation of Saccharomyces cerevisiae modified via CRISPR/Cas9 as a cellulosic platform microorganism in simultaneously saccharification and fermentation processes.},
journal = {Bioprocess and biosystems engineering},
volume = {46},
number = {8},
pages = {1111-1119},
pmid = {35932337},
issn = {1615-7605},
support = {001//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 307014/2020-7//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 2015/20630-4//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2016/04602-3//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2019/08542-7//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; },
mesh = {*Cellulose/metabolism ; Fermentation ; Saccharomyces cerevisiae/genetics/metabolism ; CRISPR-Cas Systems ; *Saccharum/metabolism ; Ethanol/metabolism ; },
abstract = {The nonrenewable character and deleterious effects of fossil fuels foster the need for cleaner and more inexhaustible energy sources, such as bioethanol. Especially from lignocellulosic biomasses. However, the economic viability of this product in the market depends on process optimization and cost reduction. This research applied a sequential experimental project to investigate the process of enzymatic saccharification and simultaneous fermentation to produce ethanol with sugarcane bagasse. The differential of the work was the application of the strain of Saccharomyces cerevisiae AGY001 which was improved by evolutionary engineering to become thermotolerant and by a heterologous expression based on genomic integration by CRISPR/Cas9 to produce endoglucanase and β-glucosidase (AsENDO-AsBGL). The maximum ethanol yield found was 89% of the maximum theoretical yield (released sugars), obtained at temperature concentrations, sugarcane bagasse and inoculum at 40 °C, 16.5%, and 4.0 g/L, respectively (12.5 FPU/g bagasse). The mathematical model obtained can predict approximately 83% of the data set with 95% confidence. Therefore, these findings demonstrated the potential of sugarcane bagasse and S. cerevisiae AGY001 strain (CRISPR/Cas9 modified) in bioethanol production without the need for impractical selection media on an industrial scale, in addition to providing useful insights for the development of SSF processes.},
}
@article {pmid37435043,
year = {2023},
author = {Dorset, SR and Bak, RO},
title = {The p53 challenge of hematopoietic stem cell gene editing.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {30},
number = {},
pages = {83-89},
pmid = {37435043},
issn = {2329-0501},
abstract = {Ex vivo gene editing in hematopoietic stem and progenitor cells (HSPCs) represents a promising curative treatment strategy for monogenic blood disorders. Gene editing using the homology-directed repair (HDR) pathway enables precise genetic modifications ranging from single base pair correction to replacement or insertion of large DNA segments. Hence, HDR-based gene editing could facilitate broad application of gene editing across monogenic disorders, but the technology still faces challenges for clinical translation. Among these, recent studies demonstrate induction of a DNA damage response (DDR) and p53 activation caused by DNA double-strand breaks and exposure to recombinant adeno-associated virus vector repair templates, resulting in reduced proliferation, engraftment, and clonogenic capacity of edited HSPCs. While different mitigation strategies can reduce this DDR, more research is needed on this phenomenon to ensure safe and efficient implementation of HDR-based gene editing in the clinic.},
}
@article {pmid37434184,
year = {2023},
author = {Qian, Y and Wang, D and Niu, W and Zhao, D and Li, J and Liu, Z and Gao, X and Han, Y and Lai, L and Li, Z},
title = {A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9.},
journal = {BMC biology},
volume = {21},
number = {1},
pages = {155},
pmid = {37434184},
issn = {1741-7007},
support = {31970574//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Humans ; Mice ; Adenine ; HEK293 Cells ; *Proprotein Convertase 9 ; *RNA, Guide, CRISPR-Cas Systems ; *Gene Editing ; },
abstract = {BACKGROUND: Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size.<br><br>RESULTS: We discovered that ABE8e can tolerate large single deletions in the REC2 (&#x394;174-296) and HNH (&#x394;786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes.<br><br>CONCLUSIONS: We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.},
}
@article {pmid37434066,
year = {2023},
author = {Liang, Y and Chen, F and Wang, K and Lai, L},
title = {Base editors: development and applications in biomedicine.},
journal = {Frontiers of medicine},
volume = {},
number = {},
pages = {},
pmid = {37434066},
issn = {2095-0225},
abstract = {Base editor (BE) is a gene-editing tool developed by combining the CRISPR/Cas system with an individual deaminase, enabling precise single-base substitution in DNA or RNA without generating a DNA double-strand break (DSB) or requiring donor DNA templates in living cells. Base editors offer more precise and secure genome-editing effects than other conventional artificial nuclease systems, such as CRISPR/Cas9, as the DSB induced by Cas9 will cause severe damage to the genome. Thus, base editors have important applications in the field of biomedicine, including gene function investigation, directed protein evolution, genetic lineage tracing, disease modeling, and gene therapy. Since the development of the two main base editors, cytosine base editors (CBEs) and adenine base editors (ABEs), scientists have developed more than 100 optimized base editors with improved editing efficiency, precision, specificity, targeting scope, and capacity to be delivered in vivo, greatly enhancing their application potential in biomedicine. Here, we review the recent development of base editors, summarize their applications in the biomedical field, and discuss future perspectives and challenges for therapeutic applications.},
}
@article {pmid37429890,
year = {2023},
author = {Eom, H and Choi, YJ and Nandre, R and Han, HG and Kim, S and Kim, M and Oh, YL and Nakazawa, T and Honda, Y and Ro, HS},
title = {The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments.},
journal = {Scientific reports},
volume = {13},
number = {1},
pages = {11133},
pmid = {37429890},
issn = {2045-2322},
mesh = {*Reishi/genetics ; CRISPR-Cas Systems ; Escherichia coli/genetics ; Plant Breeding ; DNA, Mitochondrial ; *Agaricales ; Ribonucleoproteins/genetics ; },
abstract = {Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) at the fourth position prior to the protospacer adjacent motif. Of the 66 edited transformants, 42 had deletions ranging from a single base to large deletions of up to 796 bp, with 30 being a single base deletion. Interestingly, the remaining 24 contained inserted sequences with variable sizes at the DSB site that originated from the fragmented host mitochondrial DNA, E. coli chromosomal DNA, and the Cas9 expression vector DNA. The latter two were thought to be contaminated DNAs that were not removed during the purification process of the Cas9 protein. Despite this unexpected finding, the study demonstrated that editing G. lucidum genes using the Cas9-gRNA complex is achievable with comparable efficiency to the plasmid-mediated editing system.},
}
@article {pmid37429857,
year = {2023},
author = {Cullot, G and Boutin, J and Fayet, S and Prat, F and Rosier, J and Cappellen, D and Lamrissi, I and Pennamen, P and Bouron, J and Amintas, S and Thibault, C and Moranvillier, I and Laharanne, E and Merlio, JP and Guyonnet-Duperat, V and Blouin, JM and Richard, E and Dabernat, S and Moreau-Gaudry, F and Bedel, A},
title = {Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9.},
journal = {Nature communications},
volume = {14},
number = {1},
pages = {4072},
pmid = {37429857},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Tumor Suppressor Protein p53/genetics ; Cell Cycle Checkpoints/genetics ; Cell Division ; Cell Separation ; RNA ; },
abstract = {The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has led to gene editing in clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site has revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and more sensitive detection methods. Here, we present two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable the detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). These tools reveal rare complex chromosomal rearrangements caused by Cas9-nuclease and show that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppresses the occurrence of LOH without compromising editing. These data are confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk by designing safer protocols.},
}
@article {pmid37306056,
year = {2023},
author = {Impens, L and Lorenzo, CD and Vandeputte, W and Wytynck, P and Debray, K and Haeghebaert, J and Herwegh, D and Jacobs, TB and Ruttink, T and Nelissen, H and Inzé, D and Pauwels, L},
title = {Combining multiplex gene editing and doubled haploid technology in maize.},
journal = {The New phytologist},
volume = {239},
number = {4},
pages = {1521-1532},
doi = {10.1111/nph.19021},
pmid = {37306056},
issn = {1469-8137},
mesh = {*Gene Editing ; *Zea mays/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant ; Haploidy ; Plants, Genetically Modified ; },
abstract = {A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.},
}
@article {pmid36596724,
year = {2023},
author = {Kim, JS and Lee, JH and Jeon, SR and Kim, Y and Jeon, SH and Wu, HG},
title = {Identification of Genes Involved in EGF-induced Apoptosis Using CRISPR/Cas9 Knockout Screening: Implications for Novel Therapeutic Targets in EGFR-Overexpressing Cancers.},
journal = {Cancer research and treatment},
volume = {55},
number = {3},
pages = {737-745},
doi = {10.4143/crt.2022.1414},
pmid = {36596724},
issn = {2005-9256},
support = {2019R1F1A1040583//National Research Foundation of Korea/ ; 2020M2D9A2092373//National Research Foundation of Korea/ ; 2021R1A2C1095168//National Research Foundation of Korea/ ; //Ministry of Science and ICT/ ; },
mesh = {Humans ; *Epidermal Growth Factor/genetics/pharmacology ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; Early Detection of Cancer ; ErbB Receptors/genetics/metabolism ; *Neoplasms/drug therapy/genetics ; Apoptosis/genetics ; Calcium-Binding Proteins/genetics/metabolism ; },
abstract = {PURPOSE: Exogenous epidermal growth factor (EGF) causes apoptosis in EGF receptor (EGFR)-overexpressing cell lines. The apoptosis-inducing factors could be a therapeutic target. We aimed to determine the mechanism of EGF-induced apoptosis using a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screen.<br><br>MATERIALS AND METHODS: Two-vector system of the human genome-scale CRISPR knockout library v2 was used to target 19,050 genes using 123,411 single guide RNAs (sgRNAs). Recombinant human EGF (100 nM) or distilled water four times was administered to the experimental and control groups, respectively. The read counts of each sgRNA obtained from next-generation sequencing were analyzed using the edgeR algorithm. We used another EGFR-overexpressing cell line (A549) and short hairpin RNAs (shRNAs) targeting five EGF-resistance genes for validation. DUSP1 expression in A431, A549, and HEK293FT cells was calculated using reverse transcription-quantitative polymerase chain reaction.<br><br>RESULTS: We found 77 enriched and 189 depleted genes in the experimental group using the CRISPR-based knockout screen and identified the top five EGF-resistance genes: DDX20, LHFP, REPS1, DUSP1,<.i> and KRTAP10-12. Transfecting shRNAs targeting these genes into A549 cells significantly increased the surviving fractions after EGF treatment, compared with those observed in the control shRNA-transfected cells. The expression ratio of DUSP1 (inhibits ERK signaling) increased in A431 and A549 cells after EGF treatment. However, DUSP1 expression remained unchanged in HEK293FT cells after EGF treatment.<br><br>CONCLUSION: The CRISPR-based knockout screen revealed 266 genes possibly responsible for EGF-induced apoptosis. DUSP1 might be a critical component of EGF-induced apoptosis and a novel target for EGFR-overexpressing cancers.},
}
@article {pmid37427945,
year = {2023},
author = {Xu, MF and Chen, J and Xu, Y and Zhang, JL and Zhou, Y and He, JJ and Wu, S and Wei, YL and She, ZY},
title = {Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {196},
pages = {},
doi = {10.3791/65476},
pmid = {37427945},
issn = {1940-087X},
mesh = {Humans ; HeLa Cells ; *Protein Serine-Threonine Kinases/genetics ; *CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism ; Kinetochores/metabolism ; Mitosis/genetics ; Spindle Apparatus/metabolism ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E[-/-] HeLa cells using the CRISPR/Cas9 system. Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors. In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.},
}
@article {pmid37427920,
year = {2023},
author = {Grausam, KB and Breunig, JJ},
title = {Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {196},
pages = {},
doi = {10.3791/65286},
pmid = {37427920},
issn = {1940-087X},
mesh = {Mice ; Animals ; *Neural Stem Cells/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Electroporation/methods ; Plasmids/genetics ; *Brain Neoplasms/genetics/therapy/metabolism ; DNA/genetics ; Mutation ; },
abstract = {Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.},
}
@article {pmid37426982,
year = {2023},
author = {Choudhary, N and Tandi, D and Verma, RK and Yadav, VK and Dhingra, N and Ghosh, T and Choudhary, M and Gaur, RK and Abdellatif, MH and Gacem, A and Eltayeb, LB and Alqahtani, MS and Yadav, KK and Jeon, BH},
title = {A comprehensive appraisal of mechanism of anti-CRISPR proteins: an advanced genome editor to amend the CRISPR gene editing.},
journal = {Frontiers in plant science},
volume = {14},
number = {},
pages = {1164461},
pmid = {37426982},
issn = {1664-462X},
abstract = {The development of precise and controlled CRISPR-Cas tools has been made possible by the discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPRs (Acrs). The Acr protein has the ability to control off-targeted mutations and impede Cas protein-editing operations. Acr can help with selective breeding, which could help plants and animals improve their valuable features. In this review, the Acr protein-based inhibitory mechanisms that have been adopted by several Acrs, such as (a) the interruption of CRISPR-Cas complex assembly, (b) interference with target DNA binding, (c) blocking of target DNA/RNA cleavage, and (d) enzymatic modification or degradation of signalling molecules, were discussed. In addition, this review emphasizes the applications of Acr proteins in the plant research.},
}
@article {pmid37343558,
year = {2023},
author = {Ahuja, N and Hwaun, E and Pungor, JR and Rafiq, R and Nemes, S and Sakmar, T and Vogt, MA and Grasse, B and Diaz Quiroz, J and Montague, TG and Null, RW and Dallis, DN and Gavriouchkina, D and Marletaz, F and Abbo, L and Rokhsar, DS and Niell, CM and Soltesz, I and Albertin, CB and Rosenthal, JJC},
title = {Creation of an albino squid line by CRISPR-Cas9 and its application for in vivo functional imaging of neural activity.},
journal = {Current biology : CB},
volume = {33},
number = {13},
pages = {2774-2783.e5},
doi = {10.1016/j.cub.2023.05.066},
pmid = {37343558},
issn = {1879-0445},
support = {R35 GM147273/GM/NIGMS NIH HHS/United States ; RF1 NS118466/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Decapodiformes/genetics ; Gene Editing/methods ; Gene Knockout Techniques ; Genome ; },
abstract = {Cephalopods are remarkable among invertebrates for their cognitive abilities, adaptive camouflage, novel structures, and propensity for recoding proteins through RNA editing. Due to the lack of genetically tractable cephalopod models, however, the mechanisms underlying these innovations are poorly understood. Genome editing tools such as CRISPR-Cas9 allow targeted mutations in diverse species to better link genes and function. One emerging cephalopod model, Euprymna berryi, produces large numbers of embryos that can be easily cultured throughout their life cycle and has a sequenced genome. As proof of principle, we used CRISPR-Cas9 in E. berryi to target the gene for tryptophan 2,3 dioxygenase (TDO), an enzyme required for the formation of ommochromes, the pigments present in the eyes and chromatophores of cephalopods. CRISPR-Cas9 ribonucleoproteins targeting tdo were injected into early embryos and then cultured to adulthood. Unexpectedly, the injected specimens were pigmented, despite verification of indels at the targeted sites by sequencing in injected animals (G0s). A homozygote knockout line for TDO, bred through multiple generations, was also pigmented. Surprisingly, a gene encoding indoleamine 2,3, dioxygenase (IDO), an enzyme that catalyzes the same reaction as TDO in vertebrates, was also present in E. berryi. Double knockouts of both tdo and ido with CRISPR-Cas9 produced an albino phenotype. We demonstrate the utility of these albinos for in vivo imaging of Ca[2+] signaling in the brain using two-photon microscopy. These data show the feasibility of making gene knockout cephalopod lines that can be used for live imaging of neural activity in these behaviorally sophisticated organisms.},
}
@article {pmid37060177,
year = {2023},
author = {Cheng, Y and Zhang, Y and Li, G and Fang, H and Sretenovic, S and Fan, A and Li, J and Xu, J and Que, Q and Qi, Y},
title = {CRISPR-Cas12a base editors confer efficient multiplexed genome editing in rice.},
journal = {Plant communications},
volume = {4},
number = {4},
pages = {100601},
doi = {10.1016/j.xplc.2023.100601},
pmid = {37060177},
issn = {2590-3462},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; *Oryza/genetics ; },
}
@article {pmid37056050,
year = {2023},
author = {Yu, L and Li, Z and Ding, X and Alariqi, M and Zhang, C and Zhu, X and Fan, S and Zhu, L and Zhang, X and Jin, S},
title = {Developing an efficient CRISPR-dCas9-TV-derived transcriptional activation system to create three novel cotton germplasm materials.},
journal = {Plant communications},
volume = {4},
number = {4},
pages = {100600},
doi = {10.1016/j.xplc.2023.100600},
pmid = {37056050},
issn = {2590-3462},
mesh = {Transcriptional Activation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems/genetics ; },
}
@article {pmid37426019,
year = {2023},
author = {Fernandes, LGV and Teixeira, AF and Nascimento, ALTO},
title = {Evaluation of Leptospira interrogans knockdown mutants for LipL32, LipL41, LipL21, and OmpL1 proteins.},
journal = {Frontiers in microbiology},
volume = {14},
number = {},
pages = {1199660},
pmid = {37426019},
issn = {1664-302X},
abstract = {INTRODUCTION: Leptospirosis is a worldwide zoonosis caused by pathogenic and virulent species of the genus Leptospira, whose pathophysiology and virulence factors remain widely unexplored. Recently, the application of CRISPR interference (CRISPRi) has allowed the specific and rapid gene silencing of major leptospiral proteins, favoring the elucidation of their role in bacterial basic biology, host-pathogen interaction and virulence. Episomally expressed dead Cas9 from the Streptococcus pyogenes CRISPR/Cas system (dCas9) and single-guide RNA recognize and block transcription of the target gene by base pairing, dictated by the sequence contained in the 5' 20-nt sequence of the sgRNA.<br><br>METHODS: In this work, we tailored plasmids for silencing the major proteins of L. interrogans serovar Copenhageni strain Fiocruz L1-130, namely LipL32, LipL41, LipL21 and OmpL1. Double- and triple-gene silencing by in tandem sgRNA cassettes were also achieved, despite plasmid instability.<br><br>RESULTS: OmpL1 silencing resulted in a lethal phenotype, in both L. interrogans and saprophyte L. biflexa, suggesting its essential role in leptospiral biology. Mutants were confirmed and evaluated regarding interaction with host molecules, including extracellular matrix (ECM) and plasma components, and despite the dominant abundance of the studied proteins in the leptospiral membrane, protein silencing mostly resulted in unaltered interactions, either because they intrinsically display low affinity to the molecules assayed or by a compensation mechanism, where other proteins could be upregulated to fill the niche left by protein silencing, a feature previously described for the LipL32 mutant. Evaluation of the mutants in the hamster model confirms the augmented virulence of the LipL32 mutant, as hinted previously. The essential role of LipL21 in acute disease was demonstrated, since the LipL21 knockdown mutants were avirulent in the animal model, and even though mutants could still colonize the kidneys, they were found in markedly lower numbers in the animals' liver. Taking advantage of higher bacterial burden in LipL32 mutant-infected organs, protein silencing was demonstrated in vivo directly in leptospires present in organ homogenates.<br><br>DISCUSSION: CRISPRi is now a well-established, attractive genetic tool that can be applied for exploring leptospiral virulence factors, leading to the rational for designing more effective subunit or even chimeric recombinant vaccines.},
}
@article {pmid37425722,
year = {2023},
author = {Pallaseni, A and Peets, EM and Girling, G and Crepaldi, L and Kuzmin, I and Raudvere, U and Peterson, H and Serçin, &#xd6; and Mardin, BR and Kosicki, M and Parts, L},
title = {The interplay of DNA repair context with target sequence predictably biasses Cas9-generated mutations.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2023.06.28.546891},
pmid = {37425722},
abstract = {The genome engineering capability of the CRISPR/Cas system depends on the DNA repair machinery to generate the final outcome. Several genes can have an impact on mutations created, but their exact function and contribution to the result of the repair are not completely characterised. This lack of knowledge has limited the ability to comprehend and regulate the editing outcomes. Here, we measure how the absence of 21 repair genes changes the mutation outcomes of Cas9-generated cuts at 2,812 synthetic target sequences in mouse embryonic stem cells. Absence of key non-homologous end joining genes Lig4, Xrcc4, and Xlf abolished small insertions and deletions, while disabling key microhomology-mediated repair genes Nbn and Polq reduced frequency of longer deletions. Complex alleles of combined insertion and deletions were preferentially generated in the absence of Xrcc6. We further discover finer structure in the outcome frequency changes for single nucleotide insertions and deletions between large microhomologies that are differentially modulated by the knockouts. We use the knowledge of the reproducible variation across repair milieus to build predictive models of Cas9 editing results that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of CRISPR/Cas9-generated mutations.},
}
@article {pmid37425362,
year = {2023},
author = {Poulalier-Delavelle, M and Baker, JP and Millard, J and Winzer, K and Minton, NP},
title = {Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {11},
number = {},
pages = {1213236},
pmid = {37425362},
issn = {2296-4185},
abstract = {Acetogenic bacteria can play a major role in achieving Net Zero through their ability to convert CO2 into industrially relevant chemicals and fuels. Full exploitation of this potential will be reliant on effective metabolic engineering tools, such as those based on the Streptococcus pyogenes CRISPR/Cas9 system. However, attempts to introduce cas9-containing vectors into Acetobacterium woodii were unsuccessful, most likely as a consequence of Cas9 nuclease toxicity and the presence of a recognition site for an endogenous A. woodii restriction-modification (R-M) system in the cas9 gene. As an alternative, this study aims to facilitate the exploitation of CRISPR/Cas endogenous systems as genome engineering tools. Accordingly, a Python script was developed to automate the prediction of protospacer adjacent motif (PAM) sequences and used to identify PAM candidates of the A. woodii Type I-B CRISPR/Cas system. The identified PAMs and the native leader sequence were characterized in vivo by interference assay and RT-qPCR, respectively. Expression of synthetic CRISPR arrays, consisting of the native leader sequence, direct repeats, and adequate spacer, along with an editing template for homologous recombination, successfully led to the creation of 300 bp and 354 bp in-frame deletions of pyrE and pheA, respectively. To further validate the method, a 3.2 kb deletion of hsdR1 was also generated, as well as the knock-in of the fluorescence-activating and absorption-shifting tag (FAST) reporter gene at the pheA locus. Homology arm length, cell density, and the amount of DNA used for transformation were found to significantly impact editing efficiencies. The devised workflow was subsequently applied to the Type I-B CRISPR/Cas system of Clostridium autoethanogenum, enabling the generation of a 561 bp in-frame deletion of pyrE with 100% editing efficiency. This is the first report of genome engineering of both A. woodii and C. autoethanogenum using their endogenous CRISPR/Cas systems.},
}
@article {pmid37424467,
year = {2023},
author = {Miguel, L and Gervais, J and Nicolas, G and Lecourtois, M},
title = {SorLA Protective Function Is Restored by Improving SorLA Protein Maturation in a Subset of Alzheimer's Disease-Associated SORL1 Missense Variants.},
journal = {Journal of Alzheimer's disease : JAD},
volume = {},
number = {},
pages = {},
doi = {10.3233/JAD-230211},
pmid = {37424467},
issn = {1875-8908},
abstract = {SORL1 loss of function is associated with Alzheimer's disease (AD) risk through increased Aβ peptide secretion. We expressed 10 maturation-defective rare missense SORL1 variants in HEK cells and showed that decreasing growing temperature led to a significant increase in the maturation of the encoded protein SorLA for 6/10. In edited hiPSC carrying two of these variants, maturation of the protein was restored partially by decreasing the culture temperature and was associated with concomitant decrease in Aβ secretion. Correcting SorLA maturation in the context of maturation-defective missense variants could thus be a relevant strategy to improve SorLA protective function against AD.},
}
@article {pmid37424252,
year = {2023},
author = {Si, XX and Zhang, Q and Luo, ZD and Zhang, ZY and Wang, Y and Shao, LP},
title = {A Rapid and Accurate CRISPR/Cas12b-Mediated Genotyping Assay for the Methylenetetrahydrofolate Reductase Gene Polymorphism C677T.},
journal = {Biomedical and environmental sciences : BES},
volume = {36},
number = {6},
pages = {561-565},
doi = {10.3967/bes2023.070},
pmid = {37424252},
issn = {2214-0190},
mesh = {Humans ; *Methylenetetrahydrofolate Reductase (NADPH2)/genetics/metabolism ; Genotype ; *CRISPR-Cas Systems ; Polymorphism, Genetic ; Genetic Predisposition to Disease ; Polymorphism, Single Nucleotide ; Gene Frequency ; },
}
@article {pmid37422628,
year = {2023},
author = {Bahrami, E and Schmid, JP and Jurinovic, V and Becker, M and Wirth, AK and Ludwig, R and Kreissig, S and Duque Angel, TV and Amend, D and Hunt, K and Öllinger, R and Rad, R and Frenz, JM and Solovey, M and Ziemann, F and Mann, M and Vick, B and Wichmann, C and Herold, T and Jayavelu, AK and Jeremias, I},
title = {Combined proteomics and CRISPR‒Cas9 screens in PDX identify ADAM10 as essential for leukemia in vivo.},
journal = {Molecular cancer},
volume = {22},
number = {1},
pages = {107},
pmid = {37422628},
issn = {1476-4598},
support = {DJCLS 10 R/2021//Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung/ ; DJCLS 15 R/2021//Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung/ ; 681524//European Research Council Consolidator Grant/ ; },
mesh = {Humans ; Mice ; Animals ; ADAM10 Protein/genetics/metabolism ; *Proteomics ; CRISPR-Cas Systems ; Membrane Proteins/genetics/metabolism ; *Leukemia/genetics ; Disease Models, Animal ; Tumor Microenvironment ; Amyloid Precursor Protein Secretases/genetics/metabolism ; },
abstract = {BACKGROUND: Acute leukemias represent deadly malignancies that require better treatment. As a challenge, treatment is counteracted by a microenvironment protecting dormant leukemia stem cells.<br><br>METHODS: To identify responsible surface proteins, we performed deep proteome profiling on minute numbers of dormant patient-derived xenograft (PDX) leukemia stem cells isolated from mice. Candidates were functionally screened by establishing a comprehensive CRISPR&#x2012;Cas9 pipeline in PDX models in vivo.<br><br>RESULTS: A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as an essential vulnerability required for the survival and growth of different types of acute leukemias in vivo, and reconstitution assays in PDX models confirmed the relevance of its sheddase activity. Of translational importance, molecular or pharmacological targeting of ADAM10 reduced PDX leukemia burden, cell homing to the murine bone marrow and stem cell frequency, and increased leukemia response to conventional chemotherapy in vivo.<br><br>CONCLUSIONS: These findings identify ADAM10 as an attractive therapeutic target for the future treatment of acute leukemias.},
}
@article {pmid37418936,
year = {2023},
author = {Guo, H and Zhang, Y and Kong, F and Wang, C and Chen, S and Wang, J and Wang, D},
title = {A Cas12a-based platform combined with gold nanoparticles for sensitive and visual detection of Alternaria solani.},
journal = {Ecotoxicology and environmental safety},
volume = {263},
number = {},
pages = {115220},
doi = {10.1016/j.ecoenv.2023.115220},
pmid = {37418936},
issn = {1090-2414},
abstract = {Alternaria solani (A. solani), the causal agent of early blight in potatoes, poses a serious and persistent threat to potato production worldwide. Therefore, developing a method that can accurately detect A. solani in the early stage to avoid further spread is urgent. However, the conventional PCR-based method is not appropriate for application in the fields. Recently, the CRISPR-Cas system has been developed for nucleic acids analysis at point-of-care. Here, we propose a gold nanoparticles-based visual assay combining loop-mediated isothermal amplification with CRISPR-Cas12a to detect A. solani. After optimization, the method could detect 10[-3] ng/μL genomic gene of A. solani. The specificity of the method was confirmed by discriminating A. solani from other three highly homologous pathogens. We also developed a portable device that could be used in the fields. By integrating with the smartphone readout, this platform holds significant potential in high-throughput detection of multiple pathogens in the fields.},
}
@article {pmid37416774,
year = {2023},
author = {Hu, M and Bodnar, B and Zhang, Y and Xie, F and Li, F and Li, S and Zhao, J and Zhao, R and Gedupoori, N and Mo, Y and Lin, L and Li, X and Meng, W and Yang, X and Wang, H and Barbe, MF and Srinivasan, S and Bethea, JR and Mo, X and Xu, H and Hu, W},
title = {Defective neurite elongation and branching in Nibp/Trappc9 deficient zebrafish and mice.},
journal = {International journal of biological sciences},
volume = {19},
number = {10},
pages = {3226-3248},
pmid = {37416774},
issn = {1449-2288},
mesh = {Animals ; Mice ; *Intellectual Disability/genetics/metabolism ; *Microcephaly/genetics/metabolism ; Neurites/physiology ; Neurons/metabolism ; Zebrafish ; },
abstract = {Loss of function in transport protein particles (TRAPP) links a new set of emerging genetic disorders called "TRAPPopathies". One such disorder is NIBP syndrome, characterized by microcephaly and intellectual disability, and caused by mutations of NIBP/TRAPPC9, a crucial and unique member of TRAPPII. To investigate the neural cellular/molecular mechanisms underlying microcephaly, we developed Nibp/Trappc9-deficient animal models using different techniques, including morpholino knockdown and CRISPR/Cas mutation in zebrafish and Cre/LoxP-mediated gene targeting in mice. Nibp/Trappc9 deficiency impaired the stability of the TRAPPII complex at actin filaments and microtubules of neurites and growth cones. This deficiency also impaired elongation and branching of neuronal dendrites and axons, without significant effects on neurite initiation or neural cell number/types in embryonic and adult brains. The positive correlation of TRAPPII stability and neurite elongation/branching suggests a potential role for TRAPPII in regulating neurite morphology. These results provide novel genetic/molecular evidence to define patients with a type of non-syndromic autosomal recessive intellectual disability and highlight the importance of developing therapeutic approaches targeting the TRAPPII complex to cure TRAPPopathies.},
}
@article {pmid37415116,
year = {2023},
author = {Hu, Y and Zhang, H and Guo, Z and Zhou, J and Zhang, W and Gong, M and Wu, J},
title = {CKM and TERT dual promoters drive CRISPR-dCas9 to specifically inhibit the malignant behavior of osteosarcoma cells.},
journal = {Cellular &amp; molecular biology letters},
volume = {28},
number = {1},
pages = {52},
pmid = {37415116},
issn = {1689-1392},
mesh = {Mice ; Animals ; Mice, Nude ; *Osteosarcoma/genetics/therapy/metabolism ; Promoter Regions, Genetic/genetics ; Muscles/metabolism/pathology ; *Bone Neoplasms/genetics/therapy/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Improvements in treatment and chemotherapy have increased the survival rate of osteosarcoma, but overall efficacy remains low, highlighting the need for new gene therapy methods. Clustered regularly interspaced short palindromic repeats-deactivated Cas9 (CRISPR-dCas9) technology offers a promising strategy, but targeting osteosarcoma cells precisely is a challenge. We designed a system to achieve specific expression of CRISPR-dCas9-KRAB in osteosarcoma cells by using the creatine kinase muscle (CKM) promoter to drive dCas9-KRAB and the telomerase reverse transcriptase (TERT) promoter to drive single guide (sg)RNA expression. We inhibited the MDM2 proto-oncogene using this system in vitro, which efficiently inhibited the malignant behavior of osteosarcoma cells and induced apoptosis without affecting normal cells. In vivo experiments demonstrated that this system effectively inhibited the growth of subcutaneously transplanted tumors in nude mice. These findings provide a new method for precise identification and intervention of osteosarcoma with significant implications for the development of gene therapy methods for other cancers. Future research should focus on optimizing this system for clinical translation.},
}
@article {pmid37286821,
year = {2023},
author = {Abid, T and Goodale, AB and Kalani, Z and Wyatt, M and Gonzalez, EM and Zhou, KN and Qian, K and Novikov, D and Condurat, AL and Bandopadhayay, P and Piccioni, F and Persky, NS and Root, DE},
title = {Genome-wide pooled CRISPR screening in neurospheres.},
journal = {Nature protocols},
volume = {18},
number = {7},
pages = {2014-2031},
pmid = {37286821},
issn = {1750-2799},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Genome ; Cell Line ; *Neoplasms ; },
abstract = {Spheroid culture systems have allowed in vitro propagation of cells unable to grow in canonical cell culturing conditions, and may capture cellular contexts that model tumor growth better than current model systems. The insights gleaned from genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening of thousands of cancer cell lines grown in conventional culture conditions illustrate the value of such CRISPR pooled screens. It is clear that similar genome-wide CRISPR screens of three-dimensional spheroid cultures will be important for future biological discovery. Here, we present a protocol for genome-wide CRISPR screening of three-dimensional neurospheres. While many in-depth protocols and discussions have been published for more typical cell lines, few detailed protocols are currently available in the literature for genome-wide screening in spheroidal cell lines. For those who want to screen such cell lines, and particularly neurospheres, we provide a step-by-step description of assay development tests to be performed before screening, as well as for the screen itself. We highlight considerations of variables that make these screens distinct from, or similar to, typical nonspheroid cell lines throughout. Finally, we illustrate typical outcomes of neurosphere genome-wide screens, and how neurosphere screens typically produce slightly more heterogeneous signal distributions than more canonical cancer cell lines. Completion of this entire protocol will take 8-12 weeks from the initial assay development tests to deconvolution of the sequencing data.},
}
@article {pmid37277562,
year = {2023},
author = {Lei, Z and Meng, H and Rao, X and Zhao, H and Yi, C},
title = {Detect-seq, a chemical labeling and biotin pull-down approach for the unbiased and genome-wide off-target evaluation of programmable cytosine base editors.},
journal = {Nature protocols},
volume = {18},
number = {7},
pages = {2221-2255},
pmid = {37277562},
issn = {1750-2799},
mesh = {*Gene Editing/methods ; *Biotin ; Cytosine ; Genome ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Programmable cytosine base editors show promising approaches for correcting pathogenic mutations; yet, their off-target effects have been of great concern. Detect-seq (dU-detection enabled by C-to-T transition during sequencing) is an unbiased, sensitive method for the off-target evaluation of programmable cytosine base editors. It profiles the editome by tracing the editing intermediate dU, which is introduced inside living cells and edited by programmable cytosine base editors. The genomic DNA is extracted, preprocessed and labeled by successive chemical and enzymatic reactions, followed by biotin pull-down to enrich the dU-containing loci for sequencing. Here, we describe a detailed protocol for performing the Detect-seq experiment, and a customized, open-source, bioinformatic pipeline for analyzing the characteristic Detect-seq data is also provided. Unlike those previous whole-genome sequencing-based methods, Detect-seq uses an enrichment strategy and hence is endowed with great sensitivity, a higher signal-to-noise ratio and no requirement for high sequencing depth. Furthermore, Detect-seq is widely applicable for both mitotic and postmitotic biological systems. The entire protocol typically takes 5 d from the genomic DNA extraction to sequencing and ~1 week for data analysis.},
}
@article {pmid37231266,
year = {2023},
author = {Han, D and Xiao, Q and Wang, Y and Zhang, H and Dong, X and Li, G and Kong, X and Wang, S and Song, J and Zhang, W and Zhou, J and Bi, L and Yuan, Y and Shi, L and Zhong, N and Yang, H and Zhou, Y},
title = {Development of miniature base editors using engineered IscB nickase.},
journal = {Nature methods},
volume = {20},
number = {7},
pages = {1029-1036},
pmid = {37231266},
issn = {1548-7105},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; *Deoxyribonuclease I/genetics/metabolism ; Gene Editing ; Cytosine ; RNA/genetics ; Mammals/genetics/metabolism ; },
abstract = {As a miniature RNA-guided endonuclease, IscB is presumed to be the ancestor of Cas9 and to share similar functions. IscB is less than half the size of Cas9 and thus more suitable for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells limits its in vivo applications. Here we describe the engineering of OgeuIscB and its corresponding ωRNA to develop an IscB system that is highly efficient in mammalian systems, named enIscB. By fusing enIscB with T5 exonuclease (T5E), we found enIscB-T5E exhibited comparable targeting efficiency to SpG Cas9 while showing reduced chromosome translocation effects in human cells. Furthermore, by fusing cytosine or adenosine deaminase with enIscB nickase, we generated miniature IscB-derived base editors (miBEs), exhibiting robust editing efficiency (up to 92%) to induce DNA base conversions. Overall, our work establishes enIscB-T5E and miBEs as versatile tools for genome editing.},
}
@article {pmid37188955,
year = {2023},
author = {Seo, SY and Min, S and Lee, S and Seo, JH and Park, J and Kim, HK and Song, M and Baek, D and Cho, SR and Kim, HH},
title = {Massively parallel evaluation and computational prediction of the activities and specificities of 17 small Cas9s.},
journal = {Nature methods},
volume = {20},
number = {7},
pages = {999-1009},
pmid = {37188955},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; },
abstract = {Recently, various small Cas9 orthologs and variants have been reported for use in in vivo delivery applications. Although small Cas9s are particularly suited for this purpose, selecting the most optimal small Cas9 for use at a specific target sequence continues to be challenging. Here, to this end, we have systematically compared the activities of 17 small Cas9s for thousands of target sequences. For each small Cas9, we have characterized the protospacer adjacent motif and determined optimal single guide RNA expression formats and scaffold sequence. High-throughput comparative analyses revealed distinct high- and low-activity groups of small Cas9s. We also developed DeepSmallCas9, a set of computational models predicting the activities of the small Cas9s at matched and mismatched target sequences. Together, this analysis and these computational models provide a useful guide for researchers to select the most suitable small Cas9 for specific applications.},
}
@article {pmid37156748,
year = {2023},
author = {Radoua, A and Pernon, B and Pernet, N and Jean, C and Elmallah, M and Guerrache, A and Constantinescu, AA and Hadj Hamou, S and Devy, J and Micheau, O},
title = {ptARgenOM-A Flexible Vector For CRISPR/CAS9 Nonviral Delivery.},
journal = {Small methods},
volume = {7},
number = {7},
pages = {e2300069},
doi = {10.1002/smtd.202300069},
pmid = {37156748},
issn = {2366-9608},
support = {ANR-22-LCV1-0005-01//Agence Nationale de la Recherche/ ; ANR-11LABX-0021-01//Agence Nationale de la Recherche/ ; ANR-15-IDEX-0003//Agence Nationale de la Recherche/ ; 777995//Horizon 2020/ ; 01U.2020//Conf&#xe9;rence de Coordination Interr&#xe9;gionale Est de la Ligue contre le Cancer, comit&#xe9; de C&#xf4;te d'Or/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Cell Line ; Mammals/genetics ; },
abstract = {Viral-mediated delivery of the CRISPR-Cas9 system is one the most commonly used techniques to modify the genome of a cell, with the aim of analyzing the function of the targeted gene product. While these approaches are rather straightforward for membrane-bound proteins, they can be laborious for intracellular proteins, given that selection of full knockout (KO) cells often requires the amplification of single-cell clones. Moreover, viral-mediated delivery systems, besides the Cas9 and gRNA, lead to the integration of unwanted genetic material, such as antibiotic resistance genes, introducing experimental biases. Here, an alternative non-viral delivery approach is presented for CRISPR/Cas9, allowing efficient and flexible selection of KO polyclonal cells. This all-in-one mammalian CRISPR-Cas9 expression vector, ptARgenOM, encodes the gRNA and the Cas9 linked to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein and the puromycin N-acetyltransferase, allowing for transient, expression-dependent selection and enrichment of isogenic KO cells. After evaluation using more than 12 distinct targets in 6 cell lines, ptARgenOM is found to be efficient in producing KO cells, reducing the time required to obtain a polyclonal isogenic cell line by 4-6 folds. Altogether ptARgenOM provides a simple, fast, and cost-effective delivery tool for genome editing.},
}
@article {pmid37415091,
year = {2023},
author = {Nguyen, MT and Kim, SA and Cheng, YY and Hong, SH and Jin, YS and Han, NS},
title = {A qPCR Method to Assay Endonuclease Activity of Cas9-sgRNA Ribonucleoprotein Complexes.},
journal = {Journal of microbiology and biotechnology},
volume = {33},
number = {9},
pages = {1-10},
doi = {10.4014/jmb.2305.05010},
pmid = {37415091},
issn = {1738-8872},
abstract = {The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/μg RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.},
}
@article {pmid37415009,
year = {2023},
author = {Kauert, DJ and Madariaga-Marcos, J and Rutkauskas, M and Wulfken, A and Songailiene, I and Sinkunas, T and Siksnys, V and Seidel, R},
title = {The energy landscape for R-loop formation by the CRISPR-Cas Cascade complex.},
journal = {Nature structural &amp; molecular biology},
volume = {},
number = {},
pages = {},
pmid = {37415009},
issn = {1545-9985},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) sequences and CRISPR-associated (Cas) genes comprise CIRSPR-Cas effector complexes, which have revolutionized gene editing with their ability to target specific genomic loci using CRISPR RNA (crRNA) complementarity. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base pairing between crRNA and the DNA target strand, forming an R-loop structure. Full R-loop extension is a prerequisite for subsequent DNA cleavage. However, the recognition of unintended sequences with multiple mismatches has limited therapeutic applications and is still poorly understood on a mechanistic level. Here we set up ultrafast DNA unwinding experiments on the basis of plasmonic DNA origami nanorotors to study R-loop formation by the Cascade effector complex in real time, close to base-pair resolution. We resolve a weak global downhill bias of the forming R-loop, followed by a steep uphill bias for the final base pairs. We also show that the energy landscape is modulated by base flips and mismatches. These findings suggest that Cascade-mediated R-loop formation occurs on short timescales in submillisecond single base-pair steps, but on longer timescales in six base-pair intermediate steps, in agreement with the structural periodicity of the crRNA-DNA hybrid.},
}
@article {pmid37413959,
year = {2023},
author = {Volke, DC and Orsi, E and Nikel, PI},
title = {Emergent CRISPR-Cas-based technologies for engineering non-model bacteria.},
journal = {Current opinion in microbiology},
volume = {75},
number = {},
pages = {102353},
doi = {10.1016/j.mib.2023.102353},
pmid = {37413959},
issn = {1879-0364},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) technologies brought a transformative change in the way bacterial genomes are edited, and a plethora of studies contributed to developing multiple tools based on these approaches. Prokaryotic biotechnology benefited from the implementation of such genome engineering strategies, with an increasing number of non-model bacterial species becoming genetically tractable. In this review, we summarize the recent trends in engineering non-model microbes using CRISPR-Cas technologies, discussing their potential in supporting cell factory design towards biotech