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Chemical transformation mediated CRISPR/Cas9 genome editing in Escherichia coli.
OBJECTIVES: To develop a convenient chemical transformation mediated CRISPR/Cas9 (CT-CRISPR/Cas9) system for genome editing in Escherichia coli. RESULTS: Here, we have constructed a CT-CRISPR/Cas9 system, which can precisely edit bacterial genome (replacing, deleting, inserting or point mutating a target gene) through chemical transformation. Compared with the traditional electroporation mediated CRISPR/Cas9 (ET-CRISPR/Cas9) system, genome editing with the CT-CRISPR/Cas9 system is much cheaper and simpler. In the CT-CRISPR/Cas9 system, we observed efficient genome editing on LB-agar plates. The CT-CRISPR/Cas9 system has successfully modified the target gene with the editing template flanked by short homologous DNA fragments (~ 50 bp) which were designed in primers. We used the lab-made CaCl2 solution to perform the CT-CRISPR/Cas9 experiment and successfully edited the genome of E. coli. Potential application of the CT-CRISPR/Cas9 system in high-throughput genome editing was evaluated in two E. coli strains by using a multiwell plate. CONCLUSIONS: Our work provides a simple and cheap genome-editing method, that is expected to be widely applied as a routine genetic engineering method.
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Development of a CRISPR/Cpf1 system for multiplex gene editing in Aspergillus oryzae.
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.
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CRISPR Variants for Gene Editing in Plants: Biosafety Risks and Future Directions.
The CRISPR genome editing technology is a crucial tool for enabling revolutionary advancements in plant genetic improvement. This review shows the latest developments in CRISPR/Cas9 genome editing system variants, discussing their benefits and limitations for plant improvement. While this technology presents immense opportunities for plant breeding, it also raises serious biosafety concerns that require careful consideration, including potential off-target effects and the unintended transfer of modified genes to other organisms. This paper highlights strategies to mitigate biosafety risks and explores innovative plant gene editing detection methods. Our review investigates the international biosafety guidelines for gene-edited crops, analyzing their broad implications for agricultural and biotechnology research and advancement. We hope to provide illuminating and refined perspectives for industry practitioners and policymakers by evaluating CRISPR genome enhancement in plants.
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CRISPR/Cas system targeting RNA and its derivative technology.
RNA editing is one of the important research directions in the field of epigenetics. With further research, scientists have discovered that the CRISPR/Cas system can target not only DNA but also RNA, thereby achieving precise gene editing at the transcriptional level. Moreover, using the CRISPR/Cas system for RNA editing can also avoid damage to genome. At present, a variety of derivative technologies based on RNA-targeting CRISPR systems have been developed, including RNA knockdown and editing, nucleic acid detection and imaging, and RNA tracking. The emergence of these derivative technologies provides powerful tools for deciphering biological genetic mechanisms and disease treatment. In this review, we summarize the structure, function, mechanisms, and derived technologies of RNA-targeting CRISPR/Cas systems, aiming to enrich people's understanding of CRISPR/Cas-mediated RNA editing.
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Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.
The fusion of genome engineering and adoptive cellular therapy holds immense promise for the treatment of genetic disease and cancer. Multiplex genome engineering using targeted nucleases can be used to increase the efficacy and broaden the application of such therapies but carries safety risks associated with unintended genomic alterations and genotoxicity. Here, we apply base editor technology for multiplex gene modification in primary human T cells in support of an allogeneic CAR-T platform and demonstrate that base editor can mediate highly efficient multiplex gene disruption with minimal double-strand break induction. Importantly, multiplex base edited T cells exhibit improved expansion and lack double strand break-induced translocations observed in T cells edited with Cas9 nuclease. Our findings highlight base editor as a powerful platform for genetic modification of therapeutically relevant primary cell types.
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[Development of a miniaturized CRISPR/Cas gene editing tool for human gut Bacteroides].
: Bacteroides, as one of the most abundant and diverse genera in the human gut, is regarded as a window into the study of gut microbiota-host interactions. Currently, CRISPR/Cas-based gene editing systems targeting Bacteroides have been widely applied, while the large size of Cas nucleases limits their potential application scenarios (such as in situ gut Bacteroides editing based on phage delivery). Therefore, this study aims to develop a compact and highly efficient genetic editing tool in Bacteroides., We developed a miniaturized CRISPR/Cas gene editing system for human gut Bacteroides. First, the editing capabilities of different miniaturized CRISPR/Cas systems, including AsCas12f, CasΦ2, and ISDge10, were evaluated in Bacteroides fragilis. Subsequently, the editing capability of AsCas12f was assessed across various Bacteroides species, and the size of this system was further optimized. The results demonstrated that the CRISPR/AsCas12f genome editing system exhibited the highest editing efficiency in B. fragilis. The CRISPR/AsCas12f system achieved efficient genome editing in B. fragilis, Bacteroides thetaiotaomicron, and Phocaeicola vulgatus. Furthermore, with a repair template of 500 bp homologous arms, the editing efficiency remained as high as 94.7%. In conclusion, CRISPR/AsCas12f can serve as a chassis tool enzyme for the development of Bacteroides-based miniature gene editors and derivative technologies, laying a foundation for the further development of gene editing technology for Bacteroides.
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Lipofection-Based Delivery of CRISPR/Cas9 Ribonucleoprotein for Gene Editing in Male Germline Stem Cells.
Gene editing in the murine germline is a valuable approach to investigate germ cell maturation and generate mouse models. Several studies demonstrated that CRISPR/Cas9 alters the genome of cultured male mouse germline stem cells delivered by electroporation of plasmids. Recently, we showed proof-of-principle that gene knockout can be effectively targeted in mouse germline stem cells by lipofecting Cas9:gRNA ribonucleoproteins. In this protocol, we describe a simple, fast, and cheap workflow for gene editing via the lipofection of non-integrative ribonucleoproteins in murine male germline stem cells.
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How CRISPR is transforming drug discovery.
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Heterotrimeric G protein are involved in the regulation of multiple agronomic traits and stress tolerance in rice.
BACKGROUND: The heterotrimeric G protein complex, consisting of Gα, Gβ, and Gγ subunits, are conserved signal transduction mechanism in eukaryotes. Recent molecular researches had demonstrated that G protein signaling participates in the regulation of yield related traits. However, the effects of G protein genes on yield components and stress tolerance are not well characterized. RESULTS: In this study, we generated heterotrimeric G protein mutants in rice using CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) gene-editing technology. The effects of heterotrimeric G proteins on the regulation of yield components and stress tolerance were investigated. The mutants of gs3 and dep1 generated preferable agronomic traits compared to the wild-type, whereas the mutants of rga1 showed an extreme dwarf phenotype, which led to a dramatic decrease in grain production. The mutants showed improved stress tolerance, especially under salinity treatment. We found four putative extra-large G proteins (PXLG)1-4 that also participate in the regulation of yield components and stress tolerance. A yeast two hybrid showed that the RGB1 might interact with PXLG2 but not with PXLG1, PXLG3 or PXLG4. CONCLUSION: These findings will not only improve our understanding of the repertoire of heterotrimeric G proteins in rice but also contribute to the application of heterotrimeric G proteins in rice breeding.
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Virus-Like Particle Mediated CRISPR/Cas9 Delivery for Efficient and Safe Genome Editing.
The discovery of designer nucleases has made genome editing much more efficient than before. The designer nucleases have been widely used for mechanistic studies, animal model generation and gene therapy development. However, potential off-targets and host immune responses are issues still need to be addressed for in vivo uses, especially clinical applications. Short term expression of the designer nucleases is necessary to reduce both risks. Currently, various delivery methods are being developed for transient expression of designer nucleases including Zinc Finger Nuclease (ZNF), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas). Recently, virus-like particles are being used for gene editing. In this review, we will talk through commonly used genome editing nucleases, discuss gene editing delivery tools and review the latest literature using virus-like particles to deliver gene editing effectors.
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