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CRISPR patent fight revived.
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[GII-derived tools link HYER to higher organisms' genome editing].
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First in vivo human genome editing trial.
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Genome editing using artificial site-specific nucleases in zebrafish.
Zebrafish is a model vertebrate suitable for genetic analysis. Forward genetic analysis via chemical mutagenesis screening has established a variety of zebrafish mutants that are defective in various types of organogenesis, and the genes responsible for the individual mutants have been identified from genome mapping. On the other hand, reverse genetic analysis via targeted gene disruption using embryonic stem (ES) cells (e.g., knockout mouse) can uncover gene functions by investigating the phenotypic effects. However, this approach is mostly limited to mice among the vertebrate models because of the difficulty in establishing ES cells. Recently, new gene targeting technologies, such as the transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, have been developed: that can directly introduce genome modifications at the targeted genomic locus. Here, we summarize these new and powerful genome editing techniques for the study of zebrafish.
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Oocyte electroporation prior to in vitro fertilization is an efficient method to generate single, double, and multiple knockout porcine embryos of interest in biomedicine and animal production.
Genetically modified pigs play a critical role in mimicking human diseases, xenotransplantation, and the development of pigs resistant to viral diseases. The use of programmable endonucleases, including the CRISPR/Cas9 system, has revolutionized the generation of genetically modified pigs. This study evaluates the efficiency of electroporation of oocytes prior to fertilization in generating edited gene embryos for different models. For single gene editing, phospholipase C zeta (PLC ζ) and fused in sarcoma (FUS) genes were used, and the concentration of sgRNA and Cas9 complexes was optimized. The results showed that increasing the concentration resulted in higher mutation rates without affecting the blastocyst rate. Electroporation produced double knockouts for the TPC1/TPC2 genes with high efficiency (79 %). In addition, resistance to viral diseases such as PRRS and swine influenza was achieved by electroporation, allowing the generation of double knockout embryo pigs (63 %). The study also demonstrated the potential for multiple gene editing in a single step using electroporation, which is relevant for xenotransplantation. The technique resulted in the simultaneous mutation of 5 genes (GGTA1, B4GALNT2, pseudo B4GALNT2, CMAH and GHR). Overall, electroporation proved to be an efficient and versatile method to generate genetically modified embryonic pigs, offering significant advances in biomedical and agricultural research, xenotransplantation, and disease resistance. Electroporation led to the processing of numerous oocytes in a single session using less expensive equipment. We confirmed the generation of gene-edited porcine embryos for single, double, or quintuple genes simultaneously without altering embryo development to the blastocyst stage. The results provide valuable insights into the optimization of gene editing protocols for different models, opening new avenues for research and applications in this field.
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Efficient generation of stable, heritable gene edits in wheat using CRISPR/Cas9.
BACKGROUND: The use of CRISPR/Cas9 systems could prove to be a valuable tool in crop research, providing the ability to fully knockout gene function in complex genomes or to precisely adjust gene function by knockout of individual alleles. RESULTS: We compare gene editing in hexaploid wheat (Triticum aestivum) with diploid barley (Hordeum vulgare), using a combination of single genome and tri-genome targeting. High efficiency gene editing, 11-17% for single genome targeted guides and 5% for tri-genome targeted guides, was achieved in wheat using stable Agrobacterium-mediated transformation. Gene editing in wheat was shown to be predominantly heterozygous, edits were inherited in a Mendelian fashion over multiple generations and no off-target effects were observed. Comparison of editing between the two species demonstrated that more stable, heritable edits were produced in wheat, whilst barley exhibited continued and somatic editing. CONCLUSION: Our work shows the potential to obtain stable edited transgene-free wheat lines in 36 weeks through only two generations and that targeted mutagenesis of individual homeologues within the wheat genome is achievable with a modest amount of effort, and without off-target mutations or the need for lengthy crossing strategies.
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Building Potent Chimeric Antigen Receptor T Cells With CRISPR Genome Editing.
Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematological and solid malignancies. However, despite the success of this field, there remain some major challenges, including accelerated T cell exhaustion, potential toxicities, and insertional oncogenesis. To overcome these limitations, recent advances in CRISPR technology have enabled targetable interventions of endogenous genes in human CAR T cells. These CRISPR genome editing approaches have unleashed the therapeutic potential of CAR T cell therapy. Here, we summarize the potential benefits, safety concerns, and difficulties in the generation of gene-edited CAR T cells using CRISPR technology.
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Crosstalk between androgen and Wnt/β-catenin leads to changes of wool density in FGF5-knockout sheep.
Fibroblast growth factor 5 (FGF5) is a famous dominant inhibitor of anagen phase of hair cycle. Mutations of FGF5 gene result in a longer wool in mice, donkeys, dogs, cats, and even in human eyelashes. Sheep is an important source of wool production. How to improve the production of wool quickly and effectively is an urgent problem to be solved. In this study, we generated five FGF5-knockout Dorper sheep by the CRISPR/Cas9 system. The expression level of FGF5 mRNA in knockout (KO) sheep decreased significantly, and all FGF5 proteins were dysfunctional. The KO sheep displayed a significant increase in fine-wool and active hair-follicle density. The crosstalk between androgen and Wnt/β-catenin signaling downstream of FGF5 gene plays a key role. We established downstream signaling cascades for the first time, including FGF5, FGFR1, androgen, AR, Wnt/β-catenin, Shh/Gli2, c-MYC, and KRTs. These findings further improved the function of FGF5 gene, and provided therapeutic ideas for androgen alopecia.
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CRISPR/Cas12a ribonucleoprotein mediated editing of tryptophan 2,3-dioxygenase of Spodoptera frugiperda.
In insect genome editing CRISPR/Cas9 is predominantly employed, while the potential of several classes of Cas enzymes such as Cas12a largely remain untested. As opposed to Cas9 which requires a GC-rich protospacer adjacent motif (PAM), Cas12a requires a T-rich PAM and causes staggered cleavage in the target DNA, opening possibilities for multiplexing. In this regard, the utility of Cas12a has been shown in only a few insect species such as fruit flies and the silkworm, but not in non-model insects such as the fall armyworm, Spodoptera frugiperda, a globally important invasive pest that defies most of the current management methods. In this regard, a more recent genetic biocontrol method known as the precision-guided sterile insect technique (pgSIT) has shown successful implementation in Drosophila melanogaster, with certain thematic adaptations required for application in agricultural pests. However, before the development of a controllable gene drive for a non-model species, it is important to validate the activity of Cas12a in that species. In the current study we have, for the first time, demonstrated the potential of Cas12a by editing an eye color gene, tryptophan 2,3-dioxygenase (TO) of S. frugiperda by microinjecting ribonucleoprotein complex into pre-blastoderm (G0) eggs. Analysis of G0 mutants revealed that all five mutants (two male and three female) exhibited distinct edits consisting of both deletion and insertion events. All five edits were further validated through in silico modeling to understand the changes at the protein level and further corroborate with the range of eye-color phenotypes observed in the present study.
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[Construction of a Sox17 activation vector based on the CRISPR/dCas9 system and its validation in sheep embryonic stem cells].
The CRISPR/dCas9 system is a gene editing tool that has proven to be highly efficient and precise. By utilizing transcriptional activators, such as VP64, p65, and Rta, the system can effectively and stably activate target genes. Sox17, a transcription factor belonging to the SOX family, plays a crucial role in the differentiation of the germ layers and the determination of cell fates during the early stages of embryonic development. Sheep embryonic stem cells (sESCs) are characterized by their capacity for self-renewal and multidirectional differentiation, serving as a significant in vitro model for studying the mechanisms of cell differentiation during early embryonic development. However, the importing of exogenous genes into sESCs is challenging due to their unique growth characteristics. The objective of this study was to investigate the conditions necessary for successfully activating Sox17 in sESCs. To this end, we employed the CRISPR/dCas9 system along with liposome transfection, lentivirus invasion, and electroporation to activate Sox17 in sESCs. The expression of Sox17 was then determined by fluorescence quantitative PCR, on the basis of which the performance of different transfection methods was compared. The results indicated that the electroporation group had the best transfection effect and the highest Sox17 expression among the three transfection methods. The efficient and stable gene activation protocol will provide a reference for embryonic stem cell research in other species, especially livestock animals, and lay the foundation for the subsequent study of gene function and realization of precise cell fate regulation by regulating gene expression in sheep embryonic stem cells.
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