期刊
CELL RESEARCH
卷 27, 期 7, 页码 933-945出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/cr.2017.81
关键词
complete gene knockout; CRISPR/Cas9; multiple sgRNAs
类别
资金
- CAS [XDB02050007, XDA01010409]
- National High-tech R&D Program (863 Program) [2015AA020307]
- National Natural Science Foundation of China [31522037, 31500825]
- China Youth Thousand Talents Program
- Break-through project of Chinese Academy of Sciences
- Shanghai Sailing Plan for the Young Scientific Talents [15YF1414700]
- National Key Technology R&D Program of China [2014BAI03B00]
- Shanghai City Committee of science and technology project [16JC1420202, 14140900100]
- CAS Hundreds of Talents Program of China
The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.
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