A recent study published in Cell Research demonstrated that single gene or multiple genes could be nearly 100% deleted in all cells of CRISPR/Cas9-injected mice and monkeys, using a modified “C-CRISPR” approach. This work was performed by researchers in Dr. YANG Hui’s Lab, Dr. XIONG Zhiqi’s and Dr. SUN Qiang’s team at the Institute of Neuroscience, Chinese Academy of Sciences. This work optimized the CRISPR/Cas9 technology by zygotic injection of Cas9 mRNA and multiple single-guide RNAs (sgRNAs) (spaced 10-200 bp apart) that target a single key exon. The approach can be particularly useful for large-scale (10-100 genes) functional screening in F0 mice and for rapidly establishing gene-edited monkey models.

　　The CRISPR/Cas9 system is an efficient gene editing method, but the majority of gene-edited animals showed mosaicism, impeding effective phenotypic analysis in the F0 generation. Furthermore, The problem of mosaicism becomes even more serious when the CRISPR/Cas9 method is applied to large animals such as macaque monkeys, which have a long breeding cycle (5-6 years) and a small litter size (1 per birth). Many previous studies have attempted to produce gene-modified animals without mosaicism in one step, especially for large animals. These include injection of Cas9 mRNA and sgRNAs into the oocyte rather than the zygote, Cas9 protein injection, or dual sgRNAs injection. However, these approaches achieved complete biallelic knockout animals with relatively low efficiency.

　　In this study, the authors first demonstrated that one or both alleles of the gene of interest could be completely knocked out in the mouse embryo by a cocktail of targeting sgRNAs using the CRISPR/Cas9 system (C-CRISPR hereafter), where a single exon is targeted with two or more closely spaced sgRNAs. Such high efficiency in complete gene knockout appears to be caused by a combination of frame-shift mutationd and exon deletiond. The authors also demonstrated the usefulness of this approach in rapid screening of gene functions in F0 mice and in the rapid generation of monkeys with complete knockout of a specific gene. Finally, they performed comprehensive off-target analysis on the animals produced by multiple-sgRNA targeting and found no obvious off-target effects.

　　The study entitled “One-step Generation of Complete Gene Knockout Mice and Monkeys by CRISPR/Cas9 - Mediated Gene Editing with Multiple sgRNAs” was published on June 6th, 2017 in Cell Research. ZUO Erwei, CAI Yi-Jun, LI Kui, WEI Yu and WANG Bang-An are the first authors with equal contribution. This work was supported by the CAS Strategic Priority Research Program (XDB02050007, XDA01010409), the National Hightech R&D Program (863 Program; 2015AA020307), the National Natural Science Foundation of China (NSFC grants 31522037 and 31500825),  Breakthrough Project of Chinese Academy of Sciences, Shanghai Sailing Plan for the Young Scientific Talents (15YF1414700), the National Key Technology R&D Program of China (2014BAI03B00 to QS), Shanghai City Committee of Science and Technology (16JC1420202 to HY; 14140900100 to QS).

　　One-step Generation of Complete Gene Knockout Mice and Monkeys by CRISPR/Cas9 - Mediated Gene Editing with Multiple sgRNAs

　　A. Schematic diagram of sgRNA-targeting sites at Tyr locus.

　　B. Co-injection of sgRNA cocktail and Cas9 mRNA into mouse zygote.

　　C. Schematic diagram of sgRNA-targeting sites at Prrt2 locus.

　　D. Co-injection of sgRNA cocktail and Cas9 mRNA into monkey zygote.