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. 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 and a small litter size.
Many studies have attempted to produce gene-modified animals without mosaicism in one step, especially for large animals. The attempts include injection of Cas9 mRNA and sgRNAs into the oocyte rather than the zygote, Cas9 protein injection, or dual sgRNAs injection. However, they achieved complete biallelic knockout animals with relatively low efficiency.
In a recent study published in Cell Research, Dr. YANG Hui’s Lab, Dr. XIONG Zhiqi’s and Dr. SUN Qiang’s team at the Institute of Neuroscience of Chinese Academy of Sciences demonstrated that using a modified “C-CRISPR” approach, single gene or multiple genes could be nearly 100% deleted in all cells of CRISPR/Cas9-injected mice and monkeys.
Using the CRISPR/Cas9 system (C-CRISPR hereafter), researchers 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, 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.
They then 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. They also performed comprehensive off-target analysis on the animals produced by multiple-sgRNA targeting and found no obvious off-target effects.
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.
This work was supported by the CAS Strategic Priority Research Program, the National Hightech R&D Program, the National Natural Science Foundation of China, China Youth Thousand Talents Program, Breakthrough Project of Chinese Academy of Sciences, etc.
Figure: One-step Generation of Complete Gene Knockout Mice and Monkeys by CRISPR/Cas9 - Mediated Gene Editing with Multiple sgRNAs (Image by YANG Hui's group)
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