One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering.

Taking advantage of the multiplexable genome engineering feature of the CRISPR/Cas9 system, we sought to generate different kinds of immunodeficient mouse strains by embryo co-microinjection of Cas9 mRNA and multiple sgRNAs targeting mouse B2m, Il2rg, Prf1, Prkdc, and Rag1. We successfully achieved multiple gene modifications, fragment deletion, double knockout of genes localizing on the same chromosome, and got different kinds of immunodeficient mouse models with different heritable genetic modifications at once, providing a one-step strategy for generating different immunodeficient mice which represents significant time-, labor-, and money-saving advantages over traditional approaches. Meanwhile, we improved the technology by optimizing the concentration of Cas9 and sgRNAs and designing two adjacent sgRNAs targeting one exon for each gene, which greatly increased the targeting efficiency and bi-allelic mutations.