Generating an organ-deficient animal model using a multi-targeted CRISPR-Cas9 system

Abstract

Gene-knockout animal models with organ-deficient phenotypes used for blastocyst complementation are generally not viable. Animals need to be maintained as heterozygous mutants, and homozygous mutant embryos yield only one-fourth of all embryos. In this study, we generated organ-deficient embryos using the CRISPR-Cas9-sgRNA^ms system that induces cell death with a single-guide RNA (sgRNA^ms) targeting multiple sites in the genome. The Cas9-sgRNA^ms system interrupted cell proliferation and induced cell ablation in vitro. The mouse model had Cas9 driven by the Foxn1 promoter with a ubiquitous expression cassette of sgRNA^ms at the Rosa26 locus (Foxn1^Cas9; Rosa26_ms). It showed an athymic phenotype similar to that of nude mice but was not hairless. Eventually, a rat cell-derived thymus in an interspecies chimera was generated by blastocyst complementation of Foxn1^Cas9; Rosa26_ms mouse embryos with rat embryonic stem cells. Theoretically, a half of the total embryos has the Cas9-sgRNA^ms system because Rosa26_ms could be maintained as homozygous.