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

Abstract

SummaryGene-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-sgRNAmssystem that induces cell death with a single-guide RNA (sgRNAms) targeting multiple sites in the genome. The Cas9-sgRNAmssystem interrupted cell proliferation and induced cell ablationin vitro. The mouse model had Cas9 driven by theFoxn1promoter with a ubiquitous expression cassette of sgRNAmsat theRosa26locus (Foxn1Cas9;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 interspecific chimera was generated by blastocyst complementation ofFoxn1Cas9;Rosa26_msmouse embryos with rat embryonic stem cells. Theoretically, half of the total embryos have the Cas9-sgRNAmssystem because Rosa26_ms could be maintained as homozygous.