The development of CRISPR/Cas9 technology has vastly sped up the process of mammalian genome editing by introducing a bacterial system that can be exploited for reverse genetics-based research. However, generating homozygous functional knockout (KO) animals using traditional CRISPR/Cas9-mediated techniques requires three generations of animals. A founder animal with a desired mutation is crossed to produce heterozygous F1 offspring which are subsequently interbred to generate homozygous F2 KO animals. This study describes an adaptation of the CRISPR/Cas9-mediated method to develop a cohort of homozygous gene-targeted KO animals in one generation. A well-characterized ethanol-responsive gene, MyD88, was chosen as a candidate gene for generation of KO mice as proof-of-concept. Previous studies have reported changes in ethanol-related behavioral outcomes in MyD88 KO mice. One-cell mouse embryos were simultaneously electroporated with four gRNAs targeting a critical Exon of MyD88 along with Cas9 protein. DNA and RNA analysis of founder mice revealed a complex mix of genetic alterations, all of which were predicted to ablate MyD88 gene function. Behavioral testing confirmed the hypothesis that successful one-generation KO of MyD88 would reproduce the decreased ethanol-induced sedative/hypnotic effects and increased ethanol consumption in males that were observed in previous studies. This study additionally compared responses of Mock treatment control mice generated through electroporation to controls purchased from a vendor. No substantial behavioral changes were noted between control cohorts. Overall, the CRISPR/Cas9 KO protocol reported here, which we call CRISPR Turbo Accelerated KnockOut (CRISPy TAKO), will be useful for reverse genetic in vivo screens of gene function in whole animals.
Keywords: CRISPR/Cas9; accelerated phenotypic screening; ethanol; functional knockout; genome editing; increased mutagenesis.