Abstract
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.
Highlights
Clustered regulatory interspaced short palindromic repeats (CRISPR) paired with CRISPR associated protein 9 (Cas9) is currently the dominant and preferred gene editing tool in scientific research
We report that our CRISPR protocol can reliably produce a large number of F0 KO animals and that the ethanol phenotype of MyD88 CRISPy TAKOs largely recapitulates results previously reported for traditional MyD88 global KOs
Preliminary testing of the CRISPy TAKO method occurred in vitro using embryos electroporated at the one-cell stage, cultured until the blastocyst stage, genotyped (Figure 2)
Summary
Clustered regulatory interspaced short palindromic repeats (CRISPR) paired with CRISPR associated protein 9 (Cas9) is currently the dominant and preferred gene editing tool in scientific research. Single generation F0 MyD88 KO animals were hypothesized to exhibit decreased ethanol-induced sedative/hypnotic effects, decreased sensitivity to ethanol-induced motor ataxia, and a male-specific increase in ethanol consumption relative to controls To further streamline this accelerated KO mouse protocol, we reasoned that for first pass screening of genes for behavioral phenotypes, isogenic animals purchased directly from a vendor could be used as a control group for comparison to KOs. one concern is that the CRISPR and/or electroporation procedures, irrespective of the gene being mutated, could hypothetically exert uncharacterized deleterious effects. We report that our CRISPR protocol can reliably produce a large number of F0 KO animals and that the ethanol phenotype of MyD88 CRISPy TAKOs largely recapitulates results previously reported for traditional MyD88 global KOs. for the behaviors tested in this study, vendor purchased mice and Mock treatment controls did not differ substantially. This method has moderately high throughput and will be especially useful for phenotypes, such as behavioral responses, that cannot be assayed in vitro
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