CRISPR/Cas9-mediated genome editing in wild-derived mice; Generation of tamed wild-derived strains by mutation of the a (nonagouti) gene

Wild-derived mice have contributed to mouse genetics by their genetic diversity that may increase the chance of identifying novel modifier genes responsible for specific phenotypes and diseases. However, gene-targeting using wild-derived mice has been unsuccessful due to the unavailability of stable embryonic stem cells. Here, we report that the CRISPR/Cas9-mediated gene-targeting can be applied to the Japanese wild-derived MSM/Ms strain. We targeted the nonagouti (a) gene encoding the agouti protein localized in hairs and the brain. We obtained three homozygous knockout mice as founders, all showing black coat color. While homozygous knockout offspring were physiologically indistinguishable from wild-type littermates, they showed specific domesticated behaviors; a high locomotion during the light period and a decline in the avoidance of a human hand. These phenotypes were consistent over the subsequent generations. Our findings support the empirical hypothesis that nonagouti is a domestication gene, which might repress aggressive behavior. Global gene expression patterns in the midbrain of wildtype and nonagouti (a) homogygous mutant MSM/Ms mice were analyzed by one-color Mouse Gene Expression 8x60K microarray. The midbrain tissues were manually isolated from 8-9 weeks old mice under dissecting microscope. Total RNA was purified from dissected midbrain using Trizol (Thermo Fisher Scientific). Purified total RNA was amplified and labeled with Cy3 using Low-Input QuickAmp Labeling Kit (Agilent Technologies). Cy3-labeled RNAs were hybridized to SurePrint G3 Mouse Gene Expression v2 8x60K Microarray Kit (Agilent Technologies) at 65 °C for 17h. After wash, the hybridized slides were scanned with DNA microarray scanner (Agilent Technologies). The scanned images were processed with Feature Extraction software (Agilent Technologies) to extract signal intensities of each probe. The extracted signal data were imported into the Gene Spring GX 13.1.1 software (Agilent Technologies) and normalized using the default settings. Two and six biological replicates were performed in wildtype and mutant group, respectively.