Figure 3: Recovery of three distinct coat-colour mutations by CRISPR/Cas.

(a) Schematic illustration of three coat-colour mutations in rats. albino (Tyr^c): SNP missense mutation in the Tyr gene. non-agouti (Asip^a): 19-bp deletion in exon 2 of the Agouti signalling protein (Asip) gene. hooded (Kit^h): integration of an 7,098-bp endogenous retrovirus (ERV) element within the first intron of the Kit gene. (b) Coat-colour phenotypes (C, a, h) recovered from albino by injecting gRNA:Tyr^c, Cas9 mRNA, and ssODN of the Tyr^C allele into F344 rat embryos (c, a, h). (c) Sequence analysis of the targeted Tyr exon 2 in the injected F344 rats. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise HDR-mediated SNP exchange of Tyr^C. (d) Recovery of the non-agouti phenotype by injecting gRNA:Asip^a, Cas9 mRNA, and ssODN of the Asip^A allele into F344 rat embryos. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise short DNA fragment integration of the Asip^A gene. (e) Recovery of the hooded mutation by injecting gRNA:Kit^h-1, gRNA:Kit^h-2, and ssODN of the Kit^H allele into F344 rat embryos. Cas9 and two gRNAs with ssODN mediated the introduction of indel mutations at the targeted Kit^h-1 locus, and the precise large deletion between the two cutting edges of Kit^H. (f) PCR analysis of the injected F344 rats using primers designed against each outer side of the two LTR sequences. M: DNA marker phiX174-HaeIII digest.