Figure 2.

Generation of Rosa26-targeted mice mediated by CAS9 and long-gRNA. (A) Schematic illustration of Rosa26 gene structure and sequences of wild-type and mutated alleles around the target locus. Arrows indicate the loci of PCR primers. The targeted locus of gRNA and PAM domain were indicated in the wild-type sequence by underline and bold letters, respectively. Examples of modified-allele sequences obtained from Rosa26 targeted pups are shown below in no particular order. Deleted and inserted nucleotides are indicated by hyphens and small letters, respectively. (B) Comparison of mutation efficiencies of Rosa26 target locus among various concentrations of CAS9 mRNA and gRNA injected into pronuclear-stage embryos. Numbers in the bars indicate the numbers of mutated pups/total pups. (C) Mutation efficiencies ofpotential off-target loci for Rosa26 target sequence. Mismatch nucleotides in examined sequences are shown by bold with underlines. N.D. indicates ‘not determined’. ‘a’ denotes concentrations of CAS9 mRNA + gRNA (µg/ml). ‘b’ denotes number of total pups. The locus information of these sequences are shown in Supplementary Figure S5.