Figure 3.

Production of the Zurich variant mouse.A, Zurich variant mouse CRISPR/Cas9 design scheme. Guide RNAs consisting of 20 base pairs each are boxed; break sites are indicated. Lower part shows repair DNA, which introduces new eight amino acids. B, PCR products of 460 bp in wild type (+/+) and 446 bp in the mutant (z/z) were generated using genotyping primers (refer to the Experimental procedures section for the sequences). Mutant PCR product had NsiI restriction site, while wild type did not. PCR products were then digested with NsiI enzyme resulting in appearance of ∼300 bp band for the mutant. Heterozygous (z/+) mice had both undigested and digested bands. C, representative Sanger sequencing of gDNA isolated from homozygous F2 Zurich variant mouse.