Fig. 8

Coinjection of Cas9 mRNA and Cas9 protein into zygotes. a Strategy for insertion of a Venus reporter into the mouse Rosa26 locus. sgRosa26-1 and Cas9 introduce a double-strand break between 1 kb and 0.8 kb fragments used as homology arms in the pR26-Venus targeting vector. The locations of PCR primers in the targeted and wildtype Rosa26 alleles are indicated. SA- splice acceptor, pA polyadenylation site. b Mouse zygotes were microinjected with pR26-Venus, sgRosa26-1 and Cas9 mRNA or Cas9 mRNA and protein. The embryos were cultured for 4 days and genomic DNA was isolated from 12 blastocysts each, for PCR-based detection of HDR or deletion events. Top panel: gel electrophoresis of PCR products. Targeted alleles (KI) are detected by amplification of a 1.3 kb genomic segment using the vector-specific primer VenusF and the R26R3 primer, located downstream of the vector homology region. The presence of integrated or nonintegrated vector DNA was tested using the R26F2/R2 primer pair, amplifying a 1.4 kb vector segment as well as 0.2 kb of the Rosa26 target region (middle panel). Lower panel: Rosa26 alleles with sequence deletions were detected by 0.2 kb of the target region (R26F2/R26R2 primers), followed by XbaI digestion and gel separation. XbaI resistant PCR products indicate the presence of sequence deletions (mut, 0.2 kb) whereas wildtype products are reduced to 0.12 kb fragments (wt). c Sequencing of PCR products amplified with primers VenusF and R26R3 (from B, top) showed the predicted recombination between the targeting vectors homology region and adjacent downstream genomic sequence