Fig. 1. Overlapping sgRNAs mediate KI of ssODN-containing loxP-mcs-loxP2272 sequences at the mouse Rosa26 locus.

a Schematic diagram of the strategy for precise KI of the ssODN including 45-nt homology arms (green bars) and a 110-nt insertion consisting of 34 nt of the loxP sequence, 34 nt of the loxP2272 sequence, and a multiple cloning site (MCS) into intron 1 of the Rosa26 locus. b After microinjection of overlapping sgRNAs, Cas9 mRNA, and ssODNs, PCR-based ssODN-mediated KI efficiency was measured and is represented as bar graphs. KI (%) indicates the ratio of KI embryos derived from total blastocysts or KI pups from total pups born. c PCR-based genotyping of pups using specific primers (wild type, 385 bp; KI, 495 bp). d Summary of the sequencing results from founder pups. The frequencies of each sequence (black letters) are shown as numbers of KI clones over the total examined clones