Fig. 1.

HypaCas9 exhibits reduced off-target effects without compromising on-target activity in the mouse zygote. a Genomic DNA sequence of the Gt(ROSA)26Sor-gRNA targeting site and off-target site, and the design of matched and singly mismatched gRNAs. The mismatched nucleotides are shown in red. The locus information is based on GRCm38.p4. b Comparison of on- and off-target activity between WT-Cas9 and HypaCas9 in the mouse zygote. Cas9 mRNA was injected into zygotes with Gt(ROSA)26Sor-targeting matched gRNA and the mutation efficiency was evaluated at the blastocyst stage. c Evaluation of single mismatch tolerance of WT-Cas9 and HypaCas9. gRNA with a single mismatch at position 1, 5, 10, or 18 was injected into mouse zygotes. In (b and c), numbers above the bar indicate the numbers of mutated embryos per total embryos from two replicate experiments