Although genetically modified mice can be generated with high efficiency by using CRISPR/Cas9-mediated genome editing in mouse zygotes, only the loci with a protospacer-adjacent motif (PAM) sequence are targetable. The present study investigated the usability of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes. In addition to the 5'-NGG sequence, SpCas9-NG recognized the 5'-NGA, 5'-NGC and 5'-NGT sequences in mouse zygotes as PAMs that were appropriate for the generation of knockout mice. Moreover, SpCas9-NG-mediated genome editing enabled the generation of knock-in mice untargetable by the conventional SpCas9 in mouse zygotes. These results suggest that SpCas9-NG-mediated genome editing in zygotes is available for the generation of knockout and knock-in mice at the locus corresponding to NGN-PAM.