Generation of genetically modified mice using SpCas9-NG engineered nuclease

Sci Rep. 2019 Sep 9;9(1):12878. doi: 10.1038/s41598-019-49394-5.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism*
  • Gene Editing / methods*
  • Genetic Engineering*
  • Mice
  • Mice, Transgenic
  • Streptococcus pyogenes / enzymology*
  • Zygote / metabolism


  • CRISPR-Associated Protein 9