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

Generation of genetically modified mice using SpCas9-NG engineered nuclease.

Author information

1
Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. awtrfj@mail.ecc.u-tokyo.ac.jp.
2
Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.

Abstract

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.

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