Generation of genetically modified mice using SpCas9-NG engineered nuclease

• Published in: Scientific reports Vol. 9; no. 1; pp. 12878 - 6
• Main Authors: Fujii, Wataru, Ito, Haruka, Kanke, Takuya, Ikeda, Arisa, Sugiura, Koji, Naito, Kunihiko
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 09.09.2019; Nature Publishing Group UK
• Subjects: Animals; Base Sequence; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; Gene Editing - methods; Genetic Engineering; Genome editing; Genomes; Mice; Mice, Transgenic; Nuclease; Rodents; Streptococcus pyogenes - enzymology; Zygote - metabolism; Zygotes
• ISSN: 2045-2322; 2045-2322
• 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.