Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes
Saori Mizuno‐Iijima, Shinya Ayabe, Kanako Kato, Shogo Matoba, Yoshihisa Ikeda, Tra Thi Huong Dinh, Hoai Thu Le, Hayate Suzuki, Ken‐ichi Nakashima, Yoshikazu Hasegawa, Yuko Hamada, Yoko Tanimoto, Yoko Daitoku, Natsumi Iki, Miyuki Ishida, Elzeftawy Abdelaziz Elsayed Ibrahim, Toshiaki Nakashiba, Michito Hamada, Kazuya Murata, Yoshihiro Miwa, Miki Okada‐Iwabu, Masato Iwabu, Ken‐ichi Yagami, Atsuo Ogura, Yuichi Obata, Satoru Takahashi, Seiya Mizuno, Atsushi Yoshiki, Fumihiro Sugiyama
Abstract
Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.