Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes

•Novel CRISPR-Cas vectors with a Cas9 fused to a part of mouse Cdt1.•Nuclear localisation of Cdt1-fused Cas9 in mammalian cells and embryos.•High efficiency in megabase-scale large deletion and knock-in mouse production. 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.