RRC ID 60802
Author Mizuno-Iijima S, Ayabe S, Kato K, Matoba S, Ikeda Y, Dinh TTH, Le HT, Suzuki H, Nakashima K, Hasegawa Y, Hamada Y, Tanimoto Y, Daitoku Y, Iki N, Ishida M, Ibrahim EAE, Nakashiba T, Hamada M, Murata K, Miwa Y, Okada-Iwabu M, Iwabu M, Yagami KI, Ogura A, Obata Y, Takahashi S, Mizuno S, Yoshiki A, Sugiyama F.
Title Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes.
Journal Methods
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.
Published 2020-4-22
DOI 10.1016/j.ymeth.2020.04.007
PII S1046-2023(19)30328-7
PMID 32334080
IF 3.782
Resource
DNA material T7-NLS hCas9-pA (RDB13130) pT7-hCas9-mC-polyA (RDB14423) T7-3xFLAG-NLS-hCAS9-NLS-pA (RDB14602) T7-3xFLAG-NLS-hCAS9-Cdt1-NLS-pA (RDB14604) px330-mC (RDB14406) pX330-Tyr-L (RDB14410) pX330-Tyr-R (RDB14411) pX330-mC-Tyr-L (RDB14412) pX330-mC-Tyr-R (RDB14413) pX330-Dmd-L (RDB16004) pX330-Dmd-R (RDB16005) pX330-mC-Dmd-L (RDB16006) pX330-mC-Dmd-R (RDB16007)