[HTML][HTML] One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering

H Wang, H Yang, CS Shivalila, MM Dawlaty… - cell, 2013 - cell.com
H Wang, H Yang, CS Shivalila, MM Dawlaty, AW Cheng, F Zhang, R Jaenisch
cell, 2013cell.com
Mice carrying mutations in multiple genes are traditionally generated by sequential
recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a
single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting
technology with the potential for multiplexed genome editing. We demonstrate that
CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1,
2, 3, Sry, Uty-8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection …
Summary
Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.
cell.com