TY - JOUR
T1 - One-step generation of mice carrying mutations in multiple genes by CRISPR/cas-mediated genome engineering
AU - Wang, Haoyi
AU - Yang, Hui
AU - Shivalila, Chikdu S.
AU - Dawlaty, Meelad M.
AU - Cheng, Albert W.
AU - Zhang, Feng
AU - Jaenisch, Rudolf
N1 - Funding Information:
We thank Ruth Flannery and Kibibi Ganz for support with animal care and experiments. We thank Jaenisch lab members for helpful discussions on the manuscript. We are also grateful to G. Grant Welstead and Daniel B. Dadon for the help of editing the manuscript. M.M.D is a Damon Runyon Postdoctoral Fellow. A.W.C. is supported by a Croucher scholarship. R.J. is an adviser to Stemgent and a cofounder of Fate Therapeutics. This work was supported by NIH grants R37-HD045022 and R01-CA084198 to RJ.
PY - 2013/5/9
Y1 - 2013/5/9
N2 - 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.
AB - 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.
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U2 - 10.1016/j.cell.2013.04.025
DO - 10.1016/j.cell.2013.04.025
M3 - Article
C2 - 23643243
AN - SCOPUS:84877707375
SN - 0092-8674
VL - 153
SP - 910
EP - 918
JO - Cell
JF - Cell
IS - 4
ER -