title: One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.
abstract: 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.
authors: Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
authors: Haoyi
authors: H
authors: Wang
authors: Hui
authors: H
authors: Yang
authors: Chikdu S
authors: CS
authors: Shivalila
authors: Meelad M
authors: MM
authors: Dawlaty
authors: Albert W
authors: AW
authors: Cheng
authors: Feng
authors: F
authors: Zhang
authors: Rudolf
authors: R
authors: Jaenisch
mesh_terms: D000818:Animals; D001483:Base Sequence; D053595:Embryonic Stem Cells / Q000378:metabolism; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D008297:Male; D051379:Mice / Q000235:genetics*; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D058727:RNA, Small Untranslated
chemical_list: D058727:RNA, Small Untranslated
references: Nat Biotechnol. 2013 Jan;31(1):23-4
references: 23302927
references: Nat Biotechnol. 2013 Mar;31(3):227-9
references: 23360964
references: Nature. 2011 Sep 04;477(7366):606-10
references: 21892189
references: Annu Rev Genet. 2011;45:247-71
references: 21910633
references: Nat Biotechnol. 2013 Mar;31(3):233-9
references: 23360965
references: Proc Natl Acad Sci U S A. 2013 Mar 5;110(10):3782-7
references: 23426636
references: Blood. 2011 Oct 27;118(17):4509-18
references: 21803851
references: Cell. 2011 Jul 22;146(2):318-31
references: 21757228
references: Dev Cell. 2013 Feb 11;24(3):310-23
references: 23352810
references: Science. 2011 Sep 30;333(6051):1843-6
references: 21960622
references: Science. 2012 Aug 17;337(6096):816-21
references: 22745249
references: Methods Mol Biol. 2008;435:63-77
references: 18370068
references: Science. 2013 Feb 15;339(6121):819-23
references: 23287718
references: Cell Res. 2013 Apr;23(4):465-72
references: 23528705
references: Nat Methods. 2011 Jul 17;8(9):753-5
references: 21765410
references: Methods Mol Biol. 2010;649:247-56
references: 20680839
references: Science. 2009 Jul 24;325(5939):433
references: 19628861
references: Nat Rev Genet. 2010 Sep;11(9):636-46
references: 20717154
references: Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2579-86
references: 22949671
references: Nature. 2012 Feb 15;482(7385):331-8
references: 22337052
references: Cell Stem Cell. 2011 Aug 5;9(2):166-75
references: 21816367
references: Science. 2010 Jan 8;327(5962):167-70
references: 20056882
references: Cell Res. 2013 May;23(5):720-3
references: 23545779
references: Nat Biotechnol. 2011 Jan;29(1):64-7
references: 21151125
references: Nat Biotechnol. 2011 Aug 05;29(8):695-6
references: 21822240
references: Genetics. 2010 Oct;186(2):451-9
references: 20628038
references: Trends Genet. 2008 Nov;24(11):529-38
references: 18809224
references: Proc Natl Acad Sci U S A. 2010 Aug 24;107(34):15022-6
references: 20686113
references: Nat Biotechnol. 2013 Mar;31(3):230-2
references: 23360966
references: Cancer Cell. 2011 Jul 12;20(1):11-24
references: 21723200
references: Nat Rev Genet. 2005 Jun;6(6):507-12
references: 15931173
references: Nat Rev Cancer. 2010 Oct;10(10):696-706
references: 20844553
references: Science. 2013 Feb 15;339(6121):823-6
references: 23287722
references: Nat Rev Cancer. 2009 Jun;9(6):389-99
references: 19461666