title: RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency.
abstract: Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.
authors.affiliation: Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Song
authors.affiliation: Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
authors.forename: Dongshan
authors.initials: D
authors.lastname: Yang
authors.affiliation: Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Xu
authors.affiliation: Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
authors.forename: Tianqing
authors.initials: T
authors.lastname: Zhu
authors.affiliation: Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
authors.forename: Y Eugene
authors.initials: YE
authors.lastname: Chen
authors.affiliation: Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
authors.forename: Jifeng
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D064113:CRISPR-Cas Systems; D059766:DNA End-Joining Repair / Q000187:drug effects*; D005260:Female; D055879:Gene Knock-In Techniques; D018390:Gene Targeting; D011743:Pyrimidines / Q000494:pharmacology*; D011817:Rabbits; D059767:Recombinational DNA Repair / Q000187:drug effects*; D012545:Schiff Bases / Q000494:pharmacology*; D013449:Sulfonamides / Q000494:pharmacology*
chemical_list: C000605335:3-((benzylamino)sulfonyl)-4-bromo-N-(4-bromophenyl)benzamide; C578537:5,6-bis(benzylideneamino)-2-mercaptopyrimidin-4-ol; D001549:Benzamides; D011743:Pyrimidines; D012545:Schiff Bases; D013449:Sulfonamides
references.citation: J Cell Biol. 2012 Dec 24;199(7):1067-81
references.pmid: 23266955
references.citation: Nat Biotechnol. 2013 Mar;31(3):227-9
references.pmid: 23360964
references.citation: Cell. 2013 May 9;153(4):910-8
references.pmid: 23643243
references.citation: Transgenic Res. 2015 Jun;24(3):381-96
references.pmid: 25603988
references.citation: Nat Biotechnol. 2013 Aug;31(8):684-6
references.pmid: 23929337
references.citation: Nat Biotechnol. 2013 Sep;31(9):827-32
references.pmid: 23873081
references.citation: Cell. 2013 Sep 12;154(6):1370-9
references.pmid: 23992847
references.citation: Cell Rep. 2013 Jul 11;4(1):220-8
references.pmid: 23827738
references.citation: Genetics. 2013 Nov;195(3):703-13
references.pmid: 23979585
references.citation: J Vis Exp. 2013;(81):e50957
references.pmid: 24301055
references.citation: J Mol Cell Biol. 2014 Feb;6(1):97-9
references.pmid: 24403564
references.citation: Sci Rep. 2014;4:4863
references.pmid: 24798879
references.citation: Methods Enzymol. 2014;546:215-50
references.pmid: 25398343
references.citation: Elife. 2014;3:e04766
references.pmid: 25497837
references.citation: Genetics. 2015 Jan;199(1):1-15
references.pmid: 25271304
references.citation: Nat Med. 2015 Feb;21(2):121-31
references.pmid: 25654603
references.citation: Cell Stem Cell. 2015 Feb 5;16(2):142-7
references.pmid: 25658371
references.citation: Nat Biotechnol. 2015 May;33(5):538-42
references.pmid: 25798939
references.citation: Nat Biotechnol. 2015 May;33(5):543-8
references.pmid: 25803306
references.citation: J Biol Chem. 2009 May 22;284(21):14326-36
references.pmid: 19329439
references.citation: Proc Natl Acad Sci U S A. 2008 Oct 14;105(41):15848-53
references.pmid: 18840682
references.citation: DNA Repair (Amst). 2008 May 3;7(5):686-93
references.pmid: 18243065
references.citation: DNA Repair (Amst). 2005 Jun 8;4(6):687-98
references.pmid: 15878310
references.citation: Cell. 2012 Dec 21;151(7):1474-87
references.pmid: 23260137
references.citation: Nucleic Acids Res. 2011 Jul;39(12):e82
references.pmid: 21493687
references.citation: Nat Biotechnol. 2011 Jan;29(1):64-7
references.pmid: 21151125
references.citation: Nat Struct Mol Biol. 2010 Oct;17(10):1255-9
references.pmid: 20871616
references.citation: Nat Protoc. 2013 Nov;8(11):2281-308
references.pmid: 24157548