title: Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.
abstract: A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-8240, USA.
authors.forename: Li-En
authors.initials: LE
authors.lastname: Jao
authors.forename: Susan R
authors.initials: SR
authors.lastname: Wente
authors.forename: Wenbiao
authors.initials: W
authors.lastname: Chen
mesh_terms: D000818:Animals; D001947:Breeding / Q000379:methods; D003851:Deoxyribonucleases / Q000235:genetics; D055786:Gene Knockout Techniques / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome / Q000235:genetics*; D055029:Inverted Repeat Sequences / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010641:Phenotype; D015027:Zebrafish / Q000235:genetics*
chemical_list: D003851:Deoxyribonucleases
keywords: RNA-guided mutagenesis; genome engineering; pigmentation
references.citation: Dev Biol. 2011 Feb 15;350(2):405-13
references.pmid: 21146516
references.citation: Microbiology (Reading). 2009 Mar;155(Pt 3):733-740
references.pmid: 19246744
references.citation: Nat Genet. 1998 Apr;18(4):345-9
references.pmid: 9537416
references.citation: Nat Rev Mol Cell Biol. 2013 Jan;14(1):49-55
references.pmid: 23169466
references.citation: Nat Biotechnol. 2013 Mar;31(3):230-2
references.pmid: 23360966
references.citation: Annu Rev Genet. 2011;45:273-97
references.pmid: 22060043
references.citation: Science. 2012 Aug 17;337(6096):816-21
references.pmid: 22745249
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 31;101(35):12792-7
references.pmid: 15256591
references.citation: Nat Biotechnol. 2013 Sep;31(9):822-6
references.pmid: 23792628
references.citation: Science. 2013 Feb 15;339(6121):819-23
references.pmid: 23287718
references.citation: Nature. 2005 Jun 2;435(7042):646-51
references.pmid: 15806097
references.citation: Curr Opin Microbiol. 2011 Jun;14(3):321-7
references.pmid: 21531607
references.citation: Science. 2005 Dec 16;310(5755):1782-6
references.pmid: 16357253
references.citation: Nat Biotechnol. 2013 Mar;31(3):227-9
references.pmid: 23360964
references.citation: Nat Rev Genet. 2003 Sep;4(9):690-700
references.pmid: 12951570
references.citation: Nat Biotechnol. 2013 Mar;31(3):233-9
references.pmid: 23360965
references.citation: Cell. 2013 May 9;153(4):910-8
references.pmid: 23643243
references.citation: J Invest Dermatol. 2004 Nov;123(5):924-9
references.pmid: 15482481
references.citation: Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2579-86
references.pmid: 22949671
references.citation: Nature. 2012 Feb 15;482(7385):331-8
references.pmid: 22337052
references.citation: Science. 1998 Nov 27;282(5394):1711-4
references.pmid: 9831563
references.citation: Genes Dev. 2011 May 15;25(10):1065-77
references.pmid: 21576266
references.citation: Development. 1997 Aug;124(16):3157-65
references.pmid: 9272956
references.citation: Development. 2012 Apr;139(7):1316-26
references.pmid: 22357925
references.citation: Development. 1999 Sep;126(17):3757-67
references.pmid: 10433906
references.citation: Science. 2007 Feb 2;315(5812):646-9
references.pmid: 17272721
references.citation: Nat Neurosci. 2004 Dec;7(12):1329-36
references.pmid: 15516923
references.citation: Nucleic Acids Res. 2013 Apr;41(7):4336-43
references.pmid: 23460208
references.citation: Science. 2013 Feb 15;339(6121):823-6
references.pmid: 23287722
references.citation: Nature. 2011 Mar 31;471(7340):602-7
references.pmid: 21455174
references.citation: Development. 2006 Sep;133(18):3563-73
references.pmid: 16914496
references.citation: Elife. 2013 Jan 29;2:e00471
references.pmid: 23386978
references.citation: Cell Res. 2013 Apr;23(4):465-72
references.pmid: 23528705