title: Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti.
abstract: The mosquito Aedes aegypti is a potent vector of the chikungunya, yellow fever, and dengue viruses, responsible for hundreds of millions of infections and over 50,000 human deaths per year. Mutagenesis in Ae. aegypti has been established with TALENs, ZFNs, and homing endonucleases, which require the engineering of DNA-binding protein domains to provide genomic target sequence specificity. Here, we describe the use of the CRISPR-Cas9 system to generate site-specific mutations in Ae. aegypti. This system relies on RNA-DNA base-pairing to generate targeting specificity, resulting in efficient and flexible genome-editing reagents. We investigate the efficiency of injection mix compositions, demonstrate the ability of CRISPR-Cas9 to generate different types of mutations via disparate repair mechanisms, and report stable germline mutations in several genomic loci. This work offers a detailed exploration into the use of CRISPR-Cas9 in Ae. aegypti that should be applicable to non-model organisms previously out of reach of genetic modification.
authors.affiliation: Laboratory of Neurogenetics and Behavior, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: Kistler
authors.affiliation: Laboratory of Neurogenetics and Behavior, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA; Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Leslie B
authors.initials: LB
authors.lastname: Vosshall
authors.affiliation: Laboratory of Neurogenetics and Behavior, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA; Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10065, USA. Electronic address: ben.matthews@rockefeller.edu.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Matthews
mesh_terms: D000330:Aedes / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D004268:DNA-Binding Proteins / Q000235:genetics*; D005818:Genetic Engineering; D005822:Genetic Vectors; D049750:Genome, Insect; D016296:Mutagenesis; D009154:Mutation; D017393:RNA Editing / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins
references.citation: Nat Biotechnol. 2014 Jul;32(7):670-6
references.pmid: 24752079
references.citation: Nat Biotechnol. 2014 Mar;32(3):279-84
references.pmid: 24463574
references.citation: Nat Biotechnol. 2012 May;30(5):460-5
references.pmid: 22484455
references.citation: Science. 2014 Nov 28;346(6213):1258096
references.pmid: 25430774
references.citation: Nucleic Acids Res. 2014 Dec 16;42(22):e168
references.pmid: 25300484
references.citation: Mob DNA. 2014 Mar 03;5(1):7
references.pmid: 24589358
references.citation: Cell Rep. 2014 Nov 6;9(3):1151-62
references.pmid: 25437567
references.citation: PLoS One. 2014;9(5):e98186
references.pmid: 24873830
references.citation: Cell Rep. 2013 Jul 11;4(1):220-8
references.pmid: 23827738
references.citation: Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3748-51
references.pmid: 9520438
references.citation: Insect Mol Biol. 2002 Apr;11(2):133-9
references.pmid: 11966878
references.citation: Nature. 2013 Apr 25;496(7446):504-7
references.pmid: 23563266
references.citation: PLoS One. 2013;8(3):e60082
references.pmid: 23555893
references.citation: PLoS Genet. 2012;8(8):e1002861
references.pmid: 22916025
references.citation: Sci Rep. 2013;3:1603
references.pmid: 23549343
references.citation: PLoS Negl Trop Dis. 2013;7(10):e2486
references.pmid: 24130914
references.citation: PLoS Negl Trop Dis. 2014;8(1):e2652
references.pmid: 24498447
references.citation: Proc Natl Acad Sci U S A. 2013 Nov 19;110(47):19012-7
references.pmid: 24191015
references.citation: Nat Biotechnol. 2013 Mar;31(3):227-9
references.pmid: 23360964
references.citation: Bioinformatics. 2010 Apr 1;26(7):873-81
references.pmid: 20147302
references.citation: Nat Protoc. 2006;1(3):1312-7
references.pmid: 17406416
references.citation: Nat Genet. 2011 May;43(5):491-8
references.pmid: 21478889
references.citation: BMC Biol. 2014;12:27
references.pmid: 24731704
references.citation: Science. 2007 Jun 22;316(5832):1718-23
references.pmid: 17510324
references.citation: Development. 2014 Nov;141(21):4042-54
references.pmid: 25336735
references.citation: Nucleic Acids Res. 2010 Jul;38(Web Server issue):W462-8
references.pmid: 20435679
references.citation: G3 (Bethesda). 2013 Apr 9;3(4):657-664
references.pmid: 23550125
references.citation: Nat Biotechnol. 2013 Sep;31(9):827-32
references.pmid: 23873081
references.citation: Annu Rev Biochem. 2014;83:409-39
references.pmid: 24606144
references.citation: Nat Biotechnol. 2014 Jun;32(6):577-82
references.pmid: 24770324
references.citation: Genetics. 2014 Apr;196(4):961-71
references.pmid: 24478335
references.citation: Nature. 2013 Jun 27;498(7455):487-91
references.pmid: 23719379
references.citation: Cell. 2014 Feb 27;156(5):1060-71
references.pmid: 24581501
references.citation: Science. 2014 Mar 14;343(6176):1247997
references.pmid: 24505130
references.citation: Methods. 2014 Aug 15;69(1):38-45
references.pmid: 24556554
references.citation: G3 (Bethesda). 2013 Oct;3(10):1717-25
references.pmid: 23979928
references.citation: Annu Rev Entomol. 2014;59:205-24
references.pmid: 24160434
references.citation: Science. 2012 Aug 17;337(6096):816-21
references.pmid: 22745249