title: Heritable CRISPR/Cas9-mediated genome editing in the yellow fever mosquito, Aedes aegypti.
abstract: In vivo targeted gene disruption is a powerful tool to study gene function. Thus far, two tools for genome editing in Aedes aegypti have been applied, zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN). As a promising alternative to ZFN and TALEN, which are difficult to produce and validate using standard molecular biological techniques, the clustered regularly interspaced short palindromic repeats/CRISPR-associated sequence 9 (CRISPR/Cas9) system has recently been discovered as a "do-it-yourself" genome editing tool. Here, we describe the use of CRISPR/Cas9 in the mosquito vector, Aedes aegypti. In a transgenic mosquito line expressing both Dsred and enhanced cyan fluorescent protein (ECFP) from the eye tissue-specific 3xP3 promoter in separated but tightly linked expression cassettes, we targeted the ECFP nucleotide sequence for disruption. When supplying the Cas9 enzyme and two sgRNAs targeting different regions of the ECFP gene as in vitro transcribed mRNAs for germline transformation, we recovered four different G1 pools (5.5% knockout efficiency) where individuals still expressed DsRed but no longer ECFP. PCR amplification, cloning, and sequencing of PCR amplicons revealed indels in the ECFP target gene ranging from 2-27 nucleotides. These results show for the first time that CRISPR/Cas9 mediated gene editing is achievable in Ae. aegypti, paving the way for further functional genomics related studies in this mosquito species.
authors: Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, United States of America.
authors: Shengzhang
authors: S
authors: Dong
authors: Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, United States of America.
authors: Jingyi
authors: J
authors: Lin
authors: Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, United States of America.
authors: Nicole L
authors: NL
authors: Held
authors: Division of Biology, Kansas State University, Manhattan, Kansas, United States of America.
authors: Rollie J
authors: RJ
authors: Clem
authors: Division of Biology, Kansas State University, Manhattan, Kansas, United States of America.
authors: A Lorena
authors: AL
authors: Passarelli
authors: Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, United States of America.
authors: Alexander W E
authors: AW
authors: Franz
mesh_terms: D000330:Aedes / Q000235:genetics* / Q000472:pathogenicity; D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D049750:Genome, Insect; D006801:Humans; D054643:INDEL Mutation; D017393:RNA Editing / Q000235:genetics; D015004:Yellow Fever / Q000235:genetics* / Q000635:transmission; D016335:Zinc Fingers / Q000235:genetics