Heritable CRISPR/Cas9-Mediated Genome Editing in the Yellow Fever Mosquito, Aedes aegypti

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dc.contributor.author Dong, S. Z.
dc.contributor.author Lin, J. Y.
dc.contributor.author Held, N. L.
dc.contributor.author Clem, Rollie J.
dc.contributor.author Passarelli, A. L.
dc.contributor.author Franz, A. W. E.
dc.date.accessioned 2016-04-04T22:13:52Z
dc.date.available 2016-04-04T22:13:52Z
dc.identifier.uri http://hdl.handle.net/2097/32248
dc.description Citation: Dong, S. Z., Lin, J. Y., Held, N. L., Clem, R. J., Passarelli, A. L., & Franz, A. W. E. (2015). Heritable CRISPR/Cas9-Mediated Genome Editing in the Yellow Fever Mosquito, Aedes aegypti. Plos One, 10(3), 13. doi:10.1371/journal.pone.0122353
dc.description 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.
dc.relation.uri https://doi.org/10.1371/journal.pone.0122353
dc.rights Attribution 4.0 International (CC BY 4.0)
dc.rights.uri http://creativecommons.org/licenses/by/4.0/
dc.subject Guided Cas9 Nuclease
dc.subject Targeted Mutagenesis
dc.subject Bombyx-Mori
dc.subject Rna
dc.subject Interference
dc.subject Drosophila
dc.title Heritable CRISPR/Cas9-Mediated Genome Editing in the Yellow Fever Mosquito, Aedes aegypti
dc.type Article
dc.date.published 2015
dc.citation.doi 10.1371/journal.pone.0122353
dc.citation.issn 1932-6203
dc.citation.issue 3
dc.citation.jtitle PLoS One
dc.citation.spage 13
dc.citation.volume 10
dc.contributor.authoreid rclem
dc.contributor.authoreid lpassar

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