TY - JOUR
T1 - Heritable CRISPR/Cas9-mediated genome editing in the yellow fever mosquito, Aedes aegypti
AU - Dong, Shengzhang
AU - Lin, Jingyi
AU - Held, Nicole L.
AU - Clem, Rollie J.
AU - Passarelli, A. Lorena
AU - Franz, Alexander W.E.
N1 - Publisher Copyright:
© 2015 Dong et al.
PY - 2015/3/27
Y1 - 2015/3/27
N2 - 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/CRISPRassociated sequence 9 (CRISPR/Cas9) system has recently been discovered as a "do-ityourself" 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 3×P3 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.
AB - 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/CRISPRassociated sequence 9 (CRISPR/Cas9) system has recently been discovered as a "do-ityourself" 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 3×P3 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.
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U2 - 10.1371/journal.pone.0122353
DO - 10.1371/journal.pone.0122353
M3 - Article
C2 - 25815482
AN - SCOPUS:84929492506
SN - 1932-6203
VL - 10
JO - PLoS One
JF - PLoS One
IS - 3
M1 - e0122353
ER -