In a recent PLoS One paper, Dong et al. (2015) describe their successful attempts to edit the genome of Aedes aegypti using CRISPR/Cas9.
As proof of principle, they aimed to excise a portion of a transgene that codes for enhanced cyan fluorescent protein (ECFP) in the Ae. aegypti transgenic line PubB2 P61. This line harbors two copies of a transgenic insert each containing DsRed and ECFP expressed under the eye-specific promoter 3xP3. Use of this construct allowed the authors to visually identify ECFP-specific knock-outs, which still expressed DsRed, from their wild-type siblings.
Ae. aegypti with stable germline modifications resulting in ECFP knockouts were obtained at an estimated rate of 5.5%. Knockout phenotypes were associated with one of three different variable length deletions, one to four nucleotides downstream from the PAM recognition site .
Along the way, the authors encountered two surprising results useful to any researcher interested in applying CRISPR to their insect system.
Germline transformation was only achieved via Cas9 RNA-injection, but not through DNA-injection of Cas9-expressing plasmids. There are multiple methods of delivery for the Cas protein and sgRNAs used for CRISPR genome editing. These include injection of plasmids containing DNA sequences that code for needed elements, injecting in vitro made mRNAs, and directly injecting purified Cas protein in the organism. None of the embryos that were injected with plasmid DNA produced transformed offspring. This was surprising because injection of plasmid DNA was sufficient for transformation in both Drosophila melanogaster and Bombyx mori, and the promoters used to drive expression of CRISPR/Cas9 had previously been successful at driving gene expression in Ae. aegypti. These results by Dong et al. (2015) were substantiated in another recent study of CRISPR/Cas9 in Ae. aegypti, where Kistler et al. (2015) were also unable to achieve germline transformation through Cas9-expressing plasmid DNA delivery methods.
Not all guide RNAs are created equal. The goal of the authors was to excise a long DNA fragment from their transgene, and so simultaneously injected two different sgRNAs targeting two different nucleotide sequences 450 bp apart. Small deletions were only produced downstream of one sgRNA. This was surprising because the authors had taken great care to ensure that the GC content, a factor known to influence sgRNA efficacy, was equal for each of their sgRNAs.
This research contributes to the vector biology community by providing time-saving insights for developing anti-pathogenic strains of mosquitoes and, more generally, can provide those working on other insects and planning to use the CRISPR/Cas9 system with a starting point for designing their experiments.
Dong S, Lin J, Held NL, Clem RJ, Passarelli AL, Franz AWE (2015) Heritable CRISPR/Cas9-mediated genome editing in the yellow fever mosquito, Aedes aegypti. PloS ONE 10(3):e0122353. doi: 10.1371/journal.pone.0122353