CRISPR/Cas9 mediated gene knockout in mosquito vector Aedes aegypti: In-silico approach

Abstract

Aedes aegypti is the principal vector of several viruses including Dengue, Chikungunya and yellow fever. The recent advent of Clustered-Regularly-Interspaced-Short- Palindromic-Repeats (CRISPR)-Cas9 has sparked significant enthusiasm for the genetic control of A. aegypti. In the current study, we hypothesized that CRISPR-cas9 mediated gene knockout of doublesex (Aedsx) and Actin-4 (AeAct-4) in A. aegypti would result in sterile intersex phenotype and flightless phenotype respectively in females. Designing effective sgRNA is the most critical part of CRISPR/Cas9 editing. Therefore, sgRNAs were designed in-silico with the aim of developing efficient measures to control A. aegypti populations. Gene sequences of Aedsx (IAAEL009114) and AeAct-4 (AAEL001951) were retrieved from sequence data available in the VectorBase database. BLASTP was performed in searching for paralogs. Phylogenetic analysis was performed for all paralogs with ≥80% amino acid similarity. Eight paralogs were identified for the AeAct-4 while none was identified for Aedsx. DNA sequences for the paralogs of AeAct-4 were aligned to identify targets for sgRNAs that were unique to AeAct-4. Exon 2 was selected for AeAct-4, as the target site. For the Aedsx gene, 5a and 5b female-specific exons were selected as the target sequence for AedsxF1 and AedsxF2 isoforms respectively. sgRNAs were designed using online tools CHOP CHOP and CRISPR guide express. Two sgRNAs for each AeAct-4 gene, AedsxF1 and AedsxF2 isoforms with minimum off-target effects and in-silico predicted efficiency > 60% were selected to maximise the on-target gene knockout in-vivo. The efficiency of gene knockout can be evaluated by microinjecting synthesized sgRNAs directly into mosquito embryos with Cas9protein.