Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using clustered regularly interspaced short palindrome repeats‐associated protein 9

Abstract

Culex quinquefasciatus is a vector of many diseases that adversely impact human and animal health; however, compared to other mosquito vectors limited genome engineering technologies have been characterized for this vector. Clustered regularly interspaced short palindrome repeats‐associated protein 9 (CRISPR‐Cas9) based technologies are a powerful tool for genome engineering and functional genetics and consequently have transformed genetic studies in many organisms. Our objective was to improve upon the limited technologies available for genome editing in C. quinquefasciatus to create a reproducible and straightforward method for CRISPR‐Cas9‐targeted mutagenesis in this vector. Here we describe methods to achieve high embryo survival and mutagenesis rates and we provide details on the injection supplies and procedures, embryo handling and guide RNA (gRNA) target designs. Through these efforts, we achieved embryo survival rates and germline mutagenesis rates that greatly exceed previously reported rates in this vector. This work is also the first to characterize the white gene marker in this species, which is a valuable phenotypic marker for future transgenesis or mutagenesis of this vector. Overall, these tools provide the framework for future functional genetic studies in this important disease vector and may support the development of future gene drive and genetic technologies that can be used to control this vector.

Evaluation of gene knock-outs by CRISPR as potential targets for the genetic engineering of the mosquitoCulex quinquefasciatus

“…We also note that our data for such a pool was derived from a single raft that might have displayed an exceptional mutagenesis rate. Our mutagenesis of the white gene also yielded slightly higher mutagenesis rates (92%-100%) compared to what was shown by Li et al (61%-86%), and contrary to the kh analysis where we picked different gRNA targets, for white we employed the same gRNA ( w3 ) and obtained 92% instead of 72% G1 mutant rate observed in the previous study [41].…”

“…For each gene we observed at least one gRNA yielding the expected phenotypes, and based on this preliminary analysis we were able to select a gRNA for each gene which was carried forward for further analysis of each mutant phenotype. Interestingly, the gRNA w4 gave comparable results to the previously validated w3 [41].…”

“…The approximate locus size is indicated for each gene from the start to the stop codon. The gRNAs w1, w2 , and w3 were previously described as active [41] and were used here as controls. Drawings are not to scale.…”

“…In order to recover and characterize homozygous mutant lines, we then performed an additional round of injection for the selected gRNAs, as well as for the previously validated w3 -gRNA targeting white as a control [41]. For each gene, we injected ∼100-400 eggs using the same protocol used in our preliminary testing and scored the mosaic phenotype in the G0 injected animal at the pupal stage ( Table 1 ).…”

“…Here, we used the validated w3 gRNA, which was chosen for its high transinfection rate and obvious phenotype, as noted in [41] and this study, and we performed a similar analysis to what was described above. For this line, ∼34% of G0 injected animals displayed a white-/white+ mosaic phenotype, and an intercross of such G0 animals led to a recovery of mutants in the G1 progeny with ∼92% rate ( Table 1 ), comparable to what was previously described above using w4 , as well as with the same gRNA in previous work [41]. We used the recovered G1 animals to establish a homozygous white mutant line (HI- white ) to be used in further steps of this project.…”