Genome Engineering with CRISPR-Cas9 in the Mosquito Aedes aegypti

Abstract

The mosquito Aedes aegypti is a potent vector of the chikungunya, yellow fever, and dengue viruses, responsible for hundreds of millions of infections and over 50,000 human deaths per year. Mutagenesis in Ae. aegypti has been established with TALENs, ZFNs, and homing endonucleases, which require the engineering of DNA-binding protein domains to provide genomic target sequence specificity. Here, we describe the use of the CRISPR-Cas9 system to generate site-specific mutations in Ae. aegypti. This system relies on RNA-DNA base-pairing to generate targeting specificity, resulting in efficient and flexible genome-editing reagents. We investigate the efficiency of injection mix compositions, demonstrate the ability of CRISPR-Cas9 to generate different types of mutations via disparate repair mechanisms, and report stable germ-line mutations in several genomic loci. This work offers a detailed exploration into the use of CRISPR-Cas9 in Ae. aegypti that should be applicable to non-model organisms previously out of reach of genetic modification.

microRNA-309 targets the Homeobox gene SIX4 and controls ovarian development in the mosquito Aedes aegypti

“…Embryos were injected with a mixture of recombinant Cas9 protein at 333 ng/μL and sgRNA at 40 ng/μL, which has been optimized to compromise between high survival rate and high mutagenesis level in A. aegypti (22). Approximately 51.5% of injected embryos survived to adulthood and were consequently collected to verify whether the miR-309 genomic locus could be effectively disrupted by using the CRISPR/Cas9 technique.…”

“…However, they were intact and visible in miR-309-silenced ovaries, implicating the critical role of miR-309 in programming correct ovarian development. To further define the regulatory roles of this miRNA, miR-309-specific mutations of mosquitoes were generated by using the CRISPR/Cas9 system, by which accurate genome editing with high efficiency has been achieved in many species, including mosquitoes (22). Herein, we successfully constructed miR-309-specific mutants in A. aegypti, contributing to deciphering its physiological function.…”

“…Microinjection into A. aegypti embryos (preblastoderm eggs) was performed with a mixture of recombinant Cas9 protein at 333 ng/μL and sgRNAs at 40 ng/μL according to protocols described (22,45). The embryos hatched at 4-5 d after injection and were reared to adults based on procedures described (22,45). Female mosquitoes were blood fed for egg production and phenotypic analysis.…”

The aerodynamics and control of free flight manoeuvres inDrosophila

“…Indeed, over the past few years, research has advanced substantially on various flight-related topics in other insects including motor planning and prey capture in dragonflies [1], nocturnal vision in hawkmoths [2,3], navigation in bees [4] and spatial memory in wasps [5]. The advent of transgenic techniques such as the CRISPR/Cas9 system [6] will soon allow researchers to pursue questions in these non-genetic model species at finer levels of granularity, as is already happening in mosquitos [7,8]. Nevertheless, the fruit fly (Drosophila melanogaster) will continue to provide unique experimental opportunities in flight research for several reasons.…”

Tools and strategies for scarless allele replacement inDrosophilausing CRISPR/Cas9

“…[31][32][33] However, currently available Cas9-and sgRNA-expression plasmids (including those used in this study) contain promoters derived from D. melanogaster, so injecting purified Cas9 protein or mRNA along with in vitro transcribed sgRNAs instead of using expression plasmids will likely give better results when working with other Drosophila species. 17,34,35 We have recently had success using commercially available Cas9 protein and in vitro transcribed sgRNAs to induce NHEJ and/or HDR in Drosophila elegans, Drosophila americana, Drosophila novamexicana, and Drosophila virils (unpublished data).…”