Digital droplet PCR and IDAA for the detection of CRISPR indel edits in the malaria species <i>Anopheles stephensi</i>

CRISPR/Cas9 technology is a powerful tool for the design of gene-drive systems to control and/or modify mosquito vector populations; however, CRISPR/Cas9-mediated nonhomologous end joining mutations can have an important impact on generating alleles resistant to the drive and thus on drive efficiency. We demonstrate and compare the insertions or deletions (indels) detection capabilities of two techniques in the malaria vector mosquito Anopheles stephensi: Indel Detection by Amplicon Analysis (IDAA™) and Droplet Digital™ PCR (ddPCR™). Both techniques showed accuracy and reproducibility for indel frequencies across mosquito samples containing different ratios of indels of various sizes. Moreover, these techniques have advantages that make them potentially better suited for high-throughput nonhomologous end joining analysis in cage trials and contained field testing of gene-drive mosquitoes

Mosquito Population Modification for Malaria Control

Malaria is a mosquito-borne disease that kills millions of people every year. Existing control tools have been insufficient to eliminate the disease in many endemic regions and additional approaches are needed. Novel vector-control strategies using genetic engineering to create malaria-resistant mosquitoes (population modification) can potentially contribute a new set of tools for mosquito control. Here we review the current mosquito control strategies and the development of transgenic mosquitoes expressing anti-parasite effector genes, highlighting the recent improvements in mosquito genome editing with CRISPR-Cas9 as an efficient and adaptable tool for gene-drive systems to effectively spread these genes into mosquito populations

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination