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

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 (TM)) and Droplet Digital (TM) PCR (ddPCR (TM)). 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. METHOD SUMMARY Mosquito DNA was extracted with the Promega Wizard (R) Genomic DNA Purification Kit protocol and quantified with Qubit (R) 3.0 following manufacturer protocols. PCR products for IDAA and ddPCR were generated with primers spanning 150-500 bp around the target site. IDAA amplicons were sent directly to COBO Technologies for analysis. ddPCR amplicons were analyzed using the Bio- Rad QX200T ddPCR system.