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Updated: Sep 24, 2025

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations
Published on: June 28, 2021
Double-tap gene drive uses iterative genome targeting to help overcome resistance alleles
Alena L Bishop1, Víctor López Del Amo1, Emily M Okamoto1
1Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, 92093, USA.
Abstract:
Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented "double-tap" strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.
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