Sequence mismatch between gene-drive and target-site flanking regions significantly impairs homing efficiency in Culex quinquefasciatus

CRISPR/Cas9-based homing gene-drives (homing-drives) hold enormous potential as control tools for mosquito disease-vectors. These genomically-encoded technologies spread themselves through target populations by creating double-stranded DNA breaks on homologous chromosomes, into which the homing-drives are copied ( homed). Homing is dependent on sequence homology between the genomic regions flanking the transgene insertion and the break site. Homing efficiency (i.e. copying rate) substantially impacts the power of these systems: less efficient homing-drives spread slower, have fewer applications and are more resistance-prone. Understanding what influences homing-drive efficiency is therefore vital to the successful use of these technologies. Here we report a novel mechanism by which a homing-drives efficiency can be significantly impaired by natural sequence variation within a population into which it is spreading. Using a kmo-targeting split homing-drive in the West Nile virus mosquito Culex quinquefasciatus, we found that target-site heterology (sequence mismatch between the genomic regions flanking the target cut-site and the homing-drive transgene) of less than 10% reduced homing efficiency by up to 54%. While substantial research effort has been dedicated to increasing homing-drive efficiency through optimisation of within-construct components, our results highlight that the real-world efficacy of these systems may in part depend on variation beyond these controllable factors.