ERP146524 PRJEB61434 faf20a11-925a-4f83-be6c-8261b312275d Capability of an anti-CRSIPR protein to control the spreading of a gene drive in the An. gambiae mosquito populations exposed to complex behavioural conditions. CRISPR-based gene drives have the potential to spread within a population and are considered promising vector control tools. A doublesex-targeting gene drive was shown effective to suppress laboratory populations in both small and large cages, and it is considered for field application. Challenges related to the field-use of gene drives and the evolving regulatory framework demand for systems able to modulate or revert the action of gene drives, as part of post-release risk-mitigation plans. We developed an improved AcrIIA4-based anti-drive strain and showed inhibition of gene drive spread, in complex feeding and reproductive behavioural conditions. A stochastic model predicted the experimentally-observed genotypes dynamics in overlapping generations in medium- and large-sized cages and further demonstrated the effectiveness of anti-drive in different release and fitness scenarios. This study provides further validation for the use of an anti-drive system in controlling the spread of gene drive in Anopheles under complex behavioural conditions. Anti-CRISPR Anopheles mosquitoes inhibit gene drive spread under challenging behavioural conditions in large cages CRISPR-based gene drives have the potential to spread within a population and are considered promising vector control tools. A doublesex-targeting gene drive was shown effective to suppress laboratory populations in both small and large cages, and it is considered for field application. Challenges related to the field-use of gene drives and the evolving regulatory framework demand for systems able to modulate or revert the action of gene drives, as part of post-release risk-mitigation plans. We developed an improved AcrIIA4-based anti-drive strain and showed inhibition of gene drive spread, in complex feeding and reproductive behavioural conditions. A stochastic model predicted the experimentally-observed genotypes dynamics in overlapping generations in medium- and large-sized cages and further demonstrated the effectiveness of anti-drive in different release and fitness scenarios. This study provides further validation for the use of an anti-drive system in controlling the spread of gene drive in Anopheles under complex behavioural conditions. ENA-FIRST-PUBLIC 2023-07-31 ENA-LAST-UPDATE 2023-07-31