Integrating multiplexing into confineable gene drives effectively overrides resistance in Anopheles stephensi

Anopheles stephensi is a major malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012 it has invaded several countries of eastern Africa, stimulating urgent efforts to develop more efficient strategies for vector control such as CRISPR/Cas9-based homing gene drives. Target site resistance due to end-joining repair is a significant challenge to the deployment of these systems. The use of multiple sgRNAs has the potential to solve this issue. Here we perform experimental crosses to assess the homing and cutting efficiency of both classical (e.g. four adjacent sgRNAs all in one construct) and additive (e.g. separate constructs each expressing a single sgRNA) multiplexing strategies targeting the cardinal locus, in the presence and absence of a resistance allele. We find resistance alleles at one sgRNA target site can be mitigated by the presence of the additional sgRNAs with either strategy, and do not significantly reduce the homing efficiency for either strategy, validating their effectiveness. Further modelling using parameters derived from the strains generated indicates that while both strategies can overcome resistance allele formation, the fitness of the drive-carrying alleles is a critical factor in determining the overall performance and persistence of a split drive.