Variants in Cas9 and nanos regulatory elements modulate activity and reduce resistance allele formation in homing gene drive

Gene drive is a novel approach for controlling vector borne disease via either population modification or suppression. Even with high efficiency, though, overall drive performance can be reduced by somatic Cas9 expression and by maternal deposition of Cas9, leading to resistance allele formation. The nanos promoter for Cas9 shows very little leaky somatic expression, but it causes high rates of embryo resistance allele formation in Drosophila melanogaster . By truncating the promoter, we reduced rates of embryo resistance to undetectable levels, but germline cutting in females decreased by over half. Germline cutting and successful drive conversion was eventually lost when only the 5′ UTR was present, though males still retained moderate germline drive efficiency. Several additional methods were tested to improve performance, including additional suppressor elements to the 3′ UTR and introns to increase expression level. The most successful of these was the addition of a second nuclear localization signal, which substantially increased activity when coupled with a full-length or truncated nanos promoter. Overall, these experiments show the potential to modulate Cas9 regulatory elements to achieve desired expression for gene drive applications, while also showcasing the difficulty of obtaining an optimal activity profile.