Assessment of a split homing based gene drive for efficient knockout of multiple genes

Abstract

Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that hostencoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate host encoded genes has not been thoroughly explored. To test this approach, here we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of its target gene during super-Mendelian propagation of split-HGD. However, maternal deposition and embryonic expression of Cas9 resulted in the generation of drive resistant alleles which can accumulate and limit the spread of such a drive. Alternative design principles are discussed that could mitigate the accumulation of resistance alleles while incorporating a GME.

The Developmental Transcriptome ofAe. albopictus,a Major Worldwide Human Disease Vector

“…In Aedes aegypti, several regulatory elements able to drive gene expression in a tissue-and temporal-specific manner have been identified through extensive study (Akbari et al 2013a) and transgenesis (Coates et al 1999;Kokoza et al 2000;Moreira et al 2000;Smith et al 2007). Future functional characterization of uncharacterized genes and regulatory elements may lead to the development of innovative genetic population control technologies such as precision guided sterile males (Kandul et al 2019b), and gene drive systems (Akbari et al 2013b(Akbari et al , 2014aChamper et al 2016;Buchman et al 2018bBuchman et al , 2018aKandul et al 2019aKandul et al , 2019bLi et al 2019) which can be linked to anti-pathogen effectors (Buchman et al , 2019b potentially providing paradigm-shifting technologies to control this worldwide human disease vector. Overall, our results provide a comprehensive snapshot of gene expression dynamics in the development of Ae.…”

Inherently confinable split-drive systems in Drosophila

“…One approach is to employ tightly-regulated germline-specific promoters to avoid somatic expression of Cas9 that can lead to NHEJ-induced repair events 9,10,12 . Some germline promoters are quite specific, at least in certain genomic contexts (e.g., nanos 13 or zpg 14 ), while others are leaky (e.g., vasa 15,16 ). Another tactic is to identify highlyconserved genomic targets as cleavage sites such that all NHEJ mutants suffer severe fitness costs 14,[17][18][19] .…”

Broad dengue neutralization in mosquitoes expressing an engineered antibody

“…Therefore, there has been increasing interest in the development of engineered gene-drive technologies, which are able to rapidly transmit themselves and any linked "cargo" genes, such as anti-pathogen effectors, through wild disease-transmitting populations [25,[32][33][34][35] such that only a few releases of modest amounts of engineered insects could drive desirable cargo genes through wild populations, making them efficient and cost effective for vector control. To achieve disease reduction, such gene-drive systems need to be linked to useful "cargo", such as effective anti-pathogen genes, and several approaches for engineering Ae.…”