A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment

Abstract

CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split-gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small-molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.

A CRISPR endonuclease gene drive reveals two distinct mechanisms of inheritance bias

“…For nup50-Cas9, the progeny were collected individually from F 1 parents (Table S7-S10). As can be seen in Fig 2a, there was considerable variation between the inheritance rate from different parents carrying the same drive, a notable feature that was reported in many other drive papers 18,24,26,35 . Due to this over-dispersal, we cannot reliably determine if there is a statistical difference in the inheritance rate between the different Cas9 regulatory elements.…”

Inherently confinable split-drive systems in Drosophila

“…Since many components of the HDR DNA repair pathway are shared between repair of damage-induced DSB and meiotic recombination, these shared features may underlie both the peculiar lack of male recombination in Drosophila and the reduced rates of HDR-mediated gene conversion resulting in the divergent sex specific sGD drive frequencies we observed. This phenomenon was also observed in other studies using a full genedrive [12] as well as in a trans-complementing framework [26]. Why this sex difference was not also observed for the prosalpha2 drive will require further analysis to address, but as discussed further below, may be related to a combination of highly-efficient copying and a particularly strong form of lethal mosaicism that eliminates nearly all lof alleles (NHEJs or damaged chromosomes) if they are ever generated.…”

“…It is also possible for the gRNA-carrying element to include a second gRNA that cuts the genome where the Cas9 source is inserted, empowering that latter element to copy as well. In such trans-complementing systems [26], each element behaves according to standard rules of Mendelian inheritance when propagated separately, but once combined, they copy as a coupled full-drive system. In all of these cases, the gene-drive carried on one chromosome converts the homolog in heterozygous germline cells, creating a strong bias for Super-Mendelian (>50%) transmission of the drive allele.…”

Targeted gene disruption by CRISPR/xCas9 system in Drosophila melanogaster

“…The sgRNA can be produced by in vitro transcription (Bassett et al, 2013; Yu et al, 2013), or expressed from a Pol III promoter derived from the U6 small nuclear RNA gene (Gratz, Cummings, et al, 2013; Kondo & Ueda, 2013; Ren et al, 2013; Sebo et al, 2014). Among all these methods, the most robust strategy was by crossing two stable transgenic fly strains: one specifically expressing the Cas9 protein in germline cells, the other ubiquitously expressing sgRNA (Lopez Del Amo et al, 2019; Champer et al, 2019; Kondo & Ueda, 2013). This strategy generated a much higher mutagenesis rate than the previous transient expression of Cas9 and sgRNAs.…”