Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

Abstract

CRISPR-based gene drives spread through populations bypassing the dictates of Mendelian genetics, offering a population-engineering tool for tackling vector-borne diseases, managing crop pests, and helping island conservation efforts; unfortunately, current technologies raise safety concerns for unintended gene propagation. Herein, we address this by splitting the two drive components, Cas9 and gRNAs, into separate alleles to form a novel trans-complementing split-genedrive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This bi-component nature allows for individual transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a smallmolecule-controlled version to investigate the biology of component inheritance and use our system to study the maternal effects on CRISPR inheritance, impaired homology on efficiency, and resistant allele formation. Lastly, mathematical modeling of tGD spread in a population shows potential advantages for improving current gene-drive technologies for field population modification. Figure 1 -The trans-complementing gene-drive system (tGD) features simultaneous super-Mendelian inheritance of two transgenes.(A) Schematic of the tGD genetic arrangement with two elements that can be kept separated as different transgenic lines. The Cas9 transgene is inserted in the genomic location targeted by the gRNA-A, while a separate cassette expressing a tandem gRNA construct (gRNA-A, gRNA-B) is inserted at the location targeted by gRNA-B. Upon genetic cross, each gRNA combines with Cas9 to generate a double-strand DNA break at each locus on the wild-type allele. Each break is then repaired by homology-directed repair (HDR) pathway using the intact chromosome carrying the transgene as a template. (B) Outline of the genetic cross used to demonstrate tGD in fruit flies. F0 males carrying a DsRed-marked Cas9 transgene inserted in the yellow locus were crossed to females carrying a GFP-marked cassette containing two gRNAs (y1-e1) inserted in the ebony coding sequence. Trans-heterozygous F1 females (carrying both Cas9 and gRNAs) were crossed to wild-type males to assess germline transmission rates of the fluorophores marking the transgenes in the F2 progeny. The conversion event is indicated by the red and green triangles in the F1 females (C) Single F1 female germline inheritance output measured as GFP and DsRed marker presence in the F2 progeny. The black bar represents the inheritance average. The blue shading represents the deviation from the expected 50% "Mendelian" inheritance. Inheritance average, standard deviation, number of samples (n) and total number of flies scored in each experiment are represented over the graph in line with the respective data.

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

“…For example, F 1 progeny with either maternally deposited Cas9 protein, or embryonically expressed Cas9 gene inherited from their fathers, both had moderate rates of HDR in addition to white and yellow LOF somatic mutations with up to 100% efficiency (Fig. 1B); consistent observations were generated in a recent work using a trans-complementing Gene Drive (tGD) system (Lopez del Amo et al, 2019). These data conclusively demonstrate that Cas9 is embryonically expressed and that embryonic expression is sufficient to generate high rates of cleavage and moderate rates of HDR.…”

“…To assess the feasibility and efficiency of utilizing a HGD to bias transmission while also expressing a GME, we designed a HGD that expressed two gRNAs (Lopez del Amo et al, 2019). The homing component of the split-HGD system, referred hereon as a Gene Drive element (GDe), encodes a gRNA targeting white (gRNA w , driver gRNA), a separate cargo GME targeting yellow (gRNA y , effector), a 3xP3-eGFP dominant marker, all together flanked by 1kb homology arms from the white target locus to direct targeted HDR mediated integration (Fig.…”

“…The genetic assembly of the Gene Drive element with gRNAs and 3xP3-eGFP (GDe) and employment of the w GDe /w GDe which was previously used in for a different purpose by (Lopez del Amo et al, 2019) to generate a split trans-complementing Gene Drive system. The assembly of BicC-Cas9 construct followed the same steps previously described for the other three Cas9 lines: nos-Cas9, vas-Cas9, and Ubi-Cas9 .…”

Gene drive and resilience through renewal with next generationCleave and Rescueselfish genetic elements

“…Transgenes, or alleles of endogenous loci, can be linked with a genetic element conferring drive, and this can promote their spread. A number of approaches to spreading traits through populations (population replacement/alteration/modification) in ways that are self-sustaining, by linking them with genetic elements that mediate drive, have been proposed (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) . Several of these, Medea (9,23) , UD mel (15) , engineered translocations (24) , and ClvR ( Cleave and Rescue ) selfish genetic elements (25) , have been implemented and shown to spread to transgene fixation in otherwise wildtype Drosophila .…”

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.…”