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

Abstract

<jats:title>Abstract</jats:title><jats:p>Gene drive-based strategies for modifying populations face the problem that genes encoding cargo and the drive mechanism are subject to separation, mutational inactivation, and loss of efficacy. Resilience, an ability to respond to these eventualities in ways that restore population modification with functional genes is needed for long-term success. Here we show that resilience can be achieved through cycles of population modification with “<jats:italic>Cleave and Rescue”</jats:italic> (<jats:italic>ClvR</jats:italic>) selfish genetic elements. <jats:italic>ClvR</jats:italic> comprises a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, and a recoded version of the essential gene resistant to cleavage. <jats:italic>ClvR</jats:italic> spreads by creating conditions in which those lacking <jats:italic>ClvR</jats:italic> die because they lack functional versions of the essential gene. Cycles of modification can in principal be carried out if two <jats:italic>ClvR</jats:italic> elements targeting different essential genes are located at the same genomic position, and one of them, <jats:italic>ClvR</jats:italic><jats:sup>n+1</jats:sup>, carries a <jats:italic>Rescue</jats:italic> transgene from an earlier element, <jats:italic>ClvR</jats:italic><jats:sup>n</jats:sup>. <jats:italic>ClvR</jats:italic><jats:sup>n+1</jats:sup> should spread within a population of <jats:italic>ClvR</jats:italic><jats:sup>n</jats:sup>, while also bringing about a decrease in its frequency. To test this hypothesis we first show that multiple <jats:italic>ClvR</jats:italic>s, each targeting a different essential gene, function when located at a common chromosomal position in <jats:italic>Drosophila</jats:italic>. We then show that when several of these also carry the <jats:italic>Rescue</jats:italic> from a different <jats:italic>ClvR</jats:italic>, they spread to transgene fixation in populations fixed for the latter, and at its expense. Therefore, genetic modifications of populations can be overwritten with new content, providing an ongoing point of control.</jats:p><jats:sec><jats:title>Significance</jats:title><jats:p>Gene drive can spread beneficial traits through populations, but will never be a one-shot project in which one genetic element provides all desired modifications, for an indefinitely long time. Here we show that gene drive mediated population modification in <jats:italic>Drosophila</jats:italic> can be overwritten with new content while eliminating old, using <jats:italic>Cleave and Rescue</jats:italic> (<jats:italic>ClvR</jats:italic>) selfish genetic elements. The ability to carry out cycles of modification that create and then leave behind a minimal genetic footprint while entering and exiting a population provides important points of control. It makes possible the replacement of broken elements, upgrades with new elements that better carry out their tasks and/or provide new functions, all while promoting the removal of modifications no longer needed.</jats:p></jats:sec>

A home and rescue gene drive efficiently spreads and persists in populations

“…The re-coded rescue strategy that we used to develop HomeR was also used in previous Drosophila toxin-antidote non-homing GDs (Champer et al, 2020a;Oberhofer et al, 2020aOberhofer et al, , 2020bOberhofer et al, , 2019 and recent HGD's in both Drosophila (Champer et al, 2020b), and Anopheles stephensi (Adolfi et al, 2020), though each of these examples suffered from potential drawbacks. For example, both the haplolethal HGD (Champer et al, 2020b) and the TARE design (Champer et al, 2020a) share similar problematic design architectures that can be unstable as they are susceptible to functional resistance alleles induced via recombination between the promoter including sequences 5' of the coding sequence and 3'UTR regions, which are identical between the re-coded sequence and the wt sequence ( Fig.…”

“…Therefore, these data indicate that the observed transmission rate of nearly ~100% was caused by ~90% conversion and ~10% "destruction" of the Pol-Ɣ35 WT alleles. In sum, the Pol-Ɣ35 HomeR1 transmission rate of nearly ~100% observed in Pol-Ɣ35 HomeR1 /+; nos-Cas9/+ trans-heterozygous females could not be simply explained by the "destruction" of all wt Pol-Ɣ35 alleles, which would result in the lethality of 50% progeny as in non-homing ClvR (Oberhofer et al, 2020a(Oberhofer et al, , 2020b(Oberhofer et al, , 2019 and TARE (Champer et al, 2020a) drives, and instead is a result of both conversion and destruction of the recipient allele at the Pol-Ɣ35 locus (Fig. 2B).…”

“…autonomous) and two-locus (aka. split-drive) versions of ClvR (Oberhofer et al, 2020a(Oberhofer et al, , 2020b(Oberhofer et al, , 2019, the one-locus TARE system from (Champer et al, 2020a) as well as a two-locus TARE configuration based on their design, a HGD targeting a nonessential gene (Gantz et al, 2015;Hammond et al, 2016), and HomeR. In each case, we first simulated population spread of each gene drive system for an ideal parameterization (see Methods for more details) and included additional simulations for HomeR under current experimentally-derived parameters (HomeR-exp, Fig.…”

“…To overcome the accumulation of drive resistance alleles, CRISPR based toxin-antidote (TA) based systems, in which embryos are essentially "poisoned" and only those embryos harboring the TA genetic cassette are rescued, were described ( Fig. S8 in (Kandul et al, 2019b)) and engineered (Champer et al, 2020a;Oberhofer et al, 2020aOberhofer et al, , 2020bOberhofer et al, , 2019. Generally these designs utilize a toxin consisting of a non-homing GD harboring multiple gRNAs targeting a vital gene, and an "addictive" antidote that is a re-coded, cleavage-immune version of the targeted gene.…”

Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement

“…A number of ways have been suggested to work around this, for example the linking of rescue-copies of essential genes to drive elements (27). Cleave and rescue elements are a related approach that does not rely on homing (28,29). However, not only could such approaches increase molecular complexity of the constructs (and likely also their regulatory approval) but it could also complicate the deployment of multiple waves or combinations of drive and effector within a population.…”