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 host encoded 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 disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, 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 both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. We also assess the efficiency of ‘shadow drive’ generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.

Regulation of gene drive expression increases invasive potential and mitigates resistance

“…Building suppression gene drives for mosquito control that are fit for purpose requires a combination of the following features: optimal target sites that show high functional constraint; the targeting of multiple sites by the same gene drive construct; fine tuning of the expression of the nuclease that serves as the gene drive's 'motor' in order that the drive shows the most efficient invasion dynamics. A number of these theorised improvements to design have been substantiated: multiplexing through the use of multiple guide RNAs in a single drive construct has been shown to improve robustness of the drive [12,20,35]; judicious choice of functionally constrained sites in essential genes has meant that gene drives targeting such sites do not generate resistant alleles that are selected, at least in the laboratory [8,35]. For the last aspect -optimising expression -we have shown here that simple changes to the promoter controlling nuclease activity can lead to drastic improvements in the speed of invasion due to two principle reasons: 1) less propensity to induce end-joining repair in the germline and 2) improved fecundity of 'carrier' females that contain a single copy of the gene drive and transmit it in a super-Mendelian fashion to their offspring.…”

“…Crucially, deposited nos and zpg mRNA concentrate at the germ plasm due to regulatory signals present on the untranslated regions, which also further restrict translation of maternal mRNAs to the germline [14,19]. The promoter region of exuperantia has been validated in Drosophila [20] and in the tiger mosquito, Aedes aegypti, has been used to drive robust expression in both male and female germlines and has recently been used to control expression of Cas9 in a split gene drive system in this mosquito [21,22]. In contrast to zpg and exu, several reports have suggested that nos is specific to the female germline in mosquitoes, however promoter fusions in Aedes aegypti and Anopheles gambiae led to low level expression in males perhaps due to incomplete recapitulation of endogenous gene expression [23][24][25].…”

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

“…(Buchman et al, 2020(Buchman et al, , 2019Hoermann et al, 2020;Isaacs et al, 2012;Marshall et al, 2019)), or population suppression (Hammond et al, 2016;Kyrou et al, 2018;Simoni et al, 2020) to reduce and eliminate wild disease transmitting populations of mosquitoes. Despite significant progress, HGDs are still an emerging technology that can suffer from the formation of resistance alleles, hindering their efficacy (Adolfi et al, 2020;Carballar-Lejarazú et al, 2020;Gantz et al, 2015;Hammond et al, 2016;Kandul et al, 2019a;Kyrou et al, 2018;Li et al, 2020;Pham et al, 2019;Simoni et al, 2020).…”

“…This enables the HGD to home, or copy, itself into the recipient allele (Champer et al, 2016;Esvelt et al, 2014) (referred to as homing from hereon). This general design for HGD was quickly adopted, and many HGDs were developed in several insect species (Gantz et al, 2015;Hammond et al, 2016;Kandul et al, 2019a;Kyrou et al, 2018;Li et al, 2020;Simoni et al, 2020). However, it soon became apparent that HGDs unintentionally promote the formation of resistance alleles through mutagenic repair.…”

“…However, it soon became apparent that HGDs unintentionally promote the formation of resistance alleles through mutagenic repair. When these alleles are positively selected they can hinder HGD spread in laboratory cage populations (Champer et al, 2017;Hammond et al, 2017;Kandul et al, 2019a;KaramiNejadRanjbar et al, 2018;Oberhofer et al, 2018), with one exception that targeted an ultra-conserved sex determination gene for population suppression (Kyrou et al, 2018;Simoni et al, 2020). This resistance arises from, in addition to HDR, Cas9/gRNA-directed DNA cuts are also repaired by non-homologous end joining (NHEJ), an alternative DNA repair pathway that occasionally introduces insertions or deletions (indels) at the target site.…”