Synthetic gene drive systems bias their own inheritance to spread throughout a population (Hay et al., 2021; Verkuijl et al., 2022; Wang et al., 2022). These systems show the potential of addressing disease transmission vectors, invasive species, and agriculture pests through two primary approaches: population suppression and population modification (also called replacement). Population suppression usually involves disrupting essential genes to reduce fitness and subsequently eliminate the population. For example, by targeting a conserved dsx female fertility exon in Anopheles mosquitoes, female drive homozygotes became sterile, leading to a decline in the vector population in a laboratory cage experiment (Kyrou et al., 2018). Population modification refers to changing the wild-type population into a transgenic population (Adolfi et al., 2020; Champer et al., 2020b; Hoermann et al., 2022; Carballar-Lejarazú et al., 2023; Green et al., 2023), which can be combined with a cargo gene or disruption of a native gene designed to inhibit transmission of pathogens such as malaria or dengue.

The advent of CRISPR technology has significantly advanced gene drive research. Homing gene drives constructed using Cas9 can precisely cut the wild-type allele on a homologous chromosome at the same genomic site as the drive. Subsequent homology-directed repair will use the drive allele as a template, resulting in the copying of the drive into the wild-type chromosome. This copying mechanism occurs in the germline, resulting in most offspring inheriting the drive, which leads to its rapid propagation throughout the population. This approach has been demonstrated in many organisms including yeast (Basgall et al., 2018; Shapiro et al., 2018; Yan and Finnigan, 2019), Drosophila (Champer et al., 2020b; Xu et al., 2020; Bishop et al., 2022; Yang et al., 2022; Yadav et al., 2023), Anopheles (Kyrou et al., 2018; Adolfi et al., 2020; Hoermann et al., 2022; Carballar-Lejarazú et al., 2023; Green et al., 2023), Aedes (Reid et al., 2022; Anderson et al., 2023), Culex (Harvey-Samuel et al., 2023), Ceratitis capitata (Meccariello et al., 2023), and Mus musculus (Weitzel et al., 2021).

One major issue that homing drives face is resistance alleles that block gRNA binding and Cas9 cleavage (Champer et al., 2017; Unckless et al., 2017; Hammond et al., 2017; Pham et al., 2019). These can be present due to natural genomic variation, but they can also form if the Cas9-induced DNA break is repaired by end-joining, which can mutate the target site, forming an indel. This mutation inhibits further recognition by the Cas9-gRNA complex, thereby preventing drive conversion of wild-type alleles. A resistance allele, especially one that carries a fitness advantage over the drive allele, can eventually outcompete the drive in the population. If the gene drive targets a specific gene, there can be two main resistance allele types: functional and nonfunctional. Functional resistance alleles represent sequences that preserve the function of the target gene. They are expected to have equal or higher fitness than drive alleles under most realistic circumstances. Nonfunctional resistance alleles are more commonly observed because any frameshift mutation or change in important amino acids is expected to disrupt the target gene’s function. These can be removed from the population if the target gene is essential. By using multiple gRNAs targeting nearby sites, high drive conversion efficiency can be preserved, and functional resistance alleles can often be prevented because each site must be repaired functionally for the entire allele to remain functional (Champer et al., 2018; Champer et al., 2020c).

Suppression gene drives can be used to reduce or eliminate a target population. They are designed to disrupt haplo-sufficient but essential genes without rescue, resulting in sterile or nonviable drive homozygotes. However, to target an essential gene with a modification drive, the drive must provide rescue for the gene. In such gene drives, a copy of the gene with its amino acids recoded to prevent gRNA cleavage (from the drive) serves as a “rescue”. The resistance alleles disrupted at the target will be removed from the population, but the drive will retain high fitness. One successful rescue drive targeted a haplolethal gene with two gRNAs, thus successfully avoiding functional resistance (Champer et al., 2020b). Because resistance alleles were haplolethal, any offspring inheriting a resistance allele were nonviable, quickly removing resistance alleles from the population. However, engineering drive alleles and rescue elements at haplolethal sites is challenging, even in model organisms (Chen et al., 2023a). This is because any end-joining repair that creates nonfunctional resistance will result in cell or organism nonviability, even if the drive was also successfully inserted. Additionally, high rates of Cas9 cleavage from maternal deposition could form embryo resistance alleles, which could remove drive alleles from the population at significant rates, impairing haplolethal gene-targeting drives. An alternate approach is to target an essential but haplosufficient gene, where only homozygous individuals for nonfunctional resistance alleles are nonviable. Though these are easier to engineer and could avoid the removal of drive alleles from embryo resistance, they are also substantially slower at removing resistance alleles and will tend not to reach 100% drive allele frequency if there are any fitness costs, though they still are expected to reach 100% drive carrier frequency (Champer et al., 2020c). Thus far, such drives have been made with one gRNA in Anopheles stephensi (Adolfi et al., 2020) and Drosophila melanogaster (Kandul et al., 2021; Terradas et al., 2021). All were somewhat successful but still formed functional resistance alleles.

Here, we constructed a homing gene drive targeting hairy with four gRNAs. This embryo development gene responsible for segmentation and neurodevelopment was selected because it is haplosufficient but essential. We used a similar insertion site as a previously successful CRISPR toxin-antidote gene drive targeting the same gene (Champer et al., 2020a). Experiments showed only moderate drive conversion efficiency of ∼ 36%, but the drive was still able to spread to a high level in cage experiments. However, the drive failed to reach 100% carrier frequency (peaking at 96%–97%) due to the formation of functional resistance alleles despite using multiple gRNAs. This was partially due to the use of an alternate start codon site in the functional resistance alleles. The drive also had a moderate fitness cost, which would limit its ability to persist in the face of functional resistance. Thus, additional care is needed for designing drives that target essential regions of a target gene, even with strategies involving multiplexed gRNAs.