Ioanna Morianou

CRISPR-based gene drives are selfish genetic elements with the potential to spread through entire insect populations for sustainable vector control. Gene drives designed to disrupt the reproductive capacity of females can suppress laboratory populations of the malaria mosquito, Anopheles gambiae . However, any suppressive intervention will inevitably exert an evolutionary pressure for resistance, and the likelihood of resistance emerging at natural population scales remains poorly defined. Here, we present a pipeline to quantify the evolutionary space for resistance, enabling accelerated discovery, engineering, and testing of both natural and drive-induced variants that could reverse gene drive spread. We applied our approach to stress-test a best-in-class suppression gene drive that has evaded resistance in all laboratory-contained releases to date, known as Ag(QFS)1. We showed that previously undetected resistant alleles can arise at low frequency, including a novel type of partially resistant alleles that can perturb drive-invasion dynamics. Integrating experimentally derived resistance rates with population genetic modeling shows that single-target suppression drives are unlikely to be robust at natural mosquito population sizes, even at highly constrained loci. Here, we engineer and validate multiplexed gene drives in Anopheles gambiae , that target multiple conserved sites, actively removing resistant alleles. Our models predict that such gene drives could supress large natural mosquito populations in the field.

Artificial intelligence (AI) systems such as AlphaFold have transformed structural biology by enabling accurate prediction of protein structures. However, their capacity to uncover new classes of macromolecular assemblies remains largely untapped. We developed the Structural Novelty Index (SNI), a quantitative framework for identifying protein complexes that diverge from canonical architectures. As one implementation of SNI, we developed SNI _NRC-Hexa , to identify unconventional resistosomes formed by nucleotide-binding, leucine-rich repeat immune receptors (NLRs). We used it to analyze AlphaFold 3 models of 637 non-redundant NRC proteins from 346 genomes representing 85 plant species. This analysis identified candidates with predicted architectures distinct from the canonical hexameric resistosomes of NRC proteins. Biochemical purification and negative-stain transmission electron microscopy of NRC7 orthologs from multiple species supported the SNI prediction and revealed an unexpected undecameric (11-mer) assembly. Our results establish SNI as a scalable approach for discovering atypical protein complexes.

CRISPR-based gene drives are selfish genetic elements with the potential to spread through entire insect populations for sustainable vector control. Gene drives designed to disrupt the reproductive capacity of females can suppress laboratory populations of the malaria mosquito. However, any suppressive intervention will inevitably exert an evolutionary pressure for resistance. Here, we present a pipeline for the accelerated discovery, engineering, and testing of both natural and drive-induced variants that could reverse gene drive spread. We applied our method to stress-test a highly effective gene drive that has evaded resistance in all laboratory-contained releases to date, known as Ag(QFS)1. We showed that previously undetected resistant alleles can arise at low frequency, and discovered novel, partially resistant alleles that can perturb drive-invasion dynamics. We then engineered next-generation gene drives that can actively remove resistant alleles by targeting several highly conserved and non-overlapping sites in the female-specific exon of the doublesex gene. Our models predict that such gene drive designs could suppress large, natural populations of the malaria mosquito in the field.

Homing-based gene drives use a germline source of nuclease to copy themselves at specific target sites in a genome and bias their inheritance. Such gene drives can be designed to spread and deliberately suppress populations of malaria mosquitoes by impairing female fertility. However, strong unintended fitness costs of the drive and a propensity to generate resistant mutations can limit a gene drive’s potential to spread.Alternative germline regulatory sequences in the drive element confer improved fecundity of carrier individuals and reduced propensity for target site resistance. This is explained by reduced rates of end-joining repair of DNA breaks from parentally deposited nuclease in the embryo, which can produce heritable mutations that reduce gene drive penetrance.We tracked the generation and selection of resistant mutations over the course of a gene drive invasion of a population. Improved gene drives show faster invasion dynamics, increased suppressive effect and later onset of target site resistance. Our results show that regulation of nuclease expression is as important as the choice of target site when developing a robust homing-based gene drive for population suppression.