Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

PLoS Genet. 2021 Oct 5;17(10):e1009740. doi: 10.1371/journal.pgen.1009740. eCollection 2021 Oct.


CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying ('homing') therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anopheles / genetics
  • Biological Evolution
  • CRISPR-Cas Systems / genetics*
  • Conserved Sequence / genetics*
  • Gene Drive Technology / methods
  • Genes, Essential / genetics
  • Genotype
  • Malaria / parasitology
  • Mosquito Control / methods
  • Mosquito Vectors / genetics

Grant support

SF, WG, AH, MG, GM, TJH, KW, NK, AB and AC are members of the Target Malaria not-for-profit research consortium which is supported by a grant from the Bill & Melinda Gates Foundation (Grant INV006610 “Target Malaria Phase II”) and the Open Philanthropy Project Fund, an advised fund of Silicon Valley Community Foundation (Grant O-77157) to AB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.