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

Fuchs, Silke, Garrood, William T, Beber, Anna, Hammond, Andrew, Galizi, Roberto, Gribble, Matthew, Morselli, Giulia, Hui, Tin-Yu J, Willis, Katie, Kranjc, Nace, Burt, Austin, Crisanti, Andrea and Nolan, Tony ORCID: https://orcid.org/0000-0002-2982-8333 (2021) 'Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation'. PLoS Genetics, Vol 17, Issue 10, e1009740.

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Abstract

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.

Item Type:	Article
Subjects:	QU Biochemistry > Genetics > QU 470 Genetic structures QX Parasitology > Insects. Other Parasites > QX 510 Mosquitoes WC Communicable Diseases > Tropical and Parasitic Diseases > WC 750 Malaria
Faculty: Department:	Biological Sciences > Vector Biology Department
Digital Object Identifer (DOI):	https://doi.org/10.1371/journal.pgen.1009740
Depositing User:	Samantha Sheldrake
Date Deposited:	07 Oct 2021 11:33
Last Modified:	07 Oct 2021 11:33
URI:	https://archive.lstmed.ac.uk/id/eprint/19105

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