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

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...

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Autores principales:	Silke Fuchs, William T Garrood, Anna Beber, Andrew Hammond, Roberto Galizi, Matthew Gribble, Giulia Morselli, Tin-Yu J Hui, Katie Willis, Nace Kranjc, Austin Burt, Andrea Crisanti, Tony Nolan
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Genetics QH426-470
Acceso en línea:	https://doaj.org/article/2a757e425d2a4c10bae85e0362ca3c77
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spelling	oai:doaj.org-article:2a757e425d2a4c10bae85e0362ca3c772021-12-02T20:03:32ZResistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.1553-73901553-740410.1371/journal.pgen.1009740https://doaj.org/article/2a757e425d2a4c10bae85e0362ca3c772021-10-01T00:00:00Zhttps://doi.org/10.1371/journal.pgen.1009740https://doaj.org/toc/1553-7390https://doaj.org/toc/1553-7404CRISPR-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.Silke FuchsWilliam T GarroodAnna BeberAndrew HammondRoberto GaliziMatthew GribbleGiulia MorselliTin-Yu J HuiKatie WillisNace KranjcAustin BurtAndrea CrisantiTony NolanPublic Library of Science (PLoS)articleGeneticsQH426-470ENPLoS Genetics, Vol 17, Iss 10, p e1009740 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Genetics QH426-470
spellingShingle	Genetics QH426-470 Silke Fuchs William T Garrood Anna Beber Andrew Hammond Roberto Galizi Matthew Gribble Giulia Morselli Tin-Yu J Hui Katie Willis Nace Kranjc Austin Burt Andrea Crisanti Tony Nolan Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.
description	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.
format	article
author	Silke Fuchs William T Garrood Anna Beber Andrew Hammond Roberto Galizi Matthew Gribble Giulia Morselli Tin-Yu J Hui Katie Willis Nace Kranjc Austin Burt Andrea Crisanti Tony Nolan
author_facet	Silke Fuchs William T Garrood Anna Beber Andrew Hammond Roberto Galizi Matthew Gribble Giulia Morselli Tin-Yu J Hui Katie Willis Nace Kranjc Austin Burt Andrea Crisanti Tony Nolan
author_sort	Silke Fuchs
title	Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.
title_short	Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.
title_full	Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.
title_fullStr	Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.
title_full_unstemmed	Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.
title_sort	resistance to a crispr-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/2a757e425d2a4c10bae85e0362ca3c77
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Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.

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