scholarly journals Gene-drive-mediated extinction is thwarted by evolution of sib mating

2019 ◽  
Author(s):  
James J Bull ◽  
Christopher H Remien ◽  
Stephen M Krone
Keyword(s):  
Genome Engineering ◽  
Random Mating ◽  
Target Sequence ◽  
Gene Drive ◽  
Resistance Evolution ◽  
Sib Mating ◽  
Population Structures ◽  
The Face ◽  
Mean Fitness ◽  
Gene Drives

AbstractGenetic engineering combined with CRISPR technology has developed to the point that gene drives can, in theory, be engineered to cause extinction in countless species. Success of extinction programs now rests on the possibility of resistance evolution, which is largely unknown. For CRISPR technology, resistance may take many forms, from mutations in the nuclease target sequence to specific types of non-random population structures that limit the drive. We develop mathematical models of various deviations from random mating to consider escapes from extinction-causing gene drives. We use a version of Maynard Smith’s haystack model to show that population structure can enable drive-free subpopulations to be maintained against gene drives. Our main emphasis, however, is sib mating in the face of recessive-lethal and Y-chromosome drives. Sib mating easily evolves in response to both kinds of gene drives and maintains mean fitness above 0, with equilibrium fitness depending on the level of inbreeding depression. Environmental determination of sib mating (as might stem from population density crashes) can also maintain mean fitness above 0. Translation of mean fitness into population size depends on ecological details, so understanding mean fitness evolution and dynamics is merely the first step in predicting extinction. Nonetheless, these results point to possible escapes from gene drive-mediated extinctions that lie beyond the control of genome engineering.

scholarly journals Gene-drive-mediated extinction is thwarted by population structure and evolution of sib mating

2019 ◽  
Vol 2019 (1) ◽  
pp. 66-81 ◽  
Author(s):  
James J Bull ◽  
Christopher H Remien ◽  
Stephen M Krone
Keyword(s):  
Population Structure ◽  
Genome Engineering ◽  
Random Mating ◽  
Structured Populations ◽  
Target Sequence ◽  
Gene Drive ◽  
Resistance Evolution ◽  
Sib Mating ◽  
Mean Fitness ◽  
Gene Drives

AbstractBackground and objectivesGenetic engineering combined with CRISPR technology has developed to the point that gene drives can, in theory, be engineered to cause extinction in countless species. Success of extinction programs now rests on the possibility of resistance evolution, which is largely unknown. Depending on the gene-drive technology, resistance may take many forms, from mutations in the nuclease target sequence (e.g. for CRISPR) to specific types of non-random population structures that limit the drive (that may block potentially any gene-drive technology).MethodologyWe develop mathematical models of various deviations from random mating to consider escapes from extinction-causing gene drives. A main emphasis here is sib mating in the face of recessive-lethal and Y-chromosome drives.ResultsSib mating easily evolves in response to both kinds of gene drives and maintains mean fitness above 0, with equilibrium fitness depending on the level of inbreeding depression. Environmental determination of sib mating (as might stem from population density crashes) can also maintain mean fitness above 0. A version of Maynard Smith’s haystack model shows that pre-existing population structure can enable drive-free subpopulations to be maintained against gene drives.Conclusions and implicationsTranslation of mean fitness into population size depends on ecological details, so understanding mean fitness evolution and dynamics is merely the first step in predicting extinction. Nonetheless, these results point to possible escapes from gene-drive-mediated extinctions that lie beyond the control of genome engineering.Lay summaryRecent gene drive technologies promise to suppress and even eradicate pests and disease vectors. Simple models of gene-drive evolution in structured populations show that extinction-causing gene drives can be thwarted both through the evolution of sib mating as well as from purely demographic processes that cluster drive-free individuals.

scholarly journals Integral Gene Drives: an “operating system” for population replacement

2018 ◽  
Author(s):  
Alexander Nash ◽  
Giulia Mignini Urdaneta ◽  
Andrea K. Beaghton ◽  
Astrid Hoermann ◽  
Philippos Aris Papathanos ◽  
...  
Keyword(s):  
Field Testing ◽  
Target Site ◽  
Gene Drive ◽  
Population Replacement ◽  
Pathogen Effector ◽  
Site Variation ◽  
The Face ◽  
Endogenous Genes ◽  
Target Site Resistance ◽  
Gene Drives

AbstractFirst generation CRISPR-based gene drives have now been tested in the laboratory in a number of organisms including malaria vector mosquitoes. A number of challenges for their use in the area-wide genetic control of vector-borne disease have been identified. These include the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and the practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. To address these challenges, we have evaluated the concept of Integral Gene Drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including both the drive and the anti-pathogen effector elements directly embedded within endogenous genes – an arrangement which we refer to as gene “hijacking”. This design would allow autonomous and non-autonomous IGD traits and strains to be generated, tested, optimized, regulated and imported independently. We performed quantitative modelling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population while hedging drive over multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target-site variation. IGD thus has the potential to yield more durable and flexible population replacement traits.

scholarly journals Threshold-Dependent Gene Drives in the Wild: Spread, Controllability, and Ecological Uncertainty

BioScience ◽  
2019 ◽  
Vol 69 (11) ◽  
pp. 900-907 ◽  
Author(s):  
Gregory A Backus ◽  
Jason A Delborne
Keyword(s):  
Critical Frequency ◽  
Wild Population ◽  
Detailed Knowledge ◽  
Gene Drive ◽  
Potential Solution ◽  
Spreading Behavior ◽  
The Face ◽  
In The Wild ◽  
Ecological Uncertainty ◽  
Gene Drives

Abstract Gene drive technology could allow the intentional spread of a desired gene throughout an entire wild population in relatively few generations. However, there are major concerns that gene drives could either fail to spread or spread without restraint beyond the targeted population. One potential solution is to use more localized threshold-dependent drives, which only spread when they are released in a population above a critical frequency. However, under certain conditions, small changes in gene drive fitness could lead to divergent outcomes in spreading behavior. In the face of ecological uncertainty, the inability to estimate gene drive fitness in a real-world context could prove problematic because gene drives designed to be localized could spread to fixation in neighboring populations if ecological conditions unexpectedly favor the gene drive. This perspective offers guidance to developers and managers because navigating gene drive spread and controllability could be risky without detailed knowledge of ecological contexts.

scholarly journals Gene drive escape from resistance depends on mechanism and ecology

2021 ◽  
Author(s):  
Forest Cook ◽  
James J Bull ◽  
Richard Gomulkiewicz
Keyword(s):  
Cleavage Site ◽  
Ecological Effects ◽  
Gene Drive ◽  
Resistance Evolution ◽  
Gene Drives ◽  
Population Suppression ◽  
Modest Effect

AbstractGene drives can potentially be used to suppress pest populations, and the advent of CRISPR technology has made it feasible to engineer them in many species, especially insects. What remains largely unknown for implementations is whether anti-drive resistance will evolve to block the population suppression. An especially serious threat to some kinds of drive is mutations in the CRISPR cleavage sequence that block the action of CRISPR, but designs have been proposed to avoid this type of resistance. Various types of resistance at loci away from the cleavage site remain a possibility, which is the focus here. It is known that modest-effect suppression drives can essentially ‘outrun’ unlinked resistance even when that resistance is present from the start. We demonstrate here how the risk of evolving (unlinked) resistance can be further reduced without compromising overall suppression by introducing multiple suppression drives or by designing drives with specific ecological effects. However, we show that even modest-effect suppression drives remain vulnerable to the evolution of extreme levels of inbreeding, which halt the spread of the drive without actually interfering with its mechanism. The landscape of resistance evolution against suppression drives is therefore complex, but avenues exist for enhancing gene drive success.

scholarly journals CRISPR with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research

2015 ◽  
Author(s):  
Fillip Port ◽  
Nadine Muschalik ◽  
Simon L Bullock
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Basic Research ◽  
Evidence Based ◽  
Gene Drive ◽  
Highly Active ◽  
Genome Modification ◽  
Target Sites ◽  
Gene Drives

CRISPR/Cas technology allows rapid, site-specific genome modification in a wide variety of organisms. CRISPR components produced by integrated transgenes have been shown to mutagenise some genomic target sites in Drosophila melanogaster with high efficiency, but whether this is a general feature of this system remains unknown. Here, we systematically evaluate available CRISPR/Cas reagents and experimental designs in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for genome engineering (Gantz & Bier, 2015) and conclude that optimised CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk.

scholarly journals Ultra-conserved sequences in the genomes of highly diverse Anopheles mosquitoes, with implications for malaria vector control

2021 ◽  
Author(s):  
Samantha M O’Loughlin ◽  
Annie J Forster ◽  
Silke Fuchs ◽  
Tania Dottorini ◽  
Tony Nolan ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Regulatory Function ◽  
Malaria Vector Control ◽  
Target Sequence ◽  
Gene Drive ◽  
Conserved Sequences ◽  
Anopheles Mosquitoes ◽  
Starting Point ◽  
And Function ◽  
Gene Drives

Abstract DNA sequences that are exactly conserved over long evolutionary time scales have been observed in a variety of taxa. Such sequences are likely under strong functional constraint and they have been useful in the field of comparative genomics for identifying genome regions with regulatory function. A potential new application for these ultra-conserved elements has emerged in the development of gene drives to control mosquito populations. Many gene drives work by recognising and inserting at a specific target sequence in the genome, often imposing a reproductive load as a consequence. They can therefore select for target sequence variants that provide resistance to the drive. Focusing on highly conserved, highly constrained sequences lowers the probability that variant, gene drive-resistant alleles can be tolerated. Here we search for conserved sequences of 18bp and over in an alignment of 21 Anopheles genomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containing Anopheles ultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenic Anopheles ultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of the Anopheles ultra-conserved elements contained no polymorphisms. Our list of Anopheles ultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria.

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

PLoS Genetics ◽  
2021 ◽  
Vol 17 (10) ◽  
pp. e1009740
Author(s):  
Silke Fuchs ◽  
William T. Garrood ◽  
Anna Beber ◽  
Andrew Hammond ◽  
Roberto Galizi ◽  
...  
Keyword(s):  
Essential Gene ◽  
Target Site ◽  
Target Sequence ◽  
Lethal Gene ◽  
Gene Drive ◽  
Continuous Sampling ◽  
Conserved Sequence ◽  
A Site ◽  
Gene Drives

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.

scholarly journals Ultra-conserved sequences in the genomes of highly diverse Anopheles mosquitoes, with implications for malaria vector control

2021 ◽  
Author(s):  
Samantha M. O’Loughlin ◽  
Annie J. Forster ◽  
Silke Fuchs ◽  
Tania Dottorini ◽  
Tony Nolan ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Regulatory Function ◽  
Malaria Vector Control ◽  
Target Sequence ◽  
Gene Drive ◽  
Conserved Sequences ◽  
Anopheles Mosquitoes ◽  
Starting Point ◽  
And Function ◽  
Gene Drives

ABSTRACTDNA sequences that are exactly conserved over long evolutionary time scales have been observed in a variety of taxa. Such sequences are likely under strong functional constraint and they have been useful in the field of comparative genomics for identifying genome regions with regulatory function. A potential new application for these ultra-conserved elements has emerged in the development of gene drives to control mosquito populations. Many gene drives work by recognising and inserting at a specific target sequence in the genome, often imposing a reproductive load as a consequence. They can therefore select for target sequence variants that provide resistance to the drive. Focusing on highly conserved, highly constrained sequences lowers the probability that variant, gene drive-resistant alleles can be tolerated.Here we search for conserved sequences of 18bp and over in an alignment of 21 Anopheles genomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containing Anopheles ultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenic Anopheles ultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of the Anopheles ultra-conserved elements contained no polymorphisms. Our list of Anopheles ultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria.

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

2021 ◽  
Author(s):  
Silke Fuchs ◽  
William T. Garrood ◽  
Anna Beber ◽  
Andrew Hammond ◽  
Roberto Galizi ◽  
...  
Keyword(s):  
Essential Gene ◽  
Target Site ◽  
Target Sequence ◽  
Gene Drive ◽  
Continuous Sampling ◽  
Conserved Sequence ◽  
Target Site Resistance ◽  
A Site ◽  
Gene Drives

CRISPR-based homing gene drives designed to disrupt essential genes whilst biasing their own inheritance can suppress 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 usually a reliable indicator that there is functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel gene drive in the malaria vector An. gambiae targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development and 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.

scholarly journals Regulation of gene drive expression increases invasive potential and mitigates resistance

2018 ◽  
Author(s):  
Andrew Hammond ◽  
Xenia Karlsson ◽  
Ioanna Morianou ◽  
Kyros Kyrou ◽  
Andrea Beaghton ◽  
...  
Keyword(s):  
First Generation ◽  
Target Site ◽  
Regulatory Sequences ◽  
Target Sequence ◽  
Mosquito Vector ◽  
Gene Drive ◽  
Dna Breaks ◽  
Target Site Resistance ◽  
The Impact ◽  
Gene Drives

AbstractCRISPR-Cas9 nuclease-based gene drives rely on inducing chromosomal breaks in the germline that are repaired in ways that lead to a biased inheritance of the drive. Gene drives designed to impair female fertility can suppress populations of the mosquito vector of malaria. However, strong unintended fitness costs, due to ectopic nuclease expression, and high levels of resistant mutations, limited the potential of the first generation of gene drives to spread.Here we show that changes to regulatory sequences in the drive element, designed to contain nuclease expression to the germline, confer improved fecundity over previous versions and generate drastically lower rates of target site resistance. We employed a genetic screen to show that this effect is explained by reduced rates of end-joining repair of DNA breaks at the target site caused by deposited nuclease in the embryo.Highlighting the impact of deposited Cas9, many of the mutations arising from this source of nuclease activity in the embryo are heritable, thereby having the potential to generate resistant target sites that reduce the penetrance of the gene drive.Finally, in cage invasion experiments these gene drives show improved invasion dynamics compared to first generation drives, resulting in greater than 90% suppression of the reproductive output and a delay in the emergence of target site resistance, even at a resistance-prone target sequence. We shed light on the dynamics of generation and selection of resistant alleles in a population by tracking, longitudinally, the frequency of resistant alleles in the face of an invading gene drive. Our results illustrate important considerations for future gene drive design and should expedite the development of gene drives robust to resistance.

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