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.

scholarly journals Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance

PLoS Genetics ◽  
2021 ◽  
Vol 17 (1) ◽  
pp. e1009321
Author(s):  
Andrew Hammond ◽  
Xenia Karlsson ◽  
Ioanna Morianou ◽  
Kyros Kyrou ◽  
Andrea Beaghton ◽  
...  
Keyword(s):  
Suppressive Effect ◽  
Target Site ◽  
Regulatory Sequences ◽  
Gene Drive ◽  
Dna Breaks ◽  
End Joining ◽  
A Genome ◽  
Target Site Resistance ◽  
Malaria Mosquitoes ◽  
Gene Drives

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.

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 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 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 Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Astrid Hoermann ◽  
Sofia Tapanelli ◽  
Paolo Capriotti ◽  
Giuseppe Del Corsano ◽  
Ellen KG Masters ◽  
...  
Keyword(s):  
Regulatory Sequences ◽  
Gene Drive ◽  
Passive Mode ◽  
Population Replacement ◽  
Guide Rnas ◽  
Efficient Gene ◽  
Genetic Modifications ◽  
Form Part ◽  
Endogenous Genes ◽  
Gene Drives

Gene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector Anopheles gambiae to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of Plasmodium falciparum and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an acceptable testing pathway of gene drives for malaria eradication.

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 Molecular safeguarding of CRISPR gene drive experiments

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Jackson Champer ◽  
Joan Chung ◽  
Yoo Lim Lee ◽  
Chen Liu ◽  
Emily Yang ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Natural Population ◽  
Early Embryo ◽  
Drive System ◽  
Target Site ◽  
Gene Drive ◽  
Gene Drives ◽  
Synthetic Target

CRISPR-based homing gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population. Here, we experimentally demonstrate the suitability of synthetic target site drives as well as split drives as flexible safeguarding strategies for gene drive experiments by showing that their performance closely resembles that of standard homing drives in Drosophila melanogaster. Using our split drive system, we further find that maternal deposition of both Cas9 and gRNA is required to form resistance alleles in the early embryo and that maternally-deposited Cas9 alone can power germline drive conversion in individuals that lack a genomic source of Cas9.

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 drives in plants: opportunities and challenges for weed control and engineered resilience

2019 ◽  
Vol 286 (1911) ◽  
pp. 20191515 ◽  
Author(s):  
Luke G. Barrett ◽  
Mathieu Legros ◽  
Nagalingam Kumaran ◽  
Donna Glassop ◽  
S. Raghu ◽  
...  
Keyword(s):  
Research Effort ◽  
Rapid Development ◽  
Crop Improvement ◽  
Wild Plant ◽  
Gene Drive ◽  
Genetic Management ◽  
Plant Populations ◽  
Drive Systems ◽  
The Impact ◽  
Gene Drives

Plant species, populations and communities are under threat from climate change, invasive pathogens, weeds and habitat fragmentation. Despite considerable research effort invested in genome engineering for crop improvement, the development of genetic tools for the management of wild plant populations has rarely been given detailed consideration. Gene drive systems that allow direct genetic management of plant populations via the spread of fitness-altering genetic modifications could be of great utility. However, despite the rapid development of synthetic tools and their enormous promise, little explicit consideration has been given to their application in plants and, to date, they remain untested. This article considers the potential utility of gene drives for the management of wild plant populations, and examines the factors that might influence the design, spread and efficacy of synthetic drives. To gain insight into optimal ways to design and deploy synthetic drive systems, we investigate the diversity of mechanisms underlying natural gene drives and their dynamics within plant populations and species. We also review potential approaches for engineering gene drives and discuss their potential application to plant genomes. We highlight the importance of considering the impact of plant life-history and genetic architecture on the dynamics of drive, investigate the potential for different types of resistance evolution, and touch on the ethical, regulatory and social challenges ahead.

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.

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