scholarly journals The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito

2017 ◽  
Author(s):  
Andrew Hammond ◽  
Kyros Kyrou ◽  
Marco Bruttini ◽  
Ace North ◽  
Roberto Galizi ◽  
...  
Keyword(s):  
Initial Increase ◽  
Gene Drive ◽  
Malaria Mosquito ◽  
Induced Mutations ◽  
Genetic Traits ◽  
The Face ◽  
Insect Populations ◽  
Emergence Of Resistance ◽  
Gene Drives ◽  
Population Suppression

ABSTRACTGene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance gene drives can rapidly introduce genetic traits even if these confer a negative fitness on the population.We have recently developed gene drives based on a CRISPR nuclease construct that is designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, in a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we noticed a gradual decrease in its frequency that was accompanied emergence of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations show rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance for future gene drive applications.

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 A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chrysanthi Taxiarchi ◽  
Andrea Beaghton ◽  
Nayomi Illansinhage Don ◽  
Kyros Kyrou ◽  
Matthew Gribble ◽  
...  
Keyword(s):  
Genetically Modified Organisms ◽  
Reproductive Capacity ◽  
Gene Drive ◽  
Vector Borne Diseases ◽  
Pathogen Effectors ◽  
Germline Expression ◽  
Vector Borne ◽  
Malaria Mosquitoes ◽  
Gene Drives ◽  
Population Suppression

AbstractCRISPR-based gene drives offer promising means to reduce the burden of pests and vector-borne diseases. These techniques consist of releasing genetically modified organisms carrying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modifications. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer fitness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes.

scholarly journals Tethered homing gene drives: a new design for spatially restricted population replacement and suppression

2018 ◽  
Author(s):  
Sumit Dhole ◽  
Alun L. Lloyd ◽  
Fred Gould
Keyword(s):  
Mathematical Model ◽  
Genetic Load ◽  
Target Population ◽  
Gene Drive ◽  
Population Replacement ◽  
Additional Release ◽  
New Gene ◽  
Gene Drives ◽  
Population Suppression ◽  
Proof Of Principle

ABSTRACTOptimism regarding potential epidemiological and conservation applications of modern gene drives is tempered by concern about the potential unintended spread of engineered organisms beyond the target population. In response, several novel gene drive approaches have been proposed that can, under certain conditions, locally alter characteristics of a population. One challenge for these gene drives is the difficulty of achieving high levels of localized population suppression without very large releases in face of gene flow. We present a new gene drive system, Tethered Homing (TH), with improved capacity for localized population alteration, especially for population suppression. The TH drive is based on driving a payload gene using a homing construct that is anchored to a spatially restricted gene drive. We use a proof of principle mathematical model to show the dynamics of a TH drive that uses engineered underdominance as an anchor. This system is composed of a split homing drive and a two-locus engineered underdominance drive linked to one part of the split drive (the Cas endonuclease). In addition to improved localization, the TH system offers the ability to gradually adjust the genetic load in a population after the initial alteration, with minimal additional release effort.

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 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 A homing suppression gene drive with multiplexed gRNAs maintains high drive conversion efficiency and avoids functional resistance alleles

2021 ◽  
Author(s):  
Emily Yang ◽  
Matthew Metzloff ◽  
Anna M. Langmüller ◽  
Andrew G. Clark ◽  
Philipp W Messer ◽  
...  
Keyword(s):  
Conversion Efficiency ◽  
Target Gene ◽  
Gene Drive ◽  
Wild Type ◽  
Homology Directed Repair ◽  
High Drive ◽  
Target Sites ◽  
Cage Population ◽  
Gene Drives ◽  
Population Suppression

Gene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive in Drosophila melanogaster that utilized multiplexed gRNAs to inhibit the formation of functional resistance alleles in its female fertility target gene. The gRNA target sites were placed close together, preventing reduction in drive conversion efficiency. The construct reached a moderate equilibrium frequency in cage populations without apparent formation of resistance alleles. However, a moderate fitness cost prevented suppression of the cage population. Nevertheless, our results experimentally demonstrate the viability of the multiplexed gRNAs strategy in homing type suppression gene drives.

scholarly journals Resistance is futile: A CRISPR homing gene drive targeting a haplolethal gene

2019 ◽  
Author(s):  
Jackson Champer ◽  
Emily Yang ◽  
Yoo Lim Lee ◽  
Jingxian Liu ◽  
Andrew G. Clark ◽  
...  
Keyword(s):  
Target Gene ◽  
Gene Drive ◽  
End Joining ◽  
Large Cage ◽  
Vector Borne Diseases ◽  
Genetic Modifications ◽  
Vector Borne ◽  
Potential Strategy ◽  
Gene Drives ◽  
Population Suppression

ABSTRACTEngineered gene drives are being explored as a potential strategy for the control of vector-borne diseases due to their ability to rapidly spread genetic modifications through a population. While an effective CRISPR homing gene drive for population suppression has recently been demonstrated in mosquitoes, formation of resistance alleles that prevent Cas9 cleavage remains the major obstacle for drive strategies aiming at population modification, rather than elimination. Here, we present a homing drive in Drosophila melanogaster that reduces resistance allele formation below detectable levels by targeting a haplolethal gene with two gRNAs while also providing a rescue allele. This is because any resistance alleles that form by end-joining repair will typically disrupt the haplolethal target gene, rendering the individuals carrying them nonviable. We demonstrate that our drive is highly efficient, with 91% of the progeny of drive heterozygotes inheriting the drive allele and with no resistance alleles observed in the remainder. In a large cage experiment, the drive allele successfully spread to all individuals. These results show that a haplolethal homing drive can be a highly effective tool for population modification.

scholarly journals Anopheles gambiae Genome Conservation as a Resource for Rational Gene Drive Target Site Selection

Insects ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 97
Author(s):  
Nace Kranjc ◽  
Andrea Crisanti ◽  
Tony Nolan ◽  
Federica Bernardini
Keyword(s):  
Anopheles Gambiae ◽  
Data Storage ◽  
Genomic Variation ◽  
Malaria Vectors ◽  
Anopheles Coluzzii ◽  
Structural Constraints ◽  
Gene Drive ◽  
Genetic Traits ◽  
A Genome ◽  
Insight Into

The increase in molecular tools for the genetic engineering of insect pests and disease vectors, such as Anopheles mosquitoes that transmit malaria, has led to an unprecedented investigation of the genomic landscape of these organisms. The understanding of genome variability in wild mosquito populations is of primary importance for vector control strategies. This is particularly the case for gene drive systems, which look to introduce genetic traits into a population by targeting specific genomic regions. Gene drive targets with functional or structural constraints are highly desirable as they are less likely to tolerate mutations that prevent targeting by the gene drive and consequent failure of the technology. In this study we describe a bioinformatic pipeline that allows the analysis of whole genome data for the identification of highly conserved regions that can point at potential functional or structural constraints. The analysis was conducted across the genomes of 22 insect species separated by more than hundred million years of evolution and includes the observed genomic variation within field caught samples of Anopheles gambiae and Anopheles coluzzii, the two most dominant malaria vectors. This study offers insight into the level of conservation at a genome-wide scale as well as at per base-pair resolution. The results of this analysis are gathered in a data storage system that allows for flexible extraction and bioinformatic manipulation. Furthermore, it represents a valuable resource that could provide insight into population structure and dynamics of the species in the complex and benefit the development and implementation of genetic strategies to tackle malaria.

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