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 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 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

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 A CRISPR homing gene drive targeting a haplolethal gene removes resistance alleles and successfully spreads through a cage population

2020 ◽  
Vol 117 (39) ◽  
pp. 24377-24383 ◽  
Author(s):  
Jackson Champer ◽  
Emily Yang ◽  
Esther Lee ◽  
Jingxian Liu ◽  
Andrew G. Clark ◽  
...  
Keyword(s):  
Gene Drive ◽  
End Joining ◽  
Large Cage ◽  
Vector Borne Diseases ◽  
Guide Rnas ◽  
Genetic Modifications ◽  
New Strategy ◽  
Cage Population ◽  
Vector Borne ◽  
Gene Drives

Engineered gene drives are being explored as a new strategy in the fight against vector-borne diseases due to their potential for rapidly spreading genetic modifications through a population. However, CRISPR-based homing gene drives proposed for this purpose have faced a major obstacle in the formation of resistance alleles that prevent Cas9 cleavage. Here, we present a homing drive in Drosophila melanogaster that reduces the prevalence of resistance alleles below detectable levels by targeting a haplolethal gene with two guide RNAs (gRNAs) while also providing a rescue allele. Resistance alleles that form by end-joining repair typically disrupt the haplolethal target gene and are thus removed from the population because individuals that carry them are nonviable. We demonstrate that our drive is highly efficient, with 91% of the progeny of drive heterozygotes inheriting the drive allele and with no functional resistance alleles observed in the remainder. In a large cage experiment, the drive allele successfully spread to all individuals within a few generations. These results show that a haplolethal homing drive can provide an effective tool for targeted genetic modification of entire 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 Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

2016 ◽  
Vol 113 (52) ◽  
pp. 14988-14993 ◽  
Author(s):  
Jason M. Wolfs ◽  
Thomas A. Hamilton ◽  
Jeremy T. Lant ◽  
Marcon Laforet ◽  
Jenny Zhang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Homing Endonuclease ◽  
Target Site ◽  
Hek293 Cells ◽  
Dna Breaks ◽  
End Joining ◽  
Minimal Processing ◽  
Gene Knockouts ◽  
Alternative Nhej ◽  
Dna Ends

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

scholarly journals Dodging silver bullets: good CRISPR gene-drive design is critical for eradicating exotic vertebrates

2017 ◽  
Vol 284 (1860) ◽  
pp. 20170799 ◽  
Author(s):  
Thomas A. A. Prowse ◽  
Phillip Cassey ◽  
Joshua V. Ross ◽  
Chandran Pfitzner ◽  
Talia A. Wittmann ◽  
...  
Keyword(s):  
Female Sterility ◽  
Gene Drive ◽  
End Joining ◽  
Guide Rna ◽  
Deleterious Alleles ◽  
Precise Knowledge ◽  
Animal Populations ◽  
Individual Based Models ◽  
Non Homologous End Joining ◽  
Gene Drives

Self-replicating gene drives that can spread deleterious alleles through animal populations have been promoted as a much needed but controversial ‘silver bullet’ for controlling invasive alien species. Homing-based drives comprise an endonuclease and a guide RNA (gRNA) that are replicated during meiosis via homologous recombination. However, their efficacy for controlling wild populations is threatened by inherent polymorphic resistance and the creation of resistance alleles via non-homologous end-joining (NHEJ)-mediated DNA repair. We used stochastic individual-based models to identify realistic gene-drive strategies capable of eradicating vertebrate pest populations (mice, rats and rabbits) on islands. One popular strategy, a sex-reversing drive that converts heterozygous females into sterile males, failed to spread and required the ongoing deployment of gene-drive carriers to achieve eradication. Under alternative strategies, multiplexed gRNAs could overcome inherent polymorphic resistance and were required for eradication success even when the probability of NHEJ was low. Strategies causing homozygotic embryonic non-viability or homozygotic female sterility produced high probabilities of eradication and were robust to NHEJ-mediated deletion of the DNA sequence between multiplexed endonuclease recognition sites. The latter two strategies also purged the gene drive when eradication failed, therefore posing lower long-term risk should animals escape beyond target islands. Multiplexing gRNAs will be necessary if this technology is to be useful for insular extirpation attempts; however, precise knowledge of homing rates will be required to design low-risk gene drives with high probabilities of eradication success.

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 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 A Two-in-One Strategy: Target and Nontarget Site Mechanisms Both Play Important Role in IMI-Resistant Weedy Rice

2021 ◽  
Vol 22 (3) ◽  
pp. 982
Author(s):  
Ru-Ann Yean ◽  
Masilamany Dilipkumar ◽  
Sadequr Rahman ◽  
Beng-Kah Song
Keyword(s):  
Genome Wide Association Study ◽  
Colorimetric Assay ◽  
Acetolactate Synthase ◽  
Target Site ◽  
Weedy Rice ◽  
Nucleotide Polymorphisms ◽  
A Genome ◽  
Key Factor ◽  
Target Site Resistance ◽  
Branched Chain

The introduction of Clearfield technology allows the use of imidazolinone (IMI) herbicides to control weedy rice. Imidazolinone herbicides stop the acetolactate synthase (ALS) enzyme from synthesizing branched-chain amino acids, resulting in the death of the plant. Since the launch of Clearfield technology in Malaysia in 2010, many farmers have replaced traditional cultivars with Clearfield (CL) rice lines (MR220-CL1 and MR220-CL2). This technology was initially effective; however, in recent years, local farmers have reported the reduced efficacy of IMI herbicides in controlling the spread of weedy rice. Under IMI herbicide treatment, in previous weedy rice studies, the target-site resistance (TSR) mechanism of the ALS gene has been suggested as a key factor conferring herbicide resistance. In our study, a combination of ALS gene sequencing, enzyme colorimetric assay, and a genome-wide association study (GWAS) highlighted that a non-target-site resistance (NTSR) can be an alternative molecular mechanism in IMI-resistant weedy rice. This is supported by a series of evidence, including a weak correlation between single nucleotide polymorphisms (SNPs) within the ALS exonic region and ALS enzyme activity. Our findings suggest that the adaptability of weedy rice in Clearfield rice fields can be more complicated than previously found in other rice strains.

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