Computational and experimental performance of CRISPR homing gene drive strategies with multiplexed gRNAs

Champer, Samuel E; Oh, Suh Yeon; Liu, Chen; Wen, Zhaoxin; Clark, Andrew G.; Messer, Philipp W.

doi:10.1126/sciadv.aaz0525

Cited by 94 publications

(129 citation statements)

References 40 publications

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“…Perhaps most importantly, TA systems should be far less vulnerable to the formation of resistance alleles than current CRISPR homing drives. Even though multiplexing can somewhat ameliorate the formation of the more critical r1 alleles in homing drives, this typically comes at the cost of reduced drive efficiency due to a variety of factors involved in homology-directed repair [18,27]. For homing drives designed for population modification, it would also be necessary to target essential genes to remove resistance alleles that disrupt the target function [28], which would open up the possibility for incomplete homology-directed repair to form r1 alleles, possibly at rates that would preclude success of the drive.…”

Section: Discussionmentioning

confidence: 99%

“…This is not only because we assumed a high proportion of repair resulting in r1 sequences, but also because the possibility for simultaneous cutting was not included in our deterministic model. However, such events should take place quite often, particularly as the number gRNAs increases because even one instance of simultaneous gRNA cleavage would likely cause a large enough deletion to prevent formation of an r1 allele [18,27]. Additionally, homologydirected repair of drive cleavage using disrupted alleles as a template would likely preclude the formation of r1 alleles, and this was not taken into account in our model.…”

Section: Resistance To Ta Systemsmentioning

confidence: 99%

“…This would require targeting a site that can be sufficiently recoded without rendering the target gene nonfunctional (i.e., the target sequence is not fully constrained). Yet at such a site, it should then also be possible for resistance alleles to maintain gene function [27].…”

Section: Introductionmentioning

confidence: 99%

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Performance analysis of novel toxin-antidote CRISPR gene drive systems

et al. 2020

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Background: CRISPR gene drive systems allow the rapid spread of a genetic construct throughout a population. Such systems promise novel strategies for the management of vector-borne diseases and invasive species by suppressing a target population or modifying it with a desired trait. However, current homing-type drives have two potential shortcomings. First, they can be thwarted by the rapid evolution of resistance. Second, they lack any mechanism for confinement to a specific target population. In this study, we conduct a comprehensive performance assessment of several new types of CRISPR-based gene drive systems employing toxin-antidote (TA) principles, which should be less prone to resistance and allow for the confinement of drives to a target population due to invasion frequency thresholds. Results: The underlying principle of the proposed CRISPR toxin-antidote gene drives is to disrupt an essential target gene while also providing rescue by a recoded version of the target as part of the drive allele. Thus, drive alleles tend to remain viable, while wild-type targets are disrupted and often rendered nonviable, thereby increasing the relative frequency of the drive allele. Using individual-based simulations, we show that Toxin-Antidote Recessive Embryo (TARE) drives targeting an haplosufficient but essential gene (lethal when both copies are disrupted) can enable the design of robust, regionally confined population modification strategies with high flexibility in choosing promoters and targets. Toxin-Antidote Dominant Embryo (TADE) drives require a haplolethal target gene and a germline-restricted promoter, but they could permit faster regional population modification and even regionally confined population suppression. Toxin-Antidote Dominant Sperm (TADS) drives can be used for population modification or suppression. These drives are expected to spread rapidly and could employ a variety of promoters, but unlike TARE and TADE, they would not be regionally confined and also require highly specific target genes. Conclusions: Overall, our results suggest that CRISPR-based TA gene drives provide promising candidates for flexible ecological engineering strategies in a variety of organisms.

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Section: Discussionmentioning

confidence: 99%

Section: Resistance To Ta Systemsmentioning

confidence: 99%

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Performance analysis of novel toxin-antidote CRISPR gene drive systems

et al. 2020

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“…In addition to multiplexing gene drives to recognise more than one target site [ 3 , 4 , 11 , 12 ], akin to combination therapy with antibiotics, it is necessary to reduce the relative contribution of the error-prone end-joining repair pathway, over HDR, since this can serve to increase the range and complexity of potential resistant alleles on which selection could act.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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“…However, contrary to early suspicions that all possible target sites would be prone to such mutations (e.g., Drury et al 2017), at least one essential genomic site has been identified in mosquitoes that is intolerant of mutations and thus appears to escape this problem (Kyrou et al 2018). Furthermore, CRISPR-based gene drives can be designed to cut at multiple sites in the same gene (any of which suffice to allow the drive to function), thus ameliorating most concerns about the inevitability of target-site resistance (Oberhofer et al 2018; Champer et al 2018, 2020c). Other forms of resistance to CRISPR-based gene drives are theoretically possible, but it is too early to tell whether they will prove to interfere with implementations: inbreeding, proteins that block CRISPR complex activity, and suppressors of CRISPR gene expression (Stanley and Maxwell 2018; Bull et al 2019).…”

Section: Introductionmentioning

confidence: 99%

Evading resistance to gene drives

Gomulkiewicz

Thies

Bull

2020

Preprint

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Gene drives offer the possibility of altering and even suppressing wild populations of countless plant and animal species, and CRISPR technology now provides the technical feasibility of engineering them. However, population-suppression gene drives are prone to select resistance, should it arise. Here we develop mathematical and computational models to identify conditions under which suppression drives will evade resistance, even if resistance is present initially. We show that linkage between the resistance and drive loci is critical to the evolution, that evolution of resistance requires (negative) linkage disequilibrium between the two loci. When the two loci are unlinked or only partially so, a suppression drive that causes limited inviability can evolve to fixation while causing only a minor increase in resistance frequency. Once fixed, the drive allele no longer selects resistance. Single drives of this type would achieve only partial population suppression, but multiple drives (perhaps delivered sequentially) would allow arbitrary levels of suppression. Given that it is now possible to engineer CRISPR-based gene drives capable of circumventing allelic resistance, this design may allow for the engineering of suppression gene drives that are effectively resistance-proof.

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Computational and experimental performance of CRISPR homing gene drive strategies with multiplexed gRNAs

Cited by 94 publications

References 40 publications

Performance analysis of novel toxin-antidote CRISPR gene drive systems

Performance analysis of novel toxin-antidote CRISPR gene drive systems

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

Evading resistance to gene drives

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