Article

Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... For most reports of synthetic homing drives, the method of quantifying inheritance bias (phenotypic scoring of progeny carrying a marker gene in the drive allele) cannot differentiate between the underlying inheritance bias mechanism. However, a small subset of reports of homing drives have had marked chromosomes [11][12][13][14][15][16] , especially pre-CRISPR [17][18][19][20] , which may allow homing and meiotic inheritance bias to be differentiated. Through the use of a coincidental chromosomal marker, we observed evidence for meiotic drive in male A. aegypti with a homing CRISPR gene drive design reported by Li et al. 12 . ...
... To our knowledge, for gene drives designed to function through homing, recipient/donor chromosome markers have been used with non-CRISPR nucleases in D. melanogaster [17][18][19] and A. gambiae 20 and with CRISPR-Cas9 in D. melanogaster 11,15 , A. aegypti 12 and Mus musculus 13,16 . There may be additional cases in which a split drive element can coincidentally act as a chromosome marker 14,57 . ...
... In D. melanogaster, some studies have noted a reduced inheritance of the recipient chromosome, however, these may be attributable to genotype-specific fitness effects instead of DNA damage-induced loss of the recipient chromosome 11,14 . Xu et al. have performed the most extensive investigation of homing drives with marked chromosomes and found a mix of homing and bias through chromosome damage 15 . ...
Article
Full-text available
CRISPR/Cas gene drives can bias transgene inheritance through different mechanisms. Homing drives are designed to replace a wild-type allele with a copy of a drive element on the homologous chromosome. In Aedes aegypti, the sex-determining locus is closely linked to the white gene, which was previously used as a target for a homing drive element (wGDe). Here, through an analysis using this linkage we show that in males inheritance bias of wGDe did not occur by homing, rather through increased propagation of the donor drive element. We test the same wGDe drive element with transgenes expressing Cas9 with germline regulatory elements sds3, bgcn, and nup50. We only find inheritance bias through homing, even with the identical nup50-Cas9 transgene. We propose that DNA repair outcomes may be more context dependent than anticipated and that other previously reported homing drives may, in fact, bias their inheritance through other mechanisms.
... Among the molecular approaches: (a) a threshold-dependent gene drive [47], (b) a safeguarding gene drive, and (c) a reverse gene drive, were first designed and tested in S. cerevisiae [9]. (d) The use of genetic neutralizing elements [88] and (e) anti-Crispr protein [89] was also two novel containment measures in gene drive research. 7 BioDesign Research later engineered in the fruit fly D. melanogaster [86] and the human disease vector, A. aegypti [87]. ...
... 7 BioDesign Research later engineered in the fruit fly D. melanogaster [86] and the human disease vector, A. aegypti [87]. In recent years, we also witnessed the design and assessment of new selfpropagating genetic-neutralizing elements in D. melanogaster that can either disable Cas9 carried by a gene drive (e-CHACR (erasing construct hitchhiking on the autocatalytic chain reaction)) or remove and substitute the gene drive (ERACR (element reversing the autocatalytic chain reaction)) [88]. Both designed elements encode only gRNAs without a Cas9. ...
... The additional gRNA(s) are designed to target, cleave, and inactivate the Cas9 component of the gene drive. On the contrary, ERACRs are usually inserted at the same genomic locus as the gene drive and encode for two gRNAs that will interact and form a complex with the Cas9 to generate cuts on both sides of the drive element to remove it and substitute it [88]. Data from this study revealed that e-CHACRs were efficiently able to inactivate Cas9 but with diverse transmission frequencies, whereas ERACRs were able to frequently delete and replace a gene-drive element. ...
Article
Full-text available
Ongoing pest and disease outbreaks pose a serious threat to human, crop, and animal lives, emphasizing the need for constant genetic discoveries that could serve as mitigation strategies. Gene drives are genetic engineering approaches discovered decades ago that may allow quick, super-Mendelian dissemination of genetic modifications in wild populations, offering hopes for medicine, agriculture, and ecology in combating diseases. Following its first discovery, several naturally occurring selfish genetic elements were identified and several gene drive mechanisms that could attain relatively high threshold population replacement have been proposed. This review provides a comprehensive overview of the recent advances in gene drive research with a particular emphasis on CRISPR-Cas gene drives, the technology that has revolutionized the process of genome engineering. Herein, we discuss the benefits and caveats of this technology and place it within the context of natural gene drives discovered to date and various synthetic drives engineered. Later, we elaborate on the strategies for designing synthetic drive systems to address resistance issues and prevent them from altering the entire wild populations. Lastly, we highlight the major applications of synthetic CRISPR-based gene drives in different living organisms, including plants, animals, and microorganisms.
... These elements are typically designed to target the Cas9 sequence within an autonomous drive that has already spread through a population. The sgRNA or sgRNAs derived from the braking element associate with the Cas9 protein produced from the drive allele, either mutating the Cas9 sequence, or replacing the drive allele with that of the reversal element (Gantz and Bier, 2016;Wu et al., 2016;Xu et al., 2020). ...
... For example, in the eCHACR (erasing construct hitchhiking on the autocatalytic chain reaction) iteration, a neutralizing element is inserted into the genome at a location independent of the drive allele, but is capable of self-copying and inactivating the Cas9 sequence of the gene drive. The eCHACR construct functions by exploiting the tendency of the Cas9 nuclease to generate alleles resistant to further cutting, resulting from erroneous repair of the double stranded break through nonhomologous end joining (NHEJ) (Xu et al., 2020). The eCHACR concept was demonstrated to be capable of neutralizing a gene drive in multi-generation cage trials. ...
... These sgRNAs may be directed at the insertion site to facilitate gene conversion and self-copying, or at the Cas9 sequence of the gene drive allele in order to inactivate it. In another design, an ERACR (element reversing the autocatalytic chain reaction) self-copying element is inserted directly into a locus in the drive allele, thereby deleting and replacing it (Xu et al., 2020). ERACRs encode two different sgRNAs, but they are directed at target sites flanking the drive element to facilitate its exchange with the ERACR transgene. ...
Article
Full-text available
CRISPR-based autonomous homing gene drives are a potentially transformative technology with the power to reduce the prevalence of, or even eliminate, vector-borne diseases, agricultural pests, and invasive species. However, there are a number of regulatory, ethical, environmental, and sociopolitical concerns surrounding the potential use of gene drives, particularly regarding the possibility for any unintended outcomes that might result from such a powerful technology. Therefore, there is an imminent need for countermeasures or technologies capable of exerting precise spatiotemporal control of gene drives, if their transformative potential is ever to be fully realized. This review summarizes the current state of the art in the development of technologies to prevent the uncontrolled spread of CRISPR-based autonomous homing gene drives.
... This also occurred in female progeny suggesting that the maternally inherited drive converted the paternally contributed functional yellow allele in the early embryo. However, later publications found substantially lower inheritance rates (76-85%) with a nearidentical constructs but including a fluorescent marker (Champer et al., 2017;Xu et al., 2020). It is probable that part of the seemingly super-Mendelian inheritance of the HEG observed in the earlier study was due to the maternal "deposition" of the Cas9:gRNA complex without inheritance of the drive expressing allele itself (Xu et al., 2020). ...
... However, later publications found substantially lower inheritance rates (76-85%) with a nearidentical constructs but including a fluorescent marker (Champer et al., 2017;Xu et al., 2020). It is probable that part of the seemingly super-Mendelian inheritance of the HEG observed in the earlier study was due to the maternal "deposition" of the Cas9:gRNA complex without inheritance of the drive expressing allele itself (Xu et al., 2020). The deposited nuclease and gRNA could cause the disruption of the yellow target gene in the absence of inheritance of the HEG itself. ...
... If the recipient chromosome is marked, inheritance bias through homing or through the loss of the recipient chromosome can be distinguished. An under representation of the recipient chromosome marker has been reported in multiple publications with an element otherwise expected to function through homing (Guichard et al., 2019;Xu et al., 2020;Terradas et al., 2021b), with in some cases meiotic drive seemingly exclusively mediating the observed inheritance bias Verkuijl et al., 2020). In one study, under representation of a restriction enzyme site nearby the I-SceI cut site on the recipient chromosome was suggested to be due to DNA repair after homing also replacing the nearby marker (termed "co-conversion" or "copy-grafting") (Windbichler et al., 2011). ...
Article
Full-text available
Making discrete and precise genetic changes to wild populations has been proposed as a means of addressing some of the world’s most pressing ecological and public health challenges caused by insect pests. Technologies that would allow this, such as synthetic gene drives, have been under development for many decades. Recently, a new generation of programmable nucleases has dramatically accelerated technological development. CRISPR-Cas9 has improved the efficiency of genetic engineering and has been used as the principal effector nuclease in different gene drive inheritance biasing mechanisms. Of these nuclease-based gene drives, homing endonuclease gene drives have been the subject of the bulk of research efforts (particularly in insects), with many different iterations having been developed upon similar core designs. We chart the history of homing gene drive development, highlighting the emergence of challenges such as unintended repair outcomes, “leaky” expression, and parental deposition. We conclude by discussing the progress made in developing strategies to increase the efficiency of homing endonuclease gene drives and mitigate or prevent unintended outcomes.
... As per the crossscheme depicted in Fig. 2C, a Cas9 expressing transgene (vasa-Cas9) was provided from an unlinked third chromosomal source. Introduction of Cas9 led to a substantial increase of y <CC|pF|> (DsRed + ) transmission in F2 progeny, compared to F2 progeny from control (-Cas9) crosses, (46.8% to 85.1% in females and 39.9% to 84.5% in males, Fig. 2D), confirming that the drive element copies with similar efficiency to other drives inserted at this same site [22][23][24][25] . The fraction of F2 w − receiver progeny, however, was not altered significantly in presence of the Cas9 source (~50% for both control and drive) ( Supplementary Fig. 4), indicating that DNA cleavage directed by gRNA-F did not result in frequent production of lethal mutations. ...
... uncleavable mutations by non-homologous end joining (NHEJ) events at the yellow locus may limit the spread of the y <CC|pF|> element as previously reported [22][23][24] . The frequency of the unlinked Cas9 (GFP+) source remained stable over 10 generations suggesting that the vasaCas9 transgene did not impose a detectable fitness cost, even when associated with the drive element ( Supplementary Fig. 6A), a phenomenon that has been observed in other contexts 28,29 . ...
... However, because gRNA-F generates few NHEJ alleles, its modest, but clean, cleavage profile supports a delayed but eventually superior drive outcome. Another factor likely to contribute to the extensive reversal in IR obtained in these drive experiments is the synergistic action of two independent selective processes 24 wherein the allelic drive promoting Super-Mendelian inheritance of the L1014 allele (e.g., mean copying efficiency to receiver chromosomes = 0.29) acts in combination with the greater relative fitness of the wild-type L1014 versus 1014F allele (e.g., median fitness cost of 1014F allele in males = 0.28) (Fig. 1C-F , Supplementary Tables 5 and 6), a feature also likely to be relevant in field contexts 5,7,9,[31][32][33][34] . We note that this synergy between the independently acting gene-drive and positive selection for the 1014 allele in the driving configuration (+Cas9) also may be reflected by the reduced variance in allelic frequencies between cages observed in generation 9 (Fig. 3F, most obvious in males) compared to the action of positive selection alone (−Cas9), a phenomenon we have observed in other studies 24 . ...
Article
Full-text available
A recurring target-site mutation identified in various pests and disease vectors alters the voltage gated sodium channel (vgsc) gene (often referred to as knockdown resistance or kdr) to confer resistance to commonly used insecticides, pyrethroids and DDT. The ubiquity of kdr mutations poses a major global threat to the continued use of insecticides as a means for vector control. In this study, we generate common kdr mutations in isogenic laboratory Drosophila strains using CRISPR/Cas9 editing. We identify differential sensitivities to permethrin and DDT versus deltamethrin among these mutants as well as contrasting physiological consequences of two different kdr mutations. Importantly, we apply a CRISPR-based allelic-drive to replace a resistant kdr mutation with a susceptible wild-type counterpart in population cages. This successful proof-of-principle opens-up numerous possibilities including targeted reversion of insecticide-resistant populations to a native susceptible state or replacement of malaria transmitting mosquitoes with those bearing naturally occurring parasite resistant alleles.
... In addition, there are cases where tight functional constraints at the gene drive target sequence may hinder the development of this type of reversal approach, such as for the dsx-targeting gene drive that we recently developed in the malaria vector Anopheles gambiae 2 . Alternative reversal strategies involve the use of CRISPR components to cleave and replace DNA sequences specific to the gene drive construct with 15,16 or without [17][18][19] the use of an additional Cas9 expression cassette. Recently, guide RNAonly systems developed in Drosophila showed capacity to inactivate or replace gene drives in caged populations 19 . ...
... Alternative reversal strategies involve the use of CRISPR components to cleave and replace DNA sequences specific to the gene drive construct with 15,16 or without [17][18][19] the use of an additional Cas9 expression cassette. Recently, guide RNAonly systems developed in Drosophila showed capacity to inactivate or replace gene drives in caged populations 19 . Although these strategies may offer the option to replace the drive with one or few "refractory alleles" or even restore the wild-type population, there are several complications attributable to the DNAcleaving nature of the reversal that remain to be addressed, such as the formation and selection of resistant alleles and/or genomic rearrangement at the drive locus targeted by the reversal nuclease. ...
... Here, gene drive inhibition is achieved via protein-protein interaction, rather than DNA cleavage of drive-specific sequences, bypassing potential unintended effects of genome editing alterations caused by alternative Cas9-gRNA or gRNA-only reversal strategies 15,[17][18][19] . Repair of CRISPR-mediated cleavage of genetic components encoding for the gene drive may alter either the activity or the specificity of action with unpredictable consequence in the long term; for example, upon modification of the targeting sequence of the driving gRNA. ...
Article
Full-text available
CRISPR-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. Technologies that can halt the spread of gene drives would be highly useful in controlling or reverting their effect. Here the authors use the anti-CRISPR protein AcrIIA4 to inactivate drives in A. gambiae.
... They could be designed to mitigate potential unintended consequences of another engineered gene drive by removing or preventing the spread of the original organism. The development of reversal gene drives is proceeding in flies and mosquitoes, and their potential use in the environment is being explored with population genetic models Vella et al., 2017;Friedman et al., 2020;Xu et al., 2020). However, it has been noted that reversal gene drives may induce further changes that may undo a phenotypic alteration caused by the initial drive, so they may not restore the original modification to the wild type or redress fully ecological effects from the original engineered gene drive (e.g. ...
... However, it has been noted that reversal gene drives may induce further changes that may undo a phenotypic alteration caused by the initial drive, so they may not restore the original modification to the wild type or redress fully ecological effects from the original engineered gene drive (e.g. Champer et al., 2016;NASEM, 2016;Vella et al., 2017;CSS-ENSSER-VDW, 2019;Rode et al., 2020;Xu et al., 2020). ...
... Neutralising genetic elements such as e-CHACR (erasing Constructs Hitchhiking on the Autocatalytic Chain Reaction) and ERACR (Element Reversing the Autocatalytic Chain Reaction) have been proposed , and subsequently developed and tested in D. melanogaster to halt the spread of an engineered gene drive by inactivating Cas9 carried by an engineered gene drive, or delete/ eliminate and replace a drive, respectively (Xu et al., 2020). While Xu et al. (2020) provide encouraging evidence for neutralising an engineered gene drive with e-CHACRs or ERACRs, the authors caution for unexpected outcomes. ...
Article
Full-text available
Advances in molecular and synthetic biology are enabling the engineering of gene drives in insects for disease vector/pest control. Engineered gene drives (that bias their own inheritance) can be designed either to suppress interbreeding target populations or modify them with a new genotype. Depending on the engineered gene drive system, theoretically, a genetic modification of interest could spread through target populations and persist indefinitely, or be restricted in its spread or persistence. While research on engineered gene drives and their applications in insects is advancing at a fast pace, it will take several years for technological developments to move to practical applications for deliberate release into the environment. Some gene drive modified insects (GDMIs) have been tested experimentally in the laboratory, but none has been assessed in small‐scale confined field trials or in open release trials as yet. There is concern that the deliberate release of GDMIs in the environment may have possible irreversible and unintended consequences. As a proactive measure, the European Food Safety Authority (EFSA) has been requested by the European Commission to review whether its previously published guidelines for the risk assessment of genetically modified animals (EFSA, 2012 and 2013), including insects (GMIs), are adequate and sufficient for GDMIs, primarily disease vectors, agricultural pests and invasive species, for deliberate release into the environment. Under this mandate, EFSA was not requested to develop risk assessment guidelines for GDMIs. In this Scientific Opinion, the Panel on Genetically Modified Organisms (GMO) concludes that EFSA's guidelines are adequate, but insufficient for the molecular characterisation (MC), environmental risk assessment (ERA) and post‐market environmental monitoring (PMEM) of GDMIs. While the MC,ERA and PMEM of GDMIs can build on the existing risk assessment framework for GMIs that do not contain engineered gene drives, there are specific areas where further guidance is needed for GDMIs.
... Thus, in future efforts to convert additional sGDs to fGDs, Cas9Hack lines inserted near the genomic integration site of the sGD element in question may prove most efficient. Upon conversion, the size of the copying cargo, with fGD being~8 kb larger than sGD, may also influence overall conversion efficiencies since modestly reduced copying has been reported for larger drives inserted into the same locus 11,21 . ...
... Detailed as these models are, the discrepancies suggest that yet more fine-scale models may be required to capture biochemical differences in Cas9/gRNA complexes: different categories of fitness costs (active drive versus cooccurrence) in the progeny of Cas9/gRNA-bearing males versus females or parents versus grandparents (or even great-grandparents), DNA accessibility during different developmental stages, and repair mechanisms available when different promoters are active. Modeling and analysis performed in this study, of similar or even greater complexity, is rapidly becoming requisite in the gene-drive toolbox, both for the analysis of experimental data 21,22 and simulation of potential results to inform conservation 23 and disease-elimination campaigns 7,24 . In particular, we demonstrate the application of a Naive-Bayes multiobservation HMM that optimizes the regular HMM in a parallel framework by incorporating phenotypic and genotypic data. ...
Article
Full-text available
The core components of CRISPR-based gene drives, Cas9 and guide RNA (gRNA), either can be linked within a self-contained single cassette (full gene-drive, fGD) or be provided in two separate elements (split gene-drive, sGD), the latter offering greater control options. We previously engineered split systems that could be converted genetically into autonomous full drives. Here, we examine such dual systems inserted at the spo11 locus that are recoded to restore gene function and thus organismic fertility. Despite minimal differences in transmission efficiency of the sGD or fGD drive elements in single generation crosses, the reconstituted spo11 fGD cassette surprisingly exhibits slower initial drive kinetics than the unlinked sGD element in multigenerational cage studies, but then eventually catches up to achieve a similar level of final introduction. These unexpected kinetic behaviors most likely reflect differing transient fitness costs associated with individuals co-inheriting Cas9 and gRNA transgenes during the drive process. CRISPR-based gene-drives can carry the Cas9 and guide RNA (gRNA) components in a single-linked cassette or in separate elements inserted into different genomic loci. Here the authors genetically transform and compare full versus split drives, with the former performing less efficiently than predicted.
... Recent attention has focused on CRISPR-based homing gene drives, for their ability to spread widely and their potential to control vector-borne diseases on a wide scale Kyrou et al. 2018); however, there are also threshold-dependent gene drive systems that can now be engineered using CRISPR, such as chromosomal translocations (Buchman et al. 2018) and various forms of underdominance (Akbari et al. 2013), as well as temporally self-limiting gene drive systems, such as split drive (Li et al. 2020), which display transient drive activity before being eliminated by virtue of a fitness cost. The CRISPR revolution has also enabled gene drive countermeasures to be engineered, such as homing-based drive remediation systems, ERACR (element for the reversal of the autocatalytic chain reaction) and e-CHACR (erasing construct hitchhiking on the autocatalytic chain reaction) (Gantz and Bier 2016;Xu et al. 2020). CRISPR-based homing gene drive systems bias inheritance in their favor by cleaving a highly specific target sequence in the host genome and copying themselves to the cut chromosome through a mechanism known as homology-directed repair (Gantz and Bier 2015;Champer et al. 2016). ...
... Gene drive countermeasures such as ERACR and e-CHACR are another option for remediation. The ERACR system consists of a second homing system with a target site corresponding to the original drive system, essentially removing the original system as it homes into it, while utilizing the Cas9 of the original system and thus removing that as well (Gantz and Bier 2016;Xu et al. 2020). The e-CHACR system uses the Cas9 from the original homing system to home into a second site in the genome in addition to the site of the original drive system, thus driving itself into the population while removing the original system and its Cas9 in the process Bier 2016, Xu et al. 2020). ...
Chapter
Full-text available
The discovery of CRISPR-based gene editing and its application to homing-based gene drive has been greeted with excitement, for its potential to control mosquito-borne diseases on a wide scale, and concern, for the invasiveness and potential irreversibility of a release. At the same time, CRISPR-based gene editing has enabled a range of self-limiting gene drive systems to be engineered with much greater ease, including (1) threshold-dependent systems, which tend to spread only when introduced above a certain threshold population frequency, and (2) temporally self-limiting systems, which display transient drive activity before being eliminated by virtue of a fitness cost. As these CRISPR-based gene drive systems are yet to be field-tested, plenty of open questions remain to be addressed, and insights can be gained from precedents set by field trials of other novel genetics-based and biological control systems, such as trials of Wolbachia-transfected mosquitoes, intended for either population replacement or suppression, and trials of genetically sterile male mosquitoes, either using the RIDL system (release of insects carrying a dominant lethal gene) or irradiation. We discuss lessons learned from these field trials and implications for a phased exploration of gene drive technology, including homing-based gene drive, chromosomal translocations, and split gene drive as a system potentially suitable for an intermediate release.
... Cassette size may also influence conversion efficiencies, which could also be relevant in the context of the fGD element as the hacking mechanism inserts the whole promoter-Cas9 cassette into the locus, increasing the fGD cassette size to ~8kb longer than the sGD. Reduced copying efficiency has also been observed for larger drives inserted into the same yellow locus 11,24 . ...
... Mathematical modeling confirmed that many of the inferred drive parameters are similar for the sGD and fGD population cage experiments. Such modeling is rapidly becoming requisite in the gene drive toolbox, both for the analysis of experimental data 24,25 and simulation of potential results to inform conservation 26 and disease-elimination campaigns 27,28 . In this work, we demonstrate the application of a Naive-Bayes multi-observation HMM that optimizes the regular HMM in a highly-parallel framework by incorporating phenotypic and genotypic data. ...
Preprint
Full-text available
Gene-drive systems offer an important new avenue for spreading beneficial traits into wild populations. Their core components, Cas9 and guide RNA (gRNA), can either be linked within a single cassette (full gene drive, fGD) or provided in two separate elements (split gene drive, sGD) wherein the gRNA-bearing element drives in the presence of an independent static source of Cas9. We previously designed a system engineered to turn split into full gene drives. Here, we provide experimental proof-of-principle for such a convertible system inserted at the spo11 locus, which is recoded to restore gene function. In multigenerational cage studies, the reconstituted spo11 fGD cassette initially drives with slower kinetics than the unlinked sGD element (using the same Mendelian vasa-Cas9 source), but eventually reaches a similar level of final introgression. Different kinetic behaviors may result from transient fitness costs associated with individuals co-inheriting Cas9 and gRNA transgenes during the drive process.
... The development of such gene drives is proceeding in insects such as mosquitoes and fruit flies (e.g. Oberhofer et al., 2020;Xu et al., 2020;Taxiarchi et al., 2021), while their potential use is explored with population genetic models (e.g. Girardin et al., 2019;Rode et al., 2020). ...
... Systems have also been designed to either turn on or turn off gene drive activity in the presence or absence of small organic molecules that can easily enter cells (Heffel and Finnigan, 2019;López Del Amo et al., 2020). While reversal and inducible engineered gene drive systems hold promise for risk management, developers themselves caution against unforeseen consequences (Xu et al., 2020), and indicate that reversal engineered gene drives may not necessarily be the first choice for remediation efforts due to the associated uncertainty of introducing another gene drive approach (Marshall and Vasquez, 2021). ...
Article
Full-text available
The ability to engineer gene drives (genetic elements that bias their own inheritance) has sparked enthusiasm and concerns. Engineered gene drives could potentially be used to address long-standing challenges in the control of insect disease vectors, agricultural pests and invasive species, or help to rescue endangered species. However, risk concerns and uncertainty associated with potential environmental release of gene drive modified insects (GDMIs) have led some stakeholders to call for a global moratorium on such releases or the application of other strict precautionary measures to mitigate perceived risk assessment and risk management challenges. Instead, we provide recommendations that may help to improve the relevance of risk assessment and risk management frameworks for environmental releases of GDMIs. These recommendations include: (1) developing additional and more practical risk assessment guidance to ensure appropriate levels of safety; (2) making policy goals and regulatory decision-making criteria operational for use in risk assessment so that what constitutes harm is clearly defined; (3) ensuring a more dynamic interplay between risk assessment and risk management to manage uncertainty through closely interlinked pre-release modelling and post-release monitoring; (4) considering potential risks against potential benefits, and comparing them with those of alternative actions to account for a wider (management) context; and (5) implementing a modular, phased approach to authorisations for incremental acceptance and management of risks and uncertainty. Along with providing stakeholder engagement opportunities in the risk analysis process, the recommendations proposed may enable risk managers to make choices that are more proportionate and adaptive to potential risks, uncertainty and benefits of GDMI applications, and socially robust.
... A range of gene drives have been designed to be less invasive by nature, or to mitigate risks to an extent. Some stop spreading after a certain number of generations and are thus self-limiting [26][27][28] , some require high introduction frequencies [29][30][31] , some can stop or remove a gene drive that is already present in a population 32,33 , and some target locally fixed alleles so that the gene drive cannot spread in non-target populations 34 . With the advance of such containable gene drives, we can start to consider gene drive technology as a potential tool for controlling invasive social insects like the Asian hornet. ...
Article
Full-text available
Social insects are very successful invasive species, and the continued increase of global trade and transportation has exacerbated this problem. The yellow-legged hornet, Vespa velutina nigrithorax (henceforth Asian hornet), is drastically expanding its range in Western Europe. As an apex insect predator, this hornet poses a serious threat to the honey bee industry and endemic pollinators. Current suppression methods have proven too inefficient and expensive to limit its spread. Gene drives might be an effective tool to control this species, but their use has not yet been thoroughly investigated in social insects. Here, we built a model that matches the hornet’s life history and modelled the effect of different gene drive scenarios on an established invasive population. To test the broader applicability and sensitivity of the model, we also incorporated the invasive European paper wasp Polistes dominula. We find that, due to the haplodiploidy of social hymenopterans, only a gene drive targeting female fertility is promising for population control. Our results show that although a gene drive can suppress a social wasp population, it can only do so under fairly stringent gene drive-specific conditions. This is due to a combination of two factors: first, the large number of surviving offspring that social wasp colonies produce make it possible that, even with very limited formation of resistance alleles, such alleles can quickly spread and rescue the population. Second, due to social wasp life history, infertile individuals do not compete with fertile ones, allowing fertile individuals to maintain a large population size even when drive alleles are widespread. Nevertheless, continued improvements in gene drive technology may make it a promising method for the control of invasive social insects in the future.
... This would negate the issues we find of premature dissociation of drive elements and phantom cutting; upstream elements should very rarely segregate away from downstream elements, even with low inheritance biasing efficiency and fidelity. However, there are a number of reports of drives designed to function through homing, also affecting the inheritance of a separate sequence located on the same or homologous chromosome [11,14,29,31,38,57]. This collateral inheritance bias could, in a daisy-chain with tightly linked elements, cause two or more elements to bias themselves (more) like a self-perpetuating drive [7]. ...
Article
Full-text available
The introgression of genetic traits through gene drive may serve as a powerful and widely applicable method of biological control. However, for many applications, a self-perpetuating gene drive that can spread beyond the specific target population may be undesirable and preclude use. Daisy-chain gene drives have been proposed as a means of tuning the invasiveness of a gene drive, allowing it to spread efficiently into the target population, but be self-limiting beyond that. Daisy-chain gene drives are made up of multiple independent drive elements, where each element, except one, biases the inheritance of another, forming a chain. Under ideal inheritance biasing conditions, the released drive elements remain linked in the same configuration, generating copies of most of their elements except for the last remaining link in the chain. Through mathematical modelling of populations connected by migration, we have evaluated the effect of resistance alleles, different fitness costs, reduction in the cut-rate, and maternal deposition on two alternative daisy-chain gene drive designs. We find that the self-limiting nature of daisy-chain gene drives makes their spread highly dependent on the efficiency and fidelity of the inheritance biasing mechanism. In particular, reductions in the cut-rate and the formation of non-lethal resistance alleles can cause drive elements to lose their linked configuration. This severely reduces the invasiveness of the drives and allows for phantom cutting, where an upstream drive element cuts a downstream target locus despite the corresponding drive element being absent, creating and biasing the inheritance of additional resistance alleles. This phantom cutting can be mitigated by an alternative indirect daisy-chain design. We further find that while dominant fitness costs and maternal deposition reduce daisy-chain invasiveness, if overcome with an increased release frequency, they can reduce the spread of the drive into a neighbouring population.
... We speculate that two cut sites may create a large gap that is repaired using the homologous chromosome or the sister chromosome in the leptotene-to-pachytene gametocyte. We see parallels to a similar form of cooperativity between dual gRNAs versus single gRNA configurations observed for so-called Element Reversing the Autocatalytic Chain Reaction (ERACR) elements that delete and replace gene-drive elements in Drosophila (Xu et al., 2020). In addition, we speculate that cutting the genome at two potential sites improves the frequency of Cas9-mediated DSB formation and thus increases the frequency of initiation of HDR. ...
Preprint
The utility of Active Genetic (AG) gene conversion systems in rats and mice holds great promise for facilitating the production of complex strains harboring multiple humanizing genes. The practical application of such systems requires the identification of a robust, reusable, and highly efficient system. By characterizing twenty-eight different promoter and target site pairs we aimed to define the parameters needed to establish an efficient conversion system in male and female rats and mice. Using three specific meiosis prophase I active promoters to drive Cas9 expression. We studied several variables, including the number of Cas9 target sites, the distance between target sites, the cis versus trans configuration in linked pairs, and the effect of Cas9 copy number. In the rat, three of twelve tested configurations provided efficient AG gene conversion in the 22% - 67% range, and four others catalyzed AG in the 0.7-1% range. The rat Ddx4 (Vasa) promoter provides higher AG efficiency than the Sycp1 promoter. In mice, ten of sixteen tested configurations, using the Sycp1 and pSycp1 promoters, provided efficiency in the 0.3% - 3.2% range. In rats, Cas9 expression levels are remarkably well correlated with AG gene conversion efficiency. The rat cis r Cyp3A1 /r Cyp3A2 locus was the most successful configuration, with gene conversion efficiencies of 0.7%-67%. This target site has a special property; the two Cas9 target sites are nearly perfectly homologous in the 100 bases around the gRNA target site. Our findings identify key parameters that improve AG efficiency, including the use of two Cas9 target sites, and efficient promoters that drive high levels of Cas9 expression, that are correctly timed during gamete development. These findings also uncover the unexpected benefit of high homology at paired gRNA target sites to promote efficiency. We provide new data to guide future efforts to develop yet further improved AG systems.
... Invasive species represent a global issue that has worsened 30 with increased global trade and transportation (1,2). Sup- 31 pression of these invasive species is often prohibitively ex- 32 pensive, labour intensive, and largely ineffective (1). One 33 such species currently invasive in Europe is the yellow- 34 legged hornet Vespa velutina nigrithorax, hereafter called the 35 Asian hornet. ...
Preprint
Full-text available
Social insects are very successful invasive species, and the continued increase of global trade and transportation has exacerbated this problem. The yellow-legged hornet, Vespa velutina nigrithorax (henceforth Asian hornet), is drastically expanding its range in Western Europe. As an apex insect predator, this hornet poses a serious threat to the honey bee industry and endemic pollinators. Current suppression methods have proven too inefficient and expensive to limit its spread. Gene drives might be an effective tool to control this species, but their use has not yet been thoroughly investigated in social insects. Here, we built a model that matches the hornet's life history and modelled the effect of different gene drive scenarios on an established invasive population. To test the broader applicability and sensitivity of the model, we also incorporated the invasive European paper wasp Polistes dominula . We find that although a gene drive can spread through a social wasp population, it can only do so under stringent gene drive-specific conditions. The main issue is that the large number of offspring that social wasp colonies produce guarantees that, even with very limited formation of resistance alleles, such alleles will quickly spread and rescue the population. Furthermore, we find that only a gene drive targeting female fertility is promising for population control due to the haplodiploidy of social insects. Nevertheless, continued improvements in gene drive technology may make it a promising method for the control of invasive social insects.
... Just as gene drives with minimal genetic modifications reduce the changes introduced into individual genomes, approaches reducing the modifications introduced to populations (to only those components necessary for global health, agriculture, or ecology, for example) have obvious appeal. Advances have been made in our ability to recall problematic gene drives by replacing or upgrading them with different transgenes [57,58]. Alternatively, transgenes could be replaced with natural alleles. ...
Article
Full-text available
Gene drives for mosquito population modification are novel tools for malaria control. Strategies to safely test antimalarial effectors in the field are required. Here, we modified the Anopheles gambiae zpg locus to host a CRISPR/Cas9 integral gene drive allele ( zpg D ) and characterized its behaviour and resistance profile. We found that zpg D dominantly sterilizes females but can induce efficient drive at other loci when it itself encounters resistance. We combined zpg D with multiple previously characterized non-autonomous payload drives and found that, as zpg D self-eliminates, it leads to conversion of mosquito cage populations at these loci. Our results demonstrate how self-eliminating drivers could allow safe testing of non-autonomous effector-traits by local population modification. They also suggest that after engendering resistance, gene drives intended for population suppression could nevertheless serve to propagate subsequently released non-autonomous payload genes, allowing modification of vector populations initially targeted for suppression.
... gene drives have now been demonstrated in yeast [8][9][10][11], flies [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26], mosquitoes [27][28][29][30][31][32][33][34][35][36], and mice [37]. Most of these are homing drives, which have successfully achieved the modification and suppression of laboratory populations [26,30,36]. ...
Article
Full-text available
Background Homing gene drives hold great promise for the genetic control of natural populations. However, current homing systems are capable of spreading uncontrollably between populations connected by even marginal levels of migration. This could represent a substantial sociopolitical barrier to the testing or deployment of such drives and may generally be undesirable when the objective is only local population control, such as suppression of an invasive species outside of its native range. Tethered drive systems, in which a locally confined gene drive provides the CRISPR nuclease needed for a homing drive, could provide a solution to this problem, offering the power of a homing drive and confinement of the supporting drive. Results Here, we demonstrate the engineering of a tethered drive system in Drosophila , using a regionally confined CRISPR Toxin-Antidote Recessive Embryo (TARE) drive to support modification and suppression homing drives. Each drive was able to bias inheritance in its favor, and the TARE drive was shown to spread only when released above a threshold frequency in experimental cage populations. After the TARE drive had established in the population, it facilitated the spread of a subsequently released split homing modification drive (to all individuals in the cage) and of a homing suppression drive (to its equilibrium frequency). Conclusions Our results show that the tethered drive strategy is a viable and easily engineered option for providing confinement of homing drives to target populations.
... The third construct, DT-tGD(y1,-w2,y1b,w2b), carries both the secondary gRNAs (y1b, w2b) driven by D.gri-U6-A and D.gri-U6-C, respectively (Fig. 1d). These different U6 promoters were chosen due to previous success in a gene-drive setting and to avoid the problematic recombination that has been shown to occur within the gene-drive element if identical sequences are used 24 . All of these gRNA-constructs were then inserted at the same location of our tGD(y1,w2) control in the white locus and similarly marked with GFP so they could be combined with the same Cas9 line as the original tGD 19 . ...
Article
Full-text available
Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented “double-tap” strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.
... Because homing drives spread rapidly, even with low release sizes, some studies have attempted to develop constructs that could be deployed to limit their spread or revert organisms to a wild type phenotype. One of these can overwrite existing drives in D. melanogaster [61], while another method in Anopheles gambiae can prevent a suppression drive from eliminating a population by reducing (though not eliminating) the genetic load [62]. Methods for use of chemicals in inducible genetic systems allow for control of Cas9 expression (and thus drive efficiency) [63] or removal of gene drives [64] in flies, allowing laboratory manipulation or potentially even use in wild populations if the inducing chemical can be safely and widely deployed. ...
Article
Full-text available
Mosquitoes bring global health problems by transmitting parasites and viruses such as malaria and dengue. Unfortunately, current insecticide-based control strategies are only moderately effective because of high cost and resistance. Thus, scalable, sustainable, and cost-effective strategies are needed for mosquito-borne disease control. Symbiont-based and genome engineeringbased approaches provide new tools that show promise for meeting these criteria, enabling modification or suppression approaches. Symbiotic bacteria like Wolbachia are maternally inherited and manipulate mosquito host reproduction to enhance their vertical transmission. Genome engineering-based gene drive methods, in which mosquitoes are genetically altered to spread drive alleles throughout wild populations, are also proving to be a potentially powerful approach in the laboratory. Here, we review the latest developments in both symbionts and gene drive-based methods. We describe some notable similarities, as well as distinctions and obstacles, relating to these promising technologies.
... In our model, asymmetrical gene flow between target and non-target populations was shown to generate an outcome of interest which we termed 'gene swamping'. This fail-safe strategy does not rely on the complexity of the gene drive construct [30][31][32], but rather on gene flow and population dynamics. Since these aspects are general features of wild populations, this approach could be readily combined with other types of fail-safe mechanisms to prevent the unintended spread of gene drive constructs. ...
Preprint
Full-text available
Gene drives are genetic constructs that can spread deleterious alleles with potential application to population suppression of harmful species. Given that a gene drive can potentially spill over to other populations or even other species, control measures and fail-safes strategies must be considered. Gene drives are designed to generate a rapid demographic decline, while at the same time generating a dynamic change in the population's genetics. Since these evolutionary and demographic processes are linked and are expected to occur at a similar time-scale during gene drive spread, feedback between these processes may significantly affect the outcome of deployment. To study this feedback and to understand how it affects gene drive spillovers, we developed a gene drive model that combines evolutionary and demographic dynamics in a two-population setting. The model demonstrates how feedback between evolutionary and demographic dynamics can generate additional outcomes to those generated by the evolutionary dynamics alone. We identify an outcome of particular interest, where the short-term suppression of the target population is followed by gene swamping and loss of the gene drive. This outcome could be useful for designing gene drive deployments that temporarily suppress the population, but ultimately do not remain in the population. Using our model, we demonstrate the robustness of this outcome to spillover and to the evolution of resistance, and suggest that it could be used as a fail-safe strategy for gene drive deployment.
... Insecticides would most likely still have to be used to protect against other mosquito-borne diseases such as dengue, Zika, or yellow fever, but were GMMs released into the environment, this would most likely lead to a decreased dependency on these insecticides (Hammond and Galizi 2017). Moreover, new genetic elements might reverse gene drives, disputing the irreversibility of the technology (Xu et al. 2020). In conclusion, as the technology improves continuously, some current arguments against gene drives will no longer be relevant. ...
Article
Approximately a quarter of a billion people around the world suffer from malaria each year. Most cases are located in sub-Saharan Africa where Anopheles gambiae mosquitoes are the principal vectors of this public health problem. With the use of CRISPR-based gene drives, the population of mosquitoes can be modified, eventually causing their extinction. First, we discuss the moral status of the organism and argue that using genetically modified mosquitoes to combat malaria should not be abandoned based on some moral value of A. gambiae. Secondly, we argue that environmental impact studies should be performed to obtain an accurate account of the possible effects of a potential eradication of the organism. However, the risks from the purposeful extinction of A. gambiae should not overtake the benefits of eradicating malaria and risk assessments should be used to determine acceptable risks. Thirdly, we argue that the eventual release of the genetically modified mosquitoes will depend on transparency, community involvement, and cooperation between different nations.
... The inheritance module is unchanged, and inheritance "cubes," describing the distribution of offspring genotypes given maternal and paternal genotypes for a given genetic element, are usable in both versions. Several new inheritance cubes have been made available, including: a) homing-based remediation systems, including ERACR (Element for Reversing the Autocatalytic Chain Reaction) and e-CHACR (Erasing Construct Hitchhiking on the Autocatalytic Chain Reaction) [25,26], and b) newly proposed drive systems capable of regional population replacement, including CleaveR (Cleave and Rescue) [27] and TARE (Toxin-Antidote Recessive Embryo) drive [28]. ...
Article
Full-text available
Interest in gene drive technology has continued to grow as promising new drive systems have been developed in the lab and discussions are moving towards implementing field trials. The prospect of field trials requires models that incorporate a significant degree of ecological detail, including parameters that change over time in response to environmental data such as temperature and rainfall, leading to seasonal patterns in mosquito population density. Epidemiological outcomes are also of growing importance, as: i) the suitability of a gene drive construct for release will depend on its expected impact on disease transmission, and ii) initial field trials are expected to have a measured entomological outcome and a modeled epidemiological outcome. We present MGDrivE 2 (Mosquito Gene Drive Explorer 2): a significant development from the MGDrivE 1 simulation framework that investigates the population dynamics of a variety of gene drive architectures and their spread through spatially-explicit mosquito populations. Key strengths and fundamental improvements of the MGDrivE 2 framework are: i) the ability of parameters to vary with time and induce seasonal population dynamics, ii) an epidemiological module accommodating reciprocal pathogen transmission between humans and mosquitoes, and iii) an implementation framework based on stochastic Petri nets that enables efficient model formulation and flexible implementation. Example MGDrivE 2 simulations are presented to demonstrate the application of the framework to a CRISPR-based split gene drive system intended to drive a disease-refractory gene into a population in a confinable and reversible manner, incorporating time-varying temperature and rainfall data. The simulations also evaluate impact on human disease incidence and prevalence. Further documentation and use examples are provided in vignettes at the project's CRAN repository. MGDrivE 2 is freely available as an open-source R package on CRAN (https://CRAN.R-project.org/package=MGDrivE2). We intend the package to provide a flexible tool capable of modeling gene drive constructs as they move closer to field application and to infer their expected impact on disease transmission.
... Additional gRNAs can be included in the same cassette to bias inheritance of a favored allelic variant at a different chromosomal location (allelic drive) [60] ( Figure 1D) or to alter secondary gene targets [61]. It is also possible to deploy dual gRNA drives to delete and replace target sequences [57,62]. ...
Article
Bacterial resistance to antibiotics has reached critical levels, skyrocketing in hospitals and the environment and posing a major threat to global public health. The complex and challenging problem of reducing antibiotic resistance (AR) requires a network of both societal and science-based solutions to preserve the most lifesaving pharmaceutical intervention known to medicine. In addition to developing new classes of antibiotics, it is essential to safeguard the clinical efficacy of existing drugs. In this review, we examine the potential application of novel CRISPR-based genetic approaches to reducing AR in both environmental and clinical settings and prolonging the utility of vital antibiotics.
... Several approaches have been recently proposed for mitigating spillovers, involving complex gene drive architectures and deployment strategies [39,[62][63][64][65][66][67][68][69], as well as for implementing countermeasures to halt an ongoing gene drive [10, [70][71][72]. A few of these genedrive architectures have been demonstrated in laboratory settings [10, 38,53,60,73]. ...
Article
Full-text available
The prospect of utilizing CRISPR-based gene-drive technology for controlling populations has generated much excitement. However, the potential for spillovers of gene-drive alleles from the target population to non-target populations has raised concerns. Here, using mathematical models, we investigate the possibility of limiting spillovers to non-target populations by designing differential-targeting gene drives, in which the expected equilibrium gene-drive allele frequencies are high in the target population but low in the non-target population. We find that achieving differential targeting is possible with certain configurations of gene drive parameters, but, in most cases, only under relatively low migration rates between populations. Under high migration, differential targeting is possible only in a narrow region of the parameter space. Because fixation of the gene drive in the non-target population could severely disrupt ecosystems, we outline possible ways to avoid this outcome. We apply our model to two potential applications of gene drives—field trials for malaria-vector gene drives and control of invasive species on islands. We discuss theoretical predictions of key requirements for differential targeting and their practical implications.
... given genetic element, are usable in both versions. Several new inheritance cubes have been made available, including: a) homing-based remediation systems, including ERACR (Element for Reversing the Autocatalytic Chain Reaction) and e-CHACR (Eracing Construct Hitchhiking on the Autocatalytic Chain Reaction) (Gantz and Bier, 2016;Xu et al., 2020), and b) newly proposed drive systems capable of regional population replacement, including CleaveR (Cleave and Rescue) (Oberhofer, Ivy and Hay, 2019) and TARE (Toxin-Antidote Recessive Embryo) drive (Champer et al., 2020). ...
Preprint
Full-text available
Interest in gene drive technology has continued to grow as promising new drive systems have been developed in the lab and discussions are moving towards implementing field trials. The prospect of field trials requires models that incorporate a significant degree of ecological detail, including parameters that change over time in response to environmental data such as temperature and rainfall, leading to seasonal patterns in mosquito population density. Epidemiological outcomes are also of growing importance, as: i) the suitability of a gene drive construct for release will depend on its expected impact on disease transmission, and ii) initial field trials are expected to have a measured entomological outcome and a modeled epidemiological outcome. We present MGDrivE 2 (Mosquito Gene Drive Explorer 2): an extension of and development from the MGDrivE 1 simulation framework that investigates the population dynamics of a variety of gene drive architectures and their spread through spatially-explicit mosquito populations. Key strengths and improvements of the MGDrivE 2 framework are: i) the ability of parameters to vary with time and induce seasonal population dynamics, ii) an epidemiological module accommodating reciprocal pathogen transmission between humans and mosquitoes, and iii) an implementation framework based on stochastic Petri nets that enables efficient model formulation and flexible implementation. Example MGDrivE 2 simulations are presented to demonstrate the application of the framework to a CRISPR-based homing gene drive system intended to drive a disease-refractory gene into a population, incorporating time-varying temperature and rainfall data, and predict impact on human disease incidence and prevalence. Further documentation and use examples are provided in vignettes at the project's CRAN repository. MGDrivE 2 is an open-source R package freely available on CRAN. We intend the package to provide a flexible tool capable of modeling gene drive constructs as they move closer to field application and to infer their expected impact on disease transmission.
Article
Full-text available
The last century has witnessed the introduction, establishment and expansion of mosquito-borne diseases into diverse new geographic ranges. Malaria is transmitted by female Anopheles mosquitoes. Despite making great strides over the past few decades in reducing the burden of malaria, transmission is now on the rise again, in part owing to the emergence of mosquito resistance to insecticides, antimalarial drug resistance and, more recently, the challenges of the COVID-19 pandemic, which resulted in the reduced implementation efficiency of various control programs. The utility of genetically engineered gene drive mosquitoes as tools to decrease the burden of malaria by controlling the disease-transmitting mosquitoes is being evaluated. To date, there has been remarkable progress in the development of CRISPR/Cas9-based homing endonuclease designs in malaria mosquitoes due to successful proof-of-principle and multigenerational experiments. In this review, we examine the lessons learnt from the development of current CRISPR/Cas9-based homing endonuclease gene drives, providing a framework for the development of gene drive systems for the targeted control of wild malaria-transmitting mosquito populations that overcome challenges such as with evolving drive-resistance. We also discuss the additional substantial works required to progress the development of gene drive systems from scientific discovery to further study and subsequent field application in endemic settings.
Article
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐based “active genetic” elements developed in 2015 bypassed the fundamental rules of traditional genetics. Inherited in a super‐Mendelian fashion, such selfish genetic entities offered a variety of potential applications including: gene‐drives to disseminate gene cassettes carrying desired traits throughout insect populations to control disease vectors or pest species, allelic drives biasing inheritance of preferred allelic variants, neutralizing genetic elements to delete and replace or to halt the spread of gene‐drives, split‐drives with the core constituent Cas9 endonuclease and guide RNA (gRNA) components inserted at separate genomic locations to accelerate assembly of complex arrays of genetic traits or to gain genetic entry into novel organisms (vertebrates, plants, bacteria), and interhomolog based copying systems in somatic cells to develop tools for treating inherited or infectious diseases. Here, we summarize the substantial advances that have been made on all of these fronts and look forward to the next phase of this rapidly expanding and impactful field. Active genetics bypasses the rules of traditional Mendelian inheritance permitting the development of powerful new tools such as gene‐drive systems to combat insect borne diseases, allelic drives to bias inheritance of preferred allelic variants, self‐amplifying systems reversing antibiotic resistance in bacteria, and systems promoting corrective interhomolog repair in somatic cells.
Article
Full-text available
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 selected gRNA target sites were 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 elimination of the cage population, showing the importance of using highly efficient drives in a suppression strategy, even if resistance can be addressed. Nevertheless, our results experimentally demonstrate the viability of the multiplexed gRNAs strategy in homing suppression gene drives.
Article
Full-text available
Promising genetics-based approaches are being developed to reduce or prevent the transmission of mosquito-vectored diseases. Less clear is how such transgenes can be removed from the environment, a concern that is particularly relevant for highly invasive gene drive transgenes. Here, we lay the groundwork for a transgene removal system based on single-strand annealing (SSA), a eukaryotic DNA repair mechanism. An SSA-based rescuer strain (kmoRG) was engineered to have direct repeat sequences (DRs) in the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene flanking the intervening transgenic cargo genes, DsRED and EGFP. Targeted induction of DNA double-strand breaks (DSBs) in the DsRED transgene successfully triggered complete elimination of the entire cargo from the kmoRG strain, restoring the wild-type kmo gene and thereby normal eye pigmentation. Our work establishes the framework for strategies to remove transgene sequences during the evaluation and testing of modified strains for genetics-based mosquito control.
Article
Full-text available
Mosquito-borne diseases such as malaria continue to pose a major global health burden, and the impact of currently-available interventions is stagnating. Consequently, there is interest in novel tools to control these diseases, including gene drive-modified mosquitoes. As these tools continue to be refined, decisions on whether to implement them in the field depend on their alignment with target product profiles (TPPs) that define product characteristics required to achieve desired entomological and epidemiological outcomes. TPPs are increasingly being used for malaria and vector control interventions, such as attractive targeted sugar baits and long-acting injectable drugs, as they progress through the development pipeline. For mosquito gene drive products, reliable predictions from mathematical models are an essential part of these analyses, as field releases could potentially be irreversible. Here, we review the prior use of mathematical models in developing TPPs for malaria and vector control tools and discuss lessons from these analyses that may apply to mosquito gene drives. We recommend that, as gene drive technology gets closer to field release, discussions regarding target outcomes engage a wide range of stakeholders and account for settings of interest and vector species present. Given the relatively large number of parameters that describe gene drive products, machine learning approaches may be useful to explore parameter space, and an emphasis on conservative fitness estimates is advisable, given the difficulty of accurately measuring these parameters prior to field studies. Modeling may also help to inform the risk, remediation and cost dimensions of mosquito gene drive TPPs.
Article
Gene drives are selfish genetic elements that are transmitted to progeny at super-Mendelian (>50%) frequencies. Recently developed CRISPR–Cas9-based gene-drive systems are highly efficient in laboratory settings, offering the potential to reduce the prevalence of vector-borne diseases, crop pests and non-native invasive species. However, concerns have been raised regarding the potential unintended impacts of gene-drive systems. This Review summarizes the phenomenal progress in this field, focusing on optimal design features for full-drive elements (drives with linked Cas9 and guide RNA components) that either suppress target mosquito populations or modify them to prevent pathogen transmission, allelic drives for updating genetic elements, mitigating strategies including trans-complementing split-drives and genetic neutralizing elements, and the adaptation of drive technology to other organisms. These scientific advances, combined with ethical and social considerations, will facilitate the transparent and responsible advancement of these technologies towards field implementation. In this Review, Ethan Bier discusses how several impactful technical advancements, particularly involving CRISPR-based methods, are providing a diverse toolkit of gene-drive systems for the control of populations such as insect vectors of disease.
Article
Full-text available
Potential future application of engineered gene drives (GDs), which bias their own inheritance and can spread genetic modifications in wild target populations, has sparked both enthusiasm and concern. Engineered GDs in insects could potentially be used to address long-standing challenges in control of disease vectors, agricultural pests and invasive species, or help to rescue endangered species, and thus provide important public benefits. However, there are concerns that the deliberate environmental release of GD modified insects may pose different or new harms to animal and human health and the wider environment, and raise novel challenges for risk assessment. Risk assessors, risk managers, developers, potential applicants and other stakeholders at many levels are currently discussing whether there is a need to develop new or additional risk assessment guidance for the environmental release of GD modified organisms, including insects. Developing new or additional guidance that is useful and practical is a challenge, especially at an international level, as risk assessors, risk managers and many other stakeholders have different, often contrasting, opinions and perspectives toward the environmental release of GD modified organisms, and on the adequacy of current risk assessment frameworks for such organisms. Here, we offer recommendations to overcome some of the challenges associated with the potential future development of new or additional risk assessment guidance for GD modified insects and provide considerations on areas where further risk assessment guidance may be required.
Article
Most current techniques to deal with invasive species are ineffective or have highly damaging side effects. To this end suppression-drives based on clustered regularly inter-spaced short palindromic repeats (CRISPR/Cas9) have been touted as a potential silver bullet for the problem, allowing for a highly focused, humane and cost-effective means of removing a target species from an environment. Suppression-drives come with serious risks, however, such that the precautionary principle seems to warrant us not deploying this technology. The focus of this paper is on one such risk – the danger of a suppression-drive escaping containment and wiping out the target species globally. Here, I argue that in most cases this risk is significant enough to warrant not using a gene-drive. In some cases, however, we can bypass the precautionary principle by using an approach that hinges on what I term the ‘Worst-Case Clause’. This clause, in turn, provides us with a litmus test that can be fruitfully used to determine what species are viable targets for suppression-drives in the wild. Using this metric in concert with other considerations, I suggest that only three species are currently possible viable targets – the European rabbit, ship rat and Caribbean Tree Frog.
Article
Full-text available
Synthetic CRISPR-based gene-drive systems have tremendous potential in public health and agriculture, such as for fighting vector-borne diseases or suppressing crop pest populations. These elements can rapidly spread in a population by breaching the inheritance limit of 50% dictated by Mendel’s law of gene segregation, making them a promising tool for population engineering. However, current technologies lack control over their propagation capacity, and there are important concerns about potential unchecked spreading. Here, we describe a gene-drive system in Drosophila that generates an analog inheritance output that can be tightly and conditionally controlled to between 50% and 100%. This technology uses a modified SpCas9 that responds to a synthetic, orally available small molecule, fine-tuning the inheritance probability. This system opens a new avenue to feasibility studies for spatial and temporal control of gene drives using small molecules.
Article
Full-text available
CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split–gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small–molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.
Article
Full-text available
Aedes aegypti, the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in Ae. aegypti. To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.
Article
Full-text available
Small laboratory cage trials of non-drive and gene-drive strains of the Asian malaria vector mosquito, Anopheles stephensi, were used to investigate release ratios and other strain properties for their impact on transgene spread during simulated population modification. We evaluated the effects of transgenes on survival, male contributions to next-generation populations, female reproductive success and the impact of accumulation of gene drive-resistant genomic target sites resulting from nonhomologous end-joining (NHEJ) mutagenesis during Cas9, guide RNA-mediated cleavage. Experiments with a non-drive, autosomally-linked malaria-resistance gene cassette showed ‘full introduction’ (100% of the insects have at least one copy of the transgene) within 8 weeks (≤ 3 generations) following weekly releases of 10:1 transgenic:wild-type males in an overlapping generation trial design. Male release ratios of 1:1 resulted in cages where mosquitoes with at least one copy of the transgene fluctuated around 50%. In comparison, two of three cages in which the malaria-resistance genes were linked to a gene-drive system in an overlapping generation, single 1:1 release reached full introduction in 6–8 generations with a third cage at ~80% within the same time. Release ratios of 0.1:1 failed to establish the transgenes. A non-overlapping generation, single-release trial of the same gene-drive strain resulted in two of three cages reaching 100% introduction within 6–12 generations following a 1:1 transgenic:wild-type male release. Two of three cages with 0.33:1 transgenic:wild-type male single releases achieved full introduction in 13–16 generations. All populations exhibiting full introduction went extinct within three generations due to a significant load on females having disruptions of both copies of the target gene, kynurenine hydroxylase. While repeated releases of high-ratio (10:1) non-drive constructs could achieve full introduction, results from the 1:1 release ratios across all experimental designs favor the use of gene drive, both for efficiency and anticipated cost of the control programs.
Article
Full-text available
Gene-drive systems in diploid organisms bias the inheritance of one allele over another. CRISPR-based gene-drive expresses a guide RNA (gRNA) into the genome at the site where the gRNA directs Cas9-mediated cleavage. In the presence of Cas9, the gRNA cassette and any linked cargo sequences are copied via homology-directed repair (HDR) onto the homologous chromosome. Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas9 cleavage site. This “pro-active” genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. Pro-AG can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine. Genedrives bias the inheritance of alleles in diploid organisms. Here, the authors develop a gene-drive analogous system for bacteria, selectively editing and clearing plasmids.
Article
Full-text available
CRISPR–Cas9–based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in Drosophila , the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms—often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention.
Preprint
Full-text available
Aedes aegypti , the principal mosquito vector for many arboviruses that causes yellow fever, dengue, Zika, and chikungunya, increasingly infects millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has recently sparked significant enthusiasm for the genetic control of mosquito populations, however no such system has been developed in Ae. aegypti . To fill this void and demonstrate efficacy in Ae. aegypti, here we develop several CRISPR-based split-gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild Ae. aegypti populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission. Significance Statement Ae. aegypti is a globally distributed arbovirus vector spreading deadly pathogens to millions of people annually. Current control methods are inadequate and therefore new technologies need to be innovated and implemented. With the aim of providing new tools for controlling this pest, here we engineered and tested several split gene drives in this species. These drives functioned at very high efficiency and may provide a tool to fill the void in controlling this vector. Taken together, our results provide compelling path forward for the feasibility of future effector-linked split-drive technologies that can contribute to the safe, sustained control and potentially the elimination of pathogens transmitted by this species.
Article
Full-text available
Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this “active genetic” approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. We also characterize a phenomenon we refer to as lethal-mosaicism that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process we refer to as “shadow-drive”, have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox.
Article
Full-text available
A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR–Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity1–4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR–Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR–Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.
Article
Full-text available
In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.
Article
Full-text available
Gene drive technology offers the promise for a high-impact, cost-effective, and durable method to control malaria transmission that would make a significant contribution to elimination. Gene drive systems, such as those based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein, have the potential to spread beneficial traits through interbreeding populations of malaria mosquitoes. However, the characteristics of this technology have raised concerns that necessitate careful consideration of the product development pathway. A multidisciplinary working group considered the implications of low-threshold gene drive systems on the development pathway described in the World Health Organization Guidance Framework for testing genetically modified (GM) mosquitoes, focusing on reduction of malaria transmission by Anopheles gambiae s.l. mosquitoes in Africa as a case study. The group developed recommendations for the safe and ethical testing of gene drive mosquitoes, drawing on prior experience with other vector control tools, GM organisms, and biocontrol agents. These recommendations are organized according to a testing plan that seeks to maximize safety by incrementally increasing the degree of human and environmental exposure to the investigational product. As with biocontrol agents, emphasis is placed on safety evaluation at the end of physically confined laboratory testing as a major decision point for whether to enter field testing. Progression through the testing pathway is based on fulfillment of safety and efficacy criteria, and is subject to regulatory and ethical approvals, as well as social acceptance. The working group identified several resources that were considered important to support responsible field testing of gene drive mosquitoes.
Article
Full-text available
The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these 'in locus' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis.
Article
Full-text available
A gene drive biases inheritance of a gene so that it increases in frequency within a population even when the gene confers no fitness benefit. There has been renewed interest in environmental releases of engineered gene drives due to recent proof of principle experiments with the CRISPR-Cas9 system as a drive mechanism. Release of modified organisms, however, is controversial, especially when the drive mechanism could theoretically alter all individuals of a species. Thus, it is desirable to have countermeasures to reverse a drive if a problem arises. Several genetic mechanisms for limiting or eliminating gene drives have been proposed and/or developed, including synthetic resistance, reversal drives, and immunizing reversal drives. While predictions about efficacy of these mechanisms have been optimistic, we lack detailed analyses of their expected dynamics. We develop a discrete time model for population genetics of a drive and proposed genetic countermeasures. Efficacy of drive reversal varies between countermeasures. For some parameter values, the model predicts unexpected behavior including polymorphic equilibria and oscillatory dynamics. The timing and number of released individuals containing a genetic countermeasure can substantially impact outcomes. The choice among countermeasures by researchers and regulators will depend on specific goals and population parameters of target populations.
Article
Full-text available
Approximately two years ago, two of us (E.B. and V.G.) demonstrated the first experimental application of CRISPR–Cas9 to 'drive' a desired trait throughout a population of fruit flies. In November 2015, this same team at the University of California, San Diego, joined with A.A.J. and others at the University of California, Irvine, to develop a CRISPR-based gene drive for population modification of the malaria vector mosquito Anopheles stephensi. A month later, a group in the United Kingdom applied a CRISPR-based gene drive to another malaria vector, Anopheles gambiae.
Article
Full-text available
A functioning gene drive system could fundamentally change our strategies for the control of vector-borne diseases by facilitating rapid dissemination of transgenes that prevent pathogen transmission or reduce vector capacity. CRISPR/Cas9 gene drive promises such a mechanism, which works by converting cells that are heterozygous for the drive construct into homozygotes, thereby enabling super-Mendelian inheritance. Although CRISPR gene drive activity has already been demonstrated, a key obstacle for current systems is their propensity to generate resistance alleles, which cannot be converted to drive alleles. In this study, we developed two CRISPR gene drive constructs based on the nanos and vasa promoters that allowed us to illuminate the different mechanisms by which resistance alleles are formed in the model organism Drosophila melanogaster. We observed resistance allele formation at high rates both prior to fertilization in the germline and post-fertilization in the embryo due to maternally deposited Cas9. Assessment of drive activity in genetically diverse backgrounds further revealed substantial differences in conversion efficiency and resistance rates. Our results demonstrate that the evolution of resistance will likely impose a severe limitation to the effectiveness of current CRISPR gene drive approaches, especially when applied to diverse natural populations.
Article
Full-text available
Synthetic gene drives based on CRISPR/Cas9 have the potential to control, alter, or suppress populations of crop pests and disease vectors, but it is unclear how they will function in wild populations. Using genetic data from four populations of the flour beetle Tribolium castaneum, we show that most populations harbor genetic variants in Cas9 target sites, some of which would render them immune to drive (ITD). We show that even a rare ITD allele can reduce or eliminate the efficacy of a CRISPR/Cas9-based synthetic gene drive. This effect is equivalent to and accentuated by mild inbreeding, which is a characteristic of many disease-vectoring arthropods. We conclude that designing such drives will require characterization of genetic variability and the mating system within and among targeted populations.
Article
Full-text available
Significance Gene drive mosquitoes have tremendous potential to help eliminate malaria, and multiple gene drive approaches have recently shown promise in laboratory settings. These approaches include population suppression through fertility disruption, driving-Y chromosomes, and population replacement with genes that limit malaria transmission. Mathematical modeling is used to evaluate these approaches by simulating realistic field settings with seasonality to determine constraints on construct parameters and release strategies. Parameter variation from simulation baselines captures much of sub-Saharan African epidemiology and shows high potential for gene drive constructs to provide transformational tools to facilitate elimination of malaria, even in the most challenging settings. This analysis provides insights into performance characteristics necessary for each approach to succeed that can inform their further development.
Article
Full-text available
The CRISPR/Cas9 system has revolutionized genomic editing. The Cas9 endonuclease targets genomic DNA via an experimentally determined guide RNA (gRNA). This results in a double-strand break at the target site. We generated transgenic Drosophila melanogaster in which the CRISPR/Cas9 system was used to target a GAL4 transgene in vivo To our surprise, progeny whose genomes did not contain CRISPR/Cas9 components were still capable of mutating GAL4 sequences. We demonstrate this effect was caused by maternal deposition of Cas9 and gRNAs into the embryo, leading to extensive GAL4 mutations in both somatic and germline tissues. This serves as a cautionary observation on the effects of maternal contributions when conducting experiments using genomically encoded CRISPR/Cas9 components. These results also highlight a mode of artificial inheritance in which maternal contributions of DNA editing components lead to transmissible mutant defects even in animals whose genomes lack the editing components. We suggest calling this a dominant maternal effect to reflect it is caused by the gain of maternally contributed products. Models of CRISPR-mediated gene drive will need to incorporate dominant maternal effects in order to accurately predict the efficiency and dynamics of gene drive in a population.
Article
Full-text available
Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission.
Article
Full-text available
Significance Malaria continues to impose enormous health and economic burdens on the developing world. Novel technologies proposed to reduce the impact of the disease include the introgression of parasite-resistance genes into mosquito populations, thereby modifying the ability of the vector to transmit the pathogens. Such genes have been developed for the human malaria parasite Plasmodium falciparum . Here we provide evidence for a highly efficient gene-drive system that can spread these antimalarial genes into a target vector population. This system exploits the nuclease activity and target-site specificity of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, which, when restricted to the germ line, copies a genetic element from one chromosome to its homolog with ≥98% efficiency while maintaining the transcriptional activity of the genes being introgressed.
Article
Full-text available
An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype, whereas individuals homozygous for two copies of the allele will display a mutant phenotype. We have developed a method called the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome-editing system for generating autocatalytic mutations, to produce homozygous loss-of-function mutations. In Drosophila, we found that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome, thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms.
Article
Full-text available
Double-strand breaks (DSBs) must be accurately and efficiently repaired to maintain genome integrity. Depending on the organism receiving the break, the genomic location of the DSB, and the cell cycle phase in which it occurs, a DSB can be repaired by homologous recombination (HR), non-homologous end joining (NHEJ), or single-strand annealing (SSA). Two novel DSB repair assays were developed to determine the contributions of these repair pathways and finely resolve repair event structures in Drosophila melanogaster. Rad51-dependent homologous recombination is the preferred DSB repair pathway in mitotically dividing cells, and pathway choice between HR and SSA occurs after end resection and before Rad51-dependent strand invasion. HR events are associated with long gene conversion tracts and are both bidirectional and unidirectional, consistent with repair via the synthesis-dependent strand annealing pathway. Additionally, HR between diverged sequences is suppressed in Drosophila, similar to levels reported in human cells. Junction analyses of rare NHEJ events reveal that canonical NHEJ is utilized in this system.
Article
Full-text available
Using zinc-finger nucleases (ZFNs) to cleave the chromosomal target, we have achieved high frequencies of gene targeting in the Drosophila germline. Both local mutagenesis through nonhomologous end joining (NHEJ) and gene replacement via homologous recombination (HR) are stimulated by target cleavage. In this study we investigated the mechanisms that underlie these processes, using materials for the rosy (ry) locus. The frequency of HR dropped significantly in flies homozygous for mutations in spnA (Rad51) or okr (Rad54), two components of the invasion-mediated synthesis-dependent strand annealing (SDSA) pathway. When single-strand annealing (SSA) was also blocked by the use of a circular donor DNA, HR was completely abolished. This indicates that the majority of HR proceeds via SDSA, with a minority mediated by SSA. In flies deficient in lig4 (DNA ligase IV), a component of the major NHEJ pathway, the proportion of HR products rose significantly. This indicates that most NHEJ products are produced in a lig4-dependent process. When both spnA and lig4 were mutated and a circular donor was provided, the frequency of ry mutations was still high and no HR products were recovered. The local mutations produced in these circumstances must have arisen through an alternative, lig4-independent end-joining mechanism. These results show what repair pathways operate on double-strand breaks in this gene targeting system. They also demonstrate that the outcome can be biased toward gene replacement by disabling the major NHEJ pathway and toward simple mutagenesis by interfering with the major HR process.
Article
Full-text available
We have studied P transposase-induced events on a P[w] transgene, P[wd1], harboring the whole white gene with a 3.44-kb direct duplication of its 5' regulatory sequences (containing the ZESTE-binding region, ZBR). We have recovered mutations leading to an increase or a decrease of zeste1 repression, generally as the consequence of modifications of number of ZBR in close physical proximity and/or jumps to other sites. We describe mutants displaying deletions of the original duplicated sequence or increases in the number of repeats from two to three or four. Internal deletions are more frequent than amplifications. Both require the integrity of P-element ends. We have also observed a high frequency of double P elements localized at the original P[wd1] insertion site. These double P elements are arranged in nonrandom configurations. We discuss the frequencies and the possible mechanisms leading to the various types of derivatives, in light of the current models for P excision and transposition. We propose that the P transposase induces mainly localized events. Some of these could result from frequent changes of template during gap-repair DNA synthesis, and/or from abortive transposition.
Preprint
By surpassing the 50% inheritance limit of Mendel’s law of independent assortment, CRISPR-based gene drives have the potential to fight vector-borne diseases or suppress crop pests. However, contemporary gene drives could spread unchecked, posing safety concerns that limit their use in both laboratory and field settings. Current technologies also lack chemical control strategies, which could be applied in the field for dose, spatial and temporal control of gene drives. We describe in Drosophila the first gene-drive system controlled by an engineered Cas9 and a synthetic, orally-available small molecule. Graphical Abstract
Article
With the advent of clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) technology, researchers can construct gene drives that can bias the inheritance of edited alleles to alter entire populations. As demonstrated with the mutagenic chain reaction in Drosophila4, the CRISPR-Cas9 system can propagate genomic modification together with the genome-editing machinery itself. Although gene drives might have the potential to control insect-borne diseases and agricultural pests, substantial concerns have been raised over unanticipated ecological consequences as a result of drive use. Here we report the development of a potential Cas9-based gene drive 'brake' that remains inert in a wild-type genome but is activated by Cas9 to both cleave the genomic cas9 sequence and to convert an incoming cas9 allele into a brake. This means that the propagation of the brake is favored in a cas9-carrying population.
Article
On December 18, 2014, a yellow female fly quietly emerged from her pupal case. What made her unique was that she had only one parent carrying a mutant allele of this classic recessive locus. Then, one generation later, after mating with a wild-type male, all her offspring displayed the same recessive yellow phenotype. Further analysis of other such yellow females revealed that the construct causing the mutation was converting the opposing chromosome with 95% efficiency. These simple results, seen also in mosquitoes and yeast, open the door to a new era of genetics wherein the laws of traditional Mendelian inheritance can be bypassed for a broad variety of purposes. Here, we consider the implications of this fundamentally new form of "active genetics," its applications for gene drives, reversal and amplification strategies, its potential for contributing to cell and gene therapy strategies, and ethical/biosafety considerations associated with such active genetic elements. Also watch the Video Abstract.
Article
RNA-guided gene drives capable of spreading genomic alterations made in laboratory organisms through wild populations could be used to address environmental and public health problems. However, the possibility of unintended genome editing occurring through the escape of strains from laboratories, coupled with the prospect of unanticipated ecological change, demands caution. We report the efficacy of CRISPR-Cas9 gene drive systems in wild and laboratory strains of the yeast Saccharomyces cerevisiae. Furthermore, we address concerns surrounding accidental genome editing by developing and validating methods of molecular confinement that minimize the risk of unwanted genome editing. We also present a drive system capable of overwriting the changes introduced by an earlier gene drive. These molecular safeguards should enable the development of safe CRISPR gene drives for diverse organisms.
Article
Accurate interpretation of forward genetic screens of chromosomes exposed in mature spermatozoa to a mutagenic chemical requires understanding-incomplete to date-of how exposed chromosomes and their replicas proceed through early development stages from the fertilized ovum to establishment of the germ line of the treated male's offspring. We describe a model for early embryonic development and establishment of the germ line of Drosophila melanogaster and a model-validating experiment. Our model proposes that, barring repair, DNA strands modified by treatment with alkylating agents are stable and mutagenic. Each replication of an alkylated strand can result in misreplication and a mutant-bearing daughter nucleus. Daughter nuclei thenceforth replicate faithfully and their descendants comprise the embryonic syncytium. Of the 256 nuclei present after the eighth division, several migrate into the polar plasm at the posterior end of the embryo to found the germ line. Based upon distribution of descendants of the alkylated strands, the misreplication rate, and the number of nuclei selected as germline progenitors, the frequency of gonadal mosaicism is predictable. Experimentally, we tracked chromosomes 2 and 3 from EMS-treated sperm through a number of generations, to characterize autosomal recessive lethal mutations and infer gonadal genetic content of the sons of treated males. Over 50% of 106 sons bore germ lines that were singly, doubly, or triply mosaic for chromosome 2 or chromosome 3. These findings were consistent with our model, assuming a rate of misreplication between .65 and .80 at each replication of an alkylated strand. Crossing treated males to mismatch-repair-deficient females had no apparent effect on mutation rate. Copyright © 2015, The Genetics Society of America.
Presents observations on the courtship and mating behavior of the fly, D. ampelophila Loew. Two experiments carried out to find out the part of the body responsible for causing sexual excitement, indicate that sight was not essential in sex recognition in Drosoophilia. The tactile and olfactory senses were probably involved. Experiments with males whose wings had been removed indicate that the function of these organs in courtship was the production of sexual excitement in the female. Experiments carried out with 4 mutants (white eyes, vermilion eyes, yellow body color, and curved wings), involved observations of contested matings. It was seen that the 4 characteristics had no selective value. Concludes that it is probable that, in Drosoophilia, neither sex exercises any 'choice' in the selection of a mate. (PsycINFO Database Record (c) 2012 APA, all rights reserved)
Article
Repair of meiotic double-strand breaks (DSBs) uses the homolog and recombination to yield crossovers while alternative pathways such as nonhomologous end joining (NHEJ) are suppressed. Our results indicate that NHEJ is blocked at two steps of DSB repair during meiotic prophase: first by the activity of the MCM-like protein MEI-218, which is required for crossover formation, and, second, by Rad51-related proteins SPN-B (XRCC3) and SPN-D (RAD51C), which physically interact and promote homologous recombination (HR). We further show that the MCM-like proteins also promote the activity of the DSB repair checkpoint pathway, indicating an early requirement for these proteins in DSB processing. We propose that when a meiotic DSB is formed in the absence of both MEI-218 and SPN-B or SPN-D, a DSB substrate is generated that can enter the NHEJ repair pathway. Indeed, due to its high error rate, multiple barriers may have evolved to prevent NHEJ activity during meiosis.
Article
Homing endonuclease genes (HEGs) encode proteins that in the heterozygous state cause double-strand breaks in the homologous chromosome at the precise position opposite the HEG. If the double-strand break is repaired using the homologous chromosome, the HEG becomes homozygous, and this represents a powerful genetic drive mechanism that might be used as a tool in managing vector or pest populations. HEGs may be used to decrease population fitness to drive down population densities (possibly causing local extinction) or, in disease vectors, to knock out a gene required for pathogen transmission. The relative advantages of HEGs that target viability or fecundity, that are active in one sex or both, and whose target is expressed before or after homing are explored. The conditions under which escape mutants arise are also analyzed. A different strategy is to place HEGs on the Y chromosome that cause one or more breaks on the X chromosome and so disrupt sex ratio. This strategy can cause severe sex-ratio biases with efficiencies that depend on the details of sperm competition and zygote mortality. This strategy is probably less susceptible to escape mutants, especially when multiple X shredders are used.
Article
The optional 1143 bp intron in the yeast mitochondrial 21S rRNA gene (omega +) is nearly quantitatively inserted in genetic crosses into 21S rRNA alleles that lack it (omega -). The intron contains an open reading frame that can encode a protein of 235 amino acids, but no function has been ascribed to this sequence. We previously found an in vivo double-strand break in omega - DNA at or close to the intron insertion site only in zygotes of omega + X omega - crosses that appears with the same kinetics as intron insertion. We now show that mutations in the intron open reading frame that would alter the translation product simultaneously inhibit nonreciprocal omega recombination and the in vivo double-strand break in omega - DNA. These results provide evidence that the open reading frame encodes a protein required for intron transposition and support the role of the double-strand break in the process.
Article
CHROMOSOME translocation heterozygotes (T/+) are usually semisterile, but translocation homozygotes (T/T) if viable are usually fully fertile. If such a viable translocation were produced in an insect pest, T/T insects could be reared in captivity and released into the wild, where matings with wild types (+/+) would produce T/+ progeny.
Article
We describe here a family of P elements that we refer to as type I repressors. These elements are identified by their repressor functions and their lack of any deletion within the first two-thirds of the canonical P sequence. Elements belonging to this repressor class were isolated from P strains and were made in vitro. We found that type I repressor elements could strongly repress both a cytotype-dependent allele and P element mobility in somatic and germline tissues. These effects were very dependent on genomic position. Moreover, we observed that an element's ability to repress in one assay positively correlated with its ability to repress in either of the other two assays. The type I family of repressor elements includes both autonomous P elements and those lacking exon 3 of the P element. Fine structure deletion mapping showed that the minimal 3' boundary of a functional type I element lies between nucleotide position 1950 and 1956. None of 12 elements examined with more extreme deletions extending into exon 2 made repressor. We conclude that the type I repressors form a structurally distinct group that does not include more extensively deleted repressor elements such as the KP element described previously.
Article
Homing endonucleases confer mobility to their host intervening sequence, either an intron or intein, by catalyzing a highly specific double-strand break in a cognate allele lacking the intervening sequence. These proteins are characterized by their ability to bind long DNA target sites (14–40 bp) and their tolerance of minor sequence changes in these sites. A wealth of biochemical and structural data has been generated for these enzymes over the past few years. Herein we review our current understanding of homing endonucleases, including their diversity and evolution, DNA-binding and catalytic mechanisms, and attempts to engineer them to bind novel DNA substrates.
Article
Site-specific selfish genes exploit host functions to copy themselves into a defined target DNA sequence, and include homing endonuclease genes, group II introns and some LINE-like transposable elements. If such genes can be engineered to target new host sequences, then they can be used to manipulate natural populations, even if the number of individuals released is a small fraction of the entire population. For example, a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations, if the target is an essential host gene, the knockout is recessive and the selfish gene has an appropriate promoter. There will be selection for resistance, but several strategies are available for reducing the likelihood of it evolving. These genes may also be used to genetically engineer natural populations, by means of population-wide gene knockouts, gene replacements and genetic transformations. By targeting sex-linked loci just prior to meiosis one may skew the population sex ratio, and by changing the promoter one may limit the spread of the gene to neighbouring populations. The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.
Article
The study of DNA double-strand break (DSB) repair has been greatly facilitated by the use of rare-cutting endonucleases, which induce a break precisely at their cut sites that can be strategically placed in the genome. We previously established such a system in Drosophila and showed that the yeast I-SceI enzyme cuts efficiently in Drosophila cells and those breaks are effectively repaired by conserved mechanisms. In this study, we determined the genetic requirements for the repair of this I-SceI-induced DSB in the germline. We show that Drosophila Rad51 and Rad54 are both required for homologous repair by gene conversion, but are dispensable for single-strand annealing repair. We provided evidence suggesting that Rad51 is more stringently required than Rad54 for intersister gene conversion. We uncovered a significant role of DNA ligase IV in nonhomologous end joining. We conducted a screen for candidate mutations affecting DSB repair and discovered novel mutations in genes that include mutagen sensitive 206, single-strand annealing reducer, and others. In addition, we demonstrated an intricate balance among different repair pathways in which the cell differentially utilizes repair mechanisms in response to both changes in the genomic environment surrounding the break and deficiencies in one or the other repair pathways.
A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment
  • V Ló Pez Del Amo
  • A L Bishop
  • C Sá Nchez
  • H M Bennett
  • J B Feng
  • X Marshall
  • J M Bier
  • E Gantz
Ló pez Del Amo, V., Bishop, A.L., Sá nchez C, H.M., Bennett, J.B., Feng, X., Marshall, J.M., Bier, E., and Gantz, V.M. (2020). A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment. Nat. Commun. 11, 352.
Non-random mating between yellow-white and wild type Drosophila melanogaster
  • Diederich
Gene Drives: Pursuing Opportunities, Minimizing Risk—A Johns Hopkins University Report on Responsible Governance.
  • Warmbrod K.L.
  • Kobokovich K.
  • West R.
  • Ray G.
  • Trotochaud M.
  • Montague M.