scholarly journals Novel CRISPR/Cas9 gene drive constructs inDrosophilareveal insights into mechanisms of resistance allele formation and drive efficiency in genetically diverse populations

2017 ◽  
Author(s):  
Jackson Champer ◽  
Riona Reeves ◽  
Suh Yeon Oh ◽  
Chen Liu ◽  
Jingxian Liu ◽  
...  
Keyword(s):  
Natural Populations ◽  
Model Organism ◽  
Mendelian Inheritance ◽  
Pathogen Transmission ◽  
Resistance Allele ◽  
Gene Drive ◽  
Vector Borne Diseases ◽  
Evolution Of Resistance ◽  
Resistance Rates ◽  
Vector Capacity

ABSTRACTA 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. Though CRISPR gene drive activity has already been demonstrated, a key obstacle for current systems is their propensity to generate resistance alleles. In this study, we developed two CRISPR gene drive constructs based on thenanosandvasapromoters that allowed us to illuminate the different mechanisms by which resistance alleles are formed in the model organismDrosophila 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.

scholarly journals Reducing resistance allele formation in CRISPR gene drives

2017 ◽  
Author(s):  
Jackson Champer ◽  
Jingxian Liu ◽  
Suh Yeon Oh ◽  
Riona Reeves ◽  
Anisha Luthra ◽  
...  
Keyword(s):  
Formation Rate ◽  
Natural Populations ◽  
Mendelian Inheritance ◽  
Resistance Allele ◽  
Subsequent Formation ◽  
Vector Borne Diseases ◽  
Male Germline ◽  
Vector Borne ◽  
Resistance Rates ◽  
Gene Drives

ABSTRACTCRISPR gene drives can efficiently convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. This mechanism could be used, for example, to rapidly disseminate a genetic payload through a population, promising novel strategies for the control of vector-borne diseases. However, all CRISPR gene drives tested have produced significant quantities of resistance alleles that cannot be converted to drive alleles and would likely prevent these drives from spreading in a natural population. In this study, we assessed three strategies for reducing resistance allele formation. First, we directly compared drives with thenanosandvasapromoters, which showed that thevasadrive produced high levels of resistance alleles in somatic cells. This was not observed in thenanosdrive. Another strategy was the addition of a second gRNA to the drive, which both significantly increased the drive conversion efficiency and reduced the formation rate of resistance alleles. Finally, to minimize maternal carryover of Cas9, we assessed the performance of an autosomal drive acting in the male germline, and found no subsequent formation of resistance alleles in embryos. Our results mark a step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further reduction of resistance rates.

scholarly journals Reducing resistance allele formation in CRISPR gene drive

2018 ◽  
Vol 115 (21) ◽  
pp. 5522-5527 ◽  
Author(s):  
Jackson Champer ◽  
Jingxian Liu ◽  
Suh Yeon Oh ◽  
Riona Reeves ◽  
Anisha Luthra ◽  
...  
Keyword(s):  
Natural Populations ◽  
Mendelian Inheritance ◽  
Gene Drive ◽  
Subsequent Formation ◽  
Vector Borne Diseases ◽  
Effective Strategies ◽  
Guide Rnas ◽  
Vector Borne ◽  
Resistance Rates ◽  
Gene Drives

CRISPR homing gene drives can convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. Such a mechanism could be used, for example, to rapidly disseminate a genetic payload in a population, promising effective strategies for the control of vector-borne diseases. However, all CRISPR homing gene drives studied in insects thus far have produced significant quantities of resistance alleles that would limit their spread. In this study, we provide an experimental demonstration that multiplexing of guide RNAs can both significantly increase the drive conversion efficiency and reduce germline resistance rates of a CRISPR homing gene drive inDrosophila melanogaster. We further show that an autosomal drive can achieve drive conversion in the male germline, with no subsequent formation of resistance alleles in embryos through paternal carryover of Cas9. Finally, we find that thenanospromoter significantly lowers somatic Cas9 expression compared with thevasapromoter, suggesting thatnanosprovides a superior choice in drive strategies where gene disruption in somatic cells could have fitness costs. Comparison of drive parameters among the different constructs developed in this study and a previous study suggests that, while drive conversion and germline resistance rates are similar between different genomic targets, embryo resistance rates can vary significantly. Taken together, our results mark an important step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further control of resistance rates.

scholarly journals Polyandry blocks gene drive in a wild house mouse population

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Andri Manser ◽  
Barbara König ◽  
Anna K. Lindholm
Keyword(s):  
Natural Populations ◽  
Reproductive Behaviour ◽  
Mendelian Inheritance ◽  
House Mice ◽  
Gene Drive ◽  
Mouse Population ◽  
Drive Frequency ◽  
Wild House Mice ◽  
The Impact ◽  
Wild House Mouse

Abstract Gene drives are genetic elements that manipulate Mendelian inheritance ratios in their favour. Understanding the forces that explain drive frequency in natural populations is a long-standing focus of evolutionary research. Recently, the possibility to create artificial drive constructs to modify pest populations has exacerbated our need to understand how drive spreads in natural populations. Here, we study the impact of polyandry on a well-known gene drive, called t haplotype, in an intensively monitored population of wild house mice. First, we show that house mice are highly polyandrous: 47% of 682 litters were sired by more than one male. Second, we find that drive-carrying males are particularly compromised in sperm competition, resulting in reduced reproductive success. As a result, drive frequency decreased during the 4.5 year observation period. Overall, we provide the first direct evidence that the spread of a gene drive is hampered by reproductive behaviour in a natural population.

scholarly journals Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes

2019 ◽  
Vol 10 (2) ◽  
pp. 827-837 ◽  
Author(s):  
Nikolay P. Kandul ◽  
Junru Liu ◽  
Anna Buchman ◽  
Valentino M. Gantz ◽  
Ethan Bier ◽  
...  
Keyword(s):  
Disease Transmission ◽  
Target Genes ◽  
High Efficiency ◽  
Population Control ◽  
Natural Populations ◽  
Pathogen Transmission ◽  
Gene Drive ◽  
Guide Rnas ◽  
Cas9 Protein ◽  
Gene Drives

Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. We also assess the efficiency of ‘shadow drive’ generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.

scholarly journals Small-molecule control of super-Mendelian inheritance in gene drives

2019 ◽  
Author(s):  
Víctor López Del Amo ◽  
Brittany S. Leger ◽  
Kurt J. Cox ◽  
Shubhroz Gill ◽  
Alena L. Bishop ◽  
...  
Keyword(s):  
Small Molecule ◽  
Chemical Control ◽  
Control Strategies ◽  
Mendelian Inheritance ◽  
Drive System ◽  
Gene Drive ◽  
Crop Pests ◽  
Vector Borne Diseases ◽  
Vector Borne ◽  
Gene Drives

ABSTRACTBy 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

scholarly journals Assessment of a split homing based gene drive for efficient knockout of multiple genes

2019 ◽  
Author(s):  
Nikolay P. Kandul ◽  
Junru Liu ◽  
Anna Buchman ◽  
Valentino M. Gantz ◽  
Ethan Bier ◽  
...  
Keyword(s):  
Target Gene ◽  
Tissue Specificity ◽  
Target Genes ◽  
High Efficiency ◽  
Population Control ◽  
Natural Populations ◽  
Pathogen Transmission ◽  
Gene Drive ◽  
Guide Rnas ◽  
Gene Drives

AbstractHoming based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host-encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate host encoded genes has not been thoroughly explored. To test this approach, here we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of its target gene during super-Mendelian propagation of split-HGD. However, maternal deposition and embryonic expression of Cas9 resulted in the generation of drive resistant alleles which can accumulate and limit the spread of such a drive. Alternative design principles are discussed that could mitigate the accumulation of resistance alleles while incorporating a GME.

scholarly journals Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

2019 ◽  
Author(s):  
Víctor López Del Amo ◽  
Alena L. Bishop ◽  
Héctor M. Sánchez C. ◽  
Jared B. Bennett ◽  
Xuechun Feng ◽  
...  
Keyword(s):  
Mendelian Inheritance ◽  
Gene Drive ◽  
Island Conservation ◽  
Vector Borne Diseases ◽  
Split Gene ◽  
Mendelian Genetics ◽  
Vector Borne ◽  
Field Deployment ◽  
Flexible Application ◽  
Gene Drives

ABSTRACTCRISPR-based gene drives spread through populations bypassing the dictates of Mendelian genetics, offering a population-engineering tool for tackling vector-borne diseases, managing crop pests, and helping island conservation efforts; unfortunately, current technologies raise safety concerns for unintended gene propagation. Herein, we address this by splitting the two drive components, Cas9 and gRNAs, into separate alleles to form a novel trans-complementing split–gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This bi-component nature allows for individual 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 use our system to study the maternal effects on CRISPR inheritance, impaired homology on efficiency, and resistant allele formation. Lastly, mathematical modeling of tGD spread in a population shows potential advantages for improving current gene-drive technologies for field population modification.

scholarly journals Experts’ moral views on gene drive technologies: a qualitative interview study

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
N. de Graeff ◽  
Karin R. Jongsma ◽  
Annelien L. Bredenoord
Keyword(s):  
Invasive Species ◽  
Epistemic Uncertainty ◽  
Field Trials ◽  
Interview Study ◽  
Gene Drive ◽  
Agricultural Pests ◽  
Stepping Stones ◽  
Vector Borne Diseases ◽  
Qualitative Interview Study ◽  
The Status

Abstract Background Gene drive technologies (GDTs) promote the rapid spread of a particular genetic element within a population of non-human organisms. Potential applications of GDTs include the control of insect vectors, invasive species and agricultural pests. Whether, and if so, under what conditions, GDTs should be deployed is hotly debated. Although broad stances in this debate have been described, the convictions that inform the moral views of the experts shaping these technologies and related policies have not been examined in depth in the academic literature. Methods In this qualitative study, we interviewed GDT experts (n = 33) from different disciplines to identify and better understand their moral views regarding these technologies. The pseudonymized transcripts were analyzed thematically. Results The respondents’ moral views were principally influenced by their attitudes towards (1) the uncertainty related to GDTs; (2) the alternatives to which they should be compared; and (3) the role humans should have in nature. Respondents agreed there is epistemic uncertainty related to GDTs, identified similar knowledge gaps, and stressed the importance of realistic expectations in discussions on GDTs. They disagreed about whether uncertainty provides a rationale to refrain from field trials (‘risks of intervention’ stance) or to proceed with phased testing to obtain more knowledge given the harms of the status quo (‘risks of non-intervention’ stance). With regards to alternatives to tackle vector-borne diseases, invasive species and agricultural pests, respondents disagreed about which alternatives should be considered (un)feasible and (in)sufficiently explored: conventional strategies (‘downstream solutions’ stance) or systematic changes to health care, political and agricultural systems (‘upstream solutions’ stance). Finally, respondents held different views on nature and whether the use of GDTs is compatible with humans’ role in nature (‘interference’ stance) or not (‘non-interference stance’). Conclusions This interview study helps to disentangle the debate on GDTs by providing a better understanding of the moral views of GDT experts. The obtained insights provide valuable stepping-stones for a constructive debate about underlying value conflicts and call attention to topics that deserve further (normative) reflection. Further evaluation of these issues can facilitate the debate on and responsible development of GDTs.

scholarly journals Diazinon resistance, fluctuating asymmetry and fitness in the Australian sheep blowfly, lucilia cuprina.

Genetics ◽  
1988 ◽  
Vol 120 (1) ◽  
pp. 213-220 ◽  
Author(s):  
J A McKenzie ◽  
G M Clarke
Keyword(s):  
Fluctuating Asymmetry ◽  
Natural Populations ◽  
Lucilia Cuprina ◽  
Resistance Locus ◽  
Gene Complex ◽  
Subsequent Evolution ◽  
Genetic Analyses ◽  
Evolution Of Resistance ◽  
Australian Sheep ◽  
Chromosome Iii

Abstract Genetic evidence suggests that the evolution of resistance to the insecticide diazinon in Lucilia cuprina initially produced an increase in asymmetry. At that time resistant flies were presumed to be at a selective disadvantage in the absence of diazinon. Subsequent evolution in natural populations selected modifiers to ameliorate these effects. The fitness and fluctuating asymmetry levels of resistant flies are currently similar to those of susceptibles. Previous genetic analyses have shown the fitness modifier to co-segregate with the region of chromosome III marked by the white eyes, w, locus, unlinked to the diazinon resistance locus, Rop-1, on chromosome IV. This study maps the asymmetry modifier to the same region, shows, as in the case of the fitness modifier, its effect to be dominant and presents data consistent with the fitness/asymmetry modifier being the same gene (gene complex). These results suggest changes in fluctuating asymmetry reflect changes in fitness.

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

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

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

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