scholarly journals Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Astrid Hoermann ◽  
Sofia Tapanelli ◽  
Paolo Capriotti ◽  
Giuseppe Del Corsano ◽  
Ellen KG Masters ◽  
...  
Keyword(s):  
Regulatory Sequences ◽  
Gene Drive ◽  
Passive Mode ◽  
Population Replacement ◽  
Guide Rnas ◽  
Efficient Gene ◽  
Genetic Modifications ◽  
Form Part ◽  
Endogenous Genes ◽  
Gene Drives

Gene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector Anopheles gambiae to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of Plasmodium falciparum and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an acceptable testing pathway of gene drives for malaria eradication.

scholarly journals Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement

2020 ◽  
Author(s):  
Astrid Hoermann ◽  
Sofia Tapanelli ◽  
Paolo Capriotti ◽  
Ellen K. G. Masters ◽  
Tibebu Habtewold ◽  
...  
Keyword(s):  
Regulatory Sequences ◽  
Gene Drive ◽  
Passive Mode ◽  
Population Replacement ◽  
Guide Rnas ◽  
Efficient Gene ◽  
Genetic Modifications ◽  
Form Part ◽  
Endogenous Genes ◽  
Gene Drives

AbstractGene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector Anopheles gambiae to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns, that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of Plasmodium falciparum and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an accepted testing pathway of gene drives for malaria eradication.

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

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

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

scholarly journals Integral Gene Drives: an “operating system” for population replacement

2018 ◽  
Author(s):  
Alexander Nash ◽  
Giulia Mignini Urdaneta ◽  
Andrea K. Beaghton ◽  
Astrid Hoermann ◽  
Philippos Aris Papathanos ◽  
...  
Keyword(s):  
Field Testing ◽  
Target Site ◽  
Gene Drive ◽  
Population Replacement ◽  
Pathogen Effector ◽  
Site Variation ◽  
The Face ◽  
Endogenous Genes ◽  
Target Site Resistance ◽  
Gene Drives

AbstractFirst generation CRISPR-based gene drives have now been tested in the laboratory in a number of organisms including malaria vector mosquitoes. A number of challenges for their use in the area-wide genetic control of vector-borne disease have been identified. These include the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and the practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. To address these challenges, we have evaluated the concept of Integral Gene Drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including both the drive and the anti-pathogen effector elements directly embedded within endogenous genes – an arrangement which we refer to as gene “hijacking”. This design would allow autonomous and non-autonomous IGD traits and strains to be generated, tested, optimized, regulated and imported independently. We performed quantitative modelling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population while hedging drive over multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target-site variation. IGD thus has the potential to yield more durable and flexible population replacement traits.

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

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

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

scholarly journals 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 Overcoming evolved resistance to population-suppressing homing-based gene drives

2016 ◽  
Author(s):  
John M. Marshall ◽  
Anna Buchman ◽  
Héctor M. Sánchez C. ◽  
Omar S. Akbari
Keyword(s):  
Mosquito Species ◽  
Gene Drive ◽  
Resistant Allele ◽  
Guide Rnas ◽  
Continental Scale ◽  
Fitness Advantage ◽  
Health Related ◽  
Drive Systems ◽  
Gene Drives

AbstractThe use of homing-based gene drive systems to modify or suppress wild populations of a given species has been proposed as a solution to a number of significant ecological and public health related problems, including the control of mosquito-borne diseases. The recent development of a CRISPR-Cas9-based homing system for the suppression ofAnopheles gambiae, the main African malaria vector, is encouraging for this approach; however, with current designs, the slow emergence of homing-resistant alleles is expected to result in suppressed populations rapidly rebounding, as homing-resistant alleles have a significant fitness advantage over functional, population-suppressing homing alleles. To explore this concern, we develop a mathematical model to estimate tolerable rates of homing-resistant allele generation to suppress a wild population of a given size. Our results suggest that, to achieve meaningful population suppression, tolerable rates of resistance allele generation are orders of magnitude smaller than those observed for current designs for CRISPR-Cas9-based homing systems. To remedy this, we propose a homing system architecture in which guide RNAs (gRNAs) are multiplexed, increasing the effective homing rate and decreasing the effective resistant allele generation rate. Modeling results suggest that the size of the population that can be suppressed increases exponentially with the number of multiplexed gRNAs and that, with six multiplexed gRNAs, a mosquito species could potentially be suppressed on a continental scale. We also demonstrate successful multiplexingin vivoinDrosophila melanogasterusing a ribozyme-gRNA-ribozyme (RGR) approach – a strategy that could readily be adapted to engineer stable, homing-based suppression drives in relevant organisms.

scholarly journals Gene Drives Across Engineered Fitness Valleys: Modeling a Design to Prevent Drive Spillover

2020 ◽  
Author(s):  
Frederik J.H. de Haas ◽  
Sarah P. Otto
Keyword(s):  
Local Population ◽  
Target Population ◽  
Drive System ◽  
High Threshold ◽  
Gene Drive ◽  
Time Model ◽  
Population Replacement ◽  
Low Threshold ◽  
Drive Systems ◽  
Gene Drives

1AbstractEngineered gene drive techniques for population replacement and/or suppression have potential for tackling complex challenges, including reducing the spread of diseases and invasive species. Unfortunately, the self-propelled behavior of drives can lead to the spread of transgenic elements beyond the target population, which is concerning. Gene drive systems with a low threshold frequency for invasion, such as homing-based gene drive systems, require initially few transgenic individuals to spread and are therefore easy to implement. However their ease of spread presents a double-edged sword; their low threshold makes these drives much more susceptible to spread outside of the target population (spillover). We model a proposed drive system that transitions in time from a low threshold drive system (homing-based gene drive) to a high threshold drive system (underdominance) using daisy chain technology. This combination leads to a spatially restricted drive strategy, while maintaining an attainable release threshold. We develop and analyze a discrete-time model as proof of concept and find that this technique effectively generates stable local population suppression, while preventing the spread of transgenic elements beyond the target population under biologically realistic parameters.

scholarly journals Viral gene drive in herpesviruses

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Marius Walter ◽  
Eric Verdin
Keyword(s):  
Cell Culture ◽  
High Efficiency ◽  
Human Herpesvirus ◽  
Viral Gene ◽  
Gene Drive ◽  
Dna Viruses ◽  
Successful Transmission ◽  
Genetic Modifications ◽  
Gene Drives ◽  
Proof Of Principle

Abstract Gene drives are genetic modifications designed to propagate in a population with high efficiency. Current gene drive strategies rely on sexual reproduction and are thought to be restricted to sexual organisms. Here, we report on a gene drive system that allows the spread of an engineered trait in populations of DNA viruses and, in particular, herpesviruses. We describe the successful transmission of a gene drive sequence between distinct strains of human cytomegalovirus (human herpesvirus 5) and show that gene drive viruses can efficiently target and replace wildtype populations in cell culture experiments. Moreover, by targeting sequences necessary for viral replication, our results indicate that a viral gene drive can be used as a strategy to suppress a viral infection. Taken together, this work offers a proof of principle for the design of a gene drive in viruses.

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.

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