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 Viral gene drive in herpesviruses

2019 ◽  
Author(s):  
Marius Walter ◽  
Eric Verdin
Keyword(s):  
Cell Culture ◽  
Human Cytomegalovirus ◽  
Therapeutic Strategy ◽  
Human Herpesvirus ◽  
Viral Gene ◽  
Gene Drive ◽  
Dna Viruses ◽  
Successful Transmission ◽  
Gene Drives ◽  
Novel Therapeutic

AbstractHerpesviruses are ubiquitous pathogens in need of novel therapeutic solutions. Current engineered gene drive strategies rely on sexual reproduction, and are thought to be restricted to sexual organisms. Here, we report on the design of a novel 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. Our results indicate that viral gene drives can be used to suppress a viral infection and may represent a novel therapeutic strategy against herpesviruses.

scholarly journals Targeting conserved sequences circumvents the evolution of resistance in a viral gene drive against human cytomegalovirus

2021 ◽  
Author(s):  
Marius Walter ◽  
Rosalba Perrone ◽  
Eric Verdin
Keyword(s):  
Cell Culture ◽  
Human Cytomegalovirus ◽  
Viral Population ◽  
Viral Gene ◽  
Gene Drive ◽  
Conserved Sequences ◽  
Evolution Of Resistance ◽  
Gene Drives ◽  
Over Time

Gene drives are genetic systems designed to efficiently spread a modification through a population. They have been designed almost exclusively in eukaryotic species, and especially in insects. We recently developed a CRISPR-based gene drive system in herpesviruses that relies on similar mechanisms and could efficiently spread into a population of wildtype viruses. A common consequence of gene drives in insects is the appearance and selection of drive-resistant sequences that are no longer recognized by CRISPR-Cas9. Here, we analyze in cell culture experiments the evolution of resistance in a viral gene drive against human cytomegalovirus. We report that, after an initial invasion of the wildtype population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionary conserved sequences ensures that drive-resistant viruses acquire long-lasting mutations and are durably attenuated. As a consequence, and even though engineered viruses do not stably persist in the viral population, remaining viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in herpesviruses, especially for therapeutic applications. Importance The use of defective viruses that interfere with the replication of their infectious parent after co-infecting the same cells – a therapeutic strategy known as viral interference – has recently generated a lot of interest. The CRISPR-based system that we recently reported in herpesviruses represents a novel interfering strategy that causes the conversion of wildtype viruses into new recombinant viruses and drives the native viral population to extinction. In this report, we analyzed how targeted viruses evolved resistance against the technology. Through numerical simulations and cell culture experiments with human cytomegalovirus, we show that, after the initial propagation, a resistant viral population is positively selected and outcompetes engineered viruses over time. We show however that targeting evolutionary conserved sequences ensures that resistant viruses are mutated and attenuated, which leads to a long-term reduction of viral levels. This marks an important step toward the development of novel therapeutic strategies against herpesviruses.

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

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

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

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

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

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

scholarly journals 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 Targeting evolutionary conserved sequences circumvents the evolution of resistance in a viral gene drive against human cytomegalovirus

2021 ◽  
Author(s):  
Marius Walter ◽  
Rosalba Perrone ◽  
Eric Verdin
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Gene ◽  
Gene Drive ◽  
Eukaryotic Species ◽  
Resistant Population ◽  
Evolution Of Resistance ◽  
Gene Drives ◽  
Over Time ◽  
Selection Of

Gene drives are genetic systems designed to efficiently spread a modification through a population. Most engineered gene drives rely on CRISPR-Cas9 and were designed in insects or other eukaryotic species. We recently developed a viral gene drive in herpesviruses that efficiently spread into a population of wildtype viruses. A common consequence of gene drives is the appearance and selection of drive-resistant sequences that are no longer recognized by CRISPR-Cas9. Here, we analyze in cell culture experiments the evolution of resistance in a gene drive against human cytomegalovirus. We report that after an initial invasion of the wildtype population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionary conserved regions ensures that drive-resistant viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in viruses, especially for therapeutic applications.

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 CRISPR with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research

2015 ◽  
Author(s):  
Fillip Port ◽  
Nadine Muschalik ◽  
Simon L Bullock
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Basic Research ◽  
Evidence Based ◽  
Gene Drive ◽  
Highly Active ◽  
Genome Modification ◽  
Target Sites ◽  
Gene Drives

CRISPR/Cas technology allows rapid, site-specific genome modification in a wide variety of organisms. CRISPR components produced by integrated transgenes have been shown to mutagenise some genomic target sites in Drosophila melanogaster with high efficiency, but whether this is a general feature of this system remains unknown. Here, we systematically evaluate available CRISPR/Cas reagents and experimental designs in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for genome engineering (Gantz & Bier, 2015) and conclude that optimised CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk.

scholarly journals Control of malaria-transmitting mosquitoes using gene drives

2020 ◽  
Vol 376 (1818) ◽  
pp. 20190803 ◽  
Author(s):  
Tony Nolan
Keyword(s):  
Malaria Control ◽  
Control Strategies ◽  
Technical Development ◽  
Gene Drive ◽  
Theme Issue ◽  
Genetic Elements ◽  
Selfish Genetic Elements ◽  
Gene Drives ◽  
Transmit Disease ◽  
Proof Of Principle

Gene drives are selfish genetic elements that can be re-designed to invade a population and they hold tremendous potential for the control of mosquitoes that transmit disease. Much progress has been made recently in demonstrating proof of principle for gene drives able to suppress populations of malarial mosquitoes, or to make them refractory to the Plasmodium parasites they transmit. This has been achieved using CRISPR-based gene drives. In this article, I will discuss the relative merits of this type of gene drive, as well as barriers to its technical development and to its deployment in the field as malaria control. This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases'.

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