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

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.

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

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400985 ◽

2019 ◽

Vol 10 (2) ◽

pp. 827-837 ◽

Cited By ~ 13

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.

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Selective inheritance of target genes from only one parent of sexually reproduced F1 progeny in Arabidopsis

Nature Communications ◽

10.1038/s41467-021-24195-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Tao Zhang ◽

Michael Mudgett ◽

Ratnala Rambabu ◽

Bradley Abramson ◽

Xinhua Dai ◽

...

Keyword(s):

Target Gene ◽

Target Genes ◽

Heterozygous Mutation ◽

Gene Drive ◽

Homology Directed Repair ◽

Guide Rnas ◽

Fluorescent Markers ◽

Potential Applications ◽

Cryptochrome 1 ◽

F1 Generation

AbstractSexual reproduction constrains progeny to inherit allelic genes from both parents. Selective acquisition of target genes from only one parent in the F1 generation of plants has many potential applications including the elimination of undesired alleles and acceleration of trait stacking. CRISPR/Cas9-based gene drives can generate biased transmission of a preferred allele and convert heterozygotes to homozygotes in insects and mice, but similar strategies have not been implementable in plants because of a lack of efficient homology-directed repair (HDR). Here, we place a gene drive, which consists of cassettes that produce Cas9, guide RNAs (gRNA), and fluorescent markers, into the CRYPTOCHROME 1 (CRY1) gene through CRISPR/Cas9-mediated HDR, resulting in cry1drive lines. After crossing the cry1drive/cry1drive lines to wild type, we observe F1 plants which have DNA at the CRY1 locus from only the cry1drive/cry1drive parent. Moreover, a non-autonomous trans-acting gene drive, in which the gene drive unit and the target gene are located on different chromosomes, converts a heterozygous mutation in the target gene to homozygous. Our results demonstrate that homozygous F1 plants can be obtained through zygotic conversion using a CRISPR/Cas9-based gene drive.

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Reducing resistance allele formation in CRISPR gene drive

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1720354115 ◽

2018 ◽

Vol 115 (21) ◽

pp. 5522-5527 ◽

Cited By ~ 99

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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Experimental demonstration of tethered gene drive systems for confined population modification or suppression

10.1101/2021.05.29.446308 ◽

2021 ◽

Author(s):

Matthew Metzloff ◽

Emily Yang ◽

Sumit Dhole ◽

Andrew G. Clark ◽

Philipp W. Messer ◽

...

Keyword(s):

Population Control ◽

Local Population ◽

Natural Populations ◽

Great Promise ◽

Gene Drive ◽

Native Range ◽

Target Populations ◽

Experimental Cage ◽

Drive Systems ◽

Gene Drives

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. Here, we demonstrate the engineering of a tethered drive system in Drosophila, using a 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). Our results show that the tethered drive strategy is a viable and easily engineered option for providing confinement of homing drives to target populations.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2004373117 ◽

2020 ◽

Vol 117 (39) ◽

pp. 24377-24383 ◽

Cited By ~ 7

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

10.1101/112011 ◽

2017 ◽

Cited By ~ 9

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.

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

eLife ◽

10.7554/elife.58791 ◽

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.

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Development of a Confinable Gene-Drive System in the Human Disease Vector, Aedes aegypti

10.1101/645440 ◽

2019 ◽

Cited By ~ 10

Author(s):

Ming Li ◽

Ting Yang ◽

Nikolay P. Kandul ◽

Michelle Bui ◽

Stephanie Gamez ◽

...

Keyword(s):

Aedes Aegypti ◽

High Efficiency ◽

New Technologies ◽

Current Control ◽

Pathogen Transmission ◽

Mosquito Vector ◽

Control Methods ◽

Split Gene ◽

Pathogen Effector ◽

Gene Drives

AbstractAedes 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 StatementAe. 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.

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

10.1101/088427 ◽

2016 ◽

Cited By ~ 4

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.

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

10.1101/017384 ◽

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.

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