Lethal Gene Drive Selects Inbreeding

AbstractThe use of ‘selfish’ gene drive systems to suppress or even extinguish populations has been proposed on theoretical grounds for almost half a century. Creating these genes has recently become possible with CRISPR technology. One seemingly feasible approach, originally proposed by Burt, is to create a homing endonuclease gene (HEG) that inserts into an essential gene, enabling heterozygote viability but causing homozygote lethality. With 100% segregation distortion in gametes, such genes can cause profound population suppression if resistance does not evolve. Here, population genetic models are used to consider the evolution of inbreeding (specifically selfing) as a possible response to a recessively lethal HEG with complete segregation distortion. Numerical analyses indicate a rich set of outcomes, but selfing often evolves in response to the HEG, with a corresponding partial restoration of mean fitness. Whether selfing does indeed evolve and its effect in restoring fitness depends heavily on the magnitude of inbreeding depression. Overall, these results point toward an underappreciated evolutionary response to block the harmful effects of a selfish gene. They raise the possibility that extreme population suppression may be more difficult to achieve than currently imagined.

Gene Therapy: A Promising Therapeutic Strategy for Malaria

University of Ottawa Journal of Medicine ◽

10.18192/uojm.v8i2.3651 ◽

2018 ◽

Vol 8 (2) ◽

pp. 40-44

Author(s):

Raafay Shehzad

Keyword(s):

Gene Therapy ◽

Therapeutic Strategy ◽

Homing Endonuclease ◽

Genetic Material ◽

Gene Drive ◽

Treatment And Prevention ◽

Preventative Measures ◽

Drive Systems ◽

Embryonic Arrest ◽

Diabetes And Obesity

Malaria is a serious illness caused by the Plasmodium parasite, which places approximately 3.5 billion people at risk. Currently, preventative measures are key in combatting this disease. However, gene therapy is an emerging field that shows promising results for the treatment of malaria, by modifying cells through the delivery of genetic material. Most notable was the discovery of CRISPR-Cas9, which not only allows deleterious mutations to be repaired, but does so with specificity, speed, and simplicity. There are numerous ongoing trials focusing on gene therapy in malaria treatment and prevention. They involve different approaches such as the genetic modification of vector mosquitoes to interfere with malaria transmission, use of CRISPR-Cas9, maternal-effect dominant embryonic arrest, homing endonuclease gene drive systems, and the design of specific Morpholino oligomers to interfere with the expression of parasitic characteristics. Overall, this emerging field shows promising results to treat and prevent not just malaria, but other diseases such as cancer, diabetes, and obesity.

Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs

10.1101/289546 ◽

2018 ◽

Cited By ~ 2

Author(s):

Georg Oberhofer ◽

Tobin Ivy ◽

Bruce A. Hay

Keyword(s):

Gene Function ◽

Homing Endonuclease ◽

Marker Gene ◽

Specific Gene ◽

Gene Drive ◽

Loss Of Function ◽

Complex Activity ◽

Guide Rnas ◽

Carry Over ◽

AbstractA gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous progeny. Earlier work inDrosophilaand mosquitoes utilized HEGs consisting of Cas9 and a single gRNA that together target a specific gene for cleavage. Homing was observed, but resistant alleles, immune to cleavage, while retaining wildtype gene function, were also created through non-homologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs, targeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and guide RNAs (gRNAs) designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments the HEGs failed to spread, due to the high fitness load induced in offspring as a result of maternal carry over of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.Significance statementHEG-based gene drive can bring about population suppression when genes required for viability or fertility are targeted. However, these strategies are vulnerable to failure through mechanisms that create alleles resistant to cleavage, but that retain wildtype gene function. We show that resistance allele creation can be prevented through the use of gRNAs designed to cleave a gene at four target sites. However, homing rates were modest, and the HEGs were unstable during homing. In addition, use of a promoter active in the female germline resulted in levels of HEG carryover that compromised the viability or fertility of HEG-bearing heterozygotes, thereby preventing drive. We propose strategies that can help to overcome these problems in next generation HEG systems.

Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi

10.1101/2020.08.02.233056 ◽

2020 ◽

Cited By ~ 6

Author(s):

Adriana Adolfi ◽

Valentino M. Gantz ◽

Nijole Jasinskiene ◽

Hsu-Feng Lee ◽

Kristy Hwang ◽

...

Keyword(s):

Anopheles Stephensi ◽

Pathogen Transmission ◽

Gene Drive ◽

Initial Release ◽

Genetic Technologies ◽

Rescue System ◽

Vector Borne ◽

Kynurenine Hydroxylase ◽

Drive Systems ◽

ABSTRACTThe development of Cas9/gRNA-mediated gene-drive systems has bolstered the advancement of genetic technologies for controlling vector-borne pathogen transmission. These include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of vector insects, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. We developed a recoded gene-drive rescue system for population modification in the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and a functional resistant allele does not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.

Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805278115 ◽

2018 ◽

Vol 115 (40) ◽

pp. E9343-E9352 ◽

Cited By ~ 47

Author(s):

Georg Oberhofer ◽

Tobin Ivy ◽

Bruce A. Hay

Keyword(s):

Homing Endonuclease ◽

Marker Gene ◽

Specific Gene ◽

Gene Drive ◽

Loss Of Function ◽

End Joining ◽

Complex Activity ◽

Guide Rna ◽

Type Gene ◽

A gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific, nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous, progeny. Earlier work inDrosophilaand mosquitoes utilized HEGs consisting of Cas9 and a single guide RNA (gRNA) that together target a specific gene for cleavage. Homing was observed, but resistant alleles immune to cleavage, while retaining wild-type gene function, were also created through nonhomologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs inDrosophila melanogastertargeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and gRNAs designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments, the HEGs failed to spread due to the high fitness load induced in offspring as a result of maternal carryover of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.

Modeling the mutation and reversal of engineered underdominance gene drives

10.1101/257253 ◽

2018 ◽

Cited By ~ 1

Author(s):

Matthew P. Edgington ◽

Luke S. Alphey

Keyword(s):

Genetic Material ◽

Theoretical Work ◽

The Other ◽

Lethal Gene ◽

Gene Drive ◽

Loss Of Function ◽

Wild Type ◽

Type Population ◽

Drive Systems ◽

AbstractA range of gene drive systems have been proposed that are predicted to increase their frequency and that of associated desirable genetic material even if they confer a fitness cost on individuals carrying them. Engineered underdominance (UD) is such a system and, in one version, is based on the introduction of two independently segregating transgenic constructs each carrying a lethal gene, a suppressor for the lethal at the other locus and a desirable genetic “cargo”. Under this system individuals carrying at least one copy of each construct (or no copies of either) are viable whilst those that possess just one of the transgenic constructs are non-viable. Previous theoretical work has explored various properties of these systems, concluding that they should persist indefinitely in absence of resistance or mutation. Here we study a population genetics model of UD gene drive that relaxes past assumptions by allowing for loss-of-function mutations in each introduced gene. We demonstrate that mutations are likely to cause UD systems to break down, eventually resulting in the elimination of introduced transgenes. We then go on to investigate the potential of releasing “free suppressor” carrying individuals as a new method for reversing UD gene drives and compare this to the release of wild-types; the only previously proposed reversal strategy for UD. This reveals that while free suppressor carrying individuals may represent an inexpensive reversal strategy due to extremely small release requirements, they are not able to return a fully wild-type population as rapidly as the release of wild-types.

Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

PLoS Genetics ◽

10.1371/journal.pgen.1009740 ◽

2021 ◽

Vol 17 (10) ◽

pp. e1009740

Author(s):

Silke Fuchs ◽

William T. Garrood ◽

Anna Beber ◽

Andrew Hammond ◽

Roberto Galizi ◽

...

Keyword(s):

Essential Gene ◽

Target Site ◽

Target Sequence ◽

Lethal Gene ◽

Gene Drive ◽

Continuous Sampling ◽

Conserved Sequence ◽

A Site ◽

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying (‘homing’) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.

Gene drive that results in addiction to a temperature sensitive version of an essential gene triggers population collapse in Drosophila

10.1101/2021.07.03.451005 ◽

2021 ◽

Author(s):

Georg Oberhofer ◽

Bruce Hay ◽

Tobin Ivy

Keyword(s):

Gene Function ◽

Essential Gene ◽

Trojan Horse ◽

Temperature Sensitive ◽

Gene Drive ◽

Loss Of Function ◽

Selfish Genetic Elements ◽

Population Crash ◽

Seasonal Basis ◽

One strategy for population suppression seeks to use gene drive to spread genes that confer conditional lethality or sterility, providing a way of combining population modification with suppression. Stimuli of potential interest could be introduced by humans, such as an otherwise benign virus or chemical, or occur naturally on a seasonal basis, such as a change in temperature. Cleave and Rescue (ClvR) selfish genetic elements use Cas9 and gRNAs to disrupt endogenous versions of an essential gene, while also including a Rescue version of the essential gene resistant to disruption. ClvR spreads by creating loss-of-function alleles of the essential gene that select against those lacking it, resulting in populations in which the Rescue provides the only source of essential gene function. In consequence, if function of the Rescue, a kind of Trojan horse now omnipresent in a population, is condition-dependent, so too will be the survival of that population. To test this idea we created a ClvR in Drosophila in which Rescue activity of an essential gene, dribble, requires splicing of a temperature-sensitive intein (TS-ClvRdbe). This element spreads to transgene fixation at 23° C, but when populations now dependent on TS-ClvRdbe are shifted to 29° C death and sterility result in a rapid population crash. These results show that conditional population elimination can be achieved. A similar logic, in which Rescue activity is conditional, could also be used in HEG-based drive, and to bring about suppression and/or killing of specific individuals in response to other stimuli.

Performance analysis of novel toxin-antidote CRISPR gene drive systems

10.1101/628362 ◽

2019 ◽

Cited By ~ 10

Author(s):

Jackson Champer ◽

Isabel Kim ◽

Samuel E. Champer ◽

Andrew G. Clark ◽

Philipp W. Messer

Keyword(s):

Target Genes ◽

Essential Gene ◽

Ecological Engineering ◽

Target Area ◽

Gene Drive ◽

Potential Applications ◽

Regional Population ◽

Drive Systems ◽

High Flexibility ◽

ABSTRACTGene drives can potentially fixate in a population by biasing inheritance in their favor, opening up a variety of potential applications in areas such as disease-vector control and conservation. CRISPR homing gene drives have shown much promise for providing an effective drive mechanism, but they typically suffer from the rapid formation of resistance alleles. Even if the problem of resistance can be overcome, the utility of such drives would still be limited by their tendency to spread into all areas of a population. To provide additional options for gene drive applications that are substantially less prone to the formation of resistance alleles and could potentially remain confined to a target area, we developed several designs for CRISPR-based gene drives utilizing toxin-antidote (TA) principles. These drives target and disrupt an essential gene with the drive providing rescue. Here, we assess the performance of several types of TA gene drive systems using modeling and individual-based simulations. We show that Toxin-Antidote Recessive Embryo (TARE) drive should allow for the design of robust, regionally confined, population modification strategies with high flexibility in choosing drive promoters and recessive lethal targets. Toxin-Antidote Dominant Embryo (TADE) drive requires a haplolethal target gene and a germline-restricted promoter but should enable the design of both faster regional population modification drives and even regionally-confined population suppression drives. Toxin-antidote dominant sperm (TADS) drive can be used for population modification or suppression. It spreads nearly as quickly as a homing drive and can flexibly use a variety of promoters, but unlike the other TA systems, it is not regionally confined and requires highly specific target genes. Overall, our results suggest that CRISPR-based TA gene drives provide promising candidates for further development in a variety of organisms and may allow for flexible ecological engineering strategies.

Gene drive that results in addiction to a temperature-sensitive version of an essential gene triggers population collapse in Drosophila

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2107413118 ◽

2021 ◽

Vol 118 (49) ◽

pp. e2107413118

Author(s):

Georg Oberhofer ◽

Tobin Ivy ◽

Bruce A. Hay

Keyword(s):

Gene Function ◽

Essential Gene ◽

Temperature Sensitive ◽

Gene Drive ◽

Loss Of Function ◽

Selfish Genetic Elements ◽

Guide Rnas ◽

Population Crash ◽

Seasonal Basis ◽

One strategy for population suppression seeks to use gene drive to spread genes that confer conditional lethality or sterility, providing a way of combining population modification with suppression. Stimuli of potential interest could be introduced by humans, such as an otherwise benign virus or chemical, or occur naturally on a seasonal basis, such as a change in temperature. Cleave and Rescue (ClvR) selfish genetic elements use Cas9 and guide RNAs (gRNAs) to disrupt endogenous versions of an essential gene while also including a Rescue version of the essential gene resistant to disruption. ClvR spreads by creating loss-of-function alleles of the essential gene that select against those lacking it, resulting in populations in which the Rescue provides the only source of essential gene function. As a consequence, if function of the Rescue, a kind of Trojan horse now omnipresent in a population, is condition dependent, so too will be the survival of that population. To test this idea, we created a ClvR in Drosophila in which Rescue activity of an essential gene, dribble, requires splicing of a temperature-sensitive intein (TS-ClvRdbe). This element spreads to transgene fixation at 23 °C, but when populations now dependent on Ts-ClvRdbe are shifted to 29 °C, death and sterility result in a rapid population crash. These results show that conditional population elimination can be achieved. A similar logic, in which Rescue activity is conditional, could also be used in homing-based drive and to bring about suppression and/or killing of specific individuals in response to other stimuli.

Design and analysis of CRISPR-based underdominance toxin-antidote gene drives

10.1101/861435 ◽

2019 ◽

Cited By ~ 7

Author(s):

Jackson Champer ◽

Samuel E. Champer ◽

Isabel Kim ◽

Andrew G. Clark ◽

Philipp W. Messer

Keyword(s):

Critical Frequency ◽

Essential Gene ◽

Geographic Location ◽

Early Embryo ◽

Gene Drive ◽

Wide Range ◽

Vector Borne ◽

Drive Systems ◽

Invasion Threshold ◽

ABSTRACTCRISPR gene drive systems offer a mechanism for transmitting a desirable transgene throughout a population for purposes ranging from vector-borne disease control to invasive species suppression. In this simulation study, we assess the performance of several CRISPR-based underdominance gene drive constructs employing toxin-antidote principles. These drives disrupt the wild-type version of an essential gene using a CRISPR nuclease (the toxin) while simultaneously carrying a recoded version of the gene (the antidote). Drives of this nature allow for releases that could be potentially confined to a desired geographic location. This is because such drives have a nonzero invasion threshold frequency, referring to the critical frequency required for the drive to spread through the population. We model drives which target essential genes that are either haplosufficient or haplolethal, using nuclease promoters with expression restricted to the germline, promoters that additionally result in cleavage activity in the early embryo from maternal deposition, and promoters that have ubiquitous somatic expression. We also study several possible drive architectures, considering both “same-site” and “distant-site” systems, as well as several reciprocally targeting drives. Together, these drive variants provide a wide range of invasion threshold frequencies and options for both population modification and suppression. Our results suggest that CRISPR toxin-antidote underdominance drive systems could allow for the design of highly flexible and potentially confinable gene drive strategies.