Biology subjects

Ivy, T.

Publications and source records attributed to Ivy, T..

5 recordsLinked to original sources

bioRxiv2023

Cleave and Rescue gamete killers create conditions for gene drive in plants

Gene drive elements promote the spread of linked traits, even when their presence confers a fitness cost to carriers, and can be used to change the composition or fate of wild populations. Cleave and Rescue (ClvR) drive elements sit at a fixed chromosomal position and include a DNA sequence-modifying enzyme such as Cas9/gRNAs (the Cleaver/Toxin) that disrupts endogenous versions of an essential gene, and a recoded version of the essential gene resistant to cleavage (the Rescue/Antidote). ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. We demonstrate the essential features of ClvR gene drive in the plant Arabidopsis thaliana through killing of gametes that fail to inherit a ClvR that targets the essential gene YKT61, whose expression is required in male and female gametes for their survival. Resistant (uncleavable but functional) alleles, which can slow or prevent drive, were not observed. Modeling shows plant ClvRs are likely to be robust to certain failure modes and can be used to rapidly drive population modification or suppression. Possible applications in plant breeding, weed control, and conservation are discussed.

genetics↗

bioRxiv2021

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

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{degrees}C, but when populations now dependent on Ts-ClvRdbe are shifted to 29{degrees}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.. SIGNIFICANCE STATEMENTGene drive can be used to spread traits of interest through wild populations. In some contexts the goal is to suppress or eliminate the population. In principle, one way to achieve this goal is if the trait being spread confers on carriers conditional lethality in response to an environmental stimulus that is either introduced by humans into the target area at a specific time (a virus, otherwise benign chemical; a kind of species-specific insecticide), or that occurs naturally on a seasonal basis, such as a change in temperature. Here we show that ClvR selfish elements can be used to spread a gene that confers lethality and sterility in response to increased temperature, demonstrating that conditional population elimination can be achieved.

genetics↗

bioRxiv2019

Gene drive and resilience through renewal with next generation Cleave and Rescue selfish genetic elements

Gene drive-based strategies for modifying populations face the problem that genes encoding cargo and the drive mechanism are subject to separation, mutational inactivation, and loss of efficacy. Resilience, an ability to respond to these eventualities in ways that restore population modification with functional genes is needed for long-term success. Here we show that resilience can be achieved through cycles of population modification with "Cleave and Rescue" (ClvR) selfish genetic elements. ClvR comprises a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, and a recoded version of the essential gene resistant to cleavage. ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. Cycles of modification can in principal be carried out if two ClvR elements targeting different essential genes are located at the same genomic position, and one of them, ClvRn+1, carries a Rescue transgene from an earlier element, ClvRn. ClvRn+1 should spread within a population of ClvRn, while also bringing about a decrease in its frequency. To test this hypothesis we first show that multiple ClvRs, each targeting a different essential gene, function when located at a common chromosomal position in Drosophila. We then show that when several of these also carry the Rescue from a different ClvR, they spread to transgene fixation in populations fixed for the latter, and at its expense. Therefore, genetic modifications of populations can be overwritten with new content, providing an ongoing point of control. SignificanceGene drive can spread beneficial traits through populations, but will never be a one-shot project in which one genetic element provides all desired modifications, for an indefinitely long time. Here we show that gene drive mediated population modification in Drosophila can be overwritten with new content while eliminating old, using Cleave and Rescue (ClvR) selfish genetic elements. The ability to carry out cycles of modification that create and then leave behind a minimal genetic footprint while entering and exiting a population provides important points of control. It makes possible the replacement of broken elements, upgrades with new elements that better carry out their tasks and/or provide new functions, all while promoting the removal of modifications no longer needed.

genetics↗

bioRxiv2018

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

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 in Drosophila and 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.\n\nSignificance 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.

genetics↗

bioRxiv2016

Engineered reciprocal chromosome translocations drive high threshold, reversible population replacement in Drosophila

Replacement of wild insect populations with transgene-bearing individuals unable to transmit disease or survive under specific environmental conditions provides self-perpetuating methods of disease prevention and population suppression, respectively. Gene drive mechanisms that require the gene drive element and linked cargo exceed a high threshold frequency to spread are attractive because they offer several points of control: they bring about local, but not global population replacement; and transgenes can be eliminated by reintroducing wildtypes into the population so as to drive the frequency of transgenes below the threshold required for drive. It has long been recognized that reciprocal chromosome translocations could, in principal, be used to bring about high threshold gene drive through a form of underdominance. However, translocations able to drive population replacement have not been reported, leaving it unclear if translocation-bearing strains fit enough to mediate gene drive can easily be generated. Here we use modeling to identify a range of conditions under which translocations should spread, and the equilibrium frequencies achieved, given specific introduction frequencies, fitness costs and migration rates. We also report the creation of engineered translocation-bearing strains of Drosophila melanogaster, generated through targeted chromosomal breakage and homologous recombination. By several measures translocation-bearing strains are fit, and drive high threshold, reversible population replacement in laboratory populations. These observations, together with the generality of the tools used to generate translocations, suggest that engineered translocations may be useful for controlled population replacement in many species.

evolutionary biology↗