There is great interest in being able to spread beneficial traits throughout wild populations in ways that are self-sustaining. Here, we describe a chromosomal selfish genetic element, CleaveR Cleave ...and Rescue (ClvR), able to achieve this goal. ClvR comprises two linked chromosomal components. One, germline-expressed Cas9 and guide RNAs (gRNAs)—the Cleaver—cleaves and thereby disrupts endogenous copies of a gene whose product is essential. The other, a recoded version of the essential gene resistant to cleavage and gene conversion with cleaved copies—the Rescue—provides essential gene function. ClvR enhances its transmission, and that of linked genes, by creating conditions in which progeny lacking ClvR die because they have no functional copies of the essential gene. In contrast, those who inherit ClvR survive, resulting in an increase in ClvR frequency. ClvR is predicted to spread to fixation under diverse conditions. To test these predictions,we generated a ClvR element in Drosophila melanogaster. ClvR
tko is located on chromosome 3 and uses Cas9 and four gRNAs to disrupt melanogaster technical knockout (tko), an X-linked essential gene. Rescue activity is provided by tko from Drosophila virilis. ClvR
tko results in germline and maternal carryover-dependent inactivation of melanogaster tko (>99% per generation); lethality caused by this loss is rescued by the virilis transgene; ClvR
tko activities are robust to genetic diversity in strains from five continents; and uncleavable but functional melanogaster tko alleles were not observed. Finally, ClvR
tko spreads to transgene fixation. The simplicity of ClvR suggests it may be useful for altering populations in diverse species.
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 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 in Drosophila melanogaster targeting 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.
Insects play important roles as predators, prey, pollinators, recyclers, hosts, parasitoids, and sources of economically important products. They can also destroy crops; wound animals; and serve as ...vectors for plant, animal, and human diseases. Gene drive-a process by which genes, gene complexes, or chromosomes encoding specific traits are made to spread through wild populations, even if these traits result in a fitness cost to carriers-provides new opportunities for altering populations to benefit humanity and the environment in ways that are species specific and sustainable. Gene drive can be used to alter the genetic composition of an existing population, referred to as population modification or replacement, or to bring about population suppression or elimination. We describe technologies under consideration, progress that has been made, and remaining technological hurdles, particularly with respect to evolutionary stability and our ability to control the spread and ultimate fate of genes introduced into populations.
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 principle, be carried out if two ClvR elements targeting different essential genes are located at the same genomic position, and one of them, ClvR
n+1, carries a Rescue transgene from an earlier element, ClvRⁿ. ClvR
n+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.
Gene drive elements promote the spread of linked traits, providing methods for changing the composition or fate of wild populations. Drive mechanisms that are self-limiting are attractive because ...they allow control over the duration and extent of trait spread in time and space, and are reversible through natural selection as drive wanes. Self-sustaining Cleave and Rescue (ClvR) elements include a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, a tightly linked recoded version of the essential gene resistant to cleavage (the Rescue), and a Cargo. ClvR spreads by creating loss-of-function (LOF) conditions in which those without ClvR die because they lack functional copies of the essential gene. We use modeling to show that when the Rescue-Cargo and one or both components required for LOF allele creation (Cas9 and gRNA) reside at different locations (split ClvR), drive of Rescue-Cargo is self-limiting due to a progressive decrease in Cas9 frequency, and thus opportunities for creation of LOF alleles, as spread occurs. Importantly, drive strength and duration can be extended in a measured manner-which is still self-limiting-by moving the two components close enough to each other that they experience some degree of linkage. With linkage, Cas9 transiently experiences drive by hitchhiking with Rescue-Cargo until linkage disequilibrium between the two disappears, a function of recombination frequency and number of generations, creating a novel point of control. We implement split ClvR in Drosophila, with key elements on different chromosomes. Cargo/Rescue/gRNAs spreads to high frequency in a Cas9-dependent manner, while the frequency of Cas9 decreases. These observations show that measured, transient drive, coupled with a loss of future drive potential, can be achieved using the simple toolkit that make up ClvR elements-Cas9 and gRNAs and a Rescue/Cargo.
Mitochondrial DNA (mtDNA) often exists in a state of heteroplasmy, in which mutant mtDNA co-exists in cells with wild-type mtDNA. High frequencies of pathogenic mtDNA result in maternally inherited ...diseases; maternally and somatically acquired mutations also accumulate over time and contribute to diseases of ageing. Reducing heteroplasmy is therefore a therapeutic goal and in vivo models in post-mitotic tissues are needed to facilitate these studies. Here we describe a transgene-based model of a heteroplasmic lethal mtDNA deletion (mtDNA
) in adult Drosophila muscle. Stimulation of autophagy, activation of the PINK1/parkin pathway or decreased levels of mitofusin result in a selective decrease in mtDNA
. Decreased levels of mitofusin and increased levels of ATPIF1, an inhibitor of ATP synthase reversal-dependent mitochondrial repolarization, result in a further decrease in mtDNA
levels. These results show that an adult post-mitotic tissue can be cleansed of a deleterious genome, suggesting that therapeutic removal of mutant mtDNA can be achieved.
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.
(
) selfish genetic elements use Cas9 and guide RNAs (gRNAs) to disrupt endogenous versions of an essential gene while also including a
version of the essential gene resistant to disruption.
spreads by creating loss-of-function alleles of the essential gene that select against those lacking it, resulting in populations in which the
provides the only source of essential gene function. As a consequence, if function of the
, 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
in
in which
activity of an essential gene,
, requires splicing of a temperature-sensitive intein (TS-
). This element spreads to transgene fixation at 23 °C, but when populations now dependent on Ts-
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
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.
Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a self-perpetuating method of disease prevention but requires a gene drive mechanism ...to spread these traits to high frequency 1–3. Drive mechanisms requiring that transgenes exceed a threshold frequency in order to spread are attractive because they bring about local but not global replacement, and transgenes can be eliminated through dilution of the population with wild-type individuals 4–6. These features are likely to be important in many social and regulatory contexts 7–10. Here we describe the first creation of a synthetic threshold-dependent gene drive system, designated maternal-effect lethal underdominance (UDMEL), in which two maternally expressed toxins, located on separate chromosomes, are each linked with a zygotic antidote able to rescue maternal-effect lethality of the other toxin. We demonstrate threshold-dependent replacement in single- and two-locus configurations in Drosophila. Models suggest that transgene spread can often be limited to local environments. They also show that in a population in which single-locus UDMEL has been carried out, repeated release of wild-type males can result in population suppression, a novel method of genetic population manipulation.
► We created a threshold-dependent gene drive system, UDMEL ► Transgenes can be removed from a replaced population by dilution with wild-types ► Modeling identifies contexts in which UDMEL-dependent drive can be confined ► Releasing wild-type males into a replaced population can lead to population suppression
Population-scale genome modification can alter the composition or fate of wild populations. Synthetic gene drives provide one set of tools, but their use is complicated by scientific, regulatory, and ...social issues associated with transgene persistence and flow. Here we propose an alternative approach. An Allele Sail consists of a genome editor (the Wind) that introduces DNA sequence edits, and is inherited in a Mendelian fashion. Meanwhile, the edits (the Sail) experience an arithmetic, Super-Mendelian increase in frequency. We model this system and identify contexts in which a single, low frequency release of an editor brings edits to a very high frequency. We also identify conditions in which manipulation of sex determination can bring about population suppression. In regulatory frameworks that distinguish between transgenics (GMO) and their edited non-transgenic progeny (non-GMO) Allele Sails may prove useful since the spread and persistence of the GM component can be limited.Population-scale genome modification can alter the composition or fate of wild populations. Here the authors introduce Allele Sails as a method for spreading genetic changes throughout a population.
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