44 research outputs found

    Cleave and Rescue, a novel selfish genetic element and general strategy for gene drive

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    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. ClvRenhances 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

    Split versions of Cleave and Rescue selfish genetic elements for measured self limiting gene drive

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    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

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

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    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, ClvR^n. ClvR^(n+1) should spread within a population of ClvR^n, 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

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

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    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

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

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    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-ClvR^(dbe)). This element spreads to transgene fixation at 23 °C, but when populations now dependent on Ts-ClvR^(dbe) 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

    The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development

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    The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies

    Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

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    The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

    Implementing and Modeling Gene Drives for Population Modification and Suppression

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    Gene drive as a technology has immense potential for modifying species from the local to the global population level. While the advent of the CRISPR-Cas9 system has paved the way for many previously untenable gene drives, it has also illuminated two substantial pitfalls: homing based gene drives are particularly susceptible to generating drive breaking resistance alleles and many if not most gene drives are too powerful to be regionally contained. Lack of confinability makes such gene drives impractical for real world application where international law would be violated by their usage. We developed a new general form of gene drive known as cleave and rescue (ClvR), then built and simulated the potential of multiple variants of this drive which are capable of modifying or suppressing target populations, including variants with and without introduction thresholds for drive. We also developed scripts in Python capable of generating population dynamics simulations of a user-defined gene drive, either as a deterministic, population proportion model or a stochastic, discrete individual model. This tool provides a very useful first pass answer about a given gene drive’s ability to modify or suppress a population under varying fitness costs, drive activity rates, and release proportions

    Split versions of Cleave and Rescue selfish genetic elements for measured self limiting gene drive.

    No full text
    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

    Split versions of Cleave and Rescue selfish genetic elements for measured self limiting gene drive

    No full text
    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
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