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If they are able to spread in wild populations, CRISPR-based gene-drive elements would provide new ways to address ecological problems by altering the traits of wild organisms, but the potential for uncontrolled spread tremendously complicates ethical development and use. Here, we detail a self-exhausting form of CRISPR-based drive system comprising genetic elements arranged in a daisy chain such that each drives the next. “Daisy-drive” systems can locally duplicate any effect achievable by using an equivalent self-propagating drive system, but their capacity to spread is limited by the successive loss of nondriving elements from one end of the chain. Releasing daisy-drive organisms constituting a small fraction of the local wild population can drive a useful genetic element nearly to local fixation for a wide range of fitness parameters without self-propagating spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionarily stable daisy drive as well as self-propagating CRISPR-based gene drive. Especially when combined with threshold dependence, daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.

CRISPR-based homing gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population. Here, we experimentally demonstrate the suitability of synthetic target site drives as well as split drives as flexible safeguarding strategies for gene drive experiments by showing that their performance closely resembles that of standard homing drives in Drosophila melanogaster. Using our split drive system, we further find that maternal deposition of both Cas9 and gRNA is required to form resistance alleles in the early embryo and that maternally-deposited Cas9 alone can power germline drive conversion in individuals that lack a genomic source of Cas9. Gene drives are a new genome editing technology where artificial gene packages are designed to create a mutation that will quickly spread within a population. These packages target a specific sequence in a genome, where they could potentially add, remove or deactivate a gene. They also trigger a process known as drive conversion, which ensures the mutation will be inherited at a higher rate than normal. Within several generations, nearly every organism in the population will carry this genetic change. This technology could, for example, help us eradicate disease-carrying mosquitoes, crop pests or invasive species. However, it could also have unforeseen and dangerous consequences. It is therefore crucial to keep gene drives within laboratory walls before they are ready to be released. Even if a small numbers of genetically modified animals were to escape, they could rapidly spread the packages within a wild population. To prevent this, scientists have devised two safeguarding strategies. One, called synthetic target site gene drive, uses target sequences that have been introduced on purpose in research organisms, but which are absent in wild populations. If the gene drive were to escape, it could not spread in the genomes of wild creatures because they lack the synthetic target site. Alternatively, split drive systems can also limit risk. There, the different components required for a gene drive are not packaged together, but in separate locations in the genome. Some of these elements are inherited at a normal rate, so the gene drive fizzles out after a few generations. However, it was still unclear whether synthetic gene drives and split drive systems could be used instead of the classic approach and yield the same results in research. Champer et al. compared traditional gene drives, synthetic target site gene drives, and split drive systems in fruit flies raised in the laboratory. The experiments show that the three approaches lead to similar results, with the genetic package spreading and creating resistance in a similar way. They also confirm that, in split drive systems, both components of the drive must be genetically inherited to create the intended mutation. Synthetic gene drives and split drive systems could therefore be used in experiments on gene drives, especially in studies with large numbers of organisms. Ultimately, adopting these measures could help to keep gene drive research safe, which may encourage more scientific teams to work on this technology and exploit its potential.

Invasive rodents are a major cause of environmental damage and biodiversity loss, particularly on islands. Unlike insects, genetic biocontrol strategies including populationsuppressing gene drives with biased inheritance have not been developed in mice. Here, we demonstrate a gene drive strategy (t CRISPR) that leverages super-Mendelian transmission of the t haplotype to spread inactivating mutations in a haplosufficient female fertility gene (Prl). Using spatially explicit individual-based in silico modeling, we show that t CRISPR can eradicate island populations under a range of realistic field-based parameter values. We also engineer transgenic t CRISPR mice that, crucially, exhibit biased transmission of the modified t haplotype and Prl mutations at levels ourmodeling predicts would be sufficient for eradication. This is an example of a feasible gene drive system for invasive alien rodent population control.

Gene drives could allow for control of vector-borne diseases by directly suppressing vector populations or spreading genetic payloads designed to reduce pathogen transmission. Clustered regularly interspaced short palindromic repeat (CRISPR) homing gene drives work by cleaving wild-type alleles, which are then converted to drive alleles by homology-directed repair, increasing the frequency of the drive in a population over time. However, resistance alleles can form when end-joining repair takes place in lieu of homology-directed repair. Such alleles cannot be converted to drive alleles, which would eventually halt the spread of a drive through a population. To investigate the effects of natural genetic variation on resistance formation, we developed a CRISPR homing gene drive in Drosophila melanogaster and crossed it into the genetically diverse Drosophila Genetic Reference Panel (DGRP) lines, measuring several performance parameters. Most strikingly, resistance allele formation postfertilization in the early embryo ranged from 7 to 79% among lines and averaged 42 ± 18%. We performed a genome-wide association study using our results in the DGRP lines, and found that the resistance and conversion rates were not explained by common alleles of large effect, but instead there were several genetic polymorphisms showing weak association. RNA interference knockdown of several genes containing these polymorphisms confirmed their effect, but the small effect sizes imply that their manipulation would likely yield only modest improvements to the efficacy of gene drives.

Gene drives are selfish genetic elements that are transmitted to progeny at super-Mendelian (>50%) frequencies. Recently developed CRISPR–Cas9-based gene-drive systems are highly efficient in laboratory settings, offering the potential to reduce the prevalence of vector-borne diseases, crop pests and non-native invasive species. However, concerns have been raised regarding the potential unintended impacts of gene-drive systems. This Review summarizes the phenomenal progress in this field, focusing on optimal design features for full-drive elements (drives with linked Cas9 and guide RNA components) that either suppress target mosquito populations or modify them to prevent pathogen transmission, allelic drives for updating genetic elements, mitigating strategies including trans-complementing split-drives and genetic neutralizing elements, and the adaptation of drive technology to other organisms. These scientific advances, combined with ethical and social considerations, will facilitate the transparent and responsible advancement of these technologies towards field implementation.In this Review, Ethan Bier discusses how several impactful technical advancements, particularly involving CRISPR-based methods, are providing a diverse toolkit of gene-drive systems for the control of populations such as insect vectors of disease.

Recent advances in research on gene drives have produced genetic constructs that could theoretically spread a desired gene (payload) into all populations of a species, with a single release in one place. This attribute has advantages, but also comes with risks and ethical concerns. There has been a call for research on gene drive systems that are spatially and/or temporally self‐limiting. Here, we use a population genetics model to compare the expected characteristics of three spatially self‐limiting gene drive systems: one‐locus underdominance, two‐locus underdominance and daisy‐chain drives. We find large differences between these gene drives in the minimum release size required for successfully driving a payload into a population. The daisy‐chain system is the most efficient, requiring the smallest release, followed by the two‐locus underdominance system, and then the one‐locus underdominance system. However, when the target population exchanges migrants with a nontarget population, the gene drives requiring smaller releases suffer from higher risks of unintended spread. For payloads that incur relatively low fitness costs (up to 30%), a simple daisy‐chain drive is practically incapable of remaining localized, even with migration rates as low as 0.5% per generation. The two‐locus underdominance system can achieve localized spread under a broader range of migration rates and of payload fitness costs, while the one‐locus underdominance system largely remains localized. We also find differences in the extent of population alteration and in the permanence of the alteration achieved by the three gene drives. The two‐locus underdominance system does not always spread the payload to fixation, even after successful drive, while the daisy‐chain system can, for a small set of parameter values, achieve a temporally limited spread of the payload. These differences could affect the suitability of each gene drive for specific applications.

CRISPR/Cas9 gene drive (CGD) promises to be a highly adaptable approach for spreading genetically engineered alleles throughout a species, even if those alleles impair reproductive success. CGD has been shown to be effective in laboratory crosses of insects, yet it remains unclear to what extent potential resistance mechanisms will affect the dynamics of this process in large natural populations. Here we develop a comprehensive population genetic framework for modeling CGD dynamics, which incorporates potential resistance mechanisms as well as random genetic drift. Using this framework, we calculate the probability that resistance against CGD evolves from standing genetic variation, de novo mutation of wild-type alleles, or cleavage repair by nonhomologous end joining (NHEJ)—a likely by-product of CGD itself. We show that resistance to standard CGD approaches should evolve almost inevitably in most natural populations, unless repair of CGD-induced cleavage via NHEJ can be effectively suppressed, or resistance costs are on par with those of the driver. The key factor determining the probability that resistance evolves is the overall rate at which resistance alleles arise at the population level by mutation or NHEJ. By contrast, the conversion efficiency of the driver, its fitness cost, and its introduction frequency have only minor impact. Our results shed light on strategies that could facilitate the engineering of drivers with lower resistance potential, and motivate the possibility to embrace resistance as a possible mechanism for controlling a CGD approach. This study highlights the need for careful modeling of the population dynamics of CGD prior to the actual release of a driver construct into the wild.

Population suppression is an effective way for controlling insect pests and disease vectors, which cause significant damage to crop and spread contagious diseases to plants, animals and humans. Gene drive systems provide innovative opportunities for the insect pests population suppression by driving genes that impart fitness costs on populations of pests or disease vectors. Different gene-drive systems have been developed in insects and applied for their population suppression. Here, different categories of gene drives such as meiotic drive (MD), under-dominance (UD), homing endonuclease-based gene drive (HEGD) and especially the CRISPR/Cas9-based gene drive (CCGD) were reviewed, including the history, types, process and mechanisms. Furthermore, the advantages and limitations of applying different gene-drive systems to suppress the insect population were also summarized. This review provides a foundation for developing a specific gene-drive system for insect population suppression.

Understanding the pleiotropic consequences of gene drive systems on host fitness is essential to predict their spread through a host population. Here, we study sex-ratio (SR) X-chromosome drive in the fly Drosophila recens, where SR causes the death of Y-bearing sperm in male carriers. SR males only sire daughters, which all carry SR, thus giving the chromosome a transmission advantage. The prevalence of the SR chromosome appears stable, suggesting pleiotropic costs. It was previously shown that females homozygous for SR are sterile, and here, we test for additional fitness costs of SR.We found that females heterozygous for SR have reduced fecundity and that male SR carriers have reduced fertility in conditions of sperm competition. We then use our fitness estimates to parametrize theoretical models of SR drive and show that the decrease in fecundity and sperm competition performance can account for the observed prevalence of SR in natural populations. In addition, we found that the expected equilibrium frequency of the SR chromosome is particularly sensitive to the degree of multiple mating and performance in sperm competition. Together, our data suggest that the mating system of the organism should be carefully considered during the development of gene drive systems.