Researchers Hit Roadblocks with Gene Drives

Researchers Hit Roadblocks with Gene Drives Evolved resistance and natural variation pose challenges to halting disease spread. In 2015, exciting new research raised the prospect of eliminating harmful insects through genetic manipulation. Studies suggested that the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing technique could be used in gene drives to facilitate the rapid extirpation of populations of disease-carrying insects, such as those carrying the deadly Zika virus. “We hope that gene drives can help control disease vectors, agricultural pests, and invasive species,” says Jackson Champer, of Cornell University. The idea is that genes to reduce an organism's fitness would spread rapidly in a targeted population, leading to a decrease in its numbers and, eventually, its disappearance. Although researchers remain hopeful, ongoing experiments show that evolutionary processes may be a tough hurdle to overcome before gene drives can be used for disease eradication. Initial enthusiasm about the new technology ran high. “We have the technology to destroy all Zika mosquitoes,” a headline in the MIT Technology Review proclaimed in February 2016. The article quoted Anthony James, a leading mosquito researcher from the University of California, Irvine, promising, “We could easily have [gene drives against the Zika virus] within a year.” By September of that year, the Bill and Melinda Gates Foundation had granted $75 million to the Target Malaria project out of Imperial College London, where researchers had engineered a gene drive to suppress laboratory populations of the mosquito Anopheles gambiae, the most important vector of malaria in sub-Saharan Africa. Officially, the foundation projected the technology to be ready for field use in Africa by 2029, but Bill Gates told Forbes that he expected it to be ready within 2 years. For its part, the US Defense Advanced Research Projects Agency (DARPA) invested $100 million in gene-drive research. View largeDownload slide A female Aedis aegypti mosquito, a known vector of many deadly viruses, among them Zika, yellow fever, and malaria. Photograph: Jim Gathany, Centers for Disease Control and Prevention. View largeDownload slide A female Aedis aegypti mosquito, a known vector of many deadly viruses, among them Zika, yellow fever, and malaria. Photograph: Jim Gathany, Centers for Disease Control and Prevention. Since then, researchers have encountered several evolutionary barriers. Experiments in multiple insect species have reported both the emergence of resistance to drives and the prevalence of natural genetic variation that make the CRISPR/Cas9 mechanism ineffective for spreading the genes across populations as intended. Researchers are also conducting theoretical analyses that underscore the difficulty of designing effective gene drives against unwanted species. Box 1. What is CRISPR/Cas9? Clustered regularly interspaced short palindromic repeats (CRISPR) is a family of bacterial DNA sequences. Cas9 is a CRISPR-associated protein, often obtained from the bacterium Streptococcus pyogenes, that cleaves DNA. In bacteria, the spaces between the CRISPR repeats, called spacers, can be filled with snippets of DNA cleaved from viruses that had attacked the bacteria. The CRISPR/Cas9 system provides immunity by using RNA sequences transcribed from these spacers to recognize the virus from which it was derived in a new invasion. The Cas9 protein then destroys the virus by snipping its DNA. CRISPR/Cas9 can be used for both gene editing in individuals and propagating gene drives through populations. Researchers modify genomes by creating a molecular construct consisting of a guide RNA designed to target a particular DNA sequence and genes to be inserted. Cas9 cleaves the targeted DNA in one of the two homologous chromosomes and the construct gets inserted into it. The inserted genes now target the homologous chromosome, which is cut by Cas9 at the corresponding site. This technique enables the rapid editing of somatic cell and germline genomes, potentially including human genes. Gene drives’ early promise CRISPR/Cas9 permits the easy insertion of genes into precise locations in the genome (see box 1). One potent way to use CRISPR/Cas9 technology is in a gene drive, a mechanism to transmit a favorable gene across generations more frequently than allowed by Mendel's laws of inheritance. Such genes can then be used to control or eliminate unwanted species. In gene drives designed for the suppression of a species, for example, the inserted DNA disrupts a gene in order to reduce the average fitness of populations. In other cases, gene drives can be designed specifically to prevent pathogen transmission by a disease vector. When CRISPR/Cas9 is used to insert such genes into the germline, the mechanism enables genes introduced into one chromosome to spread first to the other chromosome—and then with increasing frequency in offspring in subsequent generations. In 2015, Ethan Bier and Valentino Gantz, of the University of California, San Diego, published an article detailing the first use of CRISPR/Cas9 for a gene drive. They targeted the recessive yellow (y) gene on the X chromosome of the fruit fly, Drosophila melanogaster, that affects the body color of the flies. When they crossed females with this construct to normal males, 95–100 percent of the progeny contained the construct, much more than the 50 percent expected from Mendelian inheritance (although this later turned out to be an overestimate). View largeDownload slide Patients waiting at the Out-Patient Department of Apac Hospital in the northern region of Uganda. The majority are mothers of children with malaria who are under 5 years old. Photograph: Toshihiro Horii, Department of Molecular Protozoology, Research Institute for Microbial Diseases, University of Osaka, Osaka, Japan. View largeDownload slide Patients waiting at the Out-Patient Department of Apac Hospital in the northern region of Uganda. The majority are mothers of children with malaria who are under 5 years old. Photograph: Toshihiro Horii, Department of Molecular Protozoology, Research Institute for Microbial Diseases, University of Osaka, Osaka, Japan. Encouraged by this result, Bier began collaborating with Anthony James, who had previously isolated genes that make mosquitoes resistant to the major malarial parasite, Plasmodium falicparum. Their collaboration resulted in a successful deployment of a gene drive in a laboratory population of the mosquito Anopheles stephensi, which is an important vector of malaria in South Asia. Again, the results were promising. The researchers reported a 99.5 percent transmission and transcription of the resistant genes to the next generation. In spite of this result, their paper (published in the Proceedings of the National Academy of Sciences) was cautious, noting that gene drives alone would not eradicate malaria, because natural populations may not mimic laboratory behavior. They underscored the value of “therapeutic drugs, vaccines, and alternate vector-control measures.” Still, they viewed the successful deployment of these gene drives as a major step toward the eventual control of malaria. Even more hope was generated from Imperial College London. A team led by Tony Nolan and Andrea Crisanti engineered a gene drive in laboratory populations of A. gambiae. They identified three different genes that halted egg production in females when disrupted, inserted a CRISPR/Cas9 construct into each of these genes, and confirmed the transmission of the construct to the next generation. For one of these genes, they carried out experiments in laboratory populations in which reduced fertility spread to 70 percent of the population over four generations. Another strategy focused on male mosquitoes, which do not bite humans or spread disease. Moreover, the spread of maleness in a population at the expense of females is a precursor to its eventual extinction. Zach Adelman and his colleagues at Virginia Tech identified a dominant gene that causes maleness in Aedes aegypti, the most important mosquito vector for dengue, chikungunya, yellow fever, and Zika. The team showed how a CRISPR/Cas9 system could be used to insert this gene into the genomes of female mosquitoes to convert them to males. Given the extent to which dengue had spread during the preceding three decades (more so than any other vector-borne disease, according to the World Health Organization) and the fear of Zika, this strategy of disease control had many proponents. Still, Adelman tried to dampen excessive enthusiasm. During a March 2016 webinar program on gene drives and Zika, organized by the American Institute of Biological Sciences, he stressed that the release of gene drives was not around the corner. Nevertheless, with such promising research, most skeptics about the use of gene drives focused not on potential technical limitations but on the need to regulate the technology. Gene drives could rapidly drive an entire species of insects or other short-lived organisms to extinction. Under what circumstances was this justified? As Crisanti, at Imperial College, had observed in 2016 to MIT Technological Review, “The gene drive is controversial for the potential to wipe out a species. So there should be a clear benefit.” Suppose a gene drive was designed for a species that had become invasive in a particular region. What if the gene drive then spread uncontrollably to the species’ native range and threatened the population's survival? Could a gene drive be halted? View largeDownload slide This drawing depicts a Zika virus capsid, the protein shell that encloses the genetic material of the virus. Zika, along with malaria, dengue, chikungunya, and yellow fever, are insect-borne diseases that affect millions of people around the globe. Scientists are hoping to soon use gene-drive technology to eradicate such diseases. Illustration: Manuel Almagro Rivas. View largeDownload slide This drawing depicts a Zika virus capsid, the protein shell that encloses the genetic material of the virus. Zika, along with malaria, dengue, chikungunya, and yellow fever, are insect-borne diseases that affect millions of people around the globe. Scientists are hoping to soon use gene-drive technology to eradicate such diseases. Illustration: Manuel Almagro Rivas. In response to such concerns, Kevin Esvelt, then at Harvard University, and others began theoretical explorations of strategies to reverse gene drives. Their experiments with yeast, published in Nature Biotechnology in 2015, showed that a new gene drive could be used to counteract the inadvertent spread of an earlier one. Meanwhile, in a 2016 National Academy of Sciences report, Gene Drives on the Horizon, the authors concluded that there was “insufficient evidence at this time to support the release of gene-drive modified organisms into the environment.” They called for further research, “highly controlled field trials,” and consultations with the wider public until a regulatory framework could be created. Evolution strikes back By 2017, the challenges for gene drives were already emerging. An array of theoretical analyses and experimental results highlighted the technical limitations of CRISPR/Cas9 gene drives in insects. In a perspective published in July in PLOS Genetics, Jim Bull, of the University of Texas, and Harmit Malik, of the Fred Hutchinson Cancer Research Center in Seattle, identified three features, all relevant to the evolutionary dynamics of populations, that act to limit the potential success of gene drives. First, all natural populations have genetic variation that can both prevent recognition of the intended target by the RNA guide of the CRISPR/Cas9 construct and also foil cleavage by Cas9. Second, evolved resistance can arise through various mutational mechanisms that would be selected for if the drive reduces fitness, as was the case, for instance, in the Imperial College experiments that disrupted fertility in female mosquitoes. And third, breeding patterns other than random mating can also slow the spread of a gene drive in a population. View largeDownload slide Researchers say vector-control measures are important to continue, as gene-drive studies are underway. An educational banner hung up in residential neighborhoods to teach residents how to prevent dengue in Singapore (l). Photograph: ProjectManhattan. In Votuporanga, Brazil, a residential neighborhood is sprayed to kill mosquitoes that carry the dengue virus (r). According to the World Health Organization, dengue has spread more than any other insect-borne disease over the last 30 years. Photograph: Prefeitura de Votuporanga. View largeDownload slide Researchers say vector-control measures are important to continue, as gene-drive studies are underway. An educational banner hung up in residential neighborhoods to teach residents how to prevent dengue in Singapore (l). Photograph: ProjectManhattan. In Votuporanga, Brazil, a residential neighborhood is sprayed to kill mosquitoes that carry the dengue virus (r). According to the World Health Organization, dengue has spread more than any other insect-borne disease over the last 30 years. Photograph: Prefeitura de Votuporanga. Box 2. Further reading. Bull JJ. 2017. Lethal gene drive selects inbreeding. Evolution, Medicine, and Public Health 1: 1–16. Champer J, Reeves R, Oh SY, Liu C, Liu J, Clark, AG, Messer PW. 2017. Novel CRISPR/Cas9 gene drive constructs reveal insights into mechanisms of resistance allele formation and drive efficiency in genetically diverse populations. PLOS Genetics 13 (art. e1006796). Hammond AM, et al. 2017. The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito. PLOS Genetics 13 (art. e1007039). Min J, Smidler AL, Najjar D, Esvelt KM. 2018. Harnessing gene drive. Journal of Responsible Innovation 5: S40–S65. Zentner GE, Wade MJ. 2017. The promise and peril of CRISPR gene drives. BioEssays 39 (art. 1,700,109). Robert L. Unckless, of the University of Kansas, along with Andrew G. Clark and Philipp W. Messer, both from Cornell University, built a theoretical model of the effects of natural variation and emerging resistance from two types of mutation: de novo mutations of wild-type alleles and DNA repair that did not successfully copy the gene-drive construct (the set of genes or piece of DNA that gets inserted into the genome) and thereby created a resistance allele (genes that stopped the spread of the drive). In a paper published in Genetics in February 2017, they reported that the resistance-allele formation rate is the dominating factor determining the success of a gene drive. A team at Cornell including Clark and Messer has also been experimentally studying the emergence and evolution of resistance to gene drives in the old workhorse of genetics, D. melanogaster. In a paper published in PLOS Genetics last July, they showed that resistance to gene drives arose easily even in the absence of selection and within one generation. Resistance can arise both prior to fertilization in the germline and after fertilization (in the early embryo). Their results suggest that resistance was rising as a result of Cas9 action. After cleavage by Cas9, instead of being converted to genes to propagate the drive, the DNA was being “misrepaired” into resistance alleles. Champer, the lead author of this study, says that in retrospect, the rapid emergence of resistance was “no surprise” and that some of the early enthusiasm about the immediate prospects of gene-drive use to combat pests was not justified. “All of the early gene-drive insect studies found resistance alleles,” he says. “Resistance alleles were just not focused on in these studies.” Later in 2017, the Imperial College team of Nolan and Crisanti and their collaborators also reported the emergence of resistance alleles. They had been optimistic in 2015 when their experiments with A. gambiae had shown the rapid spread of the gene drive over four generations. However, when the team allowed the experiment to run for 25 generations, the spread halted after six generations, and the frequency of the drive allele rapidly decreased afterwards. In a paper published in PLOS Genetics in October, they interpreted their results as indicating selection for resistance to the gene drive. Bull, Champer, and others note that the implication of this result is clear: It will be much more difficult to use gene drives to control malaria in sub-Saharan Africa than what was believed in 2015. Meanwhile, a team at Indiana University Bloomington led by Michael Wade and Gabriel Zentner analyzed the potential effect of natural genetic variation on CRISPR/Cas9 gene drives in the flour beetle Tribolium castaneum, an insect pest that is estimated to consume a fifth of the grain produced in the world each year. Their results, published in Science Advances in 2017, show how small levels of genetic variation, especially when accompanied by some inbreeding, can prevent gene drives from decreasing population sizes. They analyzed variability at three drive-relevant genes in four populations of Tribolium (from India, Peru, Spain, and Indiana). Using available sequence data, they looked at stretches of DNA in the targeted gene that are essential for cleavage by Cas9. They found that most populations harbor natural variants at such Cas9-relevant sites in sufficient frequencies to prevent gene drives from propagating successfully. Modeling showed that the strength of this effect is increased by inbreeding, which is common among insects. View largeDownload slide Researchers at Cornell University recently found that resistance to gene drives arose easily in the fruit fly, Drosophila melanogaster. Photograph: Paco Romero-Ferrero. View largeDownload slide Researchers at Cornell University recently found that resistance to gene drives arose easily in the fruit fly, Drosophila melanogaster. Photograph: Paco Romero-Ferrero. Theoretical work by Bull supports these results. His models analyzed how breeding patterns may change in plant populations in which a lethal gene is spreading through a gene drive. The results predicted the spread of inbreeding in these populations, which would slow down drives and prevent the population from being extirpated. In early 2017, Bull was among the first to emphasize the evolutionary hurdles that gene drives must cross if they are to be successfully used in campaigns against vector-borne disease. An uncertain future Can gene drives be made to work as a tool for controlling troublesome insect populations? There is widespread disagreement among biologists. However, there is some consensus that the plausibility of success with gene drives for this purpose depends on the goal. “For controlling invasive species, the goal would be to wipe them out specifically in areas [where] they are invasive,” says Champer. “For agricultural pests, the goal would be to eliminate them from crop areas, though species elimination may be considered if they don’t play an important role in natural ecosystems. For major disease vectors, we could accomplish our goal by either wiping them out or modifying them so that they can no longer spread disease to humans and other animals.” Given the different goals, biologists have begun to distinguish sharply between gene drives to suppress populations and those that carry and insert a payload of genes into the genome. In the case of disease vectors, the payload is typically intended to be genetically encoded resistance to pathogen transmission into the insect. Bull is among those who find the suppression strategy to be less promising. “Trying to wipe out a species will select virtually any resistance mechanism that blocks it,” he says, because it would increase fitness. He finds the payload strategy more promising. Champer agrees: “Population modification would be easier to implement and with fewer ecosystem effects. However, pathogens could evolve resistance to a payload gene, so… if no payload is available, population suppression may be the only viable strategy.” For both Bull and Champer, the critical task is to design a gene drive that does not cause a fitness loss. Such a drive, Bull observes, would “face essentially no selection for resistance.” If they are right, efforts such as those of the University of California, Irvine, team, directed at making mosquitoes resistant to the malarial parasite, are more likely to succeed than drives that are similar to the one created at Imperial College, which attempt to suppress populations. Very few researchers say they are surprised that resistance to gene drives evolved rapidly. “Everyone expected it,” Adelman, now at Texas A&M University, says. Champer adds, “The early predictions of gene-drive use were over-optimistic. The resistance allele problem is even greater than we expected. We are several years before field tests.” Esvelt, now at the Massachusetts Institute of Technology, continues to be optimistic. But unlike Champer and Bull, he argues that “the problem of resistant alleles has always been a bit of a red herring.” Drawing on results from his modeling work from 2014, he suggests that “using CRISPR to target multiple sequences within genes important for fitness should completely avoid this problem.” This strategy, known as multiplexing, uses multiple guide RNAs within a gene drive that all target the same gene. If resistance evolves by preventing recognition by one of these RNAs, one of the others would step in to ensure that Cas9 reaches and cleaves the right target. View largeDownload slide In Cuscatlan, El Salvador, Lieutenant Commander Ebony Ferguson, a clinical nurse specialist assigned to Fort Belvoir (right), helps an Operation Blessing Medical Brigade doctor check a patient for symptoms of Zika at a temporary treatment site. The Zika epidemic helped spur intense interest in using gene drives to control disease-carrying mosquitoes. Photograph: Petty Officer 2nd Class Torrey Lee. View largeDownload slide In Cuscatlan, El Salvador, Lieutenant Commander Ebony Ferguson, a clinical nurse specialist assigned to Fort Belvoir (right), helps an Operation Blessing Medical Brigade doctor check a patient for symptoms of Zika at a temporary treatment site. The Zika epidemic helped spur intense interest in using gene drives to control disease-carrying mosquitoes. Photograph: Petty Officer 2nd Class Torrey Lee. So far, successful experimental efforts at multiplexing have been limited to two guide RNAs. In work that is under review, the Cornell group has confirmed the theoretical prediction that using two guide RNAs reduces the formation of resistance alleles (in fruit flies). Champer says that “though the improvement from a second guide RNA was not quite as much as the idealized theoretical prediction, it still shows promise for future developments.” Recent results reported by Bruce A. Hay and collaborators from the California Institute of Technology are less promising. (These are as yet unpublished but available from the bioRxiv Web depository.) They used four guide RNAs in experiments with Drosophila. Although resistance-allele formation was reduced, other problems emerged that prevented the successful incorporation of the gene drive into the germline. Even if multiplexing solves the problem of resistance alleles that arise through mutation, the problems posed by natural genetic variation will still need to be solved. There may be a silver lining to the challenges of gene drives, one that Wade emphasizes: They make it unlikely that the use of gene drives to control targeted populations will lead to the unintentional spread of modified genes across natural populations. However, even on this issue there is disagreement. Esvelt is convinced that the potential of gene drives to invade unintended populations is more significant than previously thought. Along with Martin Novak, at Harvard, he is part of a collaboration that has developed theoretical models that show that even the entry of a few individuals with gene-drive constructs can lead to a complete invasion of a population. According to Esvelt, “Our recent model… suggests that very few organisms carrying a self-propagating CRISPR-based gene drive are required to invade a new population, and that populations connected by very low gene flow rates will all be invaded by moderately effective drive systems of this type.” Unlike other researchers in the field, this team argues that gene drives remain inherently dangerous because their unplanned spread may generate a social backlash that can, for instance, harm the future use of gene drives against disease. At the very least, they argue that drives should not be field-tested in regions in which there are other neighboring populations of the species unless their potential spread to these populations, possibly across international borders, is socially and politically acceptable. Research into CRISPR/Cas9-based gene drives continues across the world as scientists seek to overcome these challenges from evolution. Nolan argues that “for gene drives, it is easy to foresee the most likely forms of resistance, and the good thing is we have many potential strategies in the locker to tackle this—such as targeting multiple sites, hitting target sites that cannot easily mutate to resistant forms.” Though it is still too early to know which type of gene drive will succeed, many researchers remain optimistic that in the future, gene drives will play an important role in controlling insect vectors of disease. © The Author(s) 2018. Published by Oxford University Press on behalf of the American Institute of Biological Sciences. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BioScience Oxford University Press

Researchers Hit Roadblocks with Gene Drives

BioScience , Volume Advance Article (7) – May 22, 2018

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Abstract

Evolved resistance and natural variation pose challenges to halting disease spread. In 2015, exciting new research raised the prospect of eliminating harmful insects through genetic manipulation. Studies suggested that the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing technique could be used in gene drives to facilitate the rapid extirpation of populations of disease-carrying insects, such as those carrying the deadly Zika virus. “We hope that gene drives can help control disease vectors, agricultural pests, and invasive species,” says Jackson Champer, of Cornell University. The idea is that genes to reduce an organism's fitness would spread rapidly in a targeted population, leading to a decrease in its numbers and, eventually, its disappearance. Although researchers remain hopeful, ongoing experiments show that evolutionary processes may be a tough hurdle to overcome before gene drives can be used for disease eradication. Initial enthusiasm about the new technology ran high. “We have the technology to destroy all Zika mosquitoes,” a headline in the MIT Technology Review proclaimed in February 2016. The article quoted Anthony James, a leading mosquito researcher from the University of California, Irvine, promising, “We could easily have [gene drives against the Zika virus] within a year.” By September of that year, the Bill and Melinda Gates Foundation had granted $75 million to the Target Malaria project out of Imperial College London, where researchers had engineered a gene drive to suppress laboratory populations of the mosquito Anopheles gambiae, the most important vector of malaria in sub-Saharan Africa. Officially, the foundation projected the technology to be ready for field use in Africa by 2029, but Bill Gates told Forbes that he expected it to be ready within 2 years. For its part, the US Defense Advanced Research Projects Agency (DARPA) invested $100 million in gene-drive research. View largeDownload slide A female Aedis aegypti mosquito, a known vector of many deadly viruses, among them Zika, yellow fever, and malaria. Photograph: Jim Gathany, Centers for Disease Control and Prevention. View largeDownload slide A female Aedis aegypti mosquito, a known vector of many deadly viruses, among them Zika, yellow fever, and malaria. Photograph: Jim Gathany, Centers for Disease Control and Prevention. Since then, researchers have encountered several evolutionary barriers. Experiments in multiple insect species have reported both the emergence of resistance to drives and the prevalence of natural genetic variation that make the CRISPR/Cas9 mechanism ineffective for spreading the genes across populations as intended. Researchers are also conducting theoretical analyses that underscore the difficulty of designing effective gene drives against unwanted species. Box 1. What is CRISPR/Cas9? Clustered regularly interspaced short palindromic repeats (CRISPR) is a family of bacterial DNA sequences. Cas9 is a CRISPR-associated protein, often obtained from the bacterium Streptococcus pyogenes, that cleaves DNA. In bacteria, the spaces between the CRISPR repeats, called spacers, can be filled with snippets of DNA cleaved from viruses that had attacked the bacteria. The CRISPR/Cas9 system provides immunity by using RNA sequences transcribed from these spacers to recognize the virus from which it was derived in a new invasion. The Cas9 protein then destroys the virus by snipping its DNA. CRISPR/Cas9 can be used for both gene editing in individuals and propagating gene drives through populations. Researchers modify genomes by creating a molecular construct consisting of a guide RNA designed to target a particular DNA sequence and genes to be inserted. Cas9 cleaves the targeted DNA in one of the two homologous chromosomes and the construct gets inserted into it. The inserted genes now target the homologous chromosome, which is cut by Cas9 at the corresponding site. This technique enables the rapid editing of somatic cell and germline genomes, potentially including human genes. Gene drives’ early promise CRISPR/Cas9 permits the easy insertion of genes into precise locations in the genome (see box 1). One potent way to use CRISPR/Cas9 technology is in a gene drive, a mechanism to transmit a favorable gene across generations more frequently than allowed by Mendel's laws of inheritance. Such genes can then be used to control or eliminate unwanted species. In gene drives designed for the suppression of a species, for example, the inserted DNA disrupts a gene in order to reduce the average fitness of populations. In other cases, gene drives can be designed specifically to prevent pathogen transmission by a disease vector. When CRISPR/Cas9 is used to insert such genes into the germline, the mechanism enables genes introduced into one chromosome to spread first to the other chromosome—and then with increasing frequency in offspring in subsequent generations. In 2015, Ethan Bier and Valentino Gantz, of the University of California, San Diego, published an article detailing the first use of CRISPR/Cas9 for a gene drive. They targeted the recessive yellow (y) gene on the X chromosome of the fruit fly, Drosophila melanogaster, that affects the body color of the flies. When they crossed females with this construct to normal males, 95–100 percent of the progeny contained the construct, much more than the 50 percent expected from Mendelian inheritance (although this later turned out to be an overestimate). View largeDownload slide Patients waiting at the Out-Patient Department of Apac Hospital in the northern region of Uganda. The majority are mothers of children with malaria who are under 5 years old. Photograph: Toshihiro Horii, Department of Molecular Protozoology, Research Institute for Microbial Diseases, University of Osaka, Osaka, Japan. View largeDownload slide Patients waiting at the Out-Patient Department of Apac Hospital in the northern region of Uganda. The majority are mothers of children with malaria who are under 5 years old. Photograph: Toshihiro Horii, Department of Molecular Protozoology, Research Institute for Microbial Diseases, University of Osaka, Osaka, Japan. Encouraged by this result, Bier began collaborating with Anthony James, who had previously isolated genes that make mosquitoes resistant to the major malarial parasite, Plasmodium falicparum. Their collaboration resulted in a successful deployment of a gene drive in a laboratory population of the mosquito Anopheles stephensi, which is an important vector of malaria in South Asia. Again, the results were promising. The researchers reported a 99.5 percent transmission and transcription of the resistant genes to the next generation. In spite of this result, their paper (published in the Proceedings of the National Academy of Sciences) was cautious, noting that gene drives alone would not eradicate malaria, because natural populations may not mimic laboratory behavior. They underscored the value of “therapeutic drugs, vaccines, and alternate vector-control measures.” Still, they viewed the successful deployment of these gene drives as a major step toward the eventual control of malaria. Even more hope was generated from Imperial College London. A team led by Tony Nolan and Andrea Crisanti engineered a gene drive in laboratory populations of A. gambiae. They identified three different genes that halted egg production in females when disrupted, inserted a CRISPR/Cas9 construct into each of these genes, and confirmed the transmission of the construct to the next generation. For one of these genes, they carried out experiments in laboratory populations in which reduced fertility spread to 70 percent of the population over four generations. Another strategy focused on male mosquitoes, which do not bite humans or spread disease. Moreover, the spread of maleness in a population at the expense of females is a precursor to its eventual extinction. Zach Adelman and his colleagues at Virginia Tech identified a dominant gene that causes maleness in Aedes aegypti, the most important mosquito vector for dengue, chikungunya, yellow fever, and Zika. The team showed how a CRISPR/Cas9 system could be used to insert this gene into the genomes of female mosquitoes to convert them to males. Given the extent to which dengue had spread during the preceding three decades (more so than any other vector-borne disease, according to the World Health Organization) and the fear of Zika, this strategy of disease control had many proponents. Still, Adelman tried to dampen excessive enthusiasm. During a March 2016 webinar program on gene drives and Zika, organized by the American Institute of Biological Sciences, he stressed that the release of gene drives was not around the corner. Nevertheless, with such promising research, most skeptics about the use of gene drives focused not on potential technical limitations but on the need to regulate the technology. Gene drives could rapidly drive an entire species of insects or other short-lived organisms to extinction. Under what circumstances was this justified? As Crisanti, at Imperial College, had observed in 2016 to MIT Technological Review, “The gene drive is controversial for the potential to wipe out a species. So there should be a clear benefit.” Suppose a gene drive was designed for a species that had become invasive in a particular region. What if the gene drive then spread uncontrollably to the species’ native range and threatened the population's survival? Could a gene drive be halted? View largeDownload slide This drawing depicts a Zika virus capsid, the protein shell that encloses the genetic material of the virus. Zika, along with malaria, dengue, chikungunya, and yellow fever, are insect-borne diseases that affect millions of people around the globe. Scientists are hoping to soon use gene-drive technology to eradicate such diseases. Illustration: Manuel Almagro Rivas. View largeDownload slide This drawing depicts a Zika virus capsid, the protein shell that encloses the genetic material of the virus. Zika, along with malaria, dengue, chikungunya, and yellow fever, are insect-borne diseases that affect millions of people around the globe. Scientists are hoping to soon use gene-drive technology to eradicate such diseases. Illustration: Manuel Almagro Rivas. In response to such concerns, Kevin Esvelt, then at Harvard University, and others began theoretical explorations of strategies to reverse gene drives. Their experiments with yeast, published in Nature Biotechnology in 2015, showed that a new gene drive could be used to counteract the inadvertent spread of an earlier one. Meanwhile, in a 2016 National Academy of Sciences report, Gene Drives on the Horizon, the authors concluded that there was “insufficient evidence at this time to support the release of gene-drive modified organisms into the environment.” They called for further research, “highly controlled field trials,” and consultations with the wider public until a regulatory framework could be created. Evolution strikes back By 2017, the challenges for gene drives were already emerging. An array of theoretical analyses and experimental results highlighted the technical limitations of CRISPR/Cas9 gene drives in insects. In a perspective published in July in PLOS Genetics, Jim Bull, of the University of Texas, and Harmit Malik, of the Fred Hutchinson Cancer Research Center in Seattle, identified three features, all relevant to the evolutionary dynamics of populations, that act to limit the potential success of gene drives. First, all natural populations have genetic variation that can both prevent recognition of the intended target by the RNA guide of the CRISPR/Cas9 construct and also foil cleavage by Cas9. Second, evolved resistance can arise through various mutational mechanisms that would be selected for if the drive reduces fitness, as was the case, for instance, in the Imperial College experiments that disrupted fertility in female mosquitoes. And third, breeding patterns other than random mating can also slow the spread of a gene drive in a population. View largeDownload slide Researchers say vector-control measures are important to continue, as gene-drive studies are underway. An educational banner hung up in residential neighborhoods to teach residents how to prevent dengue in Singapore (l). Photograph: ProjectManhattan. In Votuporanga, Brazil, a residential neighborhood is sprayed to kill mosquitoes that carry the dengue virus (r). According to the World Health Organization, dengue has spread more than any other insect-borne disease over the last 30 years. Photograph: Prefeitura de Votuporanga. View largeDownload slide Researchers say vector-control measures are important to continue, as gene-drive studies are underway. An educational banner hung up in residential neighborhoods to teach residents how to prevent dengue in Singapore (l). Photograph: ProjectManhattan. In Votuporanga, Brazil, a residential neighborhood is sprayed to kill mosquitoes that carry the dengue virus (r). According to the World Health Organization, dengue has spread more than any other insect-borne disease over the last 30 years. Photograph: Prefeitura de Votuporanga. Box 2. Further reading. Bull JJ. 2017. Lethal gene drive selects inbreeding. Evolution, Medicine, and Public Health 1: 1–16. Champer J, Reeves R, Oh SY, Liu C, Liu J, Clark, AG, Messer PW. 2017. Novel CRISPR/Cas9 gene drive constructs reveal insights into mechanisms of resistance allele formation and drive efficiency in genetically diverse populations. PLOS Genetics 13 (art. e1006796). Hammond AM, et al. 2017. The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito. PLOS Genetics 13 (art. e1007039). Min J, Smidler AL, Najjar D, Esvelt KM. 2018. Harnessing gene drive. Journal of Responsible Innovation 5: S40–S65. Zentner GE, Wade MJ. 2017. The promise and peril of CRISPR gene drives. BioEssays 39 (art. 1,700,109). Robert L. Unckless, of the University of Kansas, along with Andrew G. Clark and Philipp W. Messer, both from Cornell University, built a theoretical model of the effects of natural variation and emerging resistance from two types of mutation: de novo mutations of wild-type alleles and DNA repair that did not successfully copy the gene-drive construct (the set of genes or piece of DNA that gets inserted into the genome) and thereby created a resistance allele (genes that stopped the spread of the drive). In a paper published in Genetics in February 2017, they reported that the resistance-allele formation rate is the dominating factor determining the success of a gene drive. A team at Cornell including Clark and Messer has also been experimentally studying the emergence and evolution of resistance to gene drives in the old workhorse of genetics, D. melanogaster. In a paper published in PLOS Genetics last July, they showed that resistance to gene drives arose easily even in the absence of selection and within one generation. Resistance can arise both prior to fertilization in the germline and after fertilization (in the early embryo). Their results suggest that resistance was rising as a result of Cas9 action. After cleavage by Cas9, instead of being converted to genes to propagate the drive, the DNA was being “misrepaired” into resistance alleles. Champer, the lead author of this study, says that in retrospect, the rapid emergence of resistance was “no surprise” and that some of the early enthusiasm about the immediate prospects of gene-drive use to combat pests was not justified. “All of the early gene-drive insect studies found resistance alleles,” he says. “Resistance alleles were just not focused on in these studies.” Later in 2017, the Imperial College team of Nolan and Crisanti and their collaborators also reported the emergence of resistance alleles. They had been optimistic in 2015 when their experiments with A. gambiae had shown the rapid spread of the gene drive over four generations. However, when the team allowed the experiment to run for 25 generations, the spread halted after six generations, and the frequency of the drive allele rapidly decreased afterwards. In a paper published in PLOS Genetics in October, they interpreted their results as indicating selection for resistance to the gene drive. Bull, Champer, and others note that the implication of this result is clear: It will be much more difficult to use gene drives to control malaria in sub-Saharan Africa than what was believed in 2015. Meanwhile, a team at Indiana University Bloomington led by Michael Wade and Gabriel Zentner analyzed the potential effect of natural genetic variation on CRISPR/Cas9 gene drives in the flour beetle Tribolium castaneum, an insect pest that is estimated to consume a fifth of the grain produced in the world each year. Their results, published in Science Advances in 2017, show how small levels of genetic variation, especially when accompanied by some inbreeding, can prevent gene drives from decreasing population sizes. They analyzed variability at three drive-relevant genes in four populations of Tribolium (from India, Peru, Spain, and Indiana). Using available sequence data, they looked at stretches of DNA in the targeted gene that are essential for cleavage by Cas9. They found that most populations harbor natural variants at such Cas9-relevant sites in sufficient frequencies to prevent gene drives from propagating successfully. Modeling showed that the strength of this effect is increased by inbreeding, which is common among insects. View largeDownload slide Researchers at Cornell University recently found that resistance to gene drives arose easily in the fruit fly, Drosophila melanogaster. Photograph: Paco Romero-Ferrero. View largeDownload slide Researchers at Cornell University recently found that resistance to gene drives arose easily in the fruit fly, Drosophila melanogaster. Photograph: Paco Romero-Ferrero. Theoretical work by Bull supports these results. His models analyzed how breeding patterns may change in plant populations in which a lethal gene is spreading through a gene drive. The results predicted the spread of inbreeding in these populations, which would slow down drives and prevent the population from being extirpated. In early 2017, Bull was among the first to emphasize the evolutionary hurdles that gene drives must cross if they are to be successfully used in campaigns against vector-borne disease. An uncertain future Can gene drives be made to work as a tool for controlling troublesome insect populations? There is widespread disagreement among biologists. However, there is some consensus that the plausibility of success with gene drives for this purpose depends on the goal. “For controlling invasive species, the goal would be to wipe them out specifically in areas [where] they are invasive,” says Champer. “For agricultural pests, the goal would be to eliminate them from crop areas, though species elimination may be considered if they don’t play an important role in natural ecosystems. For major disease vectors, we could accomplish our goal by either wiping them out or modifying them so that they can no longer spread disease to humans and other animals.” Given the different goals, biologists have begun to distinguish sharply between gene drives to suppress populations and those that carry and insert a payload of genes into the genome. In the case of disease vectors, the payload is typically intended to be genetically encoded resistance to pathogen transmission into the insect. Bull is among those who find the suppression strategy to be less promising. “Trying to wipe out a species will select virtually any resistance mechanism that blocks it,” he says, because it would increase fitness. He finds the payload strategy more promising. Champer agrees: “Population modification would be easier to implement and with fewer ecosystem effects. However, pathogens could evolve resistance to a payload gene, so… if no payload is available, population suppression may be the only viable strategy.” For both Bull and Champer, the critical task is to design a gene drive that does not cause a fitness loss. Such a drive, Bull observes, would “face essentially no selection for resistance.” If they are right, efforts such as those of the University of California, Irvine, team, directed at making mosquitoes resistant to the malarial parasite, are more likely to succeed than drives that are similar to the one created at Imperial College, which attempt to suppress populations. Very few researchers say they are surprised that resistance to gene drives evolved rapidly. “Everyone expected it,” Adelman, now at Texas A&M University, says. Champer adds, “The early predictions of gene-drive use were over-optimistic. The resistance allele problem is even greater than we expected. We are several years before field tests.” Esvelt, now at the Massachusetts Institute of Technology, continues to be optimistic. But unlike Champer and Bull, he argues that “the problem of resistant alleles has always been a bit of a red herring.” Drawing on results from his modeling work from 2014, he suggests that “using CRISPR to target multiple sequences within genes important for fitness should completely avoid this problem.” This strategy, known as multiplexing, uses multiple guide RNAs within a gene drive that all target the same gene. If resistance evolves by preventing recognition by one of these RNAs, one of the others would step in to ensure that Cas9 reaches and cleaves the right target. View largeDownload slide In Cuscatlan, El Salvador, Lieutenant Commander Ebony Ferguson, a clinical nurse specialist assigned to Fort Belvoir (right), helps an Operation Blessing Medical Brigade doctor check a patient for symptoms of Zika at a temporary treatment site. The Zika epidemic helped spur intense interest in using gene drives to control disease-carrying mosquitoes. Photograph: Petty Officer 2nd Class Torrey Lee. View largeDownload slide In Cuscatlan, El Salvador, Lieutenant Commander Ebony Ferguson, a clinical nurse specialist assigned to Fort Belvoir (right), helps an Operation Blessing Medical Brigade doctor check a patient for symptoms of Zika at a temporary treatment site. The Zika epidemic helped spur intense interest in using gene drives to control disease-carrying mosquitoes. Photograph: Petty Officer 2nd Class Torrey Lee. So far, successful experimental efforts at multiplexing have been limited to two guide RNAs. In work that is under review, the Cornell group has confirmed the theoretical prediction that using two guide RNAs reduces the formation of resistance alleles (in fruit flies). Champer says that “though the improvement from a second guide RNA was not quite as much as the idealized theoretical prediction, it still shows promise for future developments.” Recent results reported by Bruce A. Hay and collaborators from the California Institute of Technology are less promising. (These are as yet unpublished but available from the bioRxiv Web depository.) They used four guide RNAs in experiments with Drosophila. Although resistance-allele formation was reduced, other problems emerged that prevented the successful incorporation of the gene drive into the germline. Even if multiplexing solves the problem of resistance alleles that arise through mutation, the problems posed by natural genetic variation will still need to be solved. There may be a silver lining to the challenges of gene drives, one that Wade emphasizes: They make it unlikely that the use of gene drives to control targeted populations will lead to the unintentional spread of modified genes across natural populations. However, even on this issue there is disagreement. Esvelt is convinced that the potential of gene drives to invade unintended populations is more significant than previously thought. Along with Martin Novak, at Harvard, he is part of a collaboration that has developed theoretical models that show that even the entry of a few individuals with gene-drive constructs can lead to a complete invasion of a population. According to Esvelt, “Our recent model… suggests that very few organisms carrying a self-propagating CRISPR-based gene drive are required to invade a new population, and that populations connected by very low gene flow rates will all be invaded by moderately effective drive systems of this type.” Unlike other researchers in the field, this team argues that gene drives remain inherently dangerous because their unplanned spread may generate a social backlash that can, for instance, harm the future use of gene drives against disease. At the very least, they argue that drives should not be field-tested in regions in which there are other neighboring populations of the species unless their potential spread to these populations, possibly across international borders, is socially and politically acceptable. Research into CRISPR/Cas9-based gene drives continues across the world as scientists seek to overcome these challenges from evolution. Nolan argues that “for gene drives, it is easy to foresee the most likely forms of resistance, and the good thing is we have many potential strategies in the locker to tackle this—such as targeting multiple sites, hitting target sites that cannot easily mutate to resistant forms.” Though it is still too early to know which type of gene drive will succeed, many researchers remain optimistic that in the future, gene drives will play an important role in controlling insect vectors of disease. © The Author(s) 2018. Published by Oxford University Press on behalf of the American Institute of Biological Sciences. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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BioScienceOxford University Press

Published: May 22, 2018

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