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Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations

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Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations FEATURE ARTICLE elifesciences.org EMERGING TECHNOLOGY Concerning RNA-guided gene drives for the alteration of wild populations Abstract Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology. DOI: 10.7554/eLife.03401.001 KEVIN M ESVELT*, ANDREA L SMIDLER, FLAMINIA CATTERUCCIA* AND GEORGE M CHURCH* Introduction agricultural and environmental damage wrought by invasive species. Despite numerous advances, the field of molec- Scientists have long known of naturally occur- ular biology has often struggled to address key ring selfish genetic elements that can increase biological problems affecting public health and the odds that they will be inherited. This advan- the environment. Until recently, editing the tage allows them to spread through popula- genomes of even model organisms has been dif- tions even if they reduce the fitness of individual *For correspondence: ficult. Moreover, altered traits typically reduce ev- organisms. Many researchers have suggested [email protected] olutionary fitness and are consequently eliminated that these elements might serve as the basis for (KME); [email protected] by natural selection. This restriction has profoundly (FC); [email protected] (GMC) limited our ability to alter ecosystems through ‘gene drives’ capable of spreading engineered molecular biology. traits through wild populations (Craig et al., 1960; Reviewing editor: Diethard If we could develop a general method of Wood et al., 1977; Sinkins and Gould, 2006; Tautz, Max Planck Institute for ensuring that engineered traits would instead be Burt and Trivers, 2009; Alphey, 2014). Austin Evolutionary Biology, Germany favored by natural selection, then those traits Burt was the first to propose gene drives based Copyright Esvelt et al. This article could spread to most members of wild populations on site-specific ‘homing’ endonuclease genes is distributed under the terms of the over many generations. This capability would over a decade ago (Burt, 2003). These genes Creative Commons Attribution License, allow us to address several major world problems, bias inheritance by cutting the homologous chro- which permits unrestricted use and redistribution provided that the original including the spread of insect-borne diseases, the mosome, inducing the cell to copy them when it author and source are credited. rise of pesticide and herbicide resistance, and the repairs the break. Several efforts have focused Esvelt et al. eLife 2014;3:e03401. DOI: 10.7554/eLife.03401 1 of 21 Feature article Emerging technology | Concerning RNA-guided gene drives for the alteration of wild populations on the possibility of using gene drives targeting his proposal. Indeed, the well-recognized poten- mosquitoes to block malaria transmission (Scott tial for gene drives to combat vector-borne dis- et al., 2002; Windbichler et al., 2007, 2008, eases such as malaria and dengue virtually ensures 2011; Li et al., 2013a; Galizi et al., 2014). that this strategy will eventually be attempted in However, development has been hindered by the mosquitoes. difficulty of engineering homing endonucleases While considerable scholarship has been to cut new target sequences (Chan et al., 2013a; devoted to the question of how gene drives Thyme et al., 2013; Takeuchi et al., 2014). might be safely utilized in mosquitoes (Scott Attempts to build gene drives with more easily et al., 2002; Touré et al., 2004; Benedict et al., retargeted zinc-finger nucleases and TALENs suf- 2008; Marshall, 2009; UNEP, 2010; Reeves fered from instability due to the repetitive nature et al., 2012; David et al., 2013; Alphey, 2014), of the genes encoding them (Simoni et al., 2014). few if any studies have examined the potential The recent discovery and development of the ecological effects of gene drives in other species. RNA-guided Cas9 nuclease has dramatically After all, constructing a drive to spread a partic- enhanced our ability to engineer the genomes of ular genomic alteration in a given species was diverse species. Originally isolated from ‘CRISPR’ simply not feasible with earlier genome editing acquired immune systems in bacteria, Cas9 is a methods. Disconcertingly, several published gene non-repetitive enzyme that can be directed to cut drive architectures could lead to extinction or almost any DNA sequence by simply expressing a other hazardous consequences if applied to ‘guide RNA’ containing that same sequence. In sensitive species, demonstrating an urgent need little more than a year following the first dem- for improved methods of controlling these ele- onstrations in human cells, it has enabled gene ments. After consulting with experts in many fields insertion, deletion, and replacement in many dif- as well as concerned environmental organizations, ferent species (Bassett et al., 2013; Cho et al., we are confident that the responsible develop- 2013; Cong et al., 2013; DiCarlo et al., 2013; ment of RNA-guided gene drive technology is Friedland et al., 2013; Gratz et al., 2013; Hu best served by full transparency and early engage- et al., 2013; Hwang et al., 2013; Jiang et al., ment with the public. 2013a, 2013b; Jinek et al., 2013; Li et al., Here we provide brief overviews of gene 2013b; Mali et al., 2013c; Tan et al., 2013; drives and Cas9-mediated genome engineering, Upadhyay et al., 2013; Wang et al., 2013b). detail the mechanistic reasons that RNA-guided Building RNA-guided gene drives based on gene drives are likely to be effective in many the Cas9 nuclease is a logical way to overcome species, and outline probable capabilities and the targeting and stability problems hindering limitations. We further propose novel gene drive gene drive development. Less obvious is the architectures that may substantially improve our extent to which the unique properties of Cas9 control over gene drives and their effects, discuss are well-suited to overcoming other molecular possible applications, and suggest guidelines and evolutionary challenges inherent to the con- for the safe development and evaluation of this struction of safe and functional gene drives. promising but as yet unrealized technology. A dis- We submit that Cas9 is highly likely to enable cussion of risk governance and regulation intended scientists to construct efficient RNA-guided gene specifically for policymakers is published separately drives not only in mosquitoes, but in many other (Oye et al., 2014). species. In addition to altering populations of insects to prevent them from spreading disease Natural gene drives (Curtis, 1968), this advance would represent an entirely new approach to ecological engineering In nature, certain genes ‘drive’ themselves through with many potential applications relevant to human populations by increasing the odds that they will health, agriculture, biodiversity, and ecological be inherited (Burt and Trivers, 2009). Examples science. include endonuclease genes that copy them- The first technical descriptions of endonuclease selves into chromosomes lacking them (Burt gene drives were provided by Austin Burt in his and Koufopanou, 2004), segregation distorters landmark proposal to engineer wild populations that destroy competing chromosomes during more than a decade ago (Burt, 2003). Any of the meiosis (Lyttle, 1991), transposons that insert rapidly expanding number of laboratories with copies of themselves elsewhere in the genome expertise in Cas9-mediated genome engineering (Charlesworth and Langley, 1989), Medea ele- could attempt to build a gene drive by substituting ments that eliminate competing siblings who do Cas9 for the homing endonucleases described in not inherit them (Beeman et al., 1992; Chen Esvelt et al. eLife 2014;3:e03401. DOI: 10.7554/eLife.03401 2 of 21 Feature article Emerging technology | Concerning RNA-guided gene drives for the alteration of wild populations et al., 2007), and heritable microbes such as Wood and Newton, 1991). In this model, the Y Wolbachia (Werren, 1997). chromosome (or its equivalent in other sex- determination systems) would encode an endo- Endonuclease gene drives nuclease that cuts and destroys the X chromosome during male meiosis, thereby ensuring that Natural homing endonuclease genes exhibit most viable sperm contain a Y chromosome drive by cutting the corresponding locus of chro- (Newton et al., 1976; Wood and Newton, mosomes lacking them. This induces the cell to 1991; Windbichler et al., 2007, 2008; Galizi repair the
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