Despite numerous advances, the field of molecular biology has often struggled to address key biological problems affecting public health and the environment. Until recently, editing the genomes of even model organisms has been difficult. Moreover, altered traits typically reduce evolutionary fitness and are consequently eliminated by natural selection. This restriction has profoundly limited our ability to alter ecosystems through molecular biology.

If we could develop a general method of ensuring that engineered traits would instead be favored by natural selection, then those traits could spread to most members of wild populations over many generations. This capability would allow us to address several major world problems, including the spread of insect-borne diseases, the rise of pesticide and herbicide resistance, and the agricultural and environmental damage wrought by invasive species.

Scientists have long known of naturally occurring selfish genetic elements that can increase the odds that they will be inherited. This advantage allows them to spread through populations even if they reduce the fitness of individual organisms. Many researchers have suggested that these elements might serve as the basis for ‘gene drives’ capable of spreading engineered traits through wild populations (Craig et al., 1960; Wood et al., 1977; Sinkins and Gould, 2006; Burt and Trivers, 2009; Alphey, 2014). Austin Burt was the first to propose gene drives based on site-specific ‘homing’ endonuclease genes over a decade ago (Burt, 2003). These genes bias inheritance by cutting the homologous chromosome, inducing the cell to copy them when it repairs the break. Several efforts have focused on the possibility of using gene drives targeting mosquitoes to block malaria transmission (Scott et al., 2002; Windbichler et al., 2007, 2008, 2011; Li et al., 2013a; Galizi et al., 2014). However, development has been hindered by the difficulty of engineering homing endonucleases to cut new target sequences (Chan et al., 2013a; Thyme et al., 2013; Takeuchi et al., 2014). Attempts to build gene drives with more easily retargeted zinc-finger nucleases and TALENs suffered from instability due to the repetitive nature of the genes encoding them (Simoni et al., 2014).

The recent discovery and development of the RNA-guided Cas9 nuclease has dramatically enhanced our ability to engineer the genomes of diverse species. Originally isolated from ‘CRISPR’ acquired immune systems in bacteria, Cas9 is a non-repetitive enzyme that can be directed to cut almost any DNA sequence by simply expressing a ‘guide RNA’ containing that same sequence. In little more than a year following the first demonstrations in human cells, it has enabled gene insertion, deletion, and replacement in many different species (Bassett et al., 2013; Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Friedland et al., 2013; Gratz et al., 2013; Hu et al., 2013; Hwang et al., 2013; Jiang et al., 2013a, 2013b; Jinek et al., 2013; Li et al., 2013b; Mali et al., 2013c; Tan et al., 2013; Upadhyay et al., 2013; Wang et al., 2013b).

Building RNA-guided gene drives based on the Cas9 nuclease is a logical way to overcome the targeting and stability problems hindering gene drive development. Less obvious is the extent to which the unique properties of Cas9 are well-suited to overcoming other molecular and evolutionary challenges inherent to the construction of safe and functional gene drives.

We submit that Cas9 is highly likely to enable scientists to construct efficient RNA-guided gene drives not only in mosquitoes, but in many other species. In addition to altering populations of insects to prevent them from spreading disease (Curtis, 1968), this advance would represent an entirely new approach to ecological engineering with many potential applications relevant to human health, agriculture, biodiversity, and ecological science.

The first technical descriptions of endonuclease gene drives were provided by Austin Burt in his landmark proposal to engineer wild populations more than a decade ago (Burt, 2003). Any of the rapidly expanding number of laboratories with expertise in Cas9-mediated genome engineering could attempt to build a gene drive by substituting Cas9 for the homing endonucleases described in his proposal. Indeed, the well-recognized potential for gene drives to combat vector-borne diseases such as malaria and dengue virtually ensures that this strategy will eventually be attempted in mosquitoes.

While considerable scholarship has been devoted to the question of how gene drives might be safely utilized in mosquitoes (Scott et al., 2002; Touré et al., 2004; Benedict et al., 2008; Marshall, 2009; UNEP, 2010; Reeves et al., 2012; David et al., 2013; Alphey, 2014), few if any studies have examined the potential ecological effects of gene drives in other species. After all, constructing a drive to spread a particular genomic alteration in a given species was simply not feasible with earlier genome editing methods. Disconcertingly, several published gene drive architectures could lead to extinction or other hazardous consequences if applied to sensitive species, demonstrating an urgent need for improved methods of controlling these elements. After consulting with experts in many fields as well as concerned environmental organizations, we are confident that the responsible development of RNA-guided gene drive technology is best served by full transparency and early engagement with the public.

Here we provide brief overviews of gene drives and Cas9-mediated genome engineering, detail the mechanistic reasons that RNA-guided gene drives are likely to be effective in many species, and outline probable capabilities and limitations. We further propose novel gene drive architectures that may substantially improve our control over gene drives and their effects, discuss possible applications, and suggest guidelines for the safe development and evaluation of this promising but as yet unrealized technology. A discussion of risk governance and regulation intended specifically for policymakers is published separately (Oye et al., 2014).

Endonuclease gene drives

Natural homing endonuclease genes exhibit drive by cutting the corresponding locus of chromosomes lacking them. This induces the cell to repair the break by copying the nuclease gene onto the damaged chromosome via homologous recombination (Figure 1A) (Burt and Koufopanou, 2004). The copying process is termed ‘homing’, while the endonuclease-containing cassette that is copied is referred to as a ‘gene drive’ or simply a ‘drive’. Because copying causes the fraction of offspring that inherit the cassette to be greater than 1/2 (Figure 1B), these genes can drive through a population even if they reduce the reproductive fitness of the individual organisms that carry them. Over many generations, this self-sustaining process can theoretically allow a gene drive to spread from a small number of individuals until it is present in all members of a population.

Figure 1

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Engineered gene drives

To build an endonuclease gene drive, an endonuclease transgene must be inserted in place of a natural sequence that it can cut. If it can efficiently cut this sequence in organisms with one transgene and one natural locus, reliably induce the cell to copy the transgene, and avoid being too costly to the organism, it will spread through susceptible wild populations.

Standard drives spread genomic changes and associated traits through populations. Burt‘s original study proposed using them to drive the spread of other transgenes or to disrupt existing genes (Figure 2A,B) (Burt, 2003). The gene drive copying step can take place immediately upon fertilization (Figure 2C) or occur only in germline cells that are immediate precursors to sperm or eggs, leaving most of the organism‘s somatic cells with only one copy of the drive (Figure 2D).

Figure 2

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Suppression drives reduce the size of the targeted population. Austin Burt outlined an elegant strategy involving the use of gene drives to disrupt genes that cause infertility or lethality only when both copies are lost (Burt, 2003). These ‘genetic load’ drives would spread rapidly through minimally impaired heterozygotes when rare, and eventually cause the population to crash or even become extinct due to the accumulated load of recessive mutations. A second approach would mimic naturally occurring ‘meiotic’ or ‘gametic’ drives that bias the sex ratio (Craig et al., 1960; Hickey and Craig, 1966; Hamilton, 1967; Newton et al., 1976; Wood and Newton, 1991). In this model, the Y chromosome (or its equivalent in other sex-determination systems) would encode an endonuclease that cuts and destroys the X chromosome during male meiosis, thereby ensuring that most viable sperm contain a Y chromosome (Newton et al., 1976; Wood and Newton, 1991; Windbichler et al., 2007, 2008; Galizi et al., 2014). The progressively dwindling number of females will culminate in a population crash or extinction (Craig et al., 1960; Lyttle, 1977; Burt, 2003; Schliekelman et al., 2005; Deredec et al., 2008, 2011; North et al., 2013; Burt, 2014).

Whether a standard gene drive will spread through a target population depends on molecular factors such as homing efficiency, fitness cost, and evolutionary stability (Marshall and Hay, 2012); only the rate of spread is determined by the mating dynamics, generation time, and other characteristics of the target population. In contrast, models suggest that the deleterious and complex effects of genetic load and sex-biasing suppression drives render them more sensitive to population-specific ecological variables such as density-dependent selection (Burt, 2003; Schliekelman et al., 2005; Huang et al., 2007; Deredec et al., 2008; Marshall, 2009; Yahara et al., 2009; Deredec et al., 2011; Alphey and Bonsall, 2014).

No engineered endonuclease gene drive capable of spreading through a wild population has yet been published. However, the Crisanti and Russell laboratories have constructed gene drives that can only spread through laboratory mosquito (Windbichler et al., 2011) and fruit fly (Chan et al., 2011; Simoni et al., 2014) populations that have been engineered to contain the endonuclease cut site. The Burt and Crisanti laboratories are attempting to build a male-biasing suppression drive using an endonuclease that serendipitously cuts a conserved sequence repeated hundreds of times in the X chromosome of the mosquito Anopheles gambiae (Windbichler et al., 2007, 2008; Galizi et al., 2014). If successful, their work promises to substantially reduce the population of this important malaria vector.

All engineered gene drives based on homing endonucleases cut the natural recognition site of the relevant enzyme. Despite early hopes, it has proven difficult to engineer homing endonucleases to cleave new target sequences. Numerous laboratories have sought to accomplish this goal for well over a decade with only a few recent successes (Chan et al., 2013a; Thyme et al., 2013; Takeuchi et al., 2014). More recently, a team constructed new versions of the fruit fly gene drive using modular zinc-finger nucleases or TALENs in place of the homing endonuclease (Simoni et al., 2014), both of which can be engineered to cut new target sequences. While initially successful at cutting and homing, both declined in effectiveness over time due to the evolutionary instability of the modular repeats inherent to those proteins.

These early attempts demonstrate that it is possible to build synthetic gene drives, but also emphasize the importance of cutting any desired gene and remaining stable during copying. The recent discovery of the RNA-guided Cas9 nuclease represents a possible solution.

RNA-guided genome editing via the Cas9 nuclease

One straightforward method of genome editing relies on the same mechanism employed by endonuclease gene drives: cut the target gene and supply an edited version for the cell to use as a template when it fixes the damage. Most eukaryotic genome engineering over the past decade was accomplished using zinc-finger nucleases (Urnov et al., 2005) or TALENs (Christian et al., 2010), both of which are modular proteins that can be redesigned or evolved to target new sequences, albeit only by specialist laboratories (Esvelt and Wang, 2013). Genome editing was democratized by the discovery and adaptation of Cas9, an enzyme that can be programmed to cut target DNA sequences specified by a guiding RNA molecule (Deltcheva et al., 2011; Jinek et al., 2012; Cho et al., 2013; Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013c).

Cas9 is a component of Type II CRISPR acquired immune systems in bacteria, which allow cells to ‘remember’ the sequences of previously encountered viral genomes and protect themselves by recognizing and cutting those sequences if encountered again. They accomplish this by incorporating DNA fragments into a memory element, transcribing it to produce RNAs with the same sequence, and directing Cas9 to cut any matching DNA sequences (Deltcheva et al., 2011). The only restriction is that Cas9 will only cut target ‘protospacer’ sequences that are flanked by a protospacer-adjacent motif (PAM) at the 3′ end. The most commonly used Cas9 ortholog has a PAM with only two required bases (NGG) and therefore can cut protospacers found approximately every 8 base pairs (Jinek et al., 2012).

Remarkably, it is possible to direct Cas9 to cut a specific protospacer in the genome using only a single guide RNA (sgRNA) less than 100 base pairs in length (Jinek et al., 2012). This guide RNA must begin with a 17-20 base pair ‘spacer’ sequence identical to the targeted protospacer sequence in the genome (Fu et al., 2014). The process of editing a target gene involves choosing protospacers within the gene, building one or more guide RNAs with matching spacers, and delivering Cas9, guide RNAs, and an edited repair template lacking those protospacers into the cell (Figure 3).

Figure 3

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Cas9 is efficient enough to cut and edit multiple genes in a single experiment (Li et al., 2013c; Wang et al., 2013a). The enzyme is active in a wide variety of organisms and is also quite specific, cutting only protospacers that are nearly identical to the spacer sequence of the guide RNA (Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013). Moreover, methods that allow Cas9 to bind but not cut enable the expression of target genes to be regulated by selectively recruiting regulatory proteins attached to Cas9 or the guide RNA (Gilbert et al., 2013; Mali et al., 2013a). All of these applications were developed within the last two years.

Because RNA-guided genome editing relies on exactly the same copying mechanism as endonuclease gene drives, it is reasonable to ask whether it might be possible to build gene drives based on Cas9. In principle, RNA-guided gene drives might be capable of spreading almost any genomic alteration that can be generated using Cas9 through sexually reproducing populations.

Although we cannot be certain until we try, current evidence suggests that RNA-guided gene drives will function in some and possibly most sexually reproducing species. Learning how to insert a drive into the germline and optimize its function in each new species will likely require months to years depending on generation length, with subsequent drives in the same species taking less time. Because inserting the drive into the germline with Cas9 involves the same molecular copying process as the drive itself will utilize, successful insertion may produce a working if not particularly efficient RNA-guided gene drive. But if population-level engineering is to become a reality, all molecular factors relevant to homing – cutting, specificity, copying, and evolutionary robustness – must be considered. Below, we provide a detailed technical analysis of the extent to which Cas9 can address each of these challenges. Capabilities, limitations, control strategies, and possible applications are discussed in subsequent sections.

Cutting

The first requirement for every endonuclease gene drive is to cut the target sequence. Incomplete cutting was a problem for the homing endonuclease drive constructed in transgenic mosquitoes (72% cutting) and also for the homing endonuclease, zinc-finger nuclease, and TALEN drives in fruit flies (37%, 86%, and 70% cutting) (Windbichler et al., 2011; Chan et al., 2013b; Simoni et al., 2014). The simplest way to increase cutting is to target multiple adjacent sequences. However, this is impractical for homing endonucleases and quite difficult for zinc-finger nucleases and TALENs, as each additional sequence requires a new nuclease protein to be engineered or evolved and then co-expressed.

In contrast, the RNA-guided Cas9 nuclease can be readily directed to cleave additional sequences by expressing additional guide RNAs (Figure 4). The sequences of these additional guide RNAs can be altered so as to avoid creating unstable repeats within the drive cassette (Nishimasu et al., 2014; Simoni et al., 2014). Including more guides has been demonstrated to improve upon already high rates of cutting. For example, fruit flies expressing both Cas9 and guide RNAs in their germline exhibited target cutting rates exceeding 85–99% in males for four out of six tested guide RNAs (Kondo and Ueda, 2013). The two least effective guide RNAs individually cut at rates exceeding 12% and 56%, but exhibited cutting rates above 91% when combined. Using more than two guide RNAs should further enhance cutting. The notable success of Cas9-based genome engineering in many different species, including studies that targeted every gene in the genome (Shalem et al., 2014; Wang et al., 2014), demonstrates that most sequences can be efficiently targeted independent of species and cell type. Thus, RNA-guided gene drives should be capable of efficiently cutting any given gene.

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Given their potentially widespread availability, it will be essential to develop a comprehensive understanding of the fundamental limitations of genetic drive systems.

First and most important, gene drives require many generations to spread through populations. Once transgenic organisms bearing the gene drive are constructed in the laboratory, they must be released into the wild to mate with wild-type individuals in order to begin the process of spreading the drive through the wild population. The total time required to spread to all members depends on the number of drive-carrying individuals that are released, the generation time of the organism, the efficiency of homing, the impact of the drive on individual fitness, and the dynamics of mating and gene flow in the population, but in general it will take several dozen generations (Burt, 2003; Huang et al., 2007; Deredec et al., 2008; Marshall, 2009; Yahara et al., 2009; Deredec et al., 2011). Thus, drives will spread very quickly in fast-reproducing species but only slowly in long-lived organisms.

Second, gene drives cannot affect species that exclusively practice asexual reproduction through clonal division or self-fertilization. This category includes all viruses and bacteria as well as most unicellular organisms. Highly efficient standard drives might be able to slowly spread through populations that employ a mix of sexual and asexual reproduction, such as certain plants, but drives intended to suppress the population would presumably force target organisms to reproduce asexually in order to avoid suppression.

Third, drive-mediated genome alterations are not permanent on an evolutionary timescale. While gene drives can spread traits through populations even if they are costly to each individual organism, harmful traits will eventually be outcompeted by more fit alleles after the drive has gone to fixation. Highly deleterious traits may be eliminated even more quickly, with non-functional versions appearing in large numbers even before the drive and its cargo can spread to all members of the population. Even when the trait is perfectly linked to the drive mechanism, the selection pressure favoring the continued function of Cas9 and the guide RNAs will relax once the drive reaches fixation. Maintaining deleterious traits within a population indefinitely is likely to require scheduled releases of new RNA-guided gene drives to periodically overwrite the broken versions in the environment.

Fourth, our current knowledge of the risk management (Scott et al., 2002; Touré et al., 2004; UNEP, 2010; McGraw and O’Neill, 2013; Alphey, 2014) and containment (Benedict et al., 2008; Marshall, 2009) issues associated with gene drives is largely due to the efforts of researchers focused on mosquito-borne illnesses. Frameworks for evaluating ecological consequences are similarly focused on mosquitoes (David et al., 2013) and the few other organisms for which alternative genetic biocontrol methods have been considered (Dana et al., 2014). While these examples provide an invaluable starting point for investigations of RNA-guided gene drives targeting other organisms, studies examining the particular drive, population, and associated ecosystem in question will be needed.

Given the potential for gene drives to alter entire wild populations and therefore ecosystems, the development of this technology must include robust safeguards and methods of control (Oye et al., 2014). Whereas existing gene drive proposals focus on adding genes (Ito et al., 2002), disrupting existing genes (Burt, 2003), or suppressing populations, RNA-guided gene drives will also be capable of replacing existing sequences with altered versions that have been recoded to remove the sites targeted by the drive (Figure 3). We hypothesize that the unique ability of RNA-guided gene drives to target any gene may allow them to control the effects of other gene drives or transgenes.

RNA-guided gene drives could reverse genome alterations that have already spread through populations. Suppose a given gene drive causes unexpected side-effects or is released without public consent. A ‘reversal’ drive released later could overwrite one or all genomic changes spread by the first drive (Figure 5A). The new sequence spread by the reversal drive must also be recoded relative to the original to keep the first drive from cutting it, but any amino acid changes introduced by the first drive could be undone. If necessary, a third drive could restore the exact wild-type sequence, leaving only the guide RNAs and the gene encoding Cas9 as signatures of past editing (Figure 5B).

Figure 5

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The ability to update or reverse genomic alterations at the speed of a drive, not just a drive-resistant allele, represents an extremely important safety feature. Reversal drives could also remove conventionally inserted transgenes that entered wild populations by cross-breeding or natural mutations that spread in response to human-induced selective pressures. However, it is important to note that even if a reversal drive were to reach all members of the population, any ecological changes caused in the interim would not necessarily be reversed.

Limiting population suppression

Population suppression may be one of the most powerful applications of gene drives. The previously described genetic load and sex-biasing drives (Burt, 2003) could potentially lead to extinction (Deredec et al., 2008, 2011). While this outcome may be necessary to achieve compelling goals such as the eradication of malaria, other situations may call for more refined methods. Here we outline a handful of alternative architectures that would provide greater control over the extent of population suppression.

Chemical approaches to population control might utilize ‘sensitizing drives’ to render target organisms vulnerable to a particular molecule using one of three strategies (Figure 6). First, a sensitizing drive might reverse known mutations that confer resistance to existing pesticides or herbicides. Second, it might carry a prodrug-converting enzyme (Schellmann et al., 2010) that would render a prodrug molecule toxic to organisms that express it. Third, it could swap a conserved gene for a version that is strongly inhibited by a particular small molecule. Because sensitizing drives would have no effect in the absence of their associated molecule – and in some cases vice versa – they could grant very fine control over the geography and extent of population suppression with minimal ecological risk.

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Temporal approaches to controlling populations would deliberately limit the lifetime of a suppression drive by rendering its effects evolutionarily unstable (Figure 6). For example, a male-determining or female-specific sterility gene carried by a standard drive on an autosome would suppress the target population, but the effect would be short-lived because any drive that acquired a loss-of-function mutation in the cargo gene would be strongly favored by natural selection due to its ability to produce fertile female offspring. Notably, turning existing female-specific sterility lines (Fu et al., 2010; Labbe et al., 2012; Alphey, 2014) into unstable drives may increase their effectiveness. Periodically releasing organisms carrying new unstable drives that are capable of replacing earlier broken versions could extend the suppression effect.

Genetic approaches to population control might initiate suppression only when two distinct ‘interacting drives’ encounter one another (Figure 6). For example, a cross between standard drives A and B might produce sterile females and fertile males that pass on the ‘sterile-daughter’ trait when crossed with females of any type. Scattering A- and B-carrying individuals throughout an existing population would produce many tiny pockets dominated by either A or B and very few organisms in between due to the infertility of AB females. Because each drive would spread from a small number of initially released individuals scattered over a wide area, this strategy may be capable of large-scale population suppression, but its effectiveness and resolution will depend on the average distance between released A and B individuals. Further suppressing the residual A and B populations could be accomplished by releasing only members of the opposite drive type. Modeling studies will be needed to determine whether this possibility is feasible for different species. Interestingly, the use of this drive type would effectively induce speciation in the affected population.

Finally, immunizing drives might protect specific subpopulations from the effects of full-scale suppression drives released elsewhere (Figure 6). Assuming some degree of gene flow, the immunized population will eventually replace the suppressed population, though this might be delayed if crossing the two drives generates a sterile-daughter effect as described above. Due to the comparatively uncontrolled spread of both drive types through the wild-type population, this method would only be suited to large geographic areas or subpopulations with limited gene flow. For example, immunization might be used to protect the native population of a species while suppressing or eradicating populations on other continents.

Box 3

What types of genes can be edited using gene drives?

Genes can be edited reliably if they are important to fitness. This is because NHEJ events that create drive-resistant alleles by deleting all the protospacer cut sites will only be harmful if they disrupt the function of important genes. NHEJ events in unimportant genes and sequences will produce drive-resistant alleles lacking the targeted sites. These will spread and interfere with propagation of the drive. As a result, unimportant genes can be reliably disrupted or deleted but not edited.

Genes that are carried as cargo will not be evolutionarily stable unless they directly contribute to the efficient function of the drive. This limits opportunities to spread transgenes unrelated to drive function, although periodically releasing new drives that overwrite earlier broken versions could potentially maintain functional cargo genes in a large fraction of the population.

RNA-guided gene drives have the potential to merge the fields of genomic and ecological engineering. They may enable us to address numerous problems in global health, agriculture, sustainability, ecological science, and many other areas (Figure 7). Of these opportunities, perhaps the most compelling involve curtailing the spread of vector-borne infectious diseases, controlling agricultural pests, and reducing populations of environmentally and economically destructive invasive species.

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Because any consequences of releasing RNA-guided gene drives into the environment would be shared by the local if not global community, research involving gene drives capable of spreading through wild-type populations should occur only after a careful and fully transparent review process. However, basic research into gene drives and methods of controlling their effects can proceed without risking this type of spread so long as appropriate ecological or molecular containment strategies are employed (Figure 8).

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A great deal of information on probable ecological outcomes can be obtained without testing or even building replication-competent gene drives. For example, early studies might examine possible ecological effects by performing contained field trials with organisms that have been engineered to contain the desired change but do not possess a functional drive to spread it. Because they do involve transgenic organisms, these experiments are not completely without risk, but such transgenes are unlikely to spread in the absence of a drive.

We recommend that all laboratories seeking to build standard gene drives capable of spreading through wild populations simultaneously create reversal drives able to restore the original phenotype. Similarly, suppression drives should be constructed in tandem with a corresponding immunizing drive. These precautions would allow the effects of an accidental release to be swiftly if partially counteracted. The prevalence of gene drives in the environment could in principle be monitored by targeted amplification or metagenomic sequencing of environmental samples. Further investigation of possible monitoring strategies will be needed.

Technologies with the potential to significantly influence the lives of the general public demand societal review and consent. As self-propagating alterations of wild populations, RNA-guided gene drives will be capable of influencing entire ecosystems for good or for ill. As such, it is imperative that all research in this nascent field operate under conditions of full transparency, including independent scientific assessments of probable impacts and thoughtful, informed, and fully inclusive public discussions.

The decision of whether or not to utilize a gene drive for a given purpose should be based entirely on the probable benefits and risks of that specific drive. That is, each drive should be judged solely by its potential outcomes, such as its ability to accomplish the intended aims, its probable effects on other species, the risk of spreading into closely related species by rare mating events, and impacts on ecosystems and human societies. As scientists developing these technologies, it will be our responsibility to make all empirical data and predictive models freely available to the public in a transparent and understandable format. Above all else, we must openly share our level of confidence in these assessments as we determine how best to proceed.

The potentially widespread implications of RNA-guided gene drives demand a thoughtful and collected response. Numerous practical difficulties must be overcome before gene drives will be in a position to address any of the suggested applications. Many of our proposals and predictions are likely to fall short simply because biological systems are complex and difficult to engineer. Even so, the current rate of scientific advancement related to Cas9 and the many outcomes accessible using the simplest of gene drives suggest that molecular biologists will soon be able to edit the genomes of wild populations, reverse or update those changes in response to field observations, and perhaps even engage in targeted population suppression.

What criteria might we use to evaluate an RNA-guided gene drive intended to address a given problem? There are compelling arguments in favor of eliminating insect-borne human diseases, developing and supporting more sustainable agricultural models, and controlling environmentally damaging invasive species. At the same time, there are valid concerns regarding our ability to accurately predict the ecological and human consequences of these interventions. By bringing these possibilities before the scientific community and the public prior to their realization in the laboratory, we hope to initiate transparent, inclusive, and well-informed discussions concerning the responsible evaluation and application of these nascent technologies (Oye et al., 2014).

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Thank you for sending your work entitled “Concerning RNA-Guided Gene Drives for the Engineering of Wild Populations” for consideration at eLife. Your article has been favorably evaluated by Detlef Weigel (Senior editor) and 3 reviewers, one of whom is another Senior editor.

The Reviewing editor, the other reviewers, and the editorial leadership discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission:

We appreciate that the overall purpose of the article is to alert the scientific community to the new capabilities offered by the CRISPR nuclease Cas 9 RNA-guided gene drives in engineering wild populations. You briefly review the concept of gene drives, the CRISPR/Cas9 technology, and current research driving advancement of gene drive and CRISPR technologies. Most of the manuscript focuses on “likely capabilities” of Cas9 mediated gene drives, including the theoretical uses for “sensitizing”, “unstable”, “interacting”, and “immunizing” drives. You briefly state that these future applications of Cas9 gene drives are associated with environmental, safety, and security concerns that should be considered before any experimentation is conducted. You conclude that Cas9 RNA-guided gene drives offer new capabilities and applications beyond what is currently available, but the implications for the environment and society as a whole should be considered. You do not, however, address any issues concerning a possible deliberately harmful use of the technique; since it does not involve complex technology, there is the possibility that it is applied outside the reaches of governmental or societal regulation.

To encourage thoughtful review of the Cas9 technology as it continues to develop and be applied in different ways, you should ensure that your manuscript is clear about which technologies have been developed, which are currently under active investigation, and which are yet to be developed. To better encourage public discussion on this complex technology, you should describe the CRISPR/Cas9 technology more clearly, as the sections describing the basic technology are difficult to read and visualize for non-specialists. It would be helpful to define CRISPR, Cas9, the DNA repeat elements, their natural functions (targeted cleavage of foreign DNA), and how they can be tailored to use as gene editing drives in multi-cellular organizations. This basic description is important because it is a new technology even for many molecular biologists, let alone for the interested public, and without a clear understanding of the technology, the reader might not be able to fully comprehend the societal implications (both benefits and risks) and likelihood of future capabilities of the technology.

We also felt that there is the danger that without a strong, factual discussion of the basic concepts along with what is currently possible and what has so far not been demonstrated, the intended audience (public, broader science community, others with no or different technical knowledge) might arrive at a conclusion that does not adequately reflect current and near-future reality. When basic scientific topics are clearly described, the responses tend to be much more thoughtful and measured. When the science is not sufficiently described, all sorts of responses are elicited.

This article will be treated as a Feature article, so it will be clear on both the web and PDF versions that it is not a Research article. This will be especially clear in the PDF, which will have a two-column layout. It is planned to be published under the heading “Emerging Technologies” and it is suggested to change the title into “Possible applications and potential concerns related to the use of RNA-guided gene drives to engineer wild populations”. The Abstract should be intelligible to a broad public. The eLife editors offer to work with you to ensure that the title and abstract accurately reflect the content of the article.

Supplementary notes cannot be used in the intended article format. Notes 2 and 4 describe important possible limitations of the technology that should be integrated into the text to ensure that it is understood by the general reader that the technology is not immediately ready for use and may even turn out to be not as powerful as it might seem at present. Notes 1 and 3 could be moved into boxes. Please ensure in all passages that a balanced discussion between possible benefits and (current) technological limitations is maintained.

To avoid any possibly distracting discussion around this article, you are asked to be particularly careful with citing previous relevant work when discussing specific issues and ideas.

We appreciate that the overall purpose of the article is to alert the scientific community to the new capabilities offered by the CRISPR nuclease Cas 9 RNA-guided gene drives in engineering wild populations. […] You conclude that Cas9 RNA-guided gene drives offer new capabilities and applications beyond what is currently available, but the implications for the environment and society as a whole should be considered. You do not, however, address any issues concerning a possible deliberately harmful use of the technique; since it does not involve complex technology, there is the possibility that it is applied outside the reaches of governmental or societal regulation.

We hope to initiate discussion concerning how this potentially powerful technology can be responsibly developed, evaluated, and used to solve major ecological problems in a way that minimizes the risk of harmful consequences. This latter purpose is best served by ensuring that control measures are developed concomitantly. We consequently devote much of the manuscript to detailing reversal and immunization drives intended to undo earlier genomic changes and protect populations against gene drives. To emphasize the importance of these novel drive architectures in this revision, we have combined the earlier figures describing these drives into a single larger and clearer Figure 5. We have also included boxes that explicitly address whether gene drives could be used to alter populations of humans, crops, or domesticated animals. The possibility that unauthorized gene drives might be deliberately misused is more relevant to regulators and consequently is discussed in greater detail in our related manuscript on preparedness and risk governance scheduled for simultaneous publication in Science.

To encourage thoughtful review of the Cas9 technology as it continues to develop and be applied in different ways, you should ensure that your manuscript is clear about which technologies have been developed, which are currently under active investigation, and which are yet to be developed. To better encourage public discussion on this complex technology, you should describe the CRISPR/Cas9 technology more clearly, as the sections describing the basic technology are difficult to read and visualize for non-specialists. It would be helpful to define CRISPR, Cas9, the DNA repeat elements, their natural functions (targeted cleavage of foreign DNA), and how they can be tailored to use as gene editing drives in multi-cellular organizations. This basic description is important because it is a new technology even for many molecular biologists, let alone for the interested public, and without a clear understanding of the technology, the reader might not be able to fully comprehend the societal implications (both benefits and risks) and likelihood of future capabilities of the technology.

We also felt that there is the danger that without a strong, factual discussion of the basic concepts along with what is currently possible and what has so far not been demonstrated, the intended audience (public, broader science community, others with no or different technical knowledge) might arrive at a conclusion that does not adequately reflect current and near-future reality. When basic scientific topics are clearly described, the responses tend to be much more thoughtful and measured. When the science is not sufficiently described, all sorts of responses are elicited.

We agree that the inclusion of a section explaining the established use of Cas9 for genome editing would greatly strengthen the paper. While the subject of CRISPR systems and Cas9-mediated genome engineering is large enough for entire review articles, we have done our best to provide a concise but thorough explanation in a new section at the end of the Introduction. We briefly detail Type II CRISPR systems as requested, but devote the bulk of the section to RNA-guided genome editing. We have additionally created a new figure detailing the mechanism of genome editing that emphasizes the similarity of this process to the homing observed in endonuclease gene drives.

This article will be treated as a Feature article, so it will be clear on both the web and pdf versions that it is not a Research article. This will be especially clear in the pdf, which will have a two-column layout. It is planned to be published under the heading “Emerging Technologies” and it is suggested to change the title into “Possible applications and potential concerns related to the use of RNA-guided gene drives to engineer wild populations”. The Abstract should be intelligible to a broad public. The eLife editors offer to work with you to ensure that the title and abstract accurately reflect the content of the article.

The Abstract and Introduction have been revised to more clearly explain the topic to a general audience. We welcome the chance to emphasize in the article format that this technology remains entirely theoretical, as our intent is to initiate discussion prior to development of this technology in the laboratory. We are grateful to eLife for establishing a new section on emerging technologies for this purpose.

While clarifying public perception is in this case an overriding reason to classify this as a Feature article rather than a Research article, we regret the implication that theoretical analysis does not constitute scientific research. Many of the most respected publications in the history of science consisted entirely of conceptual insights – some of them without any mathematical or computational component.

Supplementary notes cannot be used in the intended article format. Notes 2 and 4 describe important possible limitations of the technology that should be integrated into the text to ensure that it is understood by the general reader that the technology is not immediately ready for use and may even turn out to be not as powerful as it might seem at present. Notes 1 and 3 could be moved into boxes. Please ensure in all passages that a balanced discussion between possible benefits and (current) technological limitations is maintained.

Points relevant to the non-specialist reader that were previously covered in the Notes have been incorporated into the text and new boxes if they were not already present. Material of concern only to specialists has been transferred to new figure supplements: Figure 4—figure supplement 2, Figure 4—figure supplement 3, and Figure 7—figure supplement 1. Figure 4—figure supplement 3 specifically details existing technologies that could be applied to optimize RNA-guided gene drives and notes the challenges involved in doing so.

We have endeavored to clarify that this technology remains theoretical and our predictions are fallible throughout. Most notably, the last sentence of the Abstract describes the technology as “currently theoretical” and the section “Building Functional Gene Drives” has been prominently renamed “Will RNA-Guided Gene Drives Enable Us to Edit the Genomes of Wild Species?” It goes on to explicitly state that we cannot know for certain until we try. Combined with our cautionary statement in the Conclusion noting that, “Numerous practical difficulties must be overcome before gene drives will be in a position to address any of the suggested applications. Many of our proposals and predictions are likely to fall short simply because biological systems are complex and difficult to engineer...”, we think it unlikely that a reader will come away with a mistaken impression.

To avoid any possibly distracting discussion around this article, you are asked to be particularly careful with citing previous relevant work when discussing specific issues and ideas.

We have endeavored to cite relevant works and additionally mention several major contributors to the field of endonuclease gene drives by name.

Author details

1. Kevin M Esvelt

   Synthetic Biology Platform, Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, United States

   Contribution

   KME, Conceived of the study, performed the analysis, designed new drive types, drafted and revised the article

   For correspondence

   kevin.esvelt@wyss.harvard.edu

   Competing interests

   KME: Filed for a patent concerning RNA-guided gene drives.

   "This ORCID iD identifies the author of this article:" 0000-0001-8797-3945

2. Andrea L Smidler

   Synthetic Biology Platform, Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, United States

   Contribution

   ALS, Designed new drive types, drafted and revised the article

   Competing interests

   ALS: Filed for a patent concerning RNA-guided gene drives.

   "This ORCID iD identifies the author of this article:" 0000-0002-3281-1161

3. Flaminia Catteruccia

   Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, United States

   Contribution

   FC, Revised the article

   For correspondence

   fcatter@hsph.harvard.edu

   Competing interests

   No competing interests declared.

4. George M Church

   Synthetic Biology Platform, Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, United States

   Contribution

   GMC, Revised the article

   For correspondence

   gmc@harvard.edu

   Competing interests

   GMC: Filed for a patent concerning RNA-guided gene drives.

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