- Open Access
Functional genetics for all: engineered nucleases, CRISPR and the gene editing revolution
EvoDevo volume 5, Article number: 43 (2014)
Developmental biology, as all experimental science, is empowered by technological advances. The availability of genetic tools in some species - designated as model organisms - has driven their use as major platforms for understanding development, physiology and behavior. Extending these tools to a wider range of species determines whether (and how) we can experimentally approach developmental diversity and evolution. During the last two decades, comparative developmental biology (evo-devo) was marked by the introduction of gene knockdown and deep sequencing technologies that are applicable to a wide range of species. These approaches allowed us to test the developmental role of specific genes in diverse species, to study biological processes that are not accessible in established models and, in some cases, to conduct genome-wide screens that overcome the limitations of the candidate gene approach. The recent discovery of CRISPR/Cas as a means of precise alterations into the genome promises to revolutionize developmental genetics. In this review we describe the development of gene editing tools, from zinc-finger nucleases to TALENs and CRISPR, and examine their application in gene targeting, their limitations and the opportunities they present for evo-devo. We outline their use in gene knock-out and knock-in approaches, and in manipulating gene functions by directing molecular effectors to specific sites in the genome. The ease-of-use and efficiency of CRISPR in diverse species provide an opportunity to close the technology gap that exists between established model organisms and emerging genetically-tractable species.
Evo-devo: driven by technological advances
Our understanding of developmental mechanisms is shaped by the experimental models and approaches at hand. The powerful genetic approaches available in Drosophila, C. elegans, zebrafish, mice and Arabidopsis have largely driven developmental research during the past decades, focusing it on questions that are experimentally tractable in these species. However, biological diversity greatly surpasses what can be studied in these organisms. Phenomena such as regeneration, polyphenism and chromatin diminution challenge some of our conventional views of development, but are still poorly understood because they are not accessible in our current experimental models. Also, understanding the evolutionary paths by which diversity is generated requires that we compare developmental mechanisms among several animals, well beyond the established model organisms. Exploring these topics requires extending our genetic approaches to new species.
Establishing genetic tools in new organisms has always been a challenge for comparative developmental biology. Evo-devo started to flourish when cloning genes and studying their expression patterns in embryos could be extended to a wide range of animals, with the advent of PCR and whole-mount in situ hybridization techniques in the 1990s. These techniques allowed ‘candidate genes’ to be associated with specific developmental events in different animals and for evolutionary-developmental hypotheses to be formulated based on this information. Testing these hypotheses experimentally and exploring alternative possibilities in an unbiased fashion became, at that point, major challenges for the future of evo-devo.
Two important steps toward meeting those challenges were made since the late 1990s: the establishment of gene knockdown approaches based on RNAi and other antisense methods (see below) and the invention of low-cost deep sequencing technologies, which opened the door to unbiased genome-wide studies. Both methods could be applied to a wide range of species. We will focus here on functional genetics approaches.
The first important advance in this direction was made with the discovery of RNA interference (RNAi), a mechanism that uses small RNAs (processed from larger double-stranded precursors) to recognize and degrade specific RNA targets [1–4]. RNAi is a natural mechanism that evolved in eukaryotes to protect the genome against invading viruses and transposons . This defense mechanism can be redirected to target specific mRNAs of interest by providing double-stranded RNA matching the target sequence. Since the RNAi machinery is found naturally in most eukaryotes, RNAi-mediated gene knockdown has turned out to be widely applicable. This approach has also been complemented by other antisense methods that target RNA using different types of oligonucleotides (morpholinos, antagomirs, LNAs and others [5–8]). Together, these antisense approaches have given us the opportunity to knock down gene functions at the RNA level in a wide range of animals, including cnidarians, arthropods, nematodes, planarians, annelids, echinoderms, tunicates and vertebrates (for example, [1, 3, 9–13]). RNAi-based screens in new experimental models have allowed us to study biological problems that were genetically intractable in the past, such as tissue homeostasis and regeneration in planarians  or particular aspects of insect physiology and development in beetles (http://ibeetle.uni-goettingen.de/).
The flurry of RNAi and other antisense studies carried out at the turn of the century revealed the power of these approaches, but also their limitations. Besides technical limitations relating to delivery, toxicity and off-target effects, for which solutions and appropriate controls can often be found [15–17], there are intrinsic limitations in the type of genetic manipulation that can be carried out: interference with gene function is usually transient, unlocalized, and primarily targets mRNA. Antisense approaches do not usually allow us to achieve complete loss of gene function, to perform stable genetic modification, to pursue gain-of-function and conditional approaches, or to study cis- regulatory elements.
In some organisms, these knockdown approaches have been complemented by transgenesis [18–24], which gives access to stable genetic modification and gain-of-function experiments via gene mis-expression. Transgenesis also enables the use of reporter constructs to study cis- regulatory elements and to generate tools for live imaging, as well as opportunities to generate mosaic animals, where clones of cells can be marked, genetically modified and compared to wild-type cells in the same individual . The power of the transgenic approach in new experimental models can be seen, for example, in cell labeling and tracing experiments carried out to study regenerative progenitor cells in crustaceans and axolotls [26, 27]. The development of transgenesis requires a significant investment of time and effort, so this approach is still limited to few species.
Among the functional approaches that are applicable to a wide range of species, we can also count pharmacological treatments, which rely on the use of small molecule effectors to interfere with specific regulatory pathways .
Together, these technological advances allowed evo-devo to advance from descriptive cross-species comparisons (in the 1990s) to comparisons of gene function within a decade. In spite of this progress, most systems are still lagging far behind standard models in terms of experimental power and precision. The arrival of efficient and widely applicable gene editing approaches is set to narrow that gap, revolutionizing genetic approaches both in established models and in emerging experimental species.
Gene targeting approaches
The ability to modify a chosen sequence in its native locus offers great advantages over conventional transgenesis and RNAi-mediated knockdown, both in terms of versatility and precision. It enables us to manipulate both coding sequences and cis- regulatory elements, to perform gain- and loss-of-function experiments, and to generate reporters and sensors that accurately reflect endogenous gene expression and function. Manipulating a gene in its native context is also a more precise approach because it allows us to study gene variants within their native cis- regulatory environment, where they are expressed in biologically meaningful levels and patterns.
Conventional gene targeting has exploited the natural ability of cells to recombine DNA fragments that bear homologous sequences, copying genetic changes from an engineered template sequence to a homologous site in the genome. In practice this often involves integrating an exogenous sequence, including appropriate markers, into the locus of interest. The efficiency of this process is low, in the order of 1 in 103-107 cells receiving the template DNA, and it occurs among a high background of non-homologous integration events [29, 30]. For this reason, conventional gene targeting is workable only in systems where we are able to screen very large numbers of transfected cells and select the rare targeting events, for example, in cultured mammalian cells and in yeast [29, 31–33].
The efficiency of gene targeting, however, is strongly enhanced (100 to 10,000-fold) when the targeted locus is disrupted by a double-strand DNA break [34–38]. For example, double-strand breaks produced by the excision of transposable elements are known to induce homologous recombination around the site of excision [39, 40]. Thus, to improve the efficiency and specificity of gene targeting, much attention has focused on directing double-strand breaks to unique DNA sequences in the genome.
Double-strand breaks can elicit two types of molecular repair mechanism at the site of damage: non-homologous end joining (NHEJ) in which the broken ends are re-ligated to each other, or homology-directed repair (HDR) in which the break is repaired using a homologous DNA sequence as template (see ). NHEJ and HDR have different consequences, which are both relevant for gene editing. NHEJ is the predominant repair mechanism, but it is error-prone, resulting in the introduction of small insertions or deletions (indels) at the site of the break. Thus, NHEJ provides an efficient way to disrupt gene function (knock-out). In contrast, HDR is based on precise copying of the template and can serve to insert specific changes that have been engineered in the repair template (homology-dependent knock-in). NHEJ can also be used to ligate the broken ends to an exogenous linear DNA fragment, in the absence of sequence homology (homology-independent knock-in) [42–45]. NHEJ and HDR are almost ubiquitous in living organisms, so these targeting approaches (summarized in Figure 1) could in principle be applied in any species of interest.
One of the first approaches for generating double-strand breaks at specific sites in the genome exploited natural sequence-specific endonucleases with long recognition sequences. These so-called ‘meganucleases’ recognize sequences that are typically 15 to 30 nucleotides long, providing sufficient specificity to target unique sequences in eukaryotic genomes. Meganucleases have been successfully used in gene targeting [35, 36], but engineering these proteins to target new sequences has proven to be a major challenge . Another approach has relied on artificial triple-helix-forming oligonucleotides [54, 55], but the scope of this approach is also limited because triple-helix formation is only possible with some sequences. Endonucleases with customizable sequence specificities have been a dream of gene targeting since the 1990s.
Modular gene editing nucleases: zinc-finger nucleases and TALENs
A major breakthrough came with the realization that modular DNA recognition domains could be exploited combinatorially to generate nucleases targeting a wide range of sequences . The zinc-finger domain, which typically recognizes 3-nucleotide motifs on DNA, was the first to be exploited in this way. Artificial enzymes, called zinc-finger nucleases (ZFNs), were engineered by joining several zinc-finger domains - recognizing adjacent trinucleotide motifs - to the catalytic domain of the endonuclease FokI (reviewed in ). Sequence-specificity was further increased by engineering ZFNs in a way that requires their heterodimerization through the FokI domain for efficient cleavage . Thus, targeting an 18-nucleotide target site could (in principle) be achieved by a ZFN pair carrying 6 zinc-finger domains with the appropriate sequence specificities. To date, ZFNs have been used to target many genes in diverse organisms, exploiting both NHEJ-mediated knock-out and HDR-mediated knock-in approaches .
Despite their success in demonstrating the power of the modular approach, ZFNs suffer two major drawbacks that have limited their use. First, not all sequences can be targeted by ZFNs, because zinc-finger modules are not yet available for all possible nucleotide triplets (for example, ). Second, the sequence specificity of individual zinc-finger domains cannot always be precisely defined, and may be influenced by neighboring domains in the protein [60, 61]. This context dependence means that ZFN specificity is not easy to predict, leading to failures in targeting  and the need for costly design and testing.
The modular approach was taken a step further with the discovery of the TAL effector (TALE) DNA-binding modules of Xanthomonas bacteria, and their simple DNA recognition code [63, 64]. TAL proteins are transcription factors with a modular DNA-binding region that consists of multiple tandem repeats. Each of these repeats is a small DNA binding domain capable of recognizing a single nucleotide; two amino acid residues within each repeat determine its specificity for A, G, C or T, and this specificity is not significantly influenced by neighboring domains . Thus, using the same combinatorial logic that was applied to ZFNs, TAL effector nuclease (TALEN) heterodimers with pre-defined sequence specificities can be generated by assembling multiple TAL repeats - one per nucleotide of the target site - linked to the catalytic domain of FokI [66–68]. Thus, a 24-nucleotide sequence can be targeted by generating a TALEN heterodimer, where each monomer consists of an array of 12 TAL repeats fused to FokI.
TALENs offer three great advantages over ZFNs. First, the modularity of the TAL domains and the simplicity of their DNA recognition code mean that virtually any sequence can be targeted by TALENs. Second, the specificity of TAL domains does not appear to be as context-dependent as that of zinc fingers, which results in more accurate predictions of target specificity and a higher targeting success rate. Third, gene targeting experiments in diverse species reveal that TALENs are very efficient, yielding targeting efficiencies as high as 30 to 100% for NHEJ-mediated knock-outs and 1 to 10% for HDR-mediated knock-ins (measured as the fraction of injected individuals giving rise to progeny carrying a targeted allele). High targeting rates have been achieved in a wide range of organisms, including insects, nematodes, annelids, tunicates, vertebrates and diverse plants [69–76].
The fact that each TAL domain targets a single nucleotide means that long TAL arrays need to be assembled in order to target unique sequences in a eukaryotic genome. Ingenious protocols have been developed for this purpose [77–79], bringing TALEN technology within the reach of every competent molecular biology lab.
Simple and efficient gene editing using the RNA-guided nuclease CRISPR/Cas9
The last two years have seen the development of a new approach to build endonucleases with customized sequence specificities, which has revolutionized gene editing by its simplicity and efficiency. The approach is borrowed from a highly efficient immune mechanism of bacteria and archaea, which employs RNA-guided endonucleases to specifically target and degrade viral DNA [80–82] (reviewed in ).
The genomes of many prokaryotes possess hypervariable loci, termed clustered regularly interspaced short palindromic repeats (CRISPR), which incorporate short sequences from invading viruses and express them in the form of CRISPR-derived RNAs (crRNAs). These small RNAs associate with specific CRISPR-associated (Cas) proteins to form an active CRISPR/Cas endonuclease complex, whose specificity is determined by simple base complementarity between crRNA and the target viral DNA. Immunity to a viral infection is determined by the presence of corresponding viral sequences in CRISPR loci [80, 84, 85]. The CRISPR mechanism bears some striking analogies with eukaryotic RNAi and piRNA-mediated defense mechanismsa[83, 86].
In a ground-breaking study published in 2012, Jinek and colleagues demonstrated that this nucleotide-based recognition mechanism could provide a straightforward approach for generating customizable nucleases for gene targeting . They used the CRISPR system of Streptococcus pyogenes, which involves a single Cas protein (Cas9) and two RNAs (crRNA and trans-acting antisense RNA, also known as tracRNA) to build an active CRISPR/Cas endonuclease complex. Jinek et al. showed that it is possible to combine these two RNAs into a single chimeric guide RNA (known as gRNA or sgRNA) that can efficiently direct Cas9 activity to specific DNA targets in vitro (Figure 2). The guide RNA has a region of 20 nucleotides at its 5′ end, which binds the target DNA and determines specificity; any 20-nucleotide sequence (N20) can be placed at that siteb. The 3′ region of the guide RNA, corresponding to the bacterial tracRNA, is an invariable sequence that is required to form a complex with Cas9.
Target recognition also depends on additional interactions between Cas9 and the target DNA, which require the presence of a specific sequence motif, the ‘protospacer adjacent motif’ (PAM), immediately downstream of the 20-nucleotide sequence targeted by the guide RNA. The PAM sequence does not have a counterpart on the guide RNA (Figure 2). Streptococcus pyogenes CRISPR/Cas9 requires a PAM that is NGG; it can thus target any sequence that matches the motif N20NGG. Once a target is bound, two separate nuclease domains of Cas9 are involved in cleaving each strand of the target DNA. Cleavage occurs within the guide RNA target region, usually three nucleotides upstream of the PAM [81, 82, 87].
Within less than two years since the first demonstration of CRISPR-mediated gene editing [89–93], there has been an explosion of reports describing the application of CRISPR in diverse animal and plant species (reviewed in ). The approaches to generate knock-outs and knock-ins are similar to those previously described for ZFNs and TALENs, relying on the endonuclease to generate a double-strand break at the targeted locus and on the cell’s imprecise or template-directed mechanisms of repairing that break (Figure 1). However, compared to ZFNs and TALENs, CRISPR has radically improved the accessibility of gene targeting due to its straightforward approach for customizing sequence specificity, via target-specific guide RNAs. Its targeting efficiencies are comparable with the best efficiencies achieved using TALENs in a wide range of animals and plants (for example, [46, 51, 95, 96]), including organisms where gene targeting is not yet widely available, such as silkmoths, axolotls, Xenopus and monkeys [97–100]. Moreover, while TALEN activity is inhibited by DNA methylation , CRISPR activity does not appear to be so . Table 1 summarizes the relative benefits and drawbacks of ZFNs, TALENs and CRISPR.
CRISPR delivery and target range
Different approaches have been used to deliver gene-editing nucleases into target cells, including microinjection and transfection. CRISPR systems require the delivery of Cas9 together with guide RNA. Cas9 may be expressed from a helper plasmid carrying the coding sequence of Cas9 (fused with a nuclear localization signal and sometimes ‘codon-optimized’) under the control of an appropriate promoter. Alternatively, if a promoter is unavailable, Cas9 can be provided in the form of in vitro transcribed capped mRNA or as purified recombinant protein [104, 105]. In established models, such as Drosophila, transgenic strains have been generated that express Cas9 in the germ line [47, 48, 96].
Delivery of the guide RNA is more constrained because in vitro transcription or plasmid-derived expression impose some limitations on the sequence of the RNA and may, therefore, influence the range of potential targets. In vitro transcription of RNA is usually carried out using the RNA polymerase of bacteriophages T7, T3 or SP6, which generate transcripts that start with GG (T3 or T7 RNA polymerase) or GA (SP6 polymerase) . The alternative to in vitro synthesis is to express the guide RNA from a plasmid or transgene in vivo. Small RNAs are conventionally expressed using RNA polymerase III promoters, because they often require precisely defined initiation and termination sites and should not enter the mRNA processing pathway. Thus, guide RNAs are usually expressed via the U6 snRNA promoter [46, 89–91, 107]; U6 promoters generate transcripts that start with a G.
In principle, these sequence constraints dictate that, using Streptococcus pyogenes Cas9 (with NGG as a PAM), we can optimally target sequences that contain GGN18NGG or GAN18NGG motifs using in vitro transcribed guide RNAs and GN19NGG using the U6 promoter to drive guide RNA expression. In practice, however, it seems that mismatches at the 5′ end of the guide RNA are well tolerated, giving acceptable levels of gene targeting [51, 88]. Moreover, alternative guide RNA expression strategies are emerging, which overcome the constraints imposed by the U6 promoter [108–110]. Thus, the PAM sequence may be the only stringent limitation to CRISPR’s target range.
PAM recognition seems to play a key role in CRISPR target recognition , so the requirement for a PAM in the target sequence is likely to remain. The sequence constraints imposed by the PAM may be overcome by exploiting the natural diversity of CRISPR systems [112–114], or by rational design and artificial selection of Cas9 variants that recognize different PAM sequences.
Whether using ZFNs, TALENs or CRISPR, targeting a chosen, unique sequence in the genome may be accompanied by unintended cleavage at other loci. Several studies have investigated the specificity and potential off-targets of CRISPR [89, 102, 115–121], focusing on the stringency of base-pairing between the guide RNA and the target. These studies have established that mismatches are tolerated, especially at the 5′ end of the guide RNA, but that there are no simple rules predicting the likelihood of mis-targeting based on the number and position of mismatches. In some cases, even sequences with multiple mismatches to the guide RNA were targeted efficiently . In one study, targeting specificity deteriorated when high concentrations of CRISPR/Cas9 were used .
A recent study has also highlighted the role played by the PAM sequence as CRISPR/Cas9 interrogates complex DNA sequences to identify target sites . The study shows that the CRISPR/Cas9 complex first identifies potential targets based on the PAM sequence, and then interrogates these for sequence complementarity with the guide RNA. The complex does not appear to interact with sequences that match the guide RNA targeting motif (N20) but have no adjacent PAM. These observations suggest that off-targets will generally not include sequences that are lacking the PAM. In species where the genome sequence is known, computational tools are now routinely used to select guide RNAs and to evaluate potential off-targeting based on sequence similarity and on the presence of a PAM (see Online Resources for CRISPR in Table 2).
A number of approaches can be taken to confront the off-target problem and to mitigate its effects. First, it is often possible to control for unspecific effects through appropriate experimental design. A strategy commonly employed in RNAi studies is to examine whether consistent phenotypes are obtained by targeting different parts of a gene, using non-overlapping double-stranded RNA fragments or siRNAs . The same strategy can be easily applied in most cases of gene targeting by CRISPR, by using different guide RNAs. Guide RNAs targeting different sequences are very unlikely to share the same off-target effects. Notably, heteroallelic combinations of knock-outs generated using different guide RNAs are likely to complement off-target mutations and to give highly specific knock-out phenotypes.
Strategies for improving the specificity of CRISPR are also beginning to emerge, exploiting the combined action of pairs of CRISPR nucleases, or methods that increase the specificity of individual nucleases. The first approach relies on mutants of Cas9, known as nickases, that cleave only one of the two DNA strands. Using such mutants, a double strand break can be generated by targeting a pair of closely linked single-strand breaks (nicks) on opposite DNA strands. The requirement that these nicks coincide drastically improves targeting specificity compared to that of single CRISPR nucleases [44, 117, 119, 124]. A variant of this approach combines the CRISPR/Cas9 DNA binding activity with the FokI endonuclease, whose dimerization requirements ensure that no nicking occurs at off-target sites [109, 125]. For efficient cleavage, these ‘paired nickase’ approaches require that adjacent target sites, offset by up to 30 nucleotides, can be found on opposite DNA strands.
A second approach to increase the specificity of targeting by CRISPR relies on the observation that short recognition sequences are less forgiving in terms of allowed mismatches between the guide RNA and its targets  (similar observations on specificity and target size have been made with TALENs, ). Thus, guide RNAs with targeting sequences of 17 to 19 nucleotides show high targeting efficiencies and much reduced off-target effects compared to ones with canonical 20-nucleotide targeting sequences .
Ultimately, it may also be possible to improve the stringency of CRISPR target recognition by selecting or engineering Cas9 nucleases that are intrinsically less promiscuous.
Opportunities for evo-devo and future challenges
CRISPR-mediated gene targeting opens a wide range of opportunities, both in established model organisms and in newly emerging ones. A quick guide for applying CRISPR in new species is given in Table 2.
Knock-out approaches will surely be more widely applied in newly established experimental systems due to the extraordinary efficiency of NHEJ-mediated knock-out, which approaches 100% in some species. Generating a null-allele or disrupting a specific cis- regulatory element now seems within reach in a wide range of animals and will be primarily limited by our ability to screen for these mutations (by phenotype or by PCR) and to maintain mutant stocks.
To some extent, the high efficiency of CRISPR- and TALEN-mediated knock-out may also help to overcome the problem of stock-keeping. The high frequency of bi-allelic knock-out in injected embryos using CRISPR or TALEN has raised the possibility of carrying out ‘G0 genetics’, examining phenotypes directly in the injected embryos (for example, [48, 98, 122, 130, 131]). Phenotypic analysis without crosses could be used as a ‘quick and dirty’ approach in species that have long generation times, or for preliminary screens on a large number of candidate genes, similar to RNAi. The obvious drawback of this type of analysis is the genetic mosaicism of the organism, which is difficult to control and will lead to partial and variable phenotypes.
However, mosaicism could also be an advantage in contexts where genetic manipulation within specific cell lineages or in random cell clones is desirable to overcome lethality, or to assess cell autonomous versus non-autonomous effects of gene function. Particularly so when the extent of mosaicism can be monitored or manipulated (see ). Tissue- and stage-specific knock-outs may be achieved by manipulating the expression of Cas9 .
Knock-in approaches provide an even wider range of opportunities, including precise modification of genes in their native genomic context, and generating visible reporters for regulatory and physiological events, and drivers for transgene expression. The major challenge to overcome here is that, for any given guide RNA or TALEN, the frequency of mutagenic NHEJ repair will be much higher (by an order of magnitude) than the frequency of HDR- or NHEJ-driven knock-in (see Figure 1). A high knock-out frequency in the somatic cells of injected animals can lead to lethality that prevents knock-ins to be recovered in the next generation. This problem could be overcome in a number of ways: by using germline-specific cis-regulatory elements to restrict the activity of Cas9 to the germline [48, 123]; by targeting sites that are unlikely to be lethal if mutated, for example, targeting knock-ins to intronic sequences, with appropriate splice signals to generate functional gene fusions; by finding ways to improve the efficiency of HDR relative to NHEJ, such as by knocking down the activity of DNA ligase 4 or other factors that are essential for NHEJ [49, 50, 132]; or by developing strategies that exploit NHEJ-mediated gene knock-ins to achieve higher efficiencies [42–45] (see Figure 1).
Beyond knock-out and knock-in strategies, the ability to direct double-strand breaks to specific sites in the genome raises the prospect of chromosome engineering. Generating chromosomal deletions and inversions is presently not one of the usual tools employed in evo-devo, but one only needs to consider the enormous contribution of balancer chromosomes in Drosophila genetics to appreciate its potential value in emerging model organisms . Balancers are invaluable tools for selecting and keeping track of chromosomes bearing mutations, especially when these mutations are deleterious and not associated with a dominant marker (for example, the knock-out of an essential gene). CRISPR and TALENs now allow us to generate targeted chromosomal inversions [134–136] associated with recessive lethal mutations and, thus, to create balancers, in organisms where a genome sequence and map are available.
Last but not least, the customizable specificity of CRISPR may be harnessed to direct other molecular effectors - besides nucleases - to specific sites in the genome (reviewed in ). For example, catalytically inactive versions of Cas9 have been used to interfere with transcription [138, 139], coupled with transcriptional activators, repressors or chromatin modifiers to generate artificial transcriptional regulators [117, 139–143], or linked with fluorescent proteins to reveal chromosome dynamics . This approach allows us to manipulate the activity of regulatory elements in their native context without introducing changes in their nucleotide sequence, providing tools of unprecedented precision in our efforts to manipulate and to understand gene regulation.
CRISPR technology is young - barely two years old - and still rapidly evolving. Improvements, new applications and adaptations of the technique to new species have been published at overwhelming speed during the last year, and surely more are forthcoming. This is a true revolution for comparative studies. Practical issues, such as the delivery method and our ability to select and to propagate mutants, are still likely to limit the full deployment of CRISPR in many species. Notwithstanding these issues, targeted mutagenesis and precise gene editing are now within reach in a very wide range of organisms.
aIt is interesting to note that the adaptive diversification, specificity and efficiency of immunity mechanisms provide the basis for some of the most powerful tools in molecular biology: restriction enzymes, antibodies, RNAi and CRISPR.
bSomewhat shorter sequences may also be used (see Off-target effects).
Clustered regularly interspaced short palindromic repeats
Non-homologous end joining
Protospacer adjacent motif
TAL effector nuclease
Trans-acting antisense RNA
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998, 391: 806-811. 10.1038/35888.
Hamilton AJ: A species of small antisense RNA in posttranscriptional gene silencing in plants. Science. 1999, 286: 950-952. 10.1126/science.286.5441.950.
Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA: Targeted mRNA degradation by double-stranded RNA in vitro. Genesand Development. 1999, 13: 3191-3197. 10.1101/gad.13.24.3191.
Tijsterman M, Ketting RF, Plasterk RHA: The genetics of RNA silencing. Annual Review Of Genetics. 2002, 36: 489-519. 10.1146/annurev.genet.36.043002.091619.
Nasevicius A, Ekker SC: Effective targeted gene “knockdown” in zebrafish. Nat Genet. 2000, 26: 216-220. 10.1038/79951.
Wahlestedt C, Salmi P, Good L, Kela J, Johnsson T, Hokfelt T, Broberger C, Porreca F, Lai J, Ren K, Ossipov M, Koshkin A, Jakobsen N, Skouv J, Oerum H, Jacobsen MH, Wengel J: Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc Natl Acad Sci USA. 2000, 97: 5633-5638. 10.1073/pnas.97.10.5633.
Heasman J: Morpholino oligos: making sense of antisense?. Dev Biol. 2002, 243: 209-214. 10.1006/dbio.2001.0565.
Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M: Silencing of microRNAs in vivo with “antagomirs.”. Nature. 2005, 438: 685-689. 10.1038/nature04303.
Sanchez Alvarado A, Newmark PA: Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc Natl Acad Sci USA. 1999, 96: 5049-5054. 10.1073/pnas.96.9.5049.
Bucher G, Scholten J, Klingler M: Parental RNAi in Tribolium (Coleoptera). Curr Biol. 2002, 12: R85-R86. 10.1016/S0960-9822(02)00666-8.
Layden MJ, Röttinger E, Wolenski FS, Gilmore TD, Martindale MQ: Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis. Nat Protoc. 2013, 8: 924-934. 10.1038/nprot.2013.009.
Conzelmann M, Williams EA, Tunaru S, Randel N, Shahidi R, Asadulina A, Berger J, Offermanns S, Jékely G: Conserved MIP receptor-ligand pair regulates Platynereis larval settlement. Proc Natl Acad Sci USA. 2013, 110: 8224-8229. 10.1073/pnas.1220285110.
Yamada L: Morpholino-based gene knockdown screen of novel genes with developmental function in Ciona intestinalis. Development. 2003, 130: 6485-6495. 10.1242/dev.00847.
Reddien PW, Bermange AL, Murfitt KJ, Jennings JR, Sanchez Alvarado A: Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria. Dev Cell. 2005, 8: 635-649. 10.1016/j.devcel.2005.02.014.
Oates AC, Bruce AEE, Ho RK: Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo. Dev Biol. 2000, 224: 20-28. 10.1006/dbio.2000.9761.
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001, 411: 494-498. 10.1038/35078107.
Echeverri CJ, Beachy PA, Baum B, Boutros M, Buchholz F, Chanda SK, Downward J, Ellenberg J, Fraser AG, Hacohen N, Hahn WC, Jackson AL, Kiger A, Linsley PS, Lum L, Ma Y, Mathey-Prevot B, Root DE, Sabatini DM, Taipale J, Perrimon N, Bernards R: Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat Methods. 2006, 3: 777-779. 10.1038/nmeth1006-777.
Berghammer AJ, Klingler M, Wimmer EA: A universal marker for transgenic insects. Nature. 1999, 402: 370-371. 10.1038/46463.
Sasakura Y, Awazu S, Chiba S, Satoh N: Germ-line transgenesis of the Tc1/mariner superfamily transposon Minos in Ciona intestinalis. Proc Natl Acad Sci USA. 2003, 100: 7726-7730. 10.1073/pnas.1230736100.
Pavlopoulos A, Averof M: Establishing genetic transformation for comparative developmental studies in the crustacean Parhyale hawaiensis. Proc Natl Acad Sci USA. 2005, 102: 7888-7893. 10.1073/pnas.0501101102.
Sobkow L, Epperlein HH, Herklotz S, Straube WL, Tanaka EM: A germline GFP transgenic axolotl and its use to track cell fate: Dual origin of the fin mesenchyme during development and the fate of blood cells during regeneration. Dev Biol. 2006, 290: 386-397. 10.1016/j.ydbio.2005.11.037.
Wittlieb J, Khalturin K, Lohmann JU, Anton-Erxleben F, Bosch TCG: Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis. Proc Natl Acad Sci USA. 2006, 103: 6208-6211. 10.1073/pnas.0510163103.
Renfer E, Amon-Hassenzahl A, Steinmetz PR, Technau U: A muscle-specific transgenic reporter line of the sea anemone, Nematostella vectensis. Proc Natl Acad Sci USA. 2010, 107: 104-108. 10.1073/pnas.0909148107.
Backfisch B, Rajan VBV, Fischer RM, Lohs C, Arboleda E, Tessmar-Raible K, Raible F: Stable transgenesis in the marine annelid Platynereis dumerilii sheds new light on photoreceptor evolution. Proc Natl Acad Sci USA. 2013, 110: 193-198. 10.1073/pnas.1209657109.
Blair SS: Genetic mosaic techniques for studying Drosophila development. Development. 2003, 130: 5065-5072. 10.1242/dev.00774.
Kragl M, Knapp D, Nacu E, Khattak S, Maden M, Epperlein HH, Tanaka EM: Cells keep a memory of their tissue origin during axolotl limb regeneration. Nature. 2009, 460: 60-65. 10.1038/nature08152.
Konstantinides N, Averof M: A common cellular basis for muscle regeneration in arthropods and vertebrates. Science. 2014, 343: 788-791. 10.1126/science.1243529.
Yeh J-RJ, Crews CM: Chemical genetics adding to the developmental biology toolbox. Dev Cell. 2003, 5: 11-19. 10.1016/S1534-5807(03)00200-4.
Capecchi MR: Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet. 2005, 6: 507-512. 10.1038/nrg1619.
Deng C, Capecchi MR: Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol Cell Biol. 1992, 12: 3365-3371.
Hinnen A, Hicks JB, Fink GR: Transformation of yeast. Proc Natl Acad Sci USA. 1978, 75: 1929-1933. 10.1073/pnas.75.4.1929.
Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS: Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature. 1985, 317: 230-234. 10.1038/317230a0.
Thomas KR, Folger KR, Capecchi MR: High frequency targeting of genes to specific sites in the mammalian genome. Cell. 1986, 44: 419-428. 10.1016/0092-8674(86)90463-0.
Szostak JW, Orr-Weaver TL, Rothstein RJ, Stahl FW: The double-strand-break repair model for recombination. Cell. 1983, 33: 25-35. 10.1016/0092-8674(83)90331-8.
Rouet P, Smih F, Jasin M: Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol Cell Biol. 1994, 14: 8096-8106.
Choulika A, Perrin A, Dujon B, Nicolas JF: Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol. 1995, 15: 1968-1973.
Donoho G, Jasin M, Berg P: Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells. Mol Cell Biol. 1998, 18: 4070-4078.
Taghian DG, Nickoloff JA: Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells. Mol Cell Biol. 1997, 17: 6386-6393.
Gloor GB, Nassif NA, Johnson-Schlitz DM, Preston CR, Engels WR: Targeted gene replacement in Drosophila via P element-induced gap repair. Science. 1991, 253: 1110-1117. 10.1126/science.1653452.
Robert V, Bessereau J-L: Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks. Embo J. 2007, 26: 170-183. 10.1038/sj.emboj.7601463.
Krebs JE, Kilpatrick ST, Goldstein SE: Lewin’s Genes XI. 2014, Burlington, MA: Jones & Bartlett Publishers
Maresca M, Lin VG, Guo N, Yang Y: Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining. Genome Res. 2013, 23: 539-546. 10.1101/gr.145441.112.
Cristea S, Freyvert Y, Santiago Y, Holmes MC, Urnov FD, Gregory PD, Cost GJ: In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration. Biotechnol Bioeng. 2013, 110: 871-880. 10.1002/bit.24733.
Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F: Double nicking by RNA-Guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013, 154: 1380-1389. 10.1016/j.cell.2013.08.021.
Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F: Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res. 2014, 24: 142-153. 10.1101/gr.161638.113.
Dickinson DJ, Ward JD, Reiner DJ, Goldstein B: Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Meth. 2013, 10: 1028-1034. 10.1038/nmeth.2641.
Gratz SJ, Ukken FP, Rubinstein CD, Thiede G, Donohue LK, Cummings AM, O’Connor-Giles KM: Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila. Genetics. 2014, 196: 961-971. 10.1534/genetics.113.160713.
Port F, Chen HM, Lee T, Bullock SL: Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci USA. 2014, 111: E2967-E2976. 10.1073/pnas.1405500111.
Beumer KJ, Trautman JK, Bozas A, Liu JL, Rutter J, Gall JG, Carroll D: Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc Natl Acad Sci USA. 2008, 105: 19821-19826. 10.1073/pnas.0810475105.
Beumer KJ, Trautman JK, Mukherjee K, Carroll D: Donor DNA Utilization during Gene Targeting with Zinc-finger Nucleases. G3 (Bethesda). 2013, doi:10.1534/g3.112.005439
Hwang WY, Fu Y, Reyon D, Maeder ML, Kaini P, Sander JD, Joung JK, Peterson RT, Yeh J-RJ: Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLoS ONE. 2013, 8: e68708-10.1371/journal.pone.0068708.
Chen F, Pruett-Miller SM, Huang Y, Gjoka M, Duda K, Taunton J, Collingwood TN, Frodin M, Davis GD: High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat Meth. 2011, 8: 753-755. 10.1038/nmeth.1653.
Takeuchi R, Lambert AR, Mak AN-S, Jacoby K, Dickson RJ, Gloor GB, Scharenberg AM, Edgell DR, Stoddard BL: Tapping natural reservoirs of homing endonucleases for targeted gene modification. Proc Natl Acad Sci USA. 2011, 108: 13077-13082. 10.1073/pnas.1107719108.
Majumdar A, Muniandy PA, Liu J, Liu JL, Liu ST, Cuenoud B, Seidman MM: Targeted gene knock in and sequence modulation mediated by a psoralen-linked triplex-forming oligonucleotide. J Biol Chem. 2008, 283: 11244-11252. 10.1074/jbc.M800607200.
Pei D, Corey DR, Schultz PG: Site-specific cleavage of duplex DNA by a semisynthetic nuclease via triple-helix formation. Proc Natl Acad Sci USA. 1990, 87: 9858-9862. 10.1073/pnas.87.24.9858.
Kim YG, Cha J, Chandrasegaran S: Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA. 1996, 93: 1156-1160. 10.1073/pnas.93.3.1156.
Carroll D: Genome engineering with zinc-finger nucleases. Genetics. 2011, 188: 773-782. 10.1534/genetics.111.131433.
Miller JC, Holmes MC, Wang J, Guschin DY, Lee Y-L, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar EJ: An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol. 2007, 25: 778-785. 10.1038/nbt1319.
Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, Unger-Wallace E, Sander JD, Müller-Lerch F, Fu F, Pearlberg J, Göbel C, Dassie JP, Pruett-Miller SM, Porteus MH, Sgroi DC, Iafrate AJ, Dobbs D, McCray PB, Cathomen T, Voytas DF, Joung JK: Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell. 2008, 31: 294-301. 10.1016/j.molcel.2008.06.016.
Isalan M, Choo Y, Klug A: Synergy between adjacent zinc fingers in sequence-specific DNA recognition. Proc Natl Acad Sci USA. 1997, 94: 5617-5621. 10.1073/pnas.94.11.5617.
Sander JD, Dahlborg EJ, Goodwin MJ, Cade L, Zhang F, Cifuentes D, Curtin SJ, Blackburn JS, Thibodeau-Beganny S, Qi Y, Pierick CJ, Hoffman E, Maeder ML, Khayter C, Reyon D, Dobbs D, Langenau DM, Stupar RM, Giraldez AJ, Voytas DF, Peterson RT, Yeh J-RJ, Joung JK: Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat Meth. 2010, 8: 67-69.
Ramirez CL, Foley JE, Wright DA, Müller-Lerch F, Rahman SH, Cornu TI, Winfrey RJ, Sander JD, Fu F, Townsend JA, Cathomen T, Voytas DF, Joung JK: Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Meth. 2008, 5: 374-375. 10.1038/nmeth0508-374.
Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U: Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors. Science. 2009, 326: 1509-1512. 10.1126/science.1178811.
Moscou MJ, Bogdanove AJ: A simple cipher governs DNA recognition by TAL effectors. Science. 2009, 326: 1501-10.1126/science.1178817.
Guilinger JP, Pattanayak V, Reyon D, Tsai SQ, Sander JD, Joung JK, Liu DR: Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat Meth. 2014, 11: 429-435. 10.1038/nmeth.2845.
Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF: Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010, 186: 757-761. 10.1534/genetics.110.120717.
Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B: TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 2011, 39: 359-372. 10.1093/nar/gkq704.
Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ: A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2011, 29: 143-148. 10.1038/nbt.1755.
Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug RG, Tan W, Penheiter SG, Ma AC, Leung AYH, Fahrenkrug SC, Carlson DF, Voytas DF, Clark KJ, Essner JJ, Ekker SC: In vivo genome editing using a high-efficiency TALEN system. Nature. 2012, 491: 114-118. 10.1038/nature11537.
Watanabe T, Ochiai H, Sakuma T, Horch HW, Hamaguchi N, Nakamura T, Bando T, Ohuchi H, Yamamoto T, Noji S, Mito T: Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. Nat Commun. 2012, 3: 1017-1018.
Beumer KJ, Trautman JK, Christian M, Dahlem TJ, Lake CM, Hawley RS, Grunwald DJ, Voytas DF, Carroll D: Comparing Zinc Finger Nucleases and Transcription Activator-Like Effector Nucleases for Gene Targeting in Drosophila. G3 (Bethesda). 2013, 3: 1717-1725. 2013.
Takasu Y, Sajwan S, Daimon T, Osanai-Futahashi M, Uchino K, Sezutsu H, Tamura T, Zurovec M: Efficient TALEN construction for Bombyx mori gene targeting. PLoS ONE. 2013, 8: e73458-10.1371/journal.pone.0073458.
Lo T-W, Pickle CS, Lin S, Ralston EJ, Gurling M, Schartner CM, Bian Q, Doudna JA, Meyer BJ: Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. Genetics. 2013, 195: 331-348. 10.1534/genetics.113.155382.
Bannister S, Antonova O, Polo A, Lohs C, Hallay N, Valinciute A, Raible F, Tessmar-Raible K: TALENs mediate efficient and heritable mutation of endogenous genes in the marine annelid Platynereis dumerilii. Genetics. 2014, 197: 77-89. 10.1534/genetics.113.161091.
Yoshida K, Treen N, Hozumi A, Sakuma T, Yamamoto T, Sasakura Y: Germ cell mutations of the ascidian Ciona intestinalis with TALE nucleases. Genesis. 2014, 52: 431-439. 10.1002/dvg.22770.
Carroll D: Genome engineering with targetable nucleases. Annu Rev Biochem. 2014, 83: 409-439. 10.1146/annurev-biochem-060713-035418.
Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF: Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011, 39: e82-10.1093/nar/gkr218.
Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK: FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol. 2012, 30: 460-465. 10.1038/nbt.2170.
Sakuma T, Ochiai H, Kaneko T, Mashimo T, Tokumasu D, Sakane Y, Suzuki K-I, Miyamoto T, Sakamoto N, Matsuura S, Yamamoto T: Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Sci Rep. 2013, 3: 3379-
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P: CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science. 2007, 315: 1709-1712. 10.1126/science.1138140.
Garneau JE, Dupuis M-È, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S: The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010, 468: 67-71. 10.1038/nature09523.
Gasiunas G, Barrangou R, Horvath P, Siksnys V: Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA. 2012, 109: E2579-E2586. 10.1073/pnas.1208507109.
Wiedenheft B, Sternberg SH, Doudna JA: RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012, 482: 331-338. 10.1038/nature10886.
Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E: Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005, 60: 174-182. 10.1007/s00239-004-0046-3.
Bolotin A, Quinquis B, Sorokin A, Ehrlich SD: Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005, 151: 2551-2561. 10.1099/mic.0.28048-0.
Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV: A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct. 2006, 1: 7-10.1186/1745-6150-1-7.
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E: A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012, 337: 816-821. 10.1126/science.1225829.
Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK: Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014, 32: 279-284. 10.1038/nbt.2808.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F: Multiplex genome engineering using CRISPR/Cas systems. Science. 2013, 339: 819-823. 10.1126/science.1231143.
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM: RNA-Guided human genome engineering via Cas9. Science. 2013, 339: 823-826. 10.1126/science.1232033.
Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J, Weigel D: RNA-programmed genome editing in human cells. Elife. 2013, 2: e00471-
Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh J-RJ, Joung JK: Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 2013, 31: 227-229. 10.1038/nbt.2501.
Cho SW, Kim S, Kim JM, Kim J-S: Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013, 31: 230-232. 10.1038/nbt.2507.
Sander JD, Joung JK: CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014, 32: 347-355. 10.1038/nbt.2842.
Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V: Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods. 2013, 9: 39-10.1186/1746-4811-9-39.
Kondo S, Ueda R: Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics. 2013, 195: 715-721. 10.1534/genetics.113.156737.
Daimon T, Kiuchi T, Takasu Y: Recent progress in genome engineering techniques in the silkworm, Bombyx mori. Develop Growth Differ. 2013, 56: 14-25.
Flowers GP, Timberlake AT, Mclean KC, Monaghan JR, Crews CM: Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease. Development. 2014, 141: 2165-2171. 10.1242/dev.105072.
Guo X, Zhang T, Hu Z, Zhang Y, Shi Z, Wang Q, Cui Y, Wang F, Zhao H, Chen Y: Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis. Development. 2014, 141: 707-714. 10.1242/dev.099853.
Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J: Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos - 1–s2.0-S0092867414000798-main.pdf. Cell. 2014, 156: 836-843. 10.1016/j.cell.2014.01.027.
Valton J, Dupuy A, Daboussi F, Thomas S, Maréchal A, Macmaster R, Melliand K, Juillerat A, Duchateau P: Overcoming TALE DNA binding domain’s inability to bind methylcytosine : overcoming transcription activator-like effector (TALE) DNA binding domain sensitivity to cytosine methylation. J Biol Chem. 2012, 287: 38433-
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F: DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013, 31: 827-832. 10.1038/nbt.2647.
Kim H, Kim J-S: A guide to genome engineering with programmable nucleases. Nat Rev Genet. 2014, 15: 321-334. 10.1038/nrg3686.
Lee J-S, Kwak S-J, Kim J, Kim A-K, Noh HM, Kim J-S, Yu K: CRISPR/Cas9 mediated genome engineering in Drosophila. G3 (Bethesda). 2014, 4: 1291-1295. 2014.
Cho SW, Lee J, Carroll D, Kim J-S, Lee J: Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics. 2013, 195: 1177-1180. 10.1534/genetics.113.155853.
Imburgio D, Rong M, Ma K, McAllister WT: Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants †. Biochemistry. 2000, 39: 10419-10430. 10.1021/bi000365w.
Ren X, Sun J, Housden BE, Hu Y, Roesel C, Lin S, Liu L-P, Yang Z, Mao D, Sun L, Wu Q, Ji J-Y, Xi J, Mohr SE, Xu J, Perrimon N, Ni J-Q: Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc Natl Acad Sci USA. 2013, 110: 19012-19017. 10.1073/pnas.1318481110.
Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK: Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell. 2014, 54: 698-710. 10.1016/j.molcel.2014.04.022.
Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol. 2014, 32: 569-576. 10.1038/nbt.2908.
Ranganathan V, Wahlin K, Maruotti J, Zack DJ: Expansion of the CRISPR–Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs. Nat Commun. 2014, 5: 4516-
Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA: DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature. 2014, 507: 62-67. 10.1038/nature13011.
Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM: Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Meth. 2013, 10: 1116-1121. 10.1038/nmeth.2681.
Hou Z, Zhang Y, Propson NE, Howden SE, Chu L-F, Sontheimer EJ, Thomson JA: Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci USA. 2013, 110: 15644-15649. 10.1073/pnas.1313587110.
Fonfara I, Le Rhun A, Chylinski K, Makarova KS, Lecrivain AL, Bzdrenga J, Koonin EV, Charpentier E: Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 2014, 42: 2577-2590. 10.1093/nar/gkt1074.
Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA: RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013, 31: 233-239. 10.1038/nbt.2508.
Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD: High-frequency off-target mutagenesis induced by CrIsPr-Cas nucleases in human cells. Nat Biotechnol. 2013, 31: 822-826. 10.1038/nbt.2623.
Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM: CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013, 31: 833-838. 10.1038/nbt.2675.
Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013, 31: 839-843. 10.1038/nbt.2673.
Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim J-S: Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 2014, 24: 132-141. 10.1101/gr.162339.113.
Wu X, Scott DA, Kriz AJ, Chiu AC, Hsu PD, Dadon DB, Cheng AW, Trevino AE, Konermann S, Chen S, Jaenisch R, Zhang F, Sharp PA: Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol. 2014, 32: 670-676. 10.1038/nbt.2889.
Kuscu C, Arslan S, Singh R, Thorpe J, Adli M: Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol. 2014, 32: 677-683. 10.1038/nbt.2916.
Blitz IL, Biesinger J, Xie X, Cho KWY: Biallelic genome modification in F0 Xenopus tropicalis embryos using the CRISPR/Cas system. Genesis. 2013, 51: 827-834. 10.1002/dvg.22719.
Sebo ZL, Lee HB, Peng Y, Guo Y: A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering. Fly (Austin). 2014, 8: 52-57. 10.4161/fly.26828.
Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, Skarnes WC: Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Meth. 2014, 11: 399-402. 10.1038/nmeth.2857.
Guilinger JP, Thompson DB, Liu DR: Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol. 2014, 32: 577-582. 10.1038/nbt.2909.
Qiu P, Shandilya H, D’Alessio JM, O’Connor K, Durocher J, Gerard GF: Mutation detection using Surveyor nuclease. Biotechniques. 2004, 36: 702-707.
Kim HJ, Lee HJ, Kim H, Cho SW, Kim JS: Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res. 2009, 19: 1279-1288. 10.1101/gr.089417.108.
Bassett AR, Tibbit C, Ponting CP, Liu J-L: Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 2013, 4: 220-228. 10.1016/j.celrep.2013.06.020.
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F: Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013, 8: 2281-2308. 10.1038/nprot.2013.143.
Jao LE, Wente SR, Chen W: Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA. 2013, 110: 13904-10.1073/pnas.1308335110.
Treen N, Yoshida K, Sakuma T, Sasaki H, Kawai N, Yamamoto T, Sasakura Y: Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function in Ciona. Development. 2014, 141: 481-487. 10.1242/dev.099572.
Qi Y, Zhang Y, Zhang F, Baller JA, Cleland SC, Ryu Y, Starker CG, Voytas DF: Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways. Genome Res. 2013, 23: 547-554. 10.1101/gr.145557.112.
Greenspan RJ: Fly Pushing: The Theory and Practice of Drosophila Genetics. 2004, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press,
Xiao A, Wang Z, Hu Y, Wu Y, Luo Z, Yang Z, Zu Y, Li W, Huang P, Tong X, Zhu Z, Lin S, Zhang B: Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res. 2013, 41: e141-10.1093/nar/gkt464.
Choi PS, Meyerson M: Targeted genomic rearrangements using CRISPR/Cas technology. Nat Commun. 2014, 5: 3728-
Park C-Y, Kim J, Kweon J, Son JS, Lee JS, Yoo J-E, Cho S-R, Kim J-H, Kim J-S, Kim D-W: Targeted inversion and reversion of the blood coagulation factor 8 gene in human iPS cells using TALENs. Proc Natl Acad Sci USA. 2014, 111: 9253-9258. 10.1073/pnas.1323941111.
Mali P, Esvelt KM, Church GM: Cas9 as a versatile tool for engineering biology. Nat Meth. 2013, 10: 957-963. 10.1038/nmeth.2649.
Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA: Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013, 152: 1173-1183. 10.1016/j.cell.2013.02.022.
Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS: CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013, 154: 442-451. 10.1016/j.cell.2013.06.044.
Maeder ML, Linder SJ, Reyon D, Angstman JF, Fu Y, Sander JD, Joung JK: Robust, synergistic regulation of human gene expression using TALE activators. Nat Meth. 2013, 10: 243-245. 10.1038/nmeth.2366.
Crocker J, Stern DL: TALE-mediated modulation of transcriptional enhancers in vivo. Nat Meth. 2013, 10: 762-767. 10.1038/nmeth.2543.
Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA: RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Meth. 2013, 10: 973-976. 10.1038/nmeth.2600.
Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R: Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Research. 2013, 23: 1163-1171. 10.1038/cr.2013.122.
Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li G-W, Park J, Blackburn EH, Weissman JS, Qi LS, Huang B: Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell. 2013, 155: 1479-1491. 10.1016/j.cell.2013.12.001.
We thank Thomas Auer, Filippo del Bene, Frédéric Flamant, Martin Klingler, Nikos Konstantinides, Zacharias Kontarakis, Tassos Pavlopoulos, Miltos Tsiantis and members of our lab for reading and commenting on the manuscript. Our work is supported by grant ANR-12-CHEX-0001-01 of the Agence Nationale de la Recherche, by the Marie Curie ITN network ‘NEPTUNE’ and by a fellowship from the Université Claude Bernard Lyon 1.
The authors declare that they have no competing interests.
AFG and MA wrote the manuscript. Both authors read and approved the final manuscript.
About this article
Cite this article
Gilles, A.F., Averof, M. Functional genetics for all: engineered nucleases, CRISPR and the gene editing revolution. EvoDevo 5, 43 (2014). https://doi.org/10.1186/2041-9139-5-43
- Comparative developmental biology
- Model organisms
- Gene targeting
- Homologous recombination
- Gene-editing nucleases