SEARCH

SEARCH BY CITATION

Keywords:

  • zinc-finger nucleases;
  • transcription activator-like effector nucleases;
  • clustered regularly interspaced short palindromic repeats;
  • genome editing

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. The basics of nuclease tools: ZFN, TALEN and CRISPR/Cas
  5. Current construction methods for EENs and RGENs
  6. Diverse applications of EENs and RGENs
  7. Genome editing in animals
  8. Genome editing in cultured cells
  9. Perspectives
  10. References

Genome editing with engineered endonucleases is rapidly becoming a staple method in developmental biology studies. Engineered nucleases permit random or designed genomic modification at precise loci through the stimulation of endogenous double-strand break repair. Homology-directed repair following targeted DNA damage is mediated by co-introduction of a custom repair template, allowing the derivation of knock-out and knock-in alleles in animal models previously refractory to classic gene targeting procedures. Currently there are three main types of customizable site-specific nucleases delineated by the source mechanism of DNA binding that guides nuclease activity to a genomic target: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Among these genome engineering tools, characteristics such as the ease of design and construction, mechanism of inducing DNA damage, and DNA sequence specificity all differ, making their application complementary. By understanding the advantages and disadvantages of each method, one may make the best choice for their particular purpose.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. The basics of nuclease tools: ZFN, TALEN and CRISPR/Cas
  5. Current construction methods for EENs and RGENs
  6. Diverse applications of EENs and RGENs
  7. Genome editing in animals
  8. Genome editing in cultured cells
  9. Perspectives
  10. References

In the elucidation of gene function, modification of endogenous DNA sequences is a mainstay technology. Historically, functional genomics has made extensive use of forward genetic strategies; random mutagenesis by transgenics (viral or transposon-mediated) (Stanford et al. 2001) or chemical mutagens such as N-ethyl-N-nitrosourea (ENU) and ethylmethanesulfonate (EMS) (Acevedo-Arozena et al. 2008). By nature, random mutagenesis requires a huge investment of effort to screen for desirable mutants. In reverse genetics, intentional changes to the genome are achieved by unconstrained homologous recombination (HR) using targeting vectors (Capecchi 2005). However, effective HR in eukaryotes has been generally limited to yeast, chicken DT40 cells, and mouse embryonic stem (ES) cells. Recently, these shortcomings have been overcome by engineered endonucleases (EENs) and RNA-guided endonucleases (RGENs) (Pauwels et al. 2013). EENs and RGENs are created to recognize specific genomic loci and induce double-strand breaks (DSBs) at those target sites, stimulating DNA repair pathways that mediate gene targeting (Joung & Sander 2013).

In this article, we will provide an overview of current genome engineering technologies using EENs and RGENs. We will discuss the nature of EENs and RGENs, how to construct and utilize them for genome editing, as well as outline how their application in animals and cultured cells has so far been used to derive genetic models of differentiation and development.

The basics of nuclease tools: ZFN, TALEN and CRISPR/Cas

  1. Top of page
  2. Abstract
  3. Introduction
  4. The basics of nuclease tools: ZFN, TALEN and CRISPR/Cas
  5. Current construction methods for EENs and RGENs
  6. Diverse applications of EENs and RGENs
  7. Genome editing in animals
  8. Genome editing in cultured cells
  9. Perspectives
  10. References

The first EEN developed for targeted genome modification was the zinc-finger nuclease (ZFN), a chimeric protein composed of zinc-finger (ZF) modules and a FokI nuclease domain (Kim et al. 2011). The isolated nuclease domain, lacking DNA recognition specificities, relied on fusion with ZFs engineered to bind specific DNA sequences. As one ZF binds to three bases, a combined ZF array can recognize 9–18 bp (Fig. 1a) (Urnov et al. 2010). Similar in their conception, TALENs (transcription activator-like effector nucleases) are chimeric proteins composed of a nuclease domain and concatamerized DNA-binding modules originally discovered in plant pathogenic bacterial proteins called transcription activator-like effectors (TALEs). TALE repeat domains typically consist of 34 amino acids, where a single repeat recognizes one DNA base through the two central amino acids, called repeat-variable di-residues or RVDs (Boch et al. 2009; Moscou & Bogdanove 2009). Native TALE proteins consist of an N-terminal domain, a central segment of RVD repeat domains and a C-terminal domain including a transcriptional activation motif (Bogdanove et al. 2010). In the case of TALENs, around 15–20 RVDs are assembled in a single molecule. When repurposed as TALENs, customized repeat domains with truncated N- and C-terminal TALE domains (excluding the native transcriptional activator region) are typically fused to the same FokI nuclease domain used by ZFNs (Fig. 1b) (Mussolino & Cathomen 2012). Due to the nature of the FokI nuclease, both ZFNs and TALENs are required to work in juxtaposed pairs, where FokI dimerization induces local endonuclease activity (illustrated in Fig. 1a,b). This dimerization requirement conveys high specificity for DSB generation only at the target locus.

image

Figure 1. Schematic drawings of zinc-finger nuclease (ZFN) (a), transcription activator-like effector nuclease (TALEN) (b) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (c) enzyme structures complexed with genomic DNA. ZFNs and TALENs bind as juxtaposed pairs. Green ovals indicate ZF modules. White boxes indicate N- and C-terminal domains of TALE, while blue boxes indicate central DNA binding repeats. Black notched circles represent FokI monomers. Purple ovals represent Cas9. The gRNA is shown as a hairpin structure mediating DNA interaction. gRNA, guide RNA.

Download figure to PowerPoint

In attempts to increase both activity and specificity, several variations on the nuclease domain have been reported. A highly-active FokI mutant called ‘Sharkey’ was developed for ZFNs by directed evolution (Guo et al. 2010). Sharkey-ZFNs are certainly effective in vivo in animals (Ansai et al. 2012; Kawai et al. 2012; Ochiai et al. 2012; Watanabe et al. 2012), yet Sharkey-TALENs appear not to recapitulate increased activity (Pillay et al. 2013). In order to decrease genotoxicity by off-target activity resulting from self-dimerization, obligate heterodimers of FokI have been used for both ZFNs (Miller et al. 2007; Ramalingam et al. 2011) and TALENs (Tesson et al. 2011; Cade et al. 2012). When nuclease activity of only one of the ZFN/TALEN pair is disrupted, heterodimerizing FokI nuclease domains are prone to induce a nick on one strand, and are therefore less likely to induce a DSB (Kim et al. 2012; Ramirez et al. 2012; Wang et al. 2013). Since site-specific nickases can increase the efficiency of HR without promoting error-prone non-homologous end-joining (NHEJ), safer genome editing can be performed.

Although nuclease pairs can cooperate to promote specificity, transient co-delivery of a pair of nucleases may prove technically problematic. To resolve this issue, monomer-type ZFNs and TALENs have been developed using several strategies. One example uses single-chain FokI (scFokI); a tandemly-aligned pair of FokI nuclease domains in one protein (Minczuk et al. 2008; Mino et al. 2009; Mori et al. 2009). ZF-scFokI has proven activity in vitro and in mitochondria of cultured cells, yet there are no further reports to complement these findings. On the other hand, ZF and TALE proteins linked to a homing endonuclease, I-TevI, are active in nuclear genome editing (Kleinstiver et al. 2012; Beurdeley et al. 2013). Furthermore, TALE-TevI fusion proteins can act not only as nucleases but also nickases, and paired TALE-TevI nickases can induce DSBs at their target site (Beurdeley et al. 2013). Thus, several EEN variants may prove to be potent alternative choices for genome engineering over canonical FokI-based EENs.

Another single nuclease method guides target specificity by other means. In contrast to EENs, RGENs such as CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated) make use of short guide RNAs (gRNAs) to drive the RNA-binding Cas9 nuclease to a complementary DNA sequence for targeted DSB generation (Jinek et al. 2012; Cong et al. 2013; Mali et al. 2013a). Cas9-gRNA interaction has no DNA base recognition specificities excluding the proto-spacer adjacent motif (PAM), allowing the remainder of the gRNA to address target specificity. Current CRISPR/Cas systems are derived primarily from Streptococcus pyogenes, where the gRNA PAM sequence is 5′-NGG-3′, limiting potential targets to sequences prefixed with ‘NGG’. This limitation in design may eventually relax through increased application of Cas9/gRNA pairs from other species. For example, Streptococcus thermophilus Cas9 requires 5′-NNAGAAW-3′ (Cong et al. 2013) while Neisseria meningitidis Cas9 requires 5′-NNNNGATT-3′ (Hou et al. 2013). A plethora of analogous and completely orthogonal CRISPR/Cas systems have recently been characterized in bacteria and human cells (Esvelt et al. 2013), promising further diversity in genome engineering applications. Normally, a single gRNA and Cas9 combine to induce a DSB at the target site; however, analogous to FokI, the nuclease activity of Cas9 may also be converted to a nickase (Jinek et al. 2012). Like ZF- and TALE-nickases, Cas9-nickases induce HR over NHEJ, although off-target DSBs are not completely eliminated (Ran et al. 2013a).

Additionally, there are a variety of alternative strategies to ZFNs, TALENs and CRISPR/Cas. Engineered meganucleases have been successfully used, yet they remain refractory to customization of sequence specificity (Grizot et al. 2009; Munoz et al. 2011; Menoret et al. 2013). A DNA-based DSB-inducing molecule called ARCUT (Komiyama 2013), is another potential alternative. The possibility remains that such strategies, or as-of-yet undiscovered methods will evolve into commonplace genome editing tools in the future.

Current construction methods for EENs and RGENs

  1. Top of page
  2. Abstract
  3. Introduction
  4. The basics of nuclease tools: ZFN, TALEN and CRISPR/Cas
  5. Current construction methods for EENs and RGENs
  6. Diverse applications of EENs and RGENs
  7. Genome editing in animals
  8. Genome editing in cultured cells
  9. Perspectives
  10. References

Both EENs and RGENs can now be constructed independently by researchers using open-source plasmids available from Addgene (Cambridge, MA, USA). For ZFNs, two main construction methods can be applied: modular assembly and oligomerized pool engineering (OPEN). As modular assembly simply requires digestion and ligation of individual ZF modules from a library of plasmids, it is easy to establish (Wright et al. 2006). However, ZFNs created by this method often have different base-recognition specificities from those predicted, because of interference between tandemly-ligated ZF modules (Ramirez et al. 2008). The OPEN method, on the other hand, is a bacterial screening-based method that can directly select a functional ZF array (Maeder et al. 2009). The success rate of this method is higher than modular assembly, yet the laborious process has traditionally forced researchers away from this method.

Compared with ZFNs, construction methods for TALENs are much easier and more robust in obtaining functional EENs. Currently, five TALEN construction kits are provided by different groups through Addgene. For every kit, the principle of the construction is similar to that of ZFN modular assembly, where only the cloning method varies among them. The most widely used kit is the Bogdanove/Voytas Lab's Golden Gate TALEN and TAL Effector Kit (Cermak et al. 2011). This kit is composed of a library of individual module plasmids, intermediate vectors, and final destination vectors. In application, TALEN plasmids harboring 12–31 repeat domains can be constructed in two cloning steps (Cermak et al. 2011). Moreover, various add-on plasmids for the Voytas lab kit have been developed, including improved TALE N- and C-terminal architectures, and the Yamamoto Lab's TALEN Accessory Pack that enables enhanced construction and evaluation of custom TALENs (Sakuma et al. 2013a). In the near future, another Golden Gate cloning-based TALEN construction kit, the Platinum Gate TALEN Kit (Sakuma et al. 2013b), is expected to be provided from Addgene. TALENs made with the Platinum Gate Kit - Platinum TALENs - have been proven to be more active than the conventional Golden Gate TALENs in cultured cells and animals including frogs and rats (Sakane et al. 2013; Sakuma et al. 2013b), and also to work efficiently in nematodes (Sugi et al. 2013), sea urchins (Hosoi et al. 2013), ascidians (Treen et al. 2013), newts (Hayashi et al. 2013) and mice (Nakagawa et al. 2013).

The construction of CRISPR RGENs is far easier than that of TALENs. As Cas9 is commonly applicable to any gRNA, researchers need only to construct the specific gRNA expression vector by cloning synthesized oligonucleotides into a polIII promoter-driven expression vector (Mali et al. 2013a; Ran et al. 2013b). Furthermore, Cas9 and gRNA architectures optimized for several cells and organisms, such as bacteria, yeasts, nematodes, flies, zebrafish, and mammalian cells and embryos, have been developed and deposited in Addgene. More vector designs are expected to be added continuously.

To help in the design of EENs and RGENs, multiple online resources are available. The Zinc Finger Consortium (http://www.zincfingers.org/) offers the ZiFiT, which can be used for the design of target sequences for ZFNs, TALENs and CRISPRs (Sander et al. 2010). For off-target scanning of ZFN sites, the ZFN-Site (http://ccg.vital-it.ch/tagger/targetsearch.html) proves useful (Cradick et al. 2011). For TALEs and TALENs, TAL Effector Nucleotide Targeter (TALE-NT; https://tale-nt.cac.cornell.edu/) collection of tools is available through Cornell University (Doyle et al. 2012). Using TALE-NT, target sequences of single TALEs and paired TALENs may be determined and ranked for specificity. Potential off-target may be referenced against particular genome and promoterome databases. Alternate TALEN-designing webtools, such as idTALE (http://idtale.kaust.edu.sa/) (Li et al. 2012), Mojo Hand (http://www.talendesign.org/) (Neff et al. 2013) and E-TALEN (http://www.e-talen.org/E-TALEN/) (Heigwer et al. 2013) are available. For the design of gRNAs for the CRISPR/Cas9 system, apply the CRISPR Design Tool within CRISPR Genome Engineering Resources (http://www.genomeengineering.org/crispr/). As a comprehensive database for EENs and RGENs, EENdb (http://eendb.zfgenetics.org/) is provided by Peking University (Xiao et al. 2013a). These and other independent web-based database resources and bioinformatics tools are being constantly added and updated.

Diverse applications of EENs and RGENs

  1. Top of page
  2. Abstract
  3. Introduction
  4. The basics of nuclease tools: ZFN, TALEN and CRISPR/Cas
  5. Current construction methods for EENs and RGENs
  6. Diverse applications of EENs and RGENs
  7. Genome editing in animals
  8. Genome editing in cultured cells
  9. Perspectives
  10. References

For general mutagenesis and targeted gene disruption, EENs and RGENs may be used in a similar, or even interchangeable, manner. When EENs or RGENs induce a DSB at their target site, it will be repaired immediately by the endogenous DNA repair pathways including NHEJ, microhomology-mediated end joining (MMEJ) or HR (Podevin et al. 2013). NHEJ is an error-prone repair mechanism, resulting in insertions and/or deletions (indels) that are induced at, and extend from, the target site (Fig. 2a). MMEJ can result in short deletions defined by only 2–6 bp of homologous sequence. When EENs or RGENs are introduced with a targeting donor bearing left and right flanking homology arms, homologous recombination-mediated sequence additions or modifications can be incorporated directly into the target locus at much higher frequencies than conventional gene targeting (Fig. 2a) (Voytas 2013). As targeting donors, not only double-strand plasmid vectors but also single-strand oligonucleotides (ssODNs) have been used as templates to direct DNA repair (Fig. 2a) (Chen et al. 2011). Assuming sufficient efficiencies, ssODN targeting provides an opportunity to make more subtle modifications to the genome without the requirement for later removal of drug selection cassettes. Furthermore, simultaneous introduction of two or more EEN pairs can be used induce multiple targeted mutagenesis (Wang et al. 2013), chromosomal deletions (Lee et al. 2010), inversions (Lee et al. 2012), duplications (Lee et al. 2012) or translocations (Piganeau et al. 2013) (Fig. 2b). In this scenario, the application of CRISPR systems for multiplexing is notably advantageous, as only a single Cas9 nuclease is required to interact with any number of targeting gRNAs. Thus, promiscuity of Cas9 simplifies multiplexing.

image

Figure 2. Various genome editing strategies using engineered endonucleases (EENs) and RNA-guided endonucleases (RGENs). (a) Examples of double-strand break (DSB)-mediated genome editing at a single site. Bars indicate paired genomic DNA strands. Black triangles mark points of DSB introduction. DSB repair occurs through end joining or template mediated repair. (b) Examples of DSB-mediated genome editing using two chromosomal DSB events. Bars indicate individual chromosomes. Crosses indicate DSB events.

Download figure to PowerPoint

Complementing nuclease fusions, ZF-, TALE- and Cas9-DNA binding domains have been used in other creative manners. ZF/TALE-recombinases (Gordley et al. 2007; Gersbach et al. 2011; Mercer et al. 2012; Gaj et al. 2013) and ZF/TALE-transposases (Li et al. 2013b; Owens et al. 2013) are novel genome editing tools that do not introduce DSBs. Site-specific gene activation and repression has extended beyond the use of ZFs (Sanchez et al. 2002; Yaghmai & Cutting 2002; Liu et al. 2005) into TALEs (Tremblay et al. 2012; Crocker & Stern 2013; Maeder et al. 2013; Perez-Pinera et al. 2013) and also nuclease-inactivated Cas9 (Cheng et al. 2013; Gilbert et al. 2013; Qi et al. 2013). Recently, light-inducible transcriptional effectors and histone effectors have even been developed using TALE technology (Konermann et al. 2013). These alternate methods of site-specific genetic and epigenetic modification represent avenues of research supplementary to nuclease applications of these unique DNA binding domains.

Genome editing in animals

  1. Top of page
  2. Abstract
  3. Introduction
  4. The basics of nuclease tools: ZFN, TALEN and CRISPR/Cas
  5. Current construction methods for EENs and RGENs
  6. Diverse applications of EENs and RGENs
  7. Genome editing in animals
  8. Genome editing in cultured cells
  9. Perspectives
  10. References

Engineered endonucleases or RGENs have extended the genome engineering paradigm within, and well beyond the circle of standard animal models. Targeted mutagenesis using EENs/RGENs does not necessarily require whole genome sequence, but at a minimum, DNA sequence information around the target locus. Introduction of EENs or RGENs into animals is typically achieved by injecting in vitro transcribed RNAs directly into fertilized eggs. Lending to these flexibilities, EEN/RGEN-mediated gene targeting has been applied directly in a great number of animal organisms including nematodes (Wood et al. 2011), flies (Bibikova et al. 2002; Liu et al. 2012), mosquitos (Aryan et al. 2013; Smidler et al. 2013), butterflies (Merlin et al. 2013), crickets (Watanabe et al. 2012), silkworms (Ma et al. 2012), sea urchins (Ochiai et al. 2010), ascidians (Kawai et al. 2012), zebrafish (Doyon et al. 2008; Meng et al. 2008; Hisano et al. 2013), medaka (Ansai et al. 2012, 2013), frogs (Young et al. 2013; Suzuki et al. 2013), mice (Carbery et al. 2010; Sung et al. 2013), rats (Mashimo et al. 2010, 2013) and pigs (Hauschild et al. 2011; Carlson et al. 2012). The direct production of knock-in animals using targeting vectors, however, has not extensively reported so far. Among the rare, successful examples, targeted transgene insertion has been reported in nematodes (Dickinson et al. 2013), sea urchin (Ochiai et al. 2012), flies (Beumer et al. 2008), zebrafish (Zu et al. 2013), mice (Cui et al. 2011; Yang et al. 2013) and rats (Cui et al. 2011). On the other hand, ssODN-mediated precise gene modification has been reported more frequently (Bedell et al. 2012; Meyer et al. 2008; Gratz et al. 2013; Hwang et al. 2013; Lo et al. 2013; Wang et al. 2013; Wefers et al. 2013). More sophisticated genome editing in animals has been put into practice, such as DSB-mediated chromosomal deletion and inversion in zebrafish (Gupta et al. 2013; Xiao et al. 2013b). A current summary of animal genome editing is summarized in Table 1.

Table 1. Examples of various genome editing in animals reported so far
 ZFNTALENCRISPR/Cas
  1. 1Sakane et al. (2013); 2Jao et al. (2013); 3Wang et al. (2013); 4Li et al. 2013a; 5Meyer et al. 2013; 6Brown et al. (2013); 7Lo et al. (2013); 8Bedell et al. (2012); 9Hayashi et al. (2013); 10Wefers et al. (2013); 11Gratz et al. (2013); 12Hwang et al. (2013); 13Beumer et al. (2008); 14Ochiai et al. (2012); 15Meyer et al. (2010); 16Zu et al. (2013); 17Jones & Meisler (2013); 18Chen et al. (2013); 19Yang et al. (2013); 20Ma et al. (2012); 21Gupta et al. (2013); 22Xiao et al. (2013b); 23Fujii et al. (2013). CRISPR, clustered regularly interspaced short palindromic repeats; TALEN, transcription activator-like effector nuclease; ZFN, zinc-finger nuclease.

Targeted mutagenesisVarious animals including nematode, fly, zebrafish, frog, mouse and rat
Multiple targeted mutagenesis Frog1Zebrafish2, mouse3 and rat4
Gene knockin using ssODNMouse5 and rat6Nematode7, zebrafish8, newt9 and mouse10Fly11, zebrafish12 and mouse3
Gene knockin using targeting vectorFly13, sea urchin14, mouse15 and rat6Zebrafish16 and mouse17Nematode18 and mouse19
Chromosomal deletion or inversion Silkworm20 and zebrafish21, 22Zebrafish22 and mouse23

For animal applications, rapid and reliable screening of mutants is an important topic. Supporting phenotypic observation, many methods are available to screen mutants: DNA hybrid detection assays such as Cel-I digestion (Guschin et al. 2010), high-resolution melting analysis (HRMA) (Dahlem et al. 2012), restriction fragment length polymorphisms (RFLP) (Ochiai et al. 2010; Ansai et al. 2013; Suzuki et al. 2013), heteroduplex mobility assay (HMA) (Ota et al. 2013), or direct detection by standard Sanger or next-generation DNA sequencing (NGS). Recent comparison of multiple screening methods (phenotype observation, RFLP analysis, HMA and DNA sequencing) revealed that a combinatorial testing of these methods increases the efficiency of mutant screening (Nakagawa et al. 2013). Certainly, recent nuclease technologies are proving themselves in organisms amenable to growth and observation in the laboratory environment.

Genome editing in cultured cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. The basics of nuclease tools: ZFN, TALEN and CRISPR/Cas
  5. Current construction methods for EENs and RGENs
  6. Diverse applications of EENs and RGENs
  7. Genome editing in animals
  8. Genome editing in cultured cells
  9. Perspectives
  10. References

Development of nucleases was founded in cell culture assays, and in turn has revolutionized in vitro cell culture models. Conventional pre-screening of designer recombinant nucleases is typically performed in HEK293T (human embryonic kidney) or U2OS (osteosarcoma) cells by plasmid reporter repair assays (Sakuma et al. 2013a) or assessment of direct chromosomal DSB activity (Reyon et al. 2012). Similar to applications in embryos, HR-mediated gene targeting with donor DNA and NHEJ-mediated gene disruption may be achieved by co-introduction of either nuclease expression plasmids or in vitro transcribed RNA, where RNA transfection avoids the possibility of randomly integrating nuclease vectors. Unlike animal models, cell culture permits novel delivery of nucleases; direct transduction of ZFN protein purified from inclusion bodies can engineer HEK293T, CHO (Chinese hamster ovary), HDF (human dermal fibroblasts), THP1 (human acute monocytic leukemia), and primary CD4 + cells derived from peripheral blood (Gaj et al. 2013). Cell culture also provides unique avenues to animal derivation. Cultured mouse ES cells have been simultaneously and homozygously engineered at multiple loci using RGENs, expediting the derivation of complex mouse lines (Wang et al. 2013). Furthermore, enrichment of modified fetal fibroblasts has permitted livestock engineering once combined with somatic cell nuclear transfer (SCNT) (Tan et al. 2013).

Examples of human genome engineering in cell culture have mainly been limited to cells with extensive proliferation capacity such as transformed cell lines and adult or embryonic stem cells. ZFNs were used in K562 cells (a human erythroleukemia cell line) and primary CD4 + T-cells to sequentially disrupt and then repair both copies of the IL2RG gene – mutations in which are the principle cause of severe combined immunodeficiency (SCID) (Urnov et al. 2005). In efforts to correct genetic disorders, epithelial stem cells (Coluccio et al. 2013) and skeletal myoblasts (Ousterout et al. 2013) have been successfully modified with nucleases. In the derivation of therapeutically engineered cell lines, genome editing ex vivo provides the advantages of product quality control before delivery to the patient. T-cells (Perez et al. 2008) and CD34 + hematopoietic stem cells (Holt et al. 2010) resistant to HIV-1 infection have been derived ex vivo using genome editing, and the CRISPR system has even been used to disrupt latent HIV-1 provirus and ‘cure’ infected T-cells (Ebina et al. 2013) Moreover, the combination of gene correction with human induced pluripotent stem (iPS) cells has led to great promise in regenerative medicine and cell-based therapies by resolving questions of autologous transplantation. Although the promise exists, it is wise to abstain from human in vivo applications without an absolute assurance of tightly controlled off-target DNA-damage.

For certain biological models, in vitro cell culture has proven more flexible as an assay platform. This is certainly true for questions of human biology, where experimentation in vivo is impractical and morally questionable. The use of zinc-finger nucleases to knock out the WAS gene in K562 cells led to a human cellular model for megakaryocyte development and Wiskott-Aldrich syndrome (Toscano et al. 2013). ES and iPS cells provide massive benefits for in vitro developmental biology studies, as they can be induced to differentiate and give rise to nearly every cell found in the body, or even complex tissues (Lancaster et al. 2013). For human, this is the only option to access embryonic-like stages, or adult tissues. The advent of iPS cell technology has coincided with the rise of the genome sequencing era, leading to patient specific-disease models, and a more direct means to prove the effects of genetic contribution to disease or drug sensitivities (Tiscornia et al. 2011). Experimental strategies to modify iPS cells are, for the most part, similar to modification of other cultured cells, using EENs (Hockemeyer et al. 2009, 2011) and RGENs (Cong et al. 2013; Mali et al. 2013a). Interestingly, to effectively produce iPS cells from particular genetic diseases, such as Fanconi anemia, the nature of the mutation has made it necessary to first perform gene correction in patient somatic cells, and then reprogram to pluripotency (Raya et al. 2010).

Application of EENs or RGENs in cell culture is certainly robust, but requires unique considerations over the direct derivation of engineered animals. Cell lines provide the advantage of immediate clonal selection and genotyping, analogous to screening for progeny with desirable mutation spectra arising from mosaic founder animals. As in classic gene targeting, unmodified cells can be eliminated via gene-targeted antibiotic resistance markers (Capecchi 2005), which may be later removed by sophisticated methods (Yusa et al. 2009). Alternative to selection cassettes, clonal isolation of subtle, marker-free mutations has used single-cell plating by limiting dilutions or flow cytometric sorting. In such experiments, surrogate vectors with structure enrich for transfection and nuclease activity, highlighting cells with a high probability of genomic-modification (Kim et al. 2011). Despite these advantages for selection, cultured cells pose an interesting conundrum in that they cannot be bred to exclude secondary mutations caused by off-target cleavage. Thus, bioinformatic prediction and effective screening is required to detect such mutations. Furthermore, the inability to breed mutations to homozygosity necessitates sequential targeting or highly active EENs/RGENs that can simultaneously target both alleles to reveal recessive phenotypes. Conversely, high-level nuclease activity increases the chance of off-target effects, threatening a clear interpretation of desired phenotypes. Technologies that increase nuclease specificity or improve mutation detection are necessary areas of focus in future technology development.

Perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. The basics of nuclease tools: ZFN, TALEN and CRISPR/Cas
  5. Current construction methods for EENs and RGENs
  6. Diverse applications of EENs and RGENs
  7. Genome editing in animals
  8. Genome editing in cultured cells
  9. Perspectives
  10. References

Over just the last 3 years, EENs and RGENs have veritably revolutionized gene targeting. Although ZFNs have seen a much longer period of service, the simple construction of TALENs and CRISPRs are allowing these two newer methods to quickly take center stage in the genome engineering field. Clearly, a lingering concern for efficacious nuclease application is the potential for off-target effects, which may very well undermine precision engineering efforts. Recently, several reports have highlighted potent off-target cleavage by CRISPRs in cultured cells (Cradick et al. 2013; Fu et al. 2013; Hsu et al. 2013; Mali et al. 2013b; Pattanayak et al. 2013). The standing opinion at this time is, unfortunately, that the CRISPR system may allow for multiple mismatches in the target sequence, including PAM. This leniency in binding and cleavage will certainly lead to compounded risk in CRISPR multiplexing experiments. TALENs, on the other hand, are only active as dimers, with further limitations governed by spacer length. Moreover, even single TALEs have been shown to have relatively high specificities compared to CRISPR/Cas (Mali et al. 2013b). Considering various factors impacting the current state-of-the-art, the simple design of CRISPRs must be weighed against their tendency to tolerate mismatches. In this period of active tool development, innovative adaptations may ultimately overcome limitations in the present technology. Using the detailed examples and experiences in this issues’ collection of reports as a guide, we encourage industrious researchers to keep current, and choose the most appropriate methods in accordance with their intended applications.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. The basics of nuclease tools: ZFN, TALEN and CRISPR/Cas
  5. Current construction methods for EENs and RGENs
  6. Diverse applications of EENs and RGENs
  7. Genome editing in animals
  8. Genome editing in cultured cells
  9. Perspectives
  10. References
  • Acevedo-Arozena, A., Wells, S., Potter, P., Kelly, M., Cox, R. D. & Brown, S. D. 2008. ENU mutagenesis, a way forward to understand gene function. Annu. Rev. Genomics Hum. Genet. 9, 4969.
  • Ansai, S., Ochiai, H., Kanie, Y., Kamei, Y., Gou, Y., Kitano, T., Yamamoto, T. & Kinoshita, M. 2012. Targeted disruption of exogenous EGFP gene in medaka using zinc-finger nucleases. Dev. Growth Differ. 54, 546556.
  • Ansai, S., Sakuma, T., Yamamoto, T., Ariga, H., Uemura, N., Takahashi, R. & Kinoshita, M. 2013. Efficient targeted mutagenesis in medaka using custom-designed transcription activator-like effector nucleases. Genetics 193, 739749.
  • Aryan, A., Anderson, M. A., Myles, K. M. & Adelman, Z. N. 2013. TALEN-based gene disruption in the dengue vector Aedes aegypti. PLoS ONE 8, e60082.
  • Bedell, V. M., Wang, Y., Campbell, J. M., Poshusta, T. L., Starker, C. G., Krug, R. G. 2nd, Tan, W., Penheiter, S. G., Ma, A. C., Leung, A. Y., Fahrenkrug, S. C., Carlson, D. F., Voytas, D. F., Clark, K. J., Essner, J. J. & Ekker, S. C. 2012. In vivo genome editing using a high-efficiency TALEN system. Nature 491, 114118.
  • Beumer, K. J., Trautman, J. K., Bozas, A., Liu, J. L., Rutter, J., Gall, J. G. & Carroll, D. 2008. Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc. Natl Acad. Sci. USA 105, 1982119826.
  • Beurdeley, M., Bietz, F., Li, J., Thomas, S., Stoddard, T., Juillerat, A., Zhang, F., Voytas, D. F., Duchateau, P. & Silva, G. H. 2013. Compact designer TALENs for efficient genome engineering. Nat. Commun. 4, 1762.
  • Bibikova, M., Golic, M., Golic, K. G. & Carroll, D. 2002. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 11691175.
  • Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A. & Bonas, U. 2009. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 15091512.
  • Bogdanove, A. J., Schornack, S. & Lahaye, T. 2010. TAL effectors: finding plant genes for disease and defense. Curr. Opin. Plant Biol. 13, 394401.
  • Brown, A. J., Fisher, D. A., Kouranova, E., McCoy, A., Forbes, K., Wu, Y., Henry, R., Ji, D., Chambers, A., Warren, J., Shu, W., Weinstein, E. J. & Cui, X. 2013. Whole-rat conditional gene knockout via genome editing. Nat. Methods 10, 638640.
  • Cade, L., Reyon, D., Hwang, W. Y., Tsai, S. Q., Patel, S., Khayter, C., Joung, J. K., Sander, J. D., Peterson, R. T. & Yeh, J. R. 2012. Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs. Nucleic Acids Res. 40, 80018010.
  • Capecchi, M. R. 2005. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat. Rev. Genet. 6, 507512.
  • Carbery, I. D., Ji, D., Harrington, A., Brown, V., Weinstein, E. J., Liaw, L. & Cui, X. 2010. Targeted genome modification in mice using zinc-finger nucleases. Genetics 186, 451459.
  • Carlson, D. F., Tan, W., Lillico, S. G., Stverakova, D., Proudfoot, C., Christian, M., Voytas, D. F., Long, C. R., Whitelaw, C. B. & Fahrenkrug, S. C. 2012. Efficient TALEN-mediated gene knockout in livestock. Proc. Natl Acad. Sci. USA 109, 1738217387.
  • Cermak, T., Doyle, E. L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J. A., Somia, N. V., Bogdanove, A. J. & Voytas, D. F. 2011. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82.
  • Chen, F., Pruett-Miller, S. M., Huang, Y., Gjoka, M., Duda, K., Taunton, J., Collingwood, T. N., Frodin, M. & Davis, G. D. 2011. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat. Methods 8, 753755.
  • Chen, C., Fenk, L. A. & de Bono, M. 2013. Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination. Nucleic Acids Res. 41, e193.
  • Cheng, A. W., Wang, H., Yang, H., Shi, L., Katz, Y., Theunissen, T. W., Rangarajan, S., Shivalila, C. S., Dadon, D. B. & Jaenisch, R. 2013. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 11631171.
  • Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A. & Zhang, F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819823.
  • Cradick, T. J., Ambrosini, G., Iseli, C., Bucher, P. & McCaffrey, A. P. 2011. ZFN-site searches genomes for zinc finger nuclease target sites and off-target sites. BMC Bioinformatics 12, 152.
  • Cradick, T. J., Fine, E. J., Antico, C. J. & Bao, G. 2013. CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 95849592.
  • Crocker, J. & Stern, D. L. 2013. TALE-mediated modulation of transcriptional enhancers in vivo. Nat. Methods 10, 762767.
  • Cui, X., Ji, D., Fisher, D. A., Wu, Y., Briner, D. M. & Weinstein, E. J. 2011. Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat. Biotechnol. 29, 6467.
  • Coluccio, A., Miselli, F., Lombardo, A., Marconi, A., Malagoli Tagliazucchi, G., Gonçalves, M. A., Pincelli, C., Maruggi, G., Del Rio, M., Naldini, L., Larcher, F., Mavilio, F. & Recchia, A. 2013. Targeted gene addition in human epithelial stem cells by zinc-finger nuclease-mediated homologous recombination. Mol. Ther. 21, 16951704.
  • Dahlem, T. J., Hoshijima, K., Jurynec, M. J., Gunther, D., Starker, C. G., Locke, A. S., Weis, A. M., Voytas, D. F. & Grunwald, D. J. 2012. Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome. PLoS Genet. 8, e1002861.
  • Dickinson, D. J., Ward, J. D., Reiner, D. J. & Goldstein, B. 2013. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat. Methods 10, 10281034.
  • Doyle, E. L., Booher, N. J., Standage, D. S., Voytas, D. F., Brendel, V. P., Vandyk, J. K. & Bogdanove, A. J. 2012. TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res. 40, W117W122.
  • Doyon, Y., McCammon, J. M., Miller, J. C., Faraji, F., Ngo, C., Katibah, G. E., Amora, R., Hocking, T. D., Zhang, L., Rebar, E. J., Gregory, P. D., Urnov, F. D. & Amacher, S. L. 2008. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat. Biotechnol. 26, 702708.
  • Ebina, H., Misawa, N., Kanemura, Y. & Koyanagi, Y. 2013. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci. Rep. 3, 2510.
  • Esvelt, K. M., Mali, P., Braff, J. L., Moosburner, M., Yaung, S. J. & Church, G. M. 2013. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 11161121.
  • Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K. & Sander, J. D. 2013. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822826.
  • Fujii, W., Kawasaki, K., Sugiura, K. & Naito, K. 2013. Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res. 41, e187.
  • Gaj, T., Guo, J., Kato, Y., Sirk, S. J. & Barbas, C. F. 3rd 2012. Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat. Methods 9, 805807.
  • Gaj, T., Mercer, A. C., Sirk, S. J., Smith, H. L. & Barbas, C. F. 3rd 2013. A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells. Nucleic Acids Res. 41, 39373946.
  • Gersbach, C. A., Gaj, T., Gordley, R. M., Mercer, A. C. & Barbas, C. F. 3rd 2011. Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase. Nucleic Acids Res. 39, 78687878.
  • Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., Stern-Ginossar, N., Brandman, O., Whitehead, E. H., Doudna, J. A., Lim, W. A., Weissman, J. S. & Qi, L. S. 2013. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442451.
  • Gordley, R. M., Smith, J. D., Graslund, T. & Barbas, C. F. 3rd 2007. Evolution of programmable zinc finger-recombinases with activity in human cells. J. Mol. Biol. 367, 802813.
  • Gratz, S. J., Cummings, A. M., Nguyen, J. N., Hamm, D. C., Donohue, L. K., Harrison, M. M., Wildonger, J. & O'Connor-Giles, K. M. 2013. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194, 10291035.
  • Grizot, S., Smith, J., Daboussi, F., Prieto, J., Redondo, P., Merino, N., Villate, M., Thomas, S., Lemaire, L., Montoya, G., Blanco, F. J., Paques, F. & Duchateau, P. 2009. Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Acids Res. 37, 54055419.
  • Guo, J., Gaj, T. & Barbas, C. F. 3rd 2010. Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. J. Mol. Biol. 400, 96107.
  • Gupta, A., Hall, V. L., Kok, F. O., Shin, M., McNulty, J. C., Lawson, N. D. & Wolfe, S. A. 2013. Targeted chromosomal deletions and inversions in zebrafish. Genome Res. 23, 10081017.
  • Guschin, D. Y., Waite, A. J., Katibah, G. E., Miller, J. C., Holmes, M. C. & Rebar, E. J. 2010. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247256.
  • Hauschild, J., Petersen, B., Santiago, Y., Queisser, A. L., Carnwath, J. W., Lucas-Hahn, A., Zhang, L., Meng, X., Gregory, P. D., Schwinzer, R., Cost, G. J. & Niemann, H. 2011. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc. Natl Acad. Sci. USA 108, 1201312017.
  • Hayashi, T., Sakamoto, K., Sakuma, T., Yokotani, N., Inoue, T., Kawaguchi, E., Agata, K., Yamamoto, T. & Takeuchi, T. 2013. TALENs efficiently disrupt the target gene in Iberian ribbed newts (Pleurodeles waltl), an experimental model animal for regeneration. Dev. Growth Differ. 56, 115121.
  • Heigwer, F., Kerr, G., Walther, N., Glaeser, K., Pelz, O., Breinig, M. & Boutros, M. 2013. E-TALEN: a web tool to design TALENs for genome engineering. Nucleic Acids Res. 41, e190.
  • Hisano, Y., Ota, S., Arakawa, K., Muraki, M., Kono, N., Oshita, K., Sakuma, T., Tomita, M., Yamamoto, T., Okada, Y. & Kawahara, A. 2013. Quantitative assay for TALEN activity at endogenous genomic loci. Biol. Open 2, 363367.
  • Hockemeyer, D., Soldner, F., Beard, C., Gao, Q., Mitalipova, M., Dekelver, R. C., Katibah, G. E., Amora, R., Boydston, E. A., Zeitler, B., Meng, X., Miller, J. C., Zhang, L., Rebar, E. J., Gregory, P. D., Urnov, F. D. & Jaenisch, R. 2009. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851857.
  • Hockemeyer, D., Wang, H., Kiani, S., Lai, C. S., Gao, Q., Cassady, J. P., Cost, G. J., Zhang, L., Santiago, Y., Miller, J. C., Zeitler, B., Cherone, J. M., Meng, X., Hinkley, S. J., Rebar, E. J., Gregory, P. D., Urnov, F. D. & Jaenisch, R. 2011. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731734.
  • Holt, N., Wang, J., Kim, K., Friedman, G., Wang, X., Taupin, V., Crooks, G. M., Kohn, D. B., Gregory, P. D., Holmes, M. C. & Cannon, P. M. 2010. Human hematopoietic stem/progenitor cells modified by zinc-finger nuclease targeted to CCR5 control HIV-1 in vivo. Nat. Biotechnol. 28, 839847.
  • Hosoi, S., Sakuma, T., Sakamoto, N. & Yamamoto, T. 2013. Targeted mutagenesis in sea urchin embryos using TALENs. Dev. Growth Differ. 56, 9297.
  • Hou, Z., Zhang, Y., Propson, N. E., Howden, S. E., Chu, L. F., Sontheimer, E. J. & Thomson, J. A. 2013. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl Acad. Sci. USA 110, 1564415649.
  • Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., Cradick, T. J., Marraffini, L. A., Bao, G. & Zhang, F. 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827832.
  • Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Kaini, P., Sander, J. D., Joung, J. K., Peterson, R. T. & Yeh, J. R. 2013. Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLoS ONE 8, e68708.
  • Jao, L. E., Wente, S. R. & Chen, W. 2013. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl Acad. Sci. USA 110, 1390413909.
  • Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A. & Charpentier, E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816821.
  • Jones, J. M. & Meisler, M. H. 2013. Modeling human epilepsy by TALEN targeting of mouse sodium channel Scn8a. Genesis (in press, doi: 10.1002/dvg.22731)
  • Joung, J. K. & Sander, J. D. 2013. TALENs: a widely applicable technology for targeted genome editing. Nat. Rev. Mol. Cell Biol. 14, 4955.
  • Kawai, N., Ochiai, H., Sakuma, T., Yamada, L., Sawada, H., Yamamoto, T. & Sasakura, Y. 2012. Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases. Dev. Growth Differ. 54, 535545.
  • Kim, H., Um, E., Cho, S. R., Jung, C., Kim, H. & Kim, J. S. 2011. Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat. Methods 8, 941943.
  • Kim, E., Kim, S., Kim, D. H., Choi, B. S., Choi, I. Y. & Kim, J. S. 2012. Precision genome engineering with programmable DNA-nicking enzymes. Genome Res. 22, 13271333.
  • Kim, Y. G., Cha, J. & Chandrasegaran, S. 1996. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl Acad. Sci. USA 93, 11561160.
  • Kleinstiver, B. P., Wolfs, J. M., Kolaczyk, T., Roberts, A. K., Hu, S. X. & Edgell, D. R. 2012. Monomeric site-specific nucleases for genome editing. Proc. Natl Acad. Sci. USA 109, 80618066.
  • Komiyama, M. 2013. Cut-and-Paste of DNA Using an Artificial Restriction DNA Cutter. Int. J. Mol. Sci. 14, 33433357.
  • Konermann, S., Brigham, M. D., Trevino, A. E., Hsu, P. D., Heidenreich, M., Cong, L., Platt, R. J., Scott, D. A., Church, G. M. & Zhang, F. 2013. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472476.
  • Lancaster, M. A., Renner, M., Martin, C.-A., Wenzel, D., Bicknell, L. S., Hurles, M. E., Homfray, T., Penninger, J. M., Jackson, A. P. & Knoblich, J. A. 2013. Cerebral organoids model human brain development and microcephaly. Nature 501, 373379.
  • Lee, H. J., Kim, E. & Kim, J. S. 2010. Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res. 20, 8189.
  • Lee, H. J., Kweon, J., Kim, E., Kim, S. & Kim, J. S. 2012. Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases. Genome Res. 22, 539548.
  • Li, L., Piatek, M. J., Atef, A., Piatek, A., Wibowo, A., Fang, X., Sabir, J. S., Zhu, J. K. & Mahfouz, M. M. 2012. Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification. Plant Mol. Biol. 78, 407416.
  • Li, W., Teng, F., Li, T. & Zhou, Q. 2013a. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat. Biotechnol. 31, 684686.
  • Li, X., Burnight, E. R., Cooney, A. L., Malani, N., Brady, T., Sander, J. D., Staber, J., Wheelan, S. J., Joung, J. K., McCray, P. B. Jr, Bushman, F. D., Sinn, P. L. & Craig, N. L. 2013b. piggyBac transposase tools for genome engineering. Proc. Natl Acad. Sci. USA 110, E2279E2287.
  • Liu, P. Q., Tan, S., Mendel, M. C., Murrills, R. J., Bhat, B. M., Schlag, B., Samuel, R., Matteo, J. J., de la Rosa, R., Howes, K., Reik, A., Case, C. C., Bex, F. J., Young, K. & Gregory, P. D. 2005. Isogenic human cell lines for drug discovery: regulation of target gene expression by engineered zinc-finger protein transcription factors. J. Biomol. Screen. 10, 304313.
  • Liu, J., Li, C., Yu, Z., Huang, P., Wu, H., Wei, C., Zhu, N., Shen, Y., Chen, Y., Zhang, B., Deng, W. M. & Jiao, R. 2012. Efficient and specific modifications of the Drosophila genome by means of an easy TALEN strategy. J. Genet. Genomics 39, 209215.
  • Lo, T. W., Pickle, C. S., Lin, S., Ralston, E. J., Gurling, M., Schartner, C. M., Bian, Q., Doudna, J. A. & Meyer, B. J. 2013. Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. Genetics 195, 331348.
  • Ma, S., Zhang, S., Wang, F., Liu, Y., Liu, Y., Xu, H., Liu, C., Lin, Y., Zhao, P. & Xia, Q. 2012. Highly efficient and specific genome editing in silkworm using custom TALENs. PLoS ONE 7, e45035.
  • Maeder, M. L., Thibodeau-Beganny, S., Sander, J. D., Voytas, D. F. & Joung, J. K. 2009. Oligomerized pool engineering (OPEN): an ‘open-source’ protocol for making customized zinc-finger arrays. Nat. Protoc. 4, 14711501.
  • Maeder, M. L., Linder, S. J., Reyon, D., Angstman, J. F., Fu, Y., Sander, J. D. & Joung, J. K. 2013. Robust, synergistic regulation of human gene expression using TALE activators. Nat. Methods 10, 243245.
  • Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E. & Church, G. M. 2013a. RNA-guided human genome engineering via Cas9. Science 339, 823826.
  • Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., Yang, L. & Church, G. M. 2013b. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833838.
  • Mashimo, T., Takizawa, A., Voigt, B., Yoshimi, K., Hiai, H., Kuramoto, T. & Serikawa, T. 2010. Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases. PLoS ONE 5, e8870.
  • Mashimo, T., Kaneko, T., Sakuma, T., Kobayashi, J., Kunihiro, Y., Voigt, B., Yamamoto, T. & Serikawa, T. 2013. Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes. Sci. Rep. 3, 1253.
  • Meng, X., Noyes, M. B., Zhu, L. J., Lawson, N. D. & Wolfe, S. A. 2008. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol. 26, 695701.
  • Menoret, S., Fontaniere, S., Jantz, D., Tesson, L., Thinard, R., Remy, S., Usal, C., Ouisse, L. H., Fraichard, A. & Anegon, I. 2013. Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases. FASEB J. 27, 703711.
  • Mercer, A. C., Gaj, T., Fuller, R. P. & Barbas, C. F. 3rd 2012. Chimeric TALE recombinases with programmable DNA sequence specificity. Nucleic Acids Res. 40, 1116311172.
  • Merlin, C., Beaver, L. E., Taylor, O. R., Wolfe, S. A. & Reppert, S. M. 2013. Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases. Genome Res. 23, 159168.
  • Meyer, M., de Angelis, M. H., Wurst, W. & Kühn, R. 2010. Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc. Natl Acad. Sci. USA 107, 1502215026.
  • Meyer, M., Ortiz, O., Hrabé de Angelis, M., Wurst, W. & Kühn, R. 2012. Modeling disease mutations by gene targeting in one-cell mouse embryos. Proc. Natl Acad. Sci. USA 109, 93549359.
  • Miller, J. C., Holmes, M. C., Wang, J., Guschin, D. Y., Lee, Y. L., Rupniewski, I., Beausejour, C. M., Waite, A. J., Wang, N. S., Kim, K. A., Gregory, P. D., Pabo, C. O. & Rebar, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778785.
  • Minczuk, M., Papworth, M. A., Miller, J. C., Murphy, M. P. & Klug, A. 2008. Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA. Nucleic Acids Res. 36, 39263938.
  • Mino, T., Aoyama, Y. & Sera, T. 2009. Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer. J. Biotechnol. 140, 156161.
  • Mori, T., Kagatsume, I., Shinomiya, K., Aoyama, Y. & Sera, T. 2009. Sandwiched zinc-finger nucleases harboring a single-chain FokI dimer as a DNA-cleavage domain. Biochem. Biophys. Res. Commun. 390, 694697.
  • Moscou, M. J. & Bogdanove, A. J. 2009. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501.
  • Munoz, I. G., Prieto, J., Subramanian, S., Coloma, J., Redondo, P., Villate, M., Merino, N., Marenchino, M., D'Abramo, M., Gervasio, F. L., Grizot, S., Daboussi, F., Smith, J., Chion-Sotinel, I., Paques, F., Duchateau, P., Alibes, A., Stricher, F., Serrano, L., Blanco, F. J. & Montoya, G. 2011. Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus. Nucleic Acids Res. 39, 729743.
  • Mussolino, C. & Cathomen, T. 2012. TALE nucleases: tailored genome engineering made easy. Curr. Opin. Biotechnol. 23, 644650.
  • Nakagawa, Y., Yamamoto, T., Suzuki, K., Araki, K., Takeda, N., Ohmuraya, M. & Sakuma, T. 2013. Screening methods to identify TALEN-mediated knockout mice. Exp. Anim. 63, 7984 (in press).
  • Neff, K. L., Argue, D. P., Ma, A. C., Lee, H. B., Clark, K. J. & Ekker, S. C. 2013. Mojo Hand, a TALEN design tool for genome editing applications. BMC Bioinformatics 14, 1.
  • Ochiai, H., Fujita, K., Suzuki, K., Nishikawa, M., Shibata, T., Sakamoto, N. & Yamamoto, T. 2010. Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases. Genes Cells 15, 875885.
  • Ochiai, H., Sakamoto, N., Fujita, K., Nishikawa, M., Suzuki, K., Matsuura, S., Miyamoto, T., Sakuma, T., Shibata, T. & Yamamoto, T. 2012. Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos. Proc. Natl Acad. Sci. USA 109, 1091510920.
  • Ousterout, D. G., Perez-Pinera, P., Thakore, P. I., Kabadi, A. M., Brown, M. T., Qin, X., Fedrigo, O., Mouly, V., Tremblay, J. P. & Gersbach, C. A. 2013. Reading frame correction by targeted genome editing restores dystrophin expression in cells from duchenne muscular dystrophy patients. Mol. Ther. 21, 17181726.
  • Ota, S., Hisano, Y., Muraki, M., Hoshijima, K., Dahlem, T. J., Grunwald, D. J., Okada, Y. & Kawahara, A. 2013. Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays. Genes Cells 18, 450458.
  • Owens, J. B., Mauro, D., Stoytchev, I., Bhakta, M. S., Kim, M. S., Segal, D. J. & Moisyadi, S. 2013. Transcription activator like effector (TALE)-directed piggyBac transposition in human cells. Nucleic Acids Res. 41, 91979207.
  • Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E., Doudna, J. A. & Liu, D. R. 2013. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839843.
  • Pauwels, K., Podevin, N., Breyer, D., Carroll, D. & Herman, P. 2013. Engineering nucleases for gene targeting: safety and regulatory considerations. N. Biotechnol. 31, 1827.
  • Perez, E. E., Wang, J., Miller, J. C., Jouvenot, Y., Kim, K. A., Liu, O., Wang, N., Lee, G., Bartsevich, V. V., Lee, Y. L., Guschin, D. Y., Rupniewski, I., Waite, A. J., Carpenito, C., Carroll, R. G., Orange, J. S., Urnov, F. D., Rebar, E. J., Ando, D., Gregory, P. D., Riley, J. L., Holmes, M. C. & June, C. H. 2008. Establishment of HIV-1 resistance in CD4 +  T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808816.
  • Perez-Pinera, P., Ousterout, D. G., Brunger, J. M., Farin, A. M., Glass, K. A., Guilak, F., Crawford, G. E., Hartemink, A. J. & Gersbach, C. A. 2013. Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nat. Methods 10, 239242.
  • Piganeau, M., Ghezraoui, H., De Cian, A., Guittat, L., Tomishima, M., Perrouault, L., Rene, O., Katibah, G. E., Zhang, L., Holmes, M. C., Doyon, Y., Concordet, J. P., Giovannangeli, C., Jasin, M. & Brunet, E. 2013. Cancer translocations in human cells induced by zinc finger and TALE nucleases. Genome Res. 23, 11821193.
  • Pillay, L. M., Selland, L. G., Fleisch, V. C., Leighton, P. L., Cheng, C. S., Famulski, J. K., Ritzel, R. G., March, L. D., Wang, H., Allison, W. T. & Waskiewicz, A. J. 2013. Evaluating the mutagenic activity of targeted endonucleases containing a Sharkey FokI cleavage domain variant in zebrafish. Zebrafish 10, 353364.
  • Podevin, N., Davies, H. V., Hartung, F., Nogue, F. & Casacuberta, J. M. 2013. Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding. Trends Biotechnol. 31, 375383.
  • Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P. & Lim, W. A. 2013. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 11731183.
  • Ramalingam, S., Kandavelou, K., Rajenderan, R. & Chandrasegaran, S. 2011. Creating designed zinc-finger nucleases with minimal cytotoxicity. J. Mol. Biol. 405, 630641.
  • Ramirez, C. L., Foley, J. E., Wright, D. A., Muller-Lerch, F., Rahman, S. H., Cornu, T. I., Winfrey, R. J., Sander, J. D., Fu, F., Townsend, J. A., Cathomen, T., Voytas, D. F. & Joung, J. K. 2008. Unexpected failure rates for modular assembly of engineered zinc fingers. Nat. Methods 5, 374375.
  • Ramirez, C. L., Certo, M. T., Mussolino, C., Goodwin, M. J., Cradick, T. J., McCaffrey, A. P., Cathomen, T., Scharenberg, A. M. & Joung, J. K. 2012. Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects. Nucleic Acids Res. 40, 55605568.
  • Ran, F. A., Hsu, P. D., Lin, C. Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., Scott, D. A., Inoue, A., Matoba, S., Zhang, Y. & Zhang, F. 2013a. Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Cell 154, 13801389.
  • Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A. & Zhang, F. 2013b. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 22812308.
  • Raya, A., Rodríguez-Pizà, I., Navarro, S., Richaud-Patin, Y., Guenechea, G., Sánchez-Danés, A., Consiglio, A., Bueren, J. & Izpisúa Belmonte, J. C. 2010. A protocol describing the genetic correction of somatic human cells and subsequent generation of iPS cells. Nat. Protoc. 5, 647660.
  • Reyon, D., Tsai, S. Q., Khayter, C., Foden, J. A., Sander, J. D. & Joung, J. K. 2012. FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30, 460465.
  • Sakane, Y., Sakuma, T., Kashiwagi, K., Kashiwagi, A., Yamamoto, T. & Suzuki, K. 2013. Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases. Dev. Growth Differ. 56, 108114.
  • Sakuma, T., Hosoi, S., Woltjen, K., Suzuki, K., Kashiwagi, K., Wada, H., Ochiai, H., Miyamoto, T., Kawai, N., Sasakura, Y., Matsuura, S., Okada, Y., Kawahara, A., Hayashi, S. & Yamamoto, T. 2013a. Efficient TALEN construction and evaluation methods for human cell and animal applications. Genes Cells 18, 315326.
  • Sakuma, T., Ochiai, H., Kaneko, T., Mashimo, T., Tokumasu, D., Sakane, Y., Suzuki, K., Miyamoto, T., Sakamoto, N., Matsuura, S. & Yamamoto, T. 2013b. Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Sci. Rep. 3, 3379.
  • Sanchez, J. P., Ullman, C., Moore, M., Choo, Y. & Chua, N. H. 2002. Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol. 43, 14651472.
  • Sander, J. D., Maeder, M. L., Reyon, D., Voytas, D. F., Joung, J. K. & Dobbs, D. 2010. ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. Nucleic Acids Res. 38, W462W468.
  • Smidler, A. L., Terenzi, O., Soichot, J., Levashina, E. A. & Marois, E. 2013. Targeted mutagenesis in the malaria mosquito using TALE nucleases. PLoS ONE 8, e74511.
  • Stanford, W. L., Cohn, J. B. & Cordes, S. P. 2001. Gene-trap mutagenesis: past, present and beyond. Nat. Rev. Genet. 2, 756768.
  • Sugi, T., Sakuma, T., Ohtani, T. & Yamamoto, T. 2013. Versatile strategy for isolating TALEN-mediated knockout mutants in Caenorhabditis elegans. Dev. Growth Differ. 56, 7885.
  • Sung, Y. H., Baek, I. J., Kim, D. H., Jeon, J., Lee, J., Lee, K., Jeong, D., Kim, J. S. & Lee, H. W. 2013. Knockout mice created by TALEN-mediated gene targeting. Nat. Biotechnol. 31, 2324.
  • Suzuki, K. T., Isoyama, Y., Kashiwagi, K., Sakuma, T., Ochiai, H., Sakamoto, N., Furuno, N., Kashiwagi, A. & Yamamoto, T. 2013. High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos. Biol. Open 2, 448452.
  • Tan, W., Carlson, D. F., Lancto, C. A., Garbe, J. R., Webster, D. A., Hackett, P. B. & Fahrenkrug, S. C. 2013. Efficient nonmeiotic allele introgression in livestock using custom endonucleases. Proc. Natl Acad. Sci. USA 110, 1652616531.
  • Tesson, L., Usal, C., Menoret, S., Leung, E., Niles, B. J., Remy, S., Santiago, Y., Vincent, A. I., Meng, X., Zhang, L., Gregory, P. D., Anegon, I. & Cost, G. J. 2011. Knockout rats generated by embryo microinjection of TALENs. Nat. Biotechnol. 29, 695696.
  • Tiscornia, G., Vivas, E. L. & Belmonte, J. C. I. 2011. Diseases in a dish: modeling human genetic disorders using induced pluripotent cells. Nat. Med. 17, 15701576.
  • Toscano, M. G., Anderson, P., Muñoz, P., Lucena, G., Cobo, M., Benabdellah, K., Gregory, P. D., Holmes, M. C. & Martin, F. 2013. Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome. Dis. Model Mech. 6, 544554.
  • Treen, N., Yoshida, K., Sakuma, T., Sasaki, H., Kawai, N., Yamamoto, T. & Sasakura, Y. 2013. Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function in Ciona. Development DOI: 10.1242/dev.099572.
  • Tremblay, J. P., Chapdelaine, P., Coulombe, Z. & Rousseau, J. 2012. Transcription activator-like effector proteins induce the expression of the frataxin gene. Hum. Gene Ther. 23, 883890.
  • Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. 2010. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636646.
  • Urnov, F. D., Miller, J. C., Lee, Y. L., Beausejour, C. M., Rock, J. M., Augustus, S., Jamieson, A. C., Porteus, M. H., Gregory, P. D. & Holmes, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646651.
  • Voytas, D. F. 2013. Plant genome engineering with sequence-specific nucleases. Annu. Rev. Plant Biol. 64, 327350.
  • Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F. & Jaenisch, R. 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910918.
  • Wang, J., Friedman, G., Doyon, Y., Wang, N. S., Li, C. J., Miller, J. C., Hua, K. L., Yan, J. J., Babiarz, J. E., Gregory, P. D. & Holmes, M. C. 2012. Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme. Genome Res. 22, 13161326.
  • Watanabe, T., Ochiai, H., Sakuma, T., Horch, H. W., Hamaguchi, N., Nakamura, T., Bando, T., Ohuchi, H., Yamamoto, T., Noji, S. & Mito, T. 2012. Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. Nat. Commun. 3, 1017.
  • Wefers, B., Meyer, M., Ortiz, O., Hrabé de Angelis, M., Hansen, J., Wurst, W. & Kühn, R. 2013. Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. Proc. Natl Acad. Sci. USA 110, 37823787.
  • Wood, A. J., Lo, T. W., Zeitler, B., Pickle, C. S., Ralston, E. J., Lee, A. H., Amora, R., Miller, J. C., Leung, E., Meng, X., Zhang, L., Rebar, E. J., Gregory, P. D., Urnov, F. D. & Meyer, B. J. 2011. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307.
  • Wright, D. A., Thibodeau-Beganny, S., Sander, J. D., Winfrey, R. J., Hirsh, A. S., Eichtinger, M., Fu, F., Porteus, M. H., Dobbs, D., Voytas, D. F. & Joung, J. K. 2006. Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat. Protoc. 1, 16371652.
  • Xiao, A., Wu, Y., Yang, Z., Hu, Y., Wang, W., Zhang, Y., Kong, L., Gao, G., Zhu, Z., Lin, S. & Zhang, B. 2013a. EENdb: a database and knowledge base of ZFNs and TALENs for endonuclease engineering. Nucleic Acids Res. 41, D415D422.
  • 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. 2013b. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res. 41, e141.
  • Yaghmai, R. & Cutting, G. R. 2002. Optimized regulation of gene expression using artificial transcription factors. Mol. Ther. 5, 685694.
  • Yang, H., Wang, H., Shivalila, C. S., Cheng, A. W., Shi, L. & Jaenisch, R. 2013. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 13701379.
  • Young, J. J., Cherone, J. M., Doyon, Y., Ankoudinova, I., Faraji, F. M., Lee, A. H., Ngo, C., Guschin, D. Y., Paschon, D. E., Miller, J. C., Zhang, L., Rebar, E. J., Gregory, P. D., Urnov, F. D., Harland, R. M. & Zeitler, B. 2011. Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases. Proc. Natl Acad. Sci. USA 108, 70527057.
  • Yusa, K., Rad, R., Takeda, J. & Bradley, A. 2009. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat. Methods 6, 363369.
  • Zu, Y., Tong, X., Wang, Z., Liu, D., Pan, R., Li, Z., Hu, Y., Luo, Z., Huang, P., Wu, Q., Zhu, Z., Zhang, B. & Lin, S. 2013. TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat. Methods 10, 329331.