Researchers have long recognized that the random introduction of new DNA into the genome could result in unpredictable genetic effects due to bilateral influences between transgenic and endogenous sequences. Hence, precise editing or replacement of mutant genes has been a major goal of gene therapy since its inception. That goal looked possible with the publication of studies demonstrating that targeted cleavage of chromosomal sequences and enhanced homologous recombination (HR) could be achieved using chimeric molecules composed of a nuclease domain and separate, designer DNA-recognition domains.1Rouet P Smih F Jasin M Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.Proc Natl Acad Sci USA. 1994; 91: 6064-6068Crossref PubMed Scopus (447) Google Scholar,2Smih F Rouet P Romanienko PJ Jasin M Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells.Nucleic Acids Res. 1995; 23: 5012-5019Crossref PubMed Scopus (177) Google Scholar Zinc-finger nucleases (ZFNs) are artificial endonucleases that consist of a FokI cleavage domain tethered to engineered Cys2His2 zinc-finger, DNA-binding polypeptides.3Kim YG Cha J Chandrasegaran S Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.Proc Natl Acad Sci USA. 1996; 93: 1156-1160Crossref PubMed Scopus (1322) Google Scholar,4Porteus MH Baltimore D Chimeric nucleases stimulate gene targeting in human cells.Science. 2003; 300: 763Crossref PubMed Scopus (584) Google Scholar,5Bibikova M Beumer K Trautman JK Carroll D Enhancing gene targeting with designed zinc finger nucleases.Science. 2003; 300: 764Crossref PubMed Scopus (657) Google Scholar ZFNs have gained considerable momentum and are widely considered the most mature nuclease technology in the gene therapy field.6Rahman SH Maeder ML Joung JK Cathomen T Zinc-finger nucleases for somatic gene therapy: the next frontier.Hum Gene Ther. 2011; 22: 925-933Crossref PubMed Scopus (56) Google Scholar However, after 15 years in the spotlight, a new chimeric nuclease has emerged, Transcription Activator-Like (TAL) Effector Nucleases (TALENs).7Boch J Scholze H Schornack S Landgraf A Hahn S Kay S et al.Breaking the code of DNA binding specificity of TAL-type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1824) Google Scholar,8Boch J Bonas U Xanthomonas AvrBs3 family-type III effectors: discovery and function.Annu Rev Phytopathol. 2010; 48: 419-436Crossref PubMed Scopus (652) Google Scholar,9Moscou MJ Bogdanove AJ A simple cipher governs DNA recognition by TAL effectors.Science. 2009; 326: 1501Crossref PubMed Scopus (1400) Google Scholar,10Christian M Cermak T Doyle EL Schmidt C Zhang F Hummel A et al.Targeting DNA double-strand breaks with TAL effector nucleases.Genetics. 2010; 186: 757-761Crossref PubMed Scopus (1256) Google Scholar Here, we describe functional and design characteristics of ZFNs and TALENs and discuss their expanding role as tools for research and gene therapy. Each zinc finger (ZF) is about 30 amino acids, which form a ββα-fold stabilized by hydrophobic interactions and the chelation of a zinc ion, and generally binds to three base pairs.11Klug A The discovery of zinc fingers and their applications in gene regulation and genome manipulation.Annu Rev Biochem. 2010; 79: 213-231Crossref PubMed Scopus (480) Google Scholar Typically, arrays of 3–6 ZF modules are joined together to create a DNA-binding domain with specificity to 9–18 base pairs per ZFN monomer. Double-strand DNA cleavage requires dimerization of two FokI nuclease domains. Thus, ZFNs are used in pairs with specificity to opposing DNA strands that assemble on both sides of the targeted cleavage site (panel a of Figure 1). This enhances the specificity of ZFN targeting not only by requiring two DNA-binding events, but also by requiring precise spacing (typically 5–6 base pairs with a 4-amino acid linker between the ZF and FokI) and correct orientation between ZFNs for activity.12Händel EM Alwin S Cathomen T Expanding or restricting the target site repertoire of zinc-finger nucleases: the inter-domain linker as a major determinant of target site selectivity.Mol Ther. 2009; 17: 104-111Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar,13Urnov FD Rebar EJ Holmes MC Zhang HS Gregory PD Genome editing with engineered zinc finger nucleases.Nat Rev Genet. 2010; 11: 636-646Crossref PubMed Scopus (1470) Google Scholar The resulting double-strand breaks in a DNA sequence can be repaired by either of two mechanisms, nonhomologous end joining (NHEJ) or HR.14Urnov FD Miller JC Lee YL Beausejour CM Rock JM Augustus S et al.Highly efficient endogenous human gene correction using designed zinc-finger nucleases.Nature. 2005; 435: 646-651Crossref PubMed Scopus (1296) Google Scholar,15Porteus MH Carroll D Gene targeting using zinc finger nucleases.Nat Biotechnol. 2005; 23: 967-973Crossref PubMed Scopus (481) Google Scholar NHEJ often results in small deletions or insertions (indels) to cause missense and/or nonsense mutations that truncate or mutate the encoded protein. Consequently, NHEJ-mediated mutagenesis is used for targeted disruptions of genetic loci (e.g., gene knockout). Alternatively, HR allows for either precise modification of a target sequence or precise introduction of a specific sequence (e.g., a wild-type sequence that leads to gene repair) into the targeted site. In mammals, a double-strand DNA break can stimulate HR of an exogenous DNA sequence within about 100 base pairs of the double-stranded DNA break.16Elliott B Richardson C Winderbaum J Nickoloff JA Jasin M Gene conversion tracts from double-strand break repair in mammalian cells.Mol Cell Biol. 1998; 18: 93-101Crossref PubMed Scopus (258) Google Scholar Consequently, both targeting DNA cleavage close to a deleterious mutation and supplying either a double-stranded or single-stranded template DNA sequence can repair a damaged gene. Thus, genetic engineering has transitioned from nearly random addition of genes and expression cassettes to defined editing of a genetic material.17Carroll D Zinc-finger nucleases: a panoramic view.Curr Gene Ther. 2011; 11: 2-10Crossref PubMed Scopus (37) Google Scholar,18Cathomen T Joung JK Zinc-finger nucleases: the next generation emerges.Mol Ther. 2008; 16: 1200-1207Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar ZFNs provided, for the first time, an efficient and relatively simple platform for inducing site-specific mutations or modifications of genomes, particularly for organisms for which this technology was lacking. About 20 abstracts on ZFNs were presented at the 2011 Annual Meeting of the American Society for Gene and Cell Therapy,19Membership Abstracts of the 2011 Annual Meeting of the American Society for Gene and Cell Therapy.Mol Ther. 2011; 19 (Suppl 1): S1-S332Abstract Full Text Full Text PDF PubMed Google Scholar which indicates the considerable interest in using these targeting agents directly for gene therapy and indirectly for modifying human embryonic stem cells and induced pluripotent stem cells for cell therapy.20Zou J Maeder ML Mali P Pruett-Miller SM Thibodeau-Beganny S Chou BK et al.Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells.Cell Stem Cell. 2009; 5: 97-110Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar,21Hockemeyer D Soldner F Beard C Gao Q Mitalipova M DeKelver RC et al.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.Nat Biotechnol. 2009; 27: 851-857Crossref PubMed Scopus (809) Google Scholar,22Hockemeyer D Wang H Kiani S Lai CS Gao Q Cassady JP et al.Genetic engineering of human pluripotent cells using TALE nucleases.Nat Biotechnol. 2011; 29: 731-734Crossref PubMed Scopus (922) Google Scholar,23Soldner F Laganière J Cheng AW Hockemeyer D Gao Q Alagappan R et al.Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations.Cell. 2011; 146: 318-331Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar In terms of vertebrate models for gene and cell therapy, ZFNs have been used successfully for germ line knockout of genes by induction of DNA double-strand breaks and NHEJ in zebrafish,24Doyon Y McCammon JM Miller JC Faraji F Ngo C Katibah GE et al.Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases.Nat Biotechnol. 2008; 26: 702-708Crossref PubMed Scopus (714) Google Scholar,25Meng X Noyes MB Zhu LJ Lawson ND Wolfe SA Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases.Nat Biotechnol. 2008; 26: 695-701Crossref PubMed Scopus (617) Google Scholar mice,26Carbery ID Ji D Harrington A Brown V Weinstein EJ Liaw L et al.Targeted genome modification in mice using zinc-finger nucleases.Genetics. 2010; 186: 451-459Crossref PubMed Scopus (217) Google Scholar rats,27Geurts AM Cost GJ Freyvert Y Zeitler B Miller JC Choi VM et al.Knockout rats via embryo microinjection of zinc-finger nucleases.Science. 2009; 325: 433Crossref PubMed Scopus (727) Google Scholar,28Mashimo T Takizawa A Voigt B Yoshimi K Hiai H Kuramoto T et al.Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases.PLoS ONE. 2010; 5: e8870Crossref PubMed Scopus (198) Google Scholar rabbits,29Flisikowska T Thorey IS Offner S Ros F Lifke V Zeitler B et al.Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.PLoS ONE. 2011; 6: e21045Crossref PubMed Scopus (133) Google Scholar and pigs30Whyte JJ Zhao J Wells KD Samuel MS Whitworth KM Walters EM et al.Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs.Mol Reprod Dev. 2011; 78: 2Crossref PubMed Scopus (91) Google Scholar,31Hauschild J Petersen B Santiago Y Queisser AL Carnwath JW Lucas-Hahn A et al.Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases.Proc Natl Acad Sci USA. 2011; 108: 12013-12017Crossref PubMed Scopus (281) Google Scholar and Caenorhabditis elegans.32Wood AJ Lo TW Zeitler B Pickle CS Ralston EJ Lee AH et al.Targeted genome editing across species using ZFNs and TALENs.Science. 2011; 333: 307Crossref PubMed Scopus (469) Google Scholar These achievements are particularly important, because they suggest the possibility of achieving or improving the frequencies of gene targeting and HR that are stimulated by chromosome breaks in animal models where gene targeting is difficult. Clearly ZFNs are a very powerful resource for gene editing; however, there are some complicating issues with the design and application of ZFNs. First, some ZFNs have been associated with cytotoxicity, presumably due to cleavage at nontargeted sites.33Cornu TI Thibodeau-Beganny S Guhl E Alwin S Eichtinger M Joung JK et al.DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases.Mol Ther. 2008; 16: 352-358Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar,34Radecke S Radecke F Cathomen T Schwarz K Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications.Mol Ther. 2010; 18: 743-753Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar,35Pruett-Miller SM Connelly JP Maeder ML Joung JK Porteus MH Comparison of zinc finger nucleases for use in gene targeting in mammalian cells.Mol Ther. 2008; 16: 707-717Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar Because cleavage at off-target sites can occur when ZFN monomers form homodimers, off-target activity has been ameliorated with structure-based design of the FokI dimerization interface that blocks homodimerization.36Szczepek M Brondani V Büchel J Serrano L Segal DJ Cathomen T Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases.Nat Biotechnol. 2007; 25: 786-793Crossref PubMed Scopus (415) Google Scholar,37Miller JC Holmes MC Wang J Guschin DY Lee YL Rupniewski I et al.An improved zinc-finger nuclease architecture for highly specific genome editing.Nat Biotechnol. 2007; 25: 778-785Crossref PubMed Scopus (789) Google Scholar,38Doyon Y Vo TD Mendel MC Greenberg SG Wang J Xia DF et al.Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures.Nat Methods. 2011; 8: 74-79Crossref PubMed Scopus (310) Google Scholar Second, it was quickly appreciated that it was difficult in some cases to engineer ZFNs to have the desired binding specificities.39Ramirez CL Foley JE Wright DA Müller-Lerch F Rahman SH Cornu TI et al.Unexpected failure rates for modular assembly of engineered zinc fingers.Nat Methods. 2008; 5: 374-375Crossref PubMed Scopus (304) Google Scholar ZFNs assembled using ZFs that recognize known triplets do not always have the desired sequence specificity when assembled into arrays.40Lam KN van Bakel H Cote AG van der Ven A Hughes TR Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays.Nucleic Acids Res. 2011; 39: 4680-4690Crossref PubMed Scopus (68) Google Scholar This problem necessitated assembly of multiple ZFs that target the same base pairs followed by testing and selection of combinations for greatest ZFN specificity and efficiency. One solution is Oligomerized Pool Engineering (OPEN) in which reagents currently available can be used to create ZFNs that recognize sites about every 200 base pairs of random genomic sequence.41Maeder ML Thibodeau-Beganny S Osiak A Wright DA Anthony RM Eichtinger M et al.Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification.Mol Cell. 2008; 31: 294-301Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar,42Sander JD Reyon D Maeder ML Foley JE Thibodeau-Beganny S Li X et al.Predicting success of oligomerized pool engineering (OPEN) for zinc finger target site sequences.BMC Bioinformatics. 2010; 11: 543Crossref PubMed Scopus (21) Google Scholar Hence, there should be multiple target sites in an average gene. An alternative approach for efficient production of ZFNs is Context-Dependent Assembly (CoDA),43Sander JD Dahlborg EJ Goodwin MJ Cade L Zhang F Cifuentes D et al.Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA).Nat Methods. 2011; 8: 67-69Crossref PubMed Scopus (391) Google Scholar which uses an archive of validated two-finger units derived from selection and are known to work well when positioned adjacent to each other. With available CoDA two-finger units, ZFNs can be constructed that recognize approximately one site in every 500 base pairs of random genomic sequence. Others have identified successful combinations of naturally derived and engineered ZF modules which is used to guide modular assembly predicted to have a targeting range of 1 in 125 base pairs of random genomic sequence,44Kim 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: 1279-1288Crossref PubMed Scopus (325) Google Scholar although subsequent studies have indicated unexpected failure rates based on simple modular assembly of ZFNs.39Ramirez CL Foley JE Wright DA Müller-Lerch F Rahman SH Cornu TI et al.Unexpected failure rates for modular assembly of engineered zinc fingers.Nat Methods. 2008; 5: 374-375Crossref PubMed Scopus (304) Google Scholar The fundamental issue boils down to targeting range; that is, the precision at which efficient cleavages can be introduced in a DNA target. For inactivation of a gene via the NHEJ pathway, the site of ZFN cleavage is not as important as specification of the cleavage site for HR-directed gene correction. Hence, for applications to humans, OPEN is more versatile and looks preferable to CoDA. However, OPEN is far more arduous and time-consuming. In addition, mammalian gene conversion frequency is reduced as a function of increasing distance from a DNA double-strand break (>80% reduction 100 base pairs from the double-strand break).16Elliott B Richardson C Winderbaum J Nickoloff JA Jasin M Gene conversion tracts from double-strand break repair in mammalian cells.Mol Cell Biol. 1998; 18: 93-101Crossref PubMed Scopus (258) Google Scholar Thus, some loci of interest to gene therapists may not be modified efficiently using open source ZFN technology. Fifteen years after the introduction of ZFNs, an alternative approach for introducing chromosomal breaks at selected sites was developed. TALENs are novel fusion proteins that, like ZFNs, consist of assembled DNA-binding motifs coupled to FokI nuclease.7Boch J Scholze H Schornack S Landgraf A Hahn S Kay S et al.Breaking the code of DNA binding specificity of TAL-type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1824) Google Scholar,8Boch J Bonas U Xanthomonas AvrBs3 family-type III effectors: discovery and function.Annu Rev Phytopathol. 2010; 48: 419-436Crossref PubMed Scopus (652) Google Scholar,9Moscou MJ Bogdanove AJ A simple cipher governs DNA recognition by TAL effectors.Science. 2009; 326: 1501Crossref PubMed Scopus (1400) Google Scholar,10Christian M Cermak T Doyle EL Schmidt C Zhang F Hummel A et al.Targeting DNA double-strand breaks with TAL effector nucleases.Genetics. 2010; 186: 757-761Crossref PubMed Scopus (1256) Google Scholar,45Li T Huang S Jiang WZ Wright D Spalding MH Weeks DP et al.TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.Nucleic Acids Res. 2011; 39: 359-372Crossref PubMed Scopus (379) Google Scholar,46Mahfouz MM Li L Shamimuzzaman M Wibowo A Fang X Zhu JK De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks.Proc Natl Acad Sci USA. 2011; 108: 2623-2628Crossref PubMed Scopus (311) Google Scholar,47Miller JC Tan S Qiao G Barlow KA Wang J Xia DF et al.A TALE nuclease architecture for efficient genome editing.Nat Biotechnol. 2011; 29: 143-148Crossref PubMed Scopus (1510) Google Scholar The DNA-binding motifs come from proteins secreted by plant pathogens in the bacterial genus Xanthomonas. The proteins activate genes within infected plant cells to improve the environment for the invading pathogen.8Boch J Bonas U Xanthomonas AvrBs3 family-type III effectors: discovery and function.Annu Rev Phytopathol. 2010; 48: 419-436Crossref PubMed Scopus (652) Google Scholar The proteins, TAL effectors, have nuclear localization signals and an acidic transcription-activation domain. The DNA-binding motifs of TAL effectors consist of a tandem repeat of typically 34 amino acids. Each repeat appears to bind to a single base pair based on a simple cipher7Boch J Scholze H Schornack S Landgraf A Hahn S Kay S et al.Breaking the code of DNA binding specificity of TAL-type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1824) Google Scholar,9Moscou MJ Bogdanove AJ A simple cipher governs DNA recognition by TAL effectors.Science. 2009; 326: 1501Crossref PubMed Scopus (1400) Google Scholar shown in panel b of the Figure 1. The cipher can be used to predict the specificity of a TAL effector polypeptide. Residues 12 and 13 of the 34-amino acid repeats, referred to as repeat variable diresidues (RVDs), define binding to a specific base.7Boch J Scholze H Schornack S Landgraf A Hahn S Kay S et al.Breaking the code of DNA binding specificity of TAL-type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1824) Google Scholar,9Moscou MJ Bogdanove AJ A simple cipher governs DNA recognition by TAL effectors.Science. 2009; 326: 1501Crossref PubMed Scopus (1400) Google Scholar This code was deciphered by Boch et al.7Boch J Scholze H Schornack S Landgraf A Hahn S Kay S et al.Breaking the code of DNA binding specificity of TAL-type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1824) Google Scholar where it was demonstrated that artificial TAL effectors targeted to novel sequences could activate transcription, thereby opening the door to a variety of TAL effector-based genome engineering applications. Since then, sequence-specific DNA-binding proteins with predicted binding specificities have been generated economically in a matter of days, using molecular biology methods practiced by most laboratories.48Cermak T Doyle EL Christian M Wang L Zhang Y Schmidt C et al.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic Acids Res. 2011; 39: e82Crossref PubMed Scopus (1512) Google Scholar,49Morbitzer R Elsaesser J Hausner J Lahaye T Assembly of custom TALE-type DNA binding domains by modular cloning.Nucleic Acids Res. 2011; 39: 5790-5799Crossref PubMed Scopus (156) Google Scholar,50Li T Huang S Zhao S Wright DA Carpenter S Spalding MH et al.Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes.Nucleic Acids Res. 2011; 39: 6315-6325Crossref PubMed Scopus (314) Google Scholar,51Huang P Xiao A Zhou M Zhu Z Lin S Zhang B Heritable gene targeting in zebrafish using customized TALENs.Nat Biotechnol. 2011; 29: 699-700Crossref PubMed Scopus (538) Google Scholar,52Sander JD Yeh JR Peterson RT Joung JK Engineering zinc finger nucleases for targeted mutagenesis of zebrafish.Methods Cell Biol. 2011; 104: 51-58Crossref PubMed Scopus (26) Google Scholar,53Geissler R Scholze H Hahn S Streubel J Bonas U Behrens SE et al.Transcriptional activators of human genes with programmable DNA-specificity.PLoS ONE. 2011; 6: e19509Crossref PubMed Scopus (48) Google Scholar,54Zhang F Cong L Lodato S Kosuri S Church GM Arlotta P Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription.Nat Biotechnol. 2011; 29: 149-153Crossref PubMed Scopus (600) Google Scholar,55Weber E Gruetzner R Werner S Engler C Marillonnet S Assembly of designer TAL effectors by Golden Gate cloning.PLoS ONE. 2011; 6: e19722Crossref PubMed Scopus (157) Google Scholar The activities of custom-designed TALENs in human cells have efficiencies of NHEJ-induced mutagenesis ranging up to 45% of transfected cells.47Miller JC Tan S Qiao G Barlow KA Wang J Xia DF et al.A TALE nuclease architecture for efficient genome editing.Nat Biotechnol. 2011; 29: 143-148Crossref PubMed Scopus (1510) Google Scholar,56Mussolino C Morbitzer R Lütge F Dannemann N Lahaye T Cathomen T A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity.Nucleic Acids Res. 2011; 39: 9283-9293Crossref PubMed Scopus (553) Google Scholar In addition, TALENs have been used to create NHEJ modifications in C. elegans,32Wood AJ Lo TW Zeitler B Pickle CS Ralston EJ Lee AH et al.Targeted genome editing across species using ZFNs and TALENs.Science. 2011; 333: 307Crossref PubMed Scopus (469) Google Scholar zebrafish,51Huang P Xiao A Zhou M Zhu Z Lin S Zhang B Heritable gene targeting in zebrafish using customized TALENs.Nat Biotechnol. 2011; 29: 699-700Crossref PubMed Scopus (538) Google Scholar,52Sander JD Yeh JR Peterson RT Joung JK Engineering zinc finger nucleases for targeted mutagenesis of zebrafish.Methods Cell Biol. 2011; 104: 51-58Crossref PubMed Scopus (26) Google Scholar and rats.57Tesson L Usal C Ménoret S Leung E Niles BJ Remy S et al.Knockout rats generated by embryo microinjection of TALENs.Nat Biotechnol. 2011; 29: 695-696Crossref PubMed Scopus (479) Google Scholar As with ZFNs, TALEN-mediated double-strand breaks also stimulated HR in human cells at levels that are similar to the levels achieved with ZFNs.22Hockemeyer D Wang H Kiani S Lai CS Gao Q Cassady JP et al.Genetic engineering of human pluripotent cells using TALE nucleases.Nat Biotechnol. 2011; 29: 731-734Crossref PubMed Scopus (922) Google Scholar,46Mahfouz MM Li L Shamimuzzaman M Wibowo A Fang X Zhu JK De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks.Proc Natl Acad Sci USA. 2011; 108: 2623-2628Crossref PubMed Scopus (311) Google Scholar TALEN-binding sites are expected to occur about once every 35 base pairs based on criteria identified by examination of naturally occurring TALEs.48Cermak T Doyle EL Christian M Wang L Zhang Y Schmidt C et al.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic Acids Res. 2011; 39: e82Crossref PubMed Scopus (1512) Google Scholar If this turns out to be the case experimentally, it will provide greater flexibility in the selection of target sites than open source ZFN platforms and thereby make TALENs very attractive for research in gene and cell therapy. Furthermore, the apparent lack of context dependence and 1:1 correspondence of repeat variable diresidues with defined single base pairs, in contrast to ZF modules for which there are a multiplicity of ZFs for a given triplet of base pairs, with complex contextual interaction, makes modular assembly of TAL effector proteins more straightforward. Remarkably, the success rate for generating active TALEN pairs using simple design parameters (i.e., spacer length and adhering to characteristics of natural TALEs) has been as high as with ZFNs using open source technology.22Hockemeyer D Wang H Kiani S Lai CS Gao Q Cassady JP et al.Genetic engineering of human pluripotent cells using TALE nucleases.Nat Biotechnol. 2011; 29: 731-734Crossref PubMed Scopus (922) Google Scholar,51Huang P Xiao A Zhou M Zhu Z Lin S Zhang B Heritable gene targeting in zebrafish using customized TALENs.Nat Biotechnol. 2011; 29: 699-700Crossref PubMed Scopus (538) Google Scholar,56Mussolino C Morbitzer R Lütge F Dannemann N Lahaye T Cathomen T A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity.Nucleic Acids Res. 2011; 39: 9283-9293Crossref PubMed Scopus (553) Google Scholar,57Tesson L Usal C Ménoret S Leung E Niles BJ Remy S et al.Knockout rats generated by embryo microinjection of TALENs.Nat Biotechnol. 2011; 29: 695-696Crossref PubMed Scopus (479) Google Scholar,58Sander JD Cade L Khayter C Reyon D Peterson RT Joung JK et al.Targeted gene disruption in somatic zebrafish cells using engineered TALENs.Nat Biotechnol. 2011; 29: 697-698Crossref PubMed Scopus (480) Google Scholar Several groups have developed reagents and protocols for simple, rapid modular assembly that make TALENs broadly available to all investigators.48Cermak T Doyle EL Christian M Wang L Zhang Y Schmidt C et al.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic Acids Res. 2011; 39: e82Crossref PubMed Scopus (1512) Google Scholar,49Morbitzer R Elsaesser J Hausner J Lahaye T Assembly of custom TALE-type DNA binding domains by modular cloning.Nucleic Acids Res. 2011; 39: 5790-5799Crossref PubMed Scopus (156) Google Scholar,50Li T Huang S Zhao S Wright DA Carpenter S Spalding MH et al.Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes.Nucleic Acids Res. 2011; 39: 6315-6325Crossref PubMed Scopus (314) Google Scholar,51Huang P Xiao A Zhou M Zhu Z Lin S Zhang B Heritable gene targeting in zebrafish using customized TALENs.Nat Biotechnol. 2011; 29: 699-700Crossref PubMed Scopus (538) Google Scholar,53Geissler R Scholze H Hahn S Streubel J Bonas U Behrens SE et al.Transcriptional activators of human genes with programmable DNA-specificity.PLoS ONE. 2011; 6: e19509Crossref PubMed Scopus (48) Google Scholar,54Zhang F Cong L Lodato S Kosuri S Church GM Arlotta P Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription.Nat Biotechnol. 2011; 29: 149-153Crossref PubMed Scopus (600) Google Scholar,55Weber E Gruetzner R Werner S Engler C Marillonnet S Assembly of designer TAL effectors by Golden Gate cloning.PLoS ONE. 2011; 6: e19722Crossref PubMed Scopus (157) Google Scholar,58Sander JD Cade L Khayter C Reyon D Peterson RT Joung JK et al.Targeted gene disruption in somatic zebrafish cells using engineered TALENs.Nat Biotechnol. 2011; 29: 697-698Crossref PubMed Scopus (480) Google Scholar Extraordinary progress in gene-editing targeting technologies and the recent emergence of TALENs as an alternative, open source gene-targeting platform, supports the prediction that gene-editing will continue to gain momentum for generation of models and therapeutics. The gene therapy community has now 10 years of accumulated experience using ZFNs for precise modification of human genomes and those of model organisms.13Urnov FD Rebar EJ Holmes MC Zhang HS Gregory PD Genome editing with engineered zinc finger nucleases.Nat Rev Genet. 2010; 11: 636-646Crossref PubMed Scopus (1470) Google Scholar At this time, three promising ZFN-based therapies have entered clinical trials for treatment of diabetic neuropathy, AIDS, and glioblastoma. Despite considerable excitement revolving around TALENs, it is too early to predict that they will replace or rival ZFNs for gene therapy. Additional studies are required to: (i) develop efficient means of delivery (TALENs are typically 1,200+ amino acids in length), (ii) define immunogenicity of TALENs, and perhaps most important, (iii) characterize the specificity of TALENs. Each TALEN monomer typically specifies 15+ base pairs for a combined 30+-base pair-target sequence, which is predicted to be unique in the human genome. However, unbiased studies of ZFN specificities have revealed shortcomings of off-target predictions using computer algorithms that search for nearest matches.59Gabriel R Lombardo A Arens A Miller JC Genovese P Kaeppel C et al.An unbiased genome-wide analysis of zinc-finger nuclease specificity.Nat Biotechnol. 2011; 29: 816-823Crossref PubMed Scopus (413) Google Scholar,60Pattanayak V Ramirez CL Joung JK Liu DR Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection.Nat Methods. 2011; 8: 765-770Crossref PubMed Scopus (343) Google Scholar As with ZFNs, early studies reveal that TALENs can bind degenerate sequences and have demonstrated activity at related off-target sites.56Mussolino C Morbitzer R Lütge F Dannemann N Lahaye T Cathomen T A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity.Nucleic Acids Res. 2011; 39: 9283-9293Crossref PubMed Scopus (553) Google Scholar,57Tesson L Usal C Ménoret S Leung E Niles BJ Remy S et al.Knockout rats generated by embryo microinjection of TALENs.Nat Biotechnol. 2011; 29: 695-696Crossref PubMed Scopus (479) Google Scholar Hence, unbiased experimental assessment of off-target sites will be necessary for all ZFNs and TALENs intended for gene therapy. Whether or not TALENs withstand the vigorous standards of clinical use remains to be seen. However, considering their ease in manufacture and reliability in function, we expect increasing adoption of TALENs by the research community. This bodes well for the development of new genetic models and effective therapies for our most prevalent congenital diseases. We thank Drs Voytas, Aronovich, Carroll, and Kim, as well as the journal reviewers and several other colleagues who attended the recent Singapore Conference on Genome Engineering for critical reading of the manuscript and making significant suggestions. We especially thank Drs Voytas and Carroll for clarification of some aspects of the molecular biology of targeted nucleases and historical and balanced perspectives on their development. The work in the authors’ labs is funded by NIH grants 1R41DK081249 (P.B.H.) and 1R41HL108440 (S.C.F.). All three authors hold stock in Recombinetics Inc., whose unfunded business plan includes genetic modification of animal genomes using site-directed nucleases such as ZFNs and TALENs. Scott Fahrenkrug and Perry Hackett are cofounders of Recombinetics and are officers of the company, CEO and CSO, respectively. Please note that in the time since the last revision, Recombinetics has signed a licensing deal with Cellectis Bioresearch, a commercial provider of engineered meganucleases and TALENs. According to the press release describing this agreement, “Recombinetics will use Cellectis Bioresearch's engineered nucleases to conceive animals with improved genetic features.”