Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34–amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.
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Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).
Moscou, M.J. & Bogdanove, A.J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).
Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).
Miller, J.C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).
Morbitzer, R., Romer, P., Boch, J. & Lahaye, T. Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors. Proc. Natl. Acad. Sci. USA 107, 21617–21622 (2010).
Weber, E., Gruetzner, R., Werner, S., Engler, C. & Marillonnet, S. Assembly of designer TAL effectors by golden gate cloning. PLoS ONE 6, e19722 (2011).
Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).
Geissler, R. et al. Transcriptional activators of human genes with programmable DNA-specificity. PLoS ONE 6, e19509 (2011).
Li, T. et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res. 39, 6315–6325 (2011).
Morbitzer, R., Elsaesser, J., Hausner, J. & Lahaye, T. Assembly of custom TALE-type DNA binding domains by modular cloning. Nucleic Acids Res. 39, 5790–5799 (2011).
Wood, A.J. et al. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307 (2011).
Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010).
Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011).
Li, T. et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 39, 359–372 (2011).
Mahfouz, M.M. et al. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc. Natl. Acad. Sci. USA 108, 2623–2628 (2011).
Boch, J. & Bonas, U. Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu. Rev. Phytopathol. 48, 419–436 (2010).
Bogdanove, A.J., Schornack, S. & Lahaye, T. TAL effectors: finding plant genes for disease and defense. Curr. Opin. Plant Biol. 13, 394–401 (2010).
Romer, P. et al. Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene. Science 318, 645–648 (2007).
Kay, S., Hahn, S., Marois, E., Hause, G. & Bonas, U. A bacterial effector acts as a plant transcription factor and induces a cell size regulator. Science 318, 648–651 (2007).
Kay, S., Hahn, S., Marois, E., Wieduwild, R. & Bonas, U. Detailed analysis of the DNA recognition motifs of the Xanthomonas type III effectors AvrBs3 and AvrBs3Deltarep16. Plant J. 59, 859–871 (2009).
Romer, P. et al. Recognition of AvrBs3-like proteins is mediated by specific binding to promoters of matching pepper Bs3 alleles. Plant Physiol. 150, 1697–1712 (2009).
Hinnen, A., Hicks, J.B. & Fink, G.R. Transformation of yeast. Proc. Natl. Acad. Sci. USA 75, 1929–1933 (1978).
Szostak, J.W., Orr-Weaver, T.L., Rothstein, R.J. & Stahl, F.W. The double-strand-break repair model for recombination. Cell 33, 25–35 (1983).
Thomas, K.R., Folger, K.R. & Capecchi, M.R. High frequency targeting of genes to specific sites in the mammalian genome. Cell 44, 419–428 (1986).
Ivics, Z., Hackett, P.B., Plasterk, R.H. & Izsvak, Z. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501–510 (1997).
Kawakami, K., Shima, A. & Kawakami, N. Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc. Natl. Acad. Sci. USA 97, 11403–11408 (2000).
Akagi, K. et al. Cre-mediated somatic site-specific recombination in mice. Nucleic Acids Res. 25, 1766–1773 (1997).
Epinat, J.C. et al. A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res. 31, 2952–2962 (2003).
Lois, C., Hong, E.J., Pease, S., Brown, E.J. & Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002).
Khan, I.F., Hirata, R.K. & Russell, D.W. AAV-mediated gene targeting methods for human cells. Nat. Protoc. 6, 482–501 (2011).
Pavletich, N.P. & Pabo, C.O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science 252, 809–817 (1991).
Klug, A. The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation. Q. Rev. Biophys. 43, 1–21 (2010).
Maeder, M.L., Thibodeau-Beganny, S., Sander, J.D., Voytas, D.F. & Joung, J.K. Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays. Nat. Protoc. 4, 1471–1501 (2009).
Kim, J.S., Lee, H.J. & Carroll, D. Genome editing with modularly assembled zinc-finger nucleases. Nat. Methods 7, 91; author reply 91–92 (2010).
Sander, J.D. et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat. Methods 8, 67–69 (2011).
Perez, E.E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008).
Keenholtz, R.A., Rowland, S.J., Boocock, M.R., Stark, W.M. & Rice, P.A. Structural basis for catalytic activation of a serine recombinase. Structure 19, 799–809 (2011).
Gersbach, C.A., Gaj, T., Gordley, R.M., Mercer, A.C. & Barbas, C.F. III. Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase. Nucleic Acids Res. 39, 7868–7878 (2011).
Gaj, T., Mercer, A.C., Gersbach, C.A., Gordley, R.M. & Barbas, C.F. III. Structure-guided reprogramming of serine recombinase DNA sequence specificity. Proc. Natl. Acad. Sci. USA 108, 498–503 (2011).
Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).
Wilson, M.H., Kaminski, J.M. & George, A.L. Jr. Functional zinc finger/sleeping beauty transposase chimeras exhibit attenuated overproduction inhibition. FEBS Lett. 579, 6205–6209 (2005).
Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3, e3647 (2008).
Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE 4, e5553 (2009).
Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. A modular cloning system for standardized assembly of multigene constructs. PLoS ONE 6, e16765 (2011).
Huertas, P. DNA resection in eukaryotes: deciding how to fix the break. Nat. Struct. Mol. Biol. 17, 11–16 (2010).
Nolan, T., Hands, R.E. & Bustin, S.A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1, 1559–1582 (2006).
Guschin, D.Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247–256 (2010).
Zhang, F. et al. High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proc. Natl. Acad. Sci. USA 107, 12028–12033 (2010).
Buzdin, A.A. in Nucleic Acids Hybridization (eds. Buzdin, A., Lukyanov, S.) 211–239 (Springer, 2007).
Till, B.J., Burtner, C., Comai, L. & Henikoff, S. Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res. 32, 2632–2641 (2004).
Babon, J.J., McKenzie, M. & Cotton, R.G. The use of resolvases T4 endonuclease VII and T7 endonuclease I in mutation detection. Mol. Biotechnol. 23, 73–81 (2003).
Yang, B. et al. Purification, cloning, and characterization of the CEL I nuclease. Biochemistry 39, 3533–3541 (2000).
Kulinski, J., Besack, D., Oleykowski, C.A., Godwin, A.K. & Yeung, A.T. CEL I enzymatic mutation detection assay. Biotechniques 29, 44–46, 48 (2000).
Oleykowski, C.A., Bronson Mullins, C.R., Godwin, A.K. & Yeung, A.T. Mutation detection using a novel plant endonuclease. Nucleic Acids Res. 26, 4597–4602 (1998).
Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001).
Murakami, M.T. et al. The repeat domain of the type III effector protein PthA shows a TPR-like structure and undergoes conformational changes upon DNA interaction. Proteins 78, 3386–3395 (2010).
Scholze, H. & Boch, J. TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47–53 (2011).
Huang, P. et al. Heritable gene targeting in zebrafish using customized TALENs. Nat. Biotechnol. 29, 699–700 (2011).
Sander, J.D. et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat. Biotechnol. 29, 697–698 (2011).
Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. Nat. Biotechnol. 29, 695–696 (2011).
We thank the entire Zhang laboratory for their support. L.C. is supported by a Howard Hughes Medical Institute International Student Research Fellowship. Y.Z. is supported by a Simons Foundation Fellowship. M.M.C. is supported by a Massachusetts Institute of Technology Undergraduate Research Opportunities scholarship. F.Z. is supported by a US National Institutes of Health Transformative R01 and by the McKnight and Simons Foundations, Robert Metcalfe and Michael Boylan.
The authors declare no competing financial interests.
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Sanjana, N., Cong, L., Zhou, Y. et al. A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7, 171–192 (2012). https://doi.org/10.1038/nprot.2011.431
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