Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription

Journal name:
Nature Biotechnology
Volume:
29,
Pages:
149–153
Year published:
DOI:
doi:10.1038/nbt.1775
Received
Accepted
Published online

The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator–like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.

At a glance

Figures

  1. Design and construction of customized artificial TALEs for use in mammalian cells.
    Figure 1: Design and construction of customized artificial TALEs for use in mammalian cells.

    (a) Schematic representation of the native TALE hax3 from Xanthomonas campestris pv. armoraciae depicting the tandem repeat domain and the two repeat variable di-residues (red) within each repeat monomer. These di-residues determine the base recognition specificity. The four most common naturally occurring di-residues used for the construction of customized artificial TAL effectors are listed together with their proposed major base specificity. NLS, nuclear localization signal; AD, activation domain of the native TAL effector. (b) Schematic of the hierarchical ligation assembly method for the construction of customized TALEs. Twelve separate PCRs are done for each of the four types of repeat monomers (NI, HD, NG and NN) to generate a set of 48 monomers to serve as assembly starting material. Each of the 12 PCR products for a given monomer type (e.g., NI) has a unique linker specifying its programmed position in the assembly (color-coded digestion and ligation adapters). After enzymatic digestion with a type IIs restriction endonuclease (e.g., BsaI), orthogonal overhangs are made by recoding each amino acid in the junction to use an alternative codon. The unique overhangs facilitate the positioning of each monomer in the ligation product. The ligation product was PCR amplified subsequently to yield the full-length repeat regions, which were then cloned into a backbone plasmid containing the N and C termini of the wild-type TALE hax3. (c) Schematic representation of the fluorescence reporter system for testing TALE-DNA recognition. The diagram illustrates the composition of the tandem repeat for a TALE and its corresponding 14-bp DNA-binding target in the fluorescent reporter plasmid. VP64, synthetic transcription activation domain; 2A, self-cleavage peptide. (d) 293FT cells co-transfected with a TALE plasmid and its corresponding reporter plasmid showed considerably greater mCherry expression compared with the reporter-only control. Scale bars, 200 μm.

  2. Functional characterization of the robustness of TALE-DNA recognition in mammalian cells and truncation analysis of TALE N- and C-termini.
    Figure 2: Functional characterization of the robustness of TALE-DNA recognition in mammalian cells and truncation analysis of TALE N- and C-termini.

    (a) Thirteen TALEs were tested with their corresponding reporter constructs. Customized repeat regions and binding site sequences are shown on the left. The activities of the TALEs on target gene expression are shown on the right as the fold induction of the mCherry reporter gene. Fold induction was determined by flow cytometry analysis of mCherry expression in transfected 293FT cells, and calculated as the ratio of the total mCherry fluorescence intensity of cells transfected with and without the specified TALE, normalized by the GFP fluorescence to control for transfection efficiency differences (Online Methods). (b) The N- and C-terminal amino acid sequence of wild-type TAL effector hax3 showing the positions of all N- and C-terminal truncation constructs tested in 293FT cells. N0 to N8 designates N-terminal truncation positions (N0 retains the full-length N terminus), and C0 to C7 designate C-terminal truncations. Amino acids representing the nuclear localization signal and the activation domain in the native hax3 protein are underlined. (c) Relative activity of each N-terminal TALE truncation construct compared to the TALE (N0-C0). TALE truncation positions are indicated in b. Error bars indicate s.e.m.; n = 3. TALE-TALE relative activity was calculated by dividing the fold induction of the construct by the fold induction of the reporter gene. Fold induction calculated as in a. (d) Relative activity of each C-terminal truncation compared to TALE(N1,C0).

  3. Activation of endogenous genes in human cells by TALEs.
    Figure 3: Activation of endogenous genes in human cells by TALEs.

    (a) TALEs designed to target the genes SOX2, KLF4, c-MYC and OCT4 facilitate activation of mCherry reporter in 293FT cells. The target sites are selected from the 200-bp proximal promoter region. Fold induction was determined by flow cytometry analysis using the same methodology as in Figure 2 and detailed in Online Methods. (b) Images of TALE-induced mCherry reporter expression in 293FT cells. Scale bar, 200 μm. (c) Levels of SOX2 and KLF4 mRNA in transfected 293FT cells, as determined by quantitative RT-PCR. Mock-treated cells received the transfection vehicle. TALE1 was used as a negative control. Error bars indicate s.e.m.; n = 3. *** indicates P < 0.005.

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Author information

  1. These authors contributed equally to this work.

    • Feng Zhang &
    • Le Cong

Affiliations

  1. Society of Fellows, Harvard University, Cambridge, Massachusetts, USA.

    • Feng Zhang
  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Feng Zhang,
    • Le Cong,
    • Sriram Kosuri &
    • George M Church
  3. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.

    • Feng Zhang,
    • Le Cong,
    • Sriram Kosuri &
    • George M Church
  4. Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, USA.

    • Le Cong
  5. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Feng Zhang,
    • Simona Lodato &
    • Paola Arlotta
  6. Center for Regenerative Medicine and Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Simona Lodato &
    • Paola Arlotta
  7. Present address: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA; McGovern Institute for Brain Research, MIT, Cambridge, Massachusetts, USA; and Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA.

    • Feng Zhang

Contributions

F.Z. and L.C. conceived the study. F.Z., L.C., S.L. and S.K. designed, performed and analyzed all experiments. P.A. supervised the work of S.L. and G.M.C. supervised the work of F.Z., L.C. and S.K. G.M.C., P.A. and F.Z. provided support for this study. F.Z., L.C. and P.A. wrote the manuscript with support from all authors. G.M.C and P.A. equally contributed to this work.

Competing financial interests

The authors declare no competing financial interests.

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    Supplementary Tables 1–3, Supplementary Figs. 1–3, Supplementary Methods and Supplementary Sequences

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