a, 13 dTALEs were tested with their corresponding reporter constructs. Customized repeat regions and binding site sequences are shown on the left. The activities of the dTALE to activate target gene expression are shown on the right as the fold induction of the mCherry reporter gene in a log scale. 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. The amino acids representing the nuclear localization signal and the activation domain in the native HAX3 protein were underlined. c, Sequential truncation at the N-terminus of dTALE1 led to decreasing levels of reporter activity. Each truncation constructs is designated by its corresponding N-terminal and C-terminal truncation positions as indicated in panel b. Cartoon representations of all truncation constructs are shown on the left; the relative activity of each dTALE truncation construct compared to the dTALE(N0-C0) is demonstrated on the right. This relative activity is calculated from the fold induction of the reporter gene. d, Sequential truncation at the C-terminus of dTALE1 was used to characterize the optimal length of the C-terminus. Truncation position is designated in panel b. The relative activity of each truncation design compared to dTALE(N1,C0) is shown on the right. All error bars indicate s.e.m.; n=3. The fold induction was determined via 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 dTALE, normalized by the GFP fluorescence to control for transfection efficiency differences, as detailed in the Online Methods.

a, Schematic representation of the native TALE hax3 from Xanthomonas campestris pv. armoraciae depicting the tandem repeat domain and the two variable repeat diresidues (red) within each repeat monomer that specify the base recognition specificity. The four most common naturally occurring diresidues that were used for the construction of customized artificial TAL effectors are listed together with their proposed major base specificity. b, Schematic of the hierarchical ligation assembly method for the construction of customized dTALEs. 12 individual PCRs are performed for each of the 4 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 individual PCR products for a given monomer type (i.e. 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 cutter (e.g. BsaI), unique overhangs (generated by leveraging the alternate codons for each amino acid in the junction) are generated. 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 dTALE-DNA recognition. The diagram illustrates the composition of the tandem repeat for a dTALE and its corresponding 14bp DNA binding target in the fluorescent reporter plasmid. NLS, nuclear localization signal; AD, activation domain of the native TAL effector; VP64, synthetic transcription activation domain; 2A, self-cleavage peptide. d, 293FT cells co-transfected with a dTALE plasmid and its corresponding reporter plasmid exhibited significant level of mCherry expression compared to the reporter-only control. Scale bar, 200μm.

a, dTALEs designed to target the pluripotency factors Sox2, Klf4, c-Myc, and Oct4 facilitate activation of mCherry reporter in 293FT cells. The target sites are selected from the 200bp proximal promoter region. The fold induction was determined via flow cytometry analysis using the same methodology as stated in Fig. 2 and detailed in Supplementary Methods. b, Images of dTALE induced mCherry reporter expression in 293FT cells. Scale bar, 200μm. c, mRNA levels of Sox2 and Klf4 in 293FT cells transfected with mock, dTALE1, Sox2-dTALE and Klf4-dTALE. Bars represent the levels of Sox2 or Klf4 mRNA in the transfected cell as determined via quantitative RT-PCR. Mock consists of cells receiving the transfection vehicle and dTALE1 is used as a negative control. All error bars indicate s.e.m.; n=3. *** p < 0.005.