Notably, many of these artificial RVDs showed a distinct preference for DNA bases compared with the 25 reported RVDs, and only a few of them start with 1 of the 2 frequently occurring amino acids, Asn and His ( Figure 1B, Supplementary information, Figures S3 and S4). Besides these 25 RVDs, we determined the DNA base-recognition preference of the remaining 375 RVDs that did not evolve naturally and have not been previously examined. In addition to NI, NG, HD and NN, all the natural RVDs and a few artificial RVDs showed base-recognition preferences that were similar to those reported previously 2, 5, 6, 7 ( Supplementary information, Table S3). The results obtained from this screening provide substantial information regarding the base preference of all theoretical RVDs. A total of 1 600 data points were summarized in 3 formats: heat map ( Figure 1B), histograms categorized by the 13 th residue (X') ( Supplementary information, Figure S3) and the 12 th residue (X) ( Supplementary information, Figure S4). We then determined the base-recognition efficiencies of the 400 diresidues. We introduced each of the 400 TALE-(XX') 3 constructs ( Supplementary information, Tables S1 and S2) individually into HEK293T cells together with 1 of the 4 EGFP reporter plasmids and measured both the EGFP and mCherry levels by FACS analysis. X and X' correspond to the 12 th and 13 th amino acids in a classical TALE module, respectively. To quantitatively measure the base preference of all theoretical RVDs, we created a library of TALE-(XX') 3 constructs, which covers a total of 400 types of RVDs, following a special protocol combined with the ULtiMATE assembly method 8 ( Supplementary information, Figure S2 and Data S1). This result is consistent with the current knowledge regarding the base preference of these 4 common RVDs, demonstrating that this artificial system is suitable for testing the DNA recognition ability of RVDs ( Supplementary information, Figure S1D-S1E). The identity of XX' determined TALE-(XX') 3 specificity on different EGFP reporters: NI, NG, HD and NN predominantly recognized A, T, C and G or A, respectively. These TALE-(XX') 3 constructs were individually introduced into HEK293T cells together with 1 of the 4 EGFP reporter plasmids to examine their specificities, which were determined by the fold induction of EGFP expression compared with the basal level ( Supplementary information, Data S1). We then constructed 4 TALE-(XX') 3 expression plasmids by placing the common RVDs (NI, NG, HD and NN) in the middle to target the 3A, 3T, 3C and 3G EGFP reporters, respectively ( Supplementary information, Figure S1C). To test this system, we made a control TALE (TALE-Ctrl) that is identical to TALE-(XX') 3 except for the repeat domain ( Supplementary information, Figure S1A), and confirmed that it could not activate any of the 4 EGFP reporters ( Supplementary information, Figure S1B), thus serving as the control for basal activity. We set up a screening platform composed of an artificial TALE-VP64-mCherry construct, which expresses RVD (XX') in 3-tandem repeat format (from 7 th to 9 th, TALE-(XX') 3), and 4 corresponding EGFP reporter constructs, in which potential TALE-(XX') 3-binding sites composed of 3 consecutive nucleotides (A, T, C or G) are located in front of a minCMV promoter and its downstream EGFP gene ( Figure 1A, Supplementary information, Figure S1A and Data S1). We thus decided to conduct a thorough investigation of potential RVDs, which cover all possible combinations of amino acid diresidues, for their DNA recognition capabilities. The rare RVDs, NK and NH, have better specificity for guanine than NN, but their affinity is relatively lower 3, 6, 7.

For instance, RVD NN has a high preference for both G and A.

The nucleotide recognition preference of the commonly used RVDs has been experimentally or computationally determined 2, 5. Two hypervariable residues at positions 12 and 13 (repeat-variable diresidues or RVDs) in each repeat bind to DNA, and this modular DNA-binding feature of TALE repeats has inspired the development of custom-designed TALE repeats for gene editing 2, 3, 4, 5. The most striking feature of a transcription activator-like effector (TALE) is the presence of a central DNA-binding region composed of tandem repeats of about 34 amino acids 1.

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