Abstract
The DNA binding domain of Transcription Activator-Like (TAL) effectors can easily be engineered to have new DNA sequence specificities. Consequently, engineered TAL effector proteins have become important reagents for manipulating genomes in vivo. DNA binding by TAL effectors is mediated by arrays of 34 amino acid repeats. In each repeat, one of two amino acids (repeat variable di-residues, RVDs) contacts a base in the DNA target. RVDs with specificity for C, T and A have been described; however, among RVDs that target G, the RVD NN also binds A, and NK is rare among naturally occurring TAL effectors. Here we show that TAL effector nucleases (TALENs) made with NK to specify G have less activity than their NN-containing counterparts: fourteen of fifteen TALEN pairs made with NN showed more activity in a yeast recombination assay than otherwise identical TALENs made with NK. Activity was assayed for three of these TALEN pairs in human cells, and the results paralleled the yeast data. The in vivo data is explained by in vitro measurements of binding affinity demonstrating that NK-containing TAL effectors have less affinity for targets with G than their NN-containing counterparts. On targets for which G was substituted with A, higher G-specificity was observed for NK-containing TALENs. TALENs with different N- and C-terminal truncations were also tested on targets that differed in the length of the spacer between the two TALEN binding sites. TALENs with C-termini of either 63 or 231 amino acids after the repeat array cleaved targets across a broad range of spacer lengths – from 14 to 33 bp. TALENs with only 18 aa after the repeat array, however, showed a clear optimum for spacers of 13 to 16 bp. The data presented here provide useful guidelines for increasing the specificity and activity of engineered TAL effector proteins.
Highlights
The ability to target proteins to specific DNA sequences makes it possible to manipulate nucleic acids in vivo, for example, by creating artificial transcriptional regulators that alter gene expression or by engineering sequence-specific nucleases that modify genetic loci of interest
Our analysis of the activity of TAL effector nucleases (TALENs) that target G with either NN or NK repeat variable diresidues (RVDs) revealed three general conclusions: 1) NNcontaining TALENs are more active than their NK-containing counterparts, which can be explained by their overall higher binding affinity for G-containing targets; 2) TALENs made with NK RVDs are more specific for G-containing targets, and 3) NK RVDs have the biggest impact on TALEN activity when they occur in the N-terminal half of the repeat array
To the best of our knowledge, the first affinity data comparing the effect of RVDs specifying G to the overall affinity of the Transcription Activator-Like (TAL) effector protein
Summary
The ability to target proteins to specific DNA sequences makes it possible to manipulate nucleic acids in vivo, for example, by creating artificial transcriptional regulators that alter gene expression or by engineering sequence-specific nucleases that modify genetic loci of interest. Targeting of TAL effectors to plant gene promoters is achieved by a simple and elegant mechanism of DNA binding [3,4]. This mechanism enables the pathogen to rapidly evolve TAL effectors with new DNA sequence specificities, and by extension, it enables scientists to engineer DNA binding domains that recognize novel sites for various genome engineering applications. DNA binding by TAL effectors is mediated by the central domain of the protein, which comprises approximately 13–28 tandem repeats of a 34 amino acid motif [5]. The most common RVD that specifies G, namely NN, interacts well with A, and this interaction is achieved by hydrogen-bonding between the second asparagine in the RVD and the N7 group on these purines [3,4,6]
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