Abstract
Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas consists of a series of repeats arranged in tandem, each repeat binding a nucleotide of the target sequence. We present here a strategy for engineering of TALE proteins with novel DNA binding specificities based on the 17.5 repeat-containing AvrBs3 TALE as a scaffold. For each of the 17 full repeats, four module types were generated, each with a distinct base preference. Using this set of 68 repeat modules, recognition domains for any 17 nucleotide DNA target sequence of choice can be constructed by assembling selected modules in a defined linear order. Assembly is performed in two successive one-pot cloning steps using the Golden Gate cloning method that allows seamless fusion of multiple DNA fragments. Applying this strategy, we assembled designer TALEs with new target specificities and tested their function in vivo.
Highlights
The development of synthetic nucleases that cleave unique genomic sequences in living cells provides powerful tools for genome engineering, allowing targeted gene knockout and gene replacement [1]
DNA binding is mediated by a central repetitive region, formed by up to 33 tandem repeats of a 33 to 35 amino acid motif, each repeat corresponding to one DNA base pair of the target sequence
In addition to the 17.5 repeats, AvrBs3 contains an N-terminally adjacent repeat 0 that is thought to be specific for a thymidine (Fig. 1B)
Summary
The development of synthetic nucleases that cleave unique genomic sequences in living cells provides powerful tools for genome engineering, allowing targeted gene knockout and gene replacement [1]. The TALEs are imported into the nucleus, where they bind to specific DNA sequences and transcriptionally activate gene expression [4,5]. Repeats with different RVDs show different DNA base pair preferences, and consecutive RVDs in a TALE correspond directly to the DNA sequence in the binding side, resulting in a simple one-repeat-to-one-base pair code [6,7]. Knowledge of this TALE recognition code has been used to predict the DNA binding specificity of native TALEs and to create designer TALEs (dTALEs) which transcriptionally activate userdefined promoter sequences [8,9]. Assembly of multiple repeats with highly identical sequences by standard cloning approaches is challenging and chemical synthesis of the entire repeat region expensive
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