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ABSTRACT
Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.
ZINC finger nucleases (ZFNs) and meganucleases cleave specificDNAtarget sequences in vivo and are powerful tools forgenomemodification(Carroll 2008; Cathomen and Joung 2008; Galetto et al. 2009). Chromosome breaks created by these engineered nucleases stimulatehomologous recombination or gene targeting: in the presence of a template for repairing the double-strand break, specific DNA sequence changes in the template become incorporated intochromosomes at or near the break site. In the absence of a repairtemplate, broken chromosomes are rejoined by nonhomologous end joining, which often introduces short DNA insertions or deletions that create targeted gene knockouts. For bothZFNsandmeganucleases, a barrier to their widespread adoption has been the challenge in engineering newDNAbinding specificities. While significant progress has been made in recent years (Urnov et al. 2005; Fajardo-Sanchez et al. 2008; Maeder et al. 2008; Kim et al. 2009), generating the necessary reagents to modify new DNA targets is still resource intensive and to some degree empirical.
A novel DNA binding domain was recently described in a family of proteins known as transcription activatorlike effectors (TALEs) (Boch et al. 2009; Moscou and Bogdanove 2009). TALEs are produced by plant pathogens in the genus Xanthomonas, which deliver the proteins to plant cells during infection through the type III secretion pathway (Bogdanove et al. 2010). Once inside the plant cell, TALEs enter the nucleus, bind effector-specific DNA sequences, and transcriptionally activate gene expression. Typically, activation of target genes increases plant susceptibility to pathogen colonization, but in some cases, it triggers plant defense. TALE binding to DNA is mediated by a central region of these proteins that contains as many as 30 tandemrepeats of a 33- to 35-amino-acid-sequence motif (Figure 1A). The amino acid sequence of each repeat is largely invariant, with the exception of two adjacent amino acids (the repeat variable diresidue or RVD). Repeats with different RVDs recognize different DNA base pairs, and there is a one-to-one correspondence between the RVDs in the repeat domain and the nucleotides...