A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes.

ABSTRACT: Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throu

SUBMITTER: Schmid-Burgk JL 

PROVIDER: S-EPMC4142318 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature