A Ligation-Independent Cloning Technique for High-Throughput Assembly of Transcription activator–like Effector Genes

Nat Biotechnol. 2013 Jan;31(1):76-81. doi: 10.1038/nbt.2460.

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 throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly of >600 TALEN genes in a single day. Given its simplicity, throughput and fidelity, LIC assembly will permit the generation of TAL effector gene libraries for large-scale functional genomics studies.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • HEK293 Cells
  • High-Throughput Screening Assays*
  • Humans
  • Molecular Sequence Data
  • RNA Editing
  • Trans-Activators / genetics*

Substances

  • Trans-Activators