Improved specificity of TALE-based genome editing using an expanded RVD repertoire

Nat Methods. 2015 May;12(5):465-71. doi: 10.1038/nmeth.3330. Epub 2015 Mar 23.

Abstract

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.

MeSH terms

  • Animals
  • Base Sequence
  • DNA / genetics
  • Enzyme-Linked Immunosorbent Assay
  • Gene Expression Regulation / physiology*
  • Genetic Markers
  • Genome*
  • RNA Editing / physiology*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*

Substances

  • Genetic Markers
  • Transcription Factors
  • DNA