Directed evolution of the TALE N-terminal domain for recognition of all 5' bases

Nucleic Acids Res. 2013 Nov;41(21):9779-85. doi: 10.1093/nar/gkt754. Epub 2013 Aug 26.

Abstract

Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5'-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5' T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5' T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Base Sequence
  • Binding Sites
  • DNA / chemistry
  • DNA / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism
  • Deoxyribonucleases / chemistry*
  • Deoxyribonucleases / metabolism
  • Directed Molecular Evolution
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinases / chemistry*
  • Recombinases / metabolism
  • Transcription Factors / chemistry*
  • Transcription Factors / metabolism

Substances

  • DNA-Binding Proteins
  • Recombinases
  • Transcription Factors
  • DNA
  • Deoxyribonucleases