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

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  • Author(s): Lamb BM;Lamb BM; Mercer AC; Barbas CF 3rd
  • Source:
    Nucleic acids research [Nucleic Acids Res] 2013 Nov; Vol. 41 (21), pp. 9779-85. Date of Electronic Publication: 2013 Aug 26.
  • Publication Type:
    Journal Article; Research Support, N.I.H., Extramural
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE
    • Publication Information:
      Publication: 1992- : Oxford : Oxford University Press
      Original Publication: London, Information Retrieval ltd.
    • Subject Terms:
      DNA-Binding Proteins/*chemistry ; Deoxyribonucleases/*chemistry ; Recombinases/*chemistry ; Transcription Factors/*chemistry; Base Sequence ; Binding Sites ; DNA/chemistry ; DNA/metabolism ; DNA-Binding Proteins/metabolism ; Deoxyribonucleases/metabolism ; Directed Molecular Evolution ; Protein Binding ; Protein Structure, Tertiary ; Recombinases/metabolism ; Transcription Factors/metabolism
    • 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.
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    • Grant Information:
      DP1 CA174426 United States CA NCI NIH HHS; DP1CA174426 United States CA NCI NIH HHS
    • Accession Number:
      0 (DNA-Binding Proteins)
      0 (Recombinases)
      0 (Transcription Factors)
      9007-49-2 (DNA)
      EC 3.1.- (Deoxyribonucleases)
    • Publication Date:
      Date Created: 20130828 Date Completed: 20140122 Latest Revision: 20211021
    • Publication Date:
      20240829
    • Accession Number:
      PMC3834825
    • Accession Number:
      10.1093/nar/gkt754
    • Accession Number:
      23980031