title: Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.
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.
authors: The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037 USA, Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037 USA and Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037 USA.
authors: Brian M
authors: BM
authors: Lamb
authors: Andrew C
authors: AC
authors: Mercer
authors: Carlos F
authors: CF
authors: Barbas
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D019020:Directed Molecular Evolution; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D045522:Recombinases / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D045522:Recombinases; D014157:Transcription Factors; D004247:DNA; D003851:Deoxyribonucleases
references: Nucleic Acids Res. 2011 Jul;39(12):e82
references: 21493687
references: Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1495-500
references: 10660690
references: Nucleic Acids Res. 2013 Apr;41(7):4118-28
references: 23408851
references: Nat Methods. 2013 Mar;10(3):239-42
references: 23377379
references: Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2758-63
references: 10077584
references: Cell Res. 2012 Dec;22(12):1716-20
references: 23147789
references: Science. 2011 Sep 30;333(6051):1843-6
references: 21960622
references: J Mol Biol. 2006 Oct 20;363(2):405-21
references: 16963084
references: New Phytol. 2013 Aug;199(3):773-86
references: 23692030
references: Science. 2009 Dec 11;326(5959):1509-12
references: 19933107
references: Science. 2012 Feb 10;335(6069):716-9
references: 22223736
references: Nat Methods. 2012 Jul 01;9(8):805-7
references: 22751204
references: Nucleic Acids Res. 2012 Nov;40(21):11163-72
references: 23019222
references: Nature. 2012 Nov 1;491(7422):114-8
references: 23000899
references: Nat Biotechnol. 2011 Feb;29(2):143-8
references: 21179091
references: Nat Biotechnol. 2012 May;30(5):460-5
references: 22484455
references: Nat Commun. 2012 Jul 24;3:968
references: 22828628
references: Nucleic Acids Res. 2012 Aug;40(15):e117
references: 22740649
references: Mol Plant Microbe Interact. 2005 Aug;18(8):838-48
references: 16134896
references: Mol Biosyst. 2012 Apr;8(4):1255-63
references: 22301904
references: Angew Chem Int Ed Engl. 2012 Jan 2;51(1):58-66
references: 22162047
references: Curr Opin Biotechnol. 2012 Aug;23(4):516-21
references: 22237015