Quantitative Analysis of TALE-DNA Interactions Suggests Polarity Effects

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2013 Feb 15

PMID 23408851

Citations 84

Authors

Joshua F Meckler

Mital S Bhakta

Moon-Soo Kim

Robert Ovadia

Chris H Habrian

Artem Zykovich

Abigail Yu

Sarah H Lockwood

Robert Morbitzer

Janett Elsaesser

Thomas Lahaye

David J Segal

Enoch P Baldwin

Affiliations

Soon will be listed here.

Abstract

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN >> NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

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References

Perez-Pinera P, Ousterout D, Gersbach C . Advances in targeted genome editing. Curr Opin Chem Biol. 2012; 16(3-4):268-77. PMC: 3424393. DOI: 10.1016/j.cbpa.2012.06.007. View

cermak T, Doyle E, Christian M, Wang L, Zhang Y, Schmidt C . Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011; 39(12):e82. PMC: 3130291. DOI: 10.1093/nar/gkr218. View

Peck L, Wang J . Sequence dependence of the helical repeat of DNA in solution. Nature. 1981; 292(5821):375-8. DOI: 10.1038/292375a0. View

Roulet E, Busso S, Camargo A, Simpson A, Mermod N, Bucher P . High-throughput SELEX SAGE method for quantitative modeling of transcription-factor binding sites. Nat Biotechnol. 2002; 20(8):831-5. DOI: 10.1038/nbt718. View

Morbitzer R, Romer P, Boch J, Lahaye T . Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors. Proc Natl Acad Sci U S A. 2010; 107(50):21617-22. PMC: 3003021. DOI: 10.1073/pnas.1013133107. View

Cong L, Zhou R, Kuo Y, Cunniff M, Zhang F . Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. Nat Commun. 2012; 3:968. PMC: 3556390. DOI: 10.1038/ncomms1962. View

Zhang F, Cong L, Lodato S, Kosuri S, Church G, Arlotta P . Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol. 2011; 29(2):149-53. PMC: 3084533. DOI: 10.1038/nbt.1775. View

Szczepek M, Brondani V, Buchel J, Serrano L, Segal D, Cathomen T . Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol. 2007; 25(7):786-93. DOI: 10.1038/nbt1317. View

Deng D, Yin P, Yan C, Pan X, Gong X, Qi S . Recognition of methylated DNA by TAL effectors. Cell Res. 2012; 22(10):1502-4. PMC: 3463267. DOI: 10.1038/cr.2012.127. View

10.

Garg A, Lohmueller J, Silver P, Armel T . Engineering synthetic TAL effectors with orthogonal target sites. Nucleic Acids Res. 2012; 40(15):7584-95. PMC: 3424557. DOI: 10.1093/nar/gks404. View

11.

Streubel J, Blucher C, Landgraf A, Boch J . TAL effector RVD specificities and efficiencies. Nat Biotechnol. 2012; 30(7):593-5. DOI: 10.1038/nbt.2304. View

12.

Deng D, Yan C, Pan X, Mahfouz M, Wang J, Zhu J . Structural basis for sequence-specific recognition of DNA by TAL effectors. Science. 2012; 335(6069):720-3. PMC: 3586824. DOI: 10.1126/science.1215670. View

13.

Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S . Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009; 326(5959):1509-12. DOI: 10.1126/science.1178811. View

14.

Mak A, Bradley P, Cernadas R, Bogdanove A, Stoddard B . The crystal structure of TAL effector PthXo1 bound to its DNA target. Science. 2012; 335(6069):716-9. PMC: 3427646. DOI: 10.1126/science.1216211. View

15.

Huang P, Xiao A, Zhou M, Zhu Z, Lin S, Zhang B . Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol. 2011; 29(8):699-700. DOI: 10.1038/nbt.1939. View

16.

Silva G, Poirot L, Galetto R, Smith J, Montoya G, Duchateau P . Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Curr Gene Ther. 2010; 11(1):11-27. PMC: 3267165. DOI: 10.2174/156652311794520111. View

17.

Mercer A, Gaj T, Fuller R, Barbas 3rd C . Chimeric TALE recombinases with programmable DNA sequence specificity. Nucleic Acids Res. 2012; 40(21):11163-72. PMC: 3510496. DOI: 10.1093/nar/gks875. View

18.

Schornack S, Meyer A, Romer P, Jordan T, Lahaye T . Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins. J Plant Physiol. 2006; 163(3):256-72. DOI: 10.1016/j.jplph.2005.12.001. View

19.

Miller J, Tan S, Qiao G, Barlow K, Wang J, Xia D . A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2010; 29(2):143-8. DOI: 10.1038/nbt.1755. View

20.

Christian M, cermak T, Doyle E, Schmidt C, Zhang F, Hummel A . Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010; 186(2):757-61. PMC: 2942870. DOI: 10.1534/genetics.110.120717. View