Engineering Synthetic TAL Effectors with Orthogonal Target Sites

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2012 May 15

PMID 22581776

Citations 79

Authors

Abhishek Garg

Jason J Lohmueller

Pamela A Silver

Thomas Z Armel

Affiliations

Soon will be listed here.

Abstract

The ability to engineer biological circuits that process and respond to complex cellular signals has the potential to impact many areas of biology and medicine. Transcriptional activator-like effectors (TALEs) have emerged as an attractive component for engineering these circuits, as TALEs can be designed de novo to target a given DNA sequence. Currently, however, the use of TALEs is limited by degeneracy in the site-specific manner by which they recognize DNA. Here, we propose an algorithm to computationally address this problem. We apply our algorithm to design 180 TALEs targeting 20 bp cognate binding sites that are at least 3 nt mismatches away from all 20 bp sequences in putative 2 kb human promoter regions. We generated eight of these synthetic TALE activators and showed that each is able to activate transcription from a targeted reporter. Importantly, we show that these proteins do not activate synthetic reporters containing mismatches similar to those present in the genome nor a set of endogenous genes predicted to be the most likely targets in vivo. Finally, we generated and characterized TALE repressors comprised of our orthogonal DNA binding domains and further combined them with shRNAs to accomplish near complete repression of target gene expression.

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References

Pfaffl M, Horgan G, Dempfle L . Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002; 30(9):e36. PMC: 113859. DOI: 10.1093/nar/30.9.e36. View

Van Den Ackerveken G, Marois E, Bonas U . Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell. Cell. 1996; 87(7):1307-16. DOI: 10.1016/s0092-8674(00)81825-5. View

Mahfouz M, Li L, Piatek M, Fang X, Mansour H, Bangarusamy D . Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein. Plant Mol Biol. 2011; 78(3):311-21. PMC: 3259320. DOI: 10.1007/s11103-011-9866-x. 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

Barrett O, Chin J . Evolved orthogonal ribosome purification for in vitro characterization. Nucleic Acids Res. 2010; 38(8):2682-91. PMC: 2860124. DOI: 10.1093/nar/gkq120. View

Moscou M, Bogdanove A . A simple cipher governs DNA recognition by TAL effectors. Science. 2009; 326(5959):1501. DOI: 10.1126/science.1178817. View

Koudritsky M, Domany E . Positional distribution of human transcription factor binding sites. Nucleic Acids Res. 2008; 36(21):6795-805. PMC: 2588498. DOI: 10.1093/nar/gkn752. View

Haynes K, Silver P . Eukaryotic systems broaden the scope of synthetic biology. J Cell Biol. 2009; 187(5):589-96. PMC: 2806586. DOI: 10.1083/jcb.200908138. View

Deans T, Cantor C, Collins J . A tunable genetic switch based on RNAi and repressor proteins for regulating gene expression in mammalian cells. Cell. 2007; 130(2):363-72. DOI: 10.1016/j.cell.2007.05.045. View

10.

Kay S, Hahn S, Marois E, Wieduwild R, Bonas U . Detailed analysis of the DNA recognition motifs of the Xanthomonas type III effectors AvrBs3 and AvrBs3Deltarep16. Plant J. 2009; 59(6):859-71. DOI: 10.1111/j.1365-313X.2009.03922.x. View

11.

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

12.

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

13.

Leisner M, Bleris L, Lohmueller J, Xie Z, Benenson Y . Rationally designed logic integration of regulatory signals in mammalian cells. Nat Nanotechnol. 2010; 5(9):666-70. PMC: 2934882. DOI: 10.1038/nnano.2010.135. View

14.

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

15.

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

16.

Andrianantoandro E, Basu S, Karig D, Weiss R . Synthetic biology: new engineering rules for an emerging discipline. Mol Syst Biol. 2006; 2:2006.0028. PMC: 1681505. DOI: 10.1038/msb4100073. View

17.

Tabor J, Salis H, Simpson Z, Chevalier A, Levskaya A, Marcotte E . A synthetic genetic edge detection program. Cell. 2009; 137(7):1272-81. PMC: 2775486. DOI: 10.1016/j.cell.2009.04.048. View

18.

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

19.

An W, Chin J . Synthesis of orthogonal transcription-translation networks. Proc Natl Acad Sci U S A. 2009; 106(21):8477-82. PMC: 2689021. DOI: 10.1073/pnas.0900267106. View

20.

MacIsaac K, Lo K, Gordon W, Motola S, Mazor T, Fraenkel E . A quantitative model of transcriptional regulation reveals the influence of binding location on expression. PLoS Comput Biol. 2010; 6(4):e1000773. PMC: 2861697. DOI: 10.1371/journal.pcbi.1000773. View