Abstract
Transcription activator-like effectors (TALEs) contain programmable DNA-binding domains that can be fused to various effectors to manipulate genetic sequences or transcriptional state. However, the construction of plasmids encoding the modular DNA-binding domain remains challenging due to their repetitive nature. Here, we describe methods for a simple TALE assembly reaction (STAR) that uses a 68-part plasmid library to create TALEs binding to 17 bp target sequences. Manual production of many tens of TALEs can be achieved using a simple 8 h protocol, with full length sequence-verified plasmids available within a few days. This simple tale assembly reaction (STAR) provides a convenient method for generating tens to hundreds of TALENs or TALE-TFs without the need for large plasmid libraries or expensive liquid handling.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ et al (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148
Mendenhall EM, Williamson KE, Reyon D, Zou JY, Ram O, Joung JK, Bernstein BE (2013) Locus-specific editing of histone modifications at endogenous enhancers. Nat Biotechnol 31:1133–1136
Boch J, Bonas U (2010) Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol 48:419–436
Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509–1512
Gao X, Gao X, Tsang JCH, Tsang JCH, Gaba F, Gaba F, Wu D, Wu D, Lu L, Lu L et al (2014) Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res 42:e155
Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39:e82
Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK (2012) FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30:460–465. https://doi.org/10.1038/nbt.2170
Briggs AW, Rios X, Chari R, Yang L, Zhang F, Mali P, Church GM (2012) Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic Acids Res 40(15):e117. https://doi.org/10.1093/nar/gks624
Schmid-Burgk JL, Schmidt T, Kaiser V, Höning K, Hornung V (2012) A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes. Nat Biotechnol 31(1):76–81. https://doi.org/10.1038/nbt.2460
Gogolok S, Garcia-Diaz C, Pollard SM (2016) STAR: a simple TAL effector assembly reaction using isothermal assembly. Sci Rep 6:33209
Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Meth 6:343–345
Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F (2012) A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7:171–192
Lundin S, Stranneheim H, Pettersson E, Klevebring D, Lundeberg J (2010) Increased throughput by parallelization of library preparation for massive sequencing. PLoS One 5:e10029
Tetsushi S, Hiroshi O, Takehito K, Tomoji M, Daisuke T, Yuto S, Ken-ichi S, Tatsuo M, Naoaki S, Shinya M, Takashi Y, (2013) Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Scientific Reports 3(1)
Acknowledgment
This work was supported by BBSRC UK mammalian Synthetic biology centre (BB/M018040/1). S.P. is a CRUK Senior Research Fellow (A17368).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Gogolok, S., Köber, U., Pollard, S.M. (2018). STAR: A Simple TAL Effector Assembly Reaction Using Isothermal Assembly. In: Braman, J. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1772. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7795-6_29
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7795-6_29
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7794-9
Online ISBN: 978-1-4939-7795-6
eBook Packages: Springer Protocols