A do-it-yourself protocol for simple transcription activator-like effector assembly
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Biological Procedures Online
METHODOLOGY
Open Access
A do-it-yourself protocol for simple transcription activator-like effector assembly Claudia Uhde-Stone1, Nilang Gor1,2, Tiffany Chin2, Joseph Huang2 and Biao Lu2*
Abstract Background: TALEs (transcription activator-like effectors) are powerful molecules that have broad applications in genetic and epigenetic manipulations. The simple design of TALEs, coupled with high binding predictability and specificity, is bringing genome engineering power to the standard molecular laboratory. Currently, however, custom TALE assembly is either costly or limited to few research centers, due to complicated assembly protocols, long set-up time and specific training requirements. Results: We streamlined a Golden Gate-based method for custom TALE assembly. First, by providing ready-made, quality-controlled monomers, we eliminated the procedures for error-prone and time-consuming set-up. Second, we optimized the protocol toward a fast, two-day assembly of custom TALEs, based on four thermocycling reactions. Third, we increased the versatility for diverse downstream applications by providing series of vector sets to generate both TALENs (TALE nucleases) and TALE-TFs (TALE-transcription factors) under the control of different promoters. Finally, we validated our system by assembling a number of TALENs and TALE-TFs with DNA sequencing confirmation. We further demonstrated that an assembled TALE-TF was able to transactivate a luciferase reporter gene and a TALEN pair was able to cut its target. Conclusions: We established and validated a do-it-yourself system that enables individual researchers to assemble TALENs and TALE-TFs within 2 days. The simplified TALE assembly combined with multiple choices of vectors will facilitate the broad use of TALE technology. Keywords: TALEN, TALE-TF, Golden Gate, Transcription-activator-like effector
Background With the recent emergence of transcription activator-like effector (TALE) technology, gene editing has entered an exciting new era [1-3]. While zinc finger nucleases have been well established for the purpose of generating targeted mutations [4], their challenging design and need for experimental optimization have restricted this technology to few, highly specialized laboratories. In contrast, TALEs are simple to design, able to target almost any DNA sequence within the genome, and promise less off-target effects compared to zinc-finger nucleases [5-7]. Native TALEs are transcription factors used by plantpathogenic bacteria in the genus Xanthomonas. They activate transcription of host genes by binding to specific sequences in the promoter region of the targeted gene [8,9]. Strikingly, the TALE DNA binding domain consists * Correspondence: [email protected] 2 System Biosciences (SBI), 265 North Whisman Rd, Mountain View, CA 94043, USA Full list of author information is available at the end of the article
of tandem 33–35 amino acid repeats, followed by a single half repeat of 20 amino acids. Interestingly, the tandem repeats are nearly ident
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