A do-it-yourself protocol for simple transcription activator-like effector assembly

Biological Procedures Online

Claudia Uhde-Stone²  Nilang Gor¹  Tiffany Chin¹  Joseph Huang¹  Biao Lu¹ 

[1] System Biosciences (SBI), 265 North Whisman Rd, Mountain View, CA, 94043, USA

[2] Department of Biological Sciences, California State University, East Bay, 25800 Carlos Bee Blvd, Hayward, CA, 94542, USA

关键词: Transcription-activator-like effector;    Golden Gate;    TALE-TF;    TALEN;   

Others  :  793112
DOI  :  10.1186/1480-9222-15-3

 received in 2013-01-03, accepted in 2013-01-05,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Uhde-Stone et al.; licensee BioMed Central Ltd.

【 图 表 】

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