Abstract
BackgroundTranscription activator-like effectors (TALEs) are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The modularity of TALEs DNA binding domain enables sequence-specific perturbation and offers broad applications in genetic and epigenetic studies. Although the efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking.ResultsWe established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP) applications.ConclusionWe have designed, constructed and validated a novel dual reporter system for assessing TALE mediated gene regulations. This system offers a robust and easy-to- use tool for real-time monitoring and quantifying gene expression in mammalian cells.
Highlights
Transcription activator-like effectors (TALEs) are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression
Because luciferase reporter can provide a cost-effective way of quantification, together this dual reporter system offers a powerful tool superior to any single reporter system
To monitor TALE-induced gene expression, a minimal promoter is required to turn on the transcription of down-stream reporters
Summary
Transcription activator-like effectors (TALEs) are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking. Zinc-finger nucleases (ZFNs) and meganucleases have been well-established as tools to edit specific sites in complex genomes [1,2]. The DNA-binding domain of zinc-finger nucleases are challenging to design and require experimental optimization, and the target sequences of meganucleases are limited. Transciption activator-like effector (TALE) technology has recently emerged as an alternative robust and efficient genome editing tool [3,4]. The native function of TALEs is to modulate host gene expression by binding to specific sequences in host gene promoters and activate transcription [5,6]. The DNA binding domain of TALEs consist of a central domain of 33–35 amino acid repeats
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