The DNA binding specificity of genome editing tools can be utilized for gene regulation systems by combining these tools with transcription-activator or -repression domains. Recent studies have shown that targeting the gene promoter sequence using multiple artificial transcription factors synergistically and efficiently regulate gene expression [1,2]. Protein associations triggered by chemicals or light are useful for regulating protein functions in a spatiotemporal manner. The availability of several inducible protein association systems has enabled the regulation of multiple genes at different loci and timings. In this study, the rapamycin-inducible system was applied for gene regulation by using multiple TALEs and dCas9. We constructed FKBP-tagged TALEs and dCas9; the effector domains with FRB, VP64 for activation and SID×4 for repression, were also prepared. The fold change in gene expression was evaluated by luciferase reporter assays. The results showed that rapamycin induced FKBP-FRB association and that gene expression was regulated according to the presence of the FRB-effector fusions. The combination of TALEs or dCas9 that showed the best increase in gene expression with FRB-VP64 was applied for controlling endogenous gene expression in 293A cells. The results showed sufficient increase in IL-1RN expression after 72 hours, as determined by quantitative reverse transcription PCR (qRT-PCR). The cells were subsequently maintained with or without rapamycin for up to 7 days, and the results revealed that the expression could be regulated by rapamycin in a time-dependent manner. The system could enable the control of gene function on cellular phenomena owing to the programmable timing of expression and differential expression of multiple genes.

Development of chemical-inducible artificial transcription factors based on sequence-specific DNA binders

254th National Meeting (2017)

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