Differential Regulation of Endogenous Genes in an Orthogonal Manner By Distinct Chemically Inducible Systems

Genome editing platforms are powerful tools for gene manipulation. These platforms offer a wide variety of promising applications in medical science and biotechnology. Induction of protein domain associations by stimuli such as light or chemicals has been widely used for the temporal control of biomolecular function such as enzymatic activity. Further, the DNA binding specificity of genome editing tools can be applied in gene regulation systems by combining them with transcription-activator or transcription-repressor domains. Targeting the gene promoter sequence using multiple artificial transcription factors enables synergistic and efficient regulation of gene expression. In addition, the use of these platforms in combination can expand the scope of targetable sequences by avoiding DNA-binding issues, such as PAM recognition for CRISPR-Cas9, 5’-T recognition for TALE domain, and complementary strand recognition for ZF domain. Furthermore, the guide RNA for Cas9 cannot distinguish alleles because of the commonly used gRNA sequence recognized by Cas9, thereby limiting the utility of this approach for orthogonal regulation of multiple genes. In this study, the rapamycin- (R-) or gibberellin- (G-) inducible system was applied for gene regulation using multiple TALEs and dCas9 to enable orthogonally inducible regulation of endogenous genes. For the R-inducible system, FKBP-tagged TALEs, and dCas9, the effector domains with FRB, VP64 for activation and SID×4 for repression, were constructed. GID-tagged TALEs and dCas9 as well as GAI-fused VP64 and SID×4 were constructed for the G-inducible system. The fold change in gene expression was evaluated by luciferase reporter assays. The results showed that gene expression was regulated by addition of chemicals, indicating rapamycin-induced FKBP-FRB association and gibberellin-induced GAI-GID association. The systems were further utilized for controlling endogenous gene expression in 293A cells. The results revealed that the expression of two genes could be differentially regulated by rapamycin and gibberellin in a time-dependent manner. This system could achieve the control of gene function in cellular phenomena owing to the programmable timing of expression and differential expression of multiple genes.