(87c) Orthogonal Regulation of Human Genes Using Chemically Induced Crispr/Cas9 Activators

Synthetic transcription factors (sTFs) represent a powerful tool for regulating endogenous gene expressions in human cells. With the rapid advancement of CRISPR technologies, catalytically dead Cas9 (dCas9) has become the go-to choice as DNA binding domain (DBD) for the creation of sTFs. dCas9 can be readily targeted to virtually any DNA sequences within the human genome, by simply changing the guide sequence of dCas9 guide RNA (gRNA). This feature makes dCas9 an ideal DBD for simultaneously regulating multiple endogenous gene targets, as is required in programming embryonic stem cell or induced pluripotent stem cell differentiation. To achieve efficient, fast and reliable programming, it is desirable to impart spatial and temporal control over endogenous genes and gene networks. Current studies directly fuse dCas9 with effector domains such as activators, repressors and epigenetic modifiers. A lack of control over these constitutive sTFs makes it difficult to reset, fine tune and orchestrate expression levels of key lineage-associated endogenous TFs, which is crucial for directed programming of cell fate. To achieve this level of regulation, we have engineered orthogonal transcription activators based on two commonly used CRISPR/Cas9 systems from Streptococcus pyogenes and Neisseria meningitidis. We have shown that these two systems are orthogonal in terms of gRNA recognition. When fusing these dCas9s and a synthetic transactivation domain to chemically induced dimerization (CID) proteins, we were able to activate both transgenes and endogenous human genes in a ligand-dependent manner. We further showed that multiple endogenous human genes can be simultaneously activated with addition of ligands. Finally, we used these two orthogonal systems to regulate the expression pattern of two different pairs of endogenous human genes. In each pair of genes, each gene was only activated in the presence of the corresponding ligand. With both ligands present, both genes could be activated. To our knowledge, this is the first example showing coordinated regulation of multiple endogenous human genes. Given the existence of various orthogonal CRISPR/Cas9 and CID systems, this study serves as the first step toward achieving precise regulation of complex gene networks and may elicit new therapeutic applications.