Regulatory Element and Cell-Type Specific Landscapes Control dCas9-Based Transcription Factor Efficacy in Human Cells

dCas9-based technologies have emerged as powerful tools to site-specifically write and erase endogenous epigenetic modifications and to modulate gene expression. However, a comprehensive comparison and benchmarking of dCas9-based transcriptional activators at cell-type specific human enhancers in different cell types is lacking. The functional evaluation of dCas9-based activators at enhancers is needed because enhancer function is tightly linked to cognate promoter activity (i.e. gene expression levels), developmental disorders, and human diseases. Here, we compare the efficacy of five state-of-the-art dCas9-based activators (dCas9-VPR, dCas9-SunTag, dCas9-SAM, dCas9-p300, and a novel dCas9-CBP fusion protein) at three testbed enhancer/promoter pairs across three human cell lines to evaluate the cell-type specific influence over their functions. Our results demonstrate that different dCas9-based activators have altered protein stabilities that vary based upon cell type, and furthermore, that the functional efficacy at enhancers and promoters is both locus and cell type specific. Furthermore, we conducted focused comparisons of theses activators spanning 1.5 kb over the OCT4 testbed promoter locus to illuminate how basal chromatin architecture impacts dCas9-based transcriptional activation, which reveals that underlying chromatin context impacts efficacy, in addition to cell-type specific milieu. Our comprehensive comparison of dCas9-based activators confirms the robust efficacy of our dCas9-CBP fusion protein at human enhancers streamlines the applicability of existing dCas9-based activators. Our work also provides insights that pave the way for future studies to define how cell-type specific cis (e.g. epigenetic context) and trans factors (e.g. protein complexes) influence both natural and synthetic transcriptional regulation.