The ability to synthetically alter gene expression in target cells is vital for discovering novel therapeutic treatments for many genetic diseases and cancers. CRISPR technology, initially discovered as a form of adaptive immunity in prokaryotes, has revolutionized biomedical research through its generalizable gene editing capability. The traditional CRISPR platform employs an active protein nuclease, Cas9, in conjunction with a single guide RNA (sgRNA) to induce mutagenesis at precise locations within the DNA. To counteract the cytotoxicity associated with double-stranded DNA breaks, an inactive version of the nuclease, dCas9, can be used instead to induce gene expression changes. CRISPR interference (CRISPRi) utilizes fusions of dCas9 and transcriptional repressor domains to achieve precise, robust repression of any target gene in human cells. Since its inception, several approaches have improved CRISPRi performance, such as fusing single repressor domains from the Krüppel-associated box (KRAB) family and performing mutagenesis screens to optimize KRAB domain activity. Despite the widespread adoption of several "gold standard" repressors that resulted from previous studies, CRISPRi still suffers from incomplete repression and high-performance variability, limiting its usefulness for studying gene-phenotype relationships. In order to enhance the repressive capability of the CRISPRi system in human cells, we sought to identify novel repressor domains, characterize their activity when fused to dCas9, and perform high-throughput screens to discover improved multi-repressor domains. With this approach, we hypothesize that we can engineer improved variants to enhance the efficacy of CRISPRi beyond what is achievable with current methods.
To accomplish this goal, we first identified twenty novel human protein domains with diverse repressive mechanisms of action from a recently published tiling library. Next, we quantified the repressive capability of each putative repressor domain using a fluorescence-based reporter assay in HEK293T cells and discovered that nearly all of our novel domains exhibit compatibility with the CRISPRi system. We also incorporated publicly available RNA-sequencing data to analyze the gene expression patterns of regulatory co-factors that interact with each putative domain to explore the basis of their repressive capabilities. In future experimentation, we will construct a library and screen novel bipartite repressor fusions composed of our top-performing novel domains and current "gold standards", evaluate potential synergistic activity between the various domains, and assess their generalizability across several human cell lines. We envision that this experimental approach will yield foundational insight into human transcriptional regulation and represent a significant step forward in applying CRISPRi to regulate gene expression in human cells.
