(337br) Engineering Next-Generation Crispri Systems for Improved Cancer Drug Discovery

The ability to turn off (silence or repress) genes in cancer cells on-demand is vital for discovering connections between gene expression and attributes such as growth rates and drug resistance. CRISPR interference (CRISPRi) technology is a new method enabling rapid, reliable, and highly specific repression of gene expression for these applications. The CRISPRi system works by delivering an engineered fusion protein (consisting of the bacterial protein dCas9 attached to additional repressor domains) to the nucleus of host cells. Once internalized within cells, dCas9 complexes with a tunable guide RNA (sgRNA) molecule, localizes to the gene of interest encoded by the sgRNA, and then reduces expression of that specific gene. Although CRISPRi holds great promise for cancer and immunological drug discovery, it suffers from incomplete gene repression and high performance variability across different cell types. Because of these limitations, we sought to discover new repressors, characterize their mechanisms of action, and assemble new repressor combinations to generate improved CRISPRi systems. To do this, we measured gene knockdown activity of potential repressor domains and identified eleven novel CRISPRi-compatible domains that recruit co-factors from diverse biological pathways. Next, we screened a combinatorial library of two-part repressor fusions and identified four unique variants each achieving ~30% improved gene silencing relative to current CRISPRi systems. We then analyzed libraries of three-part repressor fusions and discovered one variant achieving the strongest reported CRISPRi activity to-date. In addition, we confirmed that our variants exhibit best-in-class performance in a broad panel of human cancer cell lines without non-specific toxicity. We envision these findings will accelerate the adoption of CRISPRi technology in the pharmaceutical industry as a powerful tool for cancer drug discovery.