STTR Phase I: Harnessing CRISPR-mediated silencing for the one-step optimization of protein production strains

The broader/commercial impacts of this Small Business Technology Transfer (STTR) project will be the development of a technology to engineer E. coli that will simplify the removal of contaminating cellular proteins as part of protein purification. Despite major advancements in biopharmaceutical manufacturing, protein purification remains one of the most costly and restrictive aspects of production that impacts negatively impacting start-ups and Fortune 500 companies alike. This STTR project seeks to directly tackle this challenge by developing a platform to identify the cellular proteins that exert the greatest burden on downstream processing, and then shut off these genes in the production strain. This platform could be readily implemented across much of the biomanufacturing industry, with the potential to reduce costs to consumers in the ever-burgeoning healthcare market and ease the time and investment required to bring new therapeutics to market. This STTR Phase I project proposes to develop a single construct that can coordinately and tunably silence expression of the most burdensome contaminating proteins that are encountered when purifying protein therapeutics via ion exchange chromatography. The resulting construct could be readily incorporated into virtually any E. coli strain, offering a simple add-on to both commercialized and proprietary production strains. The planned outcome of the proposed Phase I work is a minimal viable product that substantially boosts the capture and purity of a representative biotherapeutic protein purified via ion-exchange chromatography. The associated construct will coordinately and completely turn off the expression of contaminating, non-essential genes, and finely tune the expression of contaminating, essential genes to improve target capture and purity while preserving cell growth. This construct will represent an important step toward a commercial product that could revolutionize how downstream processing is conducted in the expansive biopharmaceuticals industry.