SBIR Phase I: HiUGE Powered CRISPR Knock-In Library for Protein Tagging Across the Human Genome

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project impacts the ability of drug developers, translational investigators, and basic scientists to track proteins in living cells. Current approaches are time-consuming, labor-intensive and low-throughput. Investing in these approaches is therefore relatively high-risk for most investigators and are most often employed only for proteins with well-defined functions. This technology represents a rapid and scalable method for protein labeling, allowing investigators to include a broader range of proteins of interest (POIs) in a given study. By reducing the initial investment required, both academic and industry investigators are likely to expand the POIs included in a given study. Our expectation is that the scalability and flexibility of this method will accelerate drug screening and development activities, revealing more promising lead candidates. The proposed project will advance translation of the homology-independent universal genome engineering (HiUGE) method, developed as a means of evaluating a broad range of proteins in neural function and development. This project advances research in developing a number of functional knock-in vectors, allowing fluorescent proteins labeling (FluorTag), functional disruption (DisrupTag), and downstream protein purification (MassTag). Pairing these vectors with the genome-wide library under development during this project will allow the company to rapidly provide cell lines expressing proteins of interest with customized modifications, all under endogenous promoter control. The specific goal of this project is to develop a scalable, high-throughput screening platform using the technology to label every protein encoded by the human genome. Technical tasks include: identify gene specific-gRNA sequences for all coding regions, searching and prioritizing insertion sites both at the N- and C-terminal regions of all proteins using defined criteria; develop a pilot scale screening library, comprising cells with HiUGE insertion sites in 96 genes of interest; apply new automation resources to scale for all 20,000 protein-encoding genes. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.