Sterically-Modulated Affinity by RNA Triggers (SMART): a novel toolbox for the purification of CRISPR Cas nucleases

About This Grant

CRISPR is a new gene-editing technology that is revolutionizing medicine, agriculture, and biotechnology. CRISPR offers hope for treating genetic diseases and creating improved crops and foods. These applications require manufacturing the protein complexes needed for CRISPR at high purity and large scale. This project will develop an innovative technology to purify these enzyme complexes. The research team will create a purification toolkit that can efficiently separate and collect only the most active forms of CRISPR complexes, making the purification process faster, more reliable, and cost-effective. This project will speed up medical discoveries and support the growing bioeconomy. In addition, it will provide hands-on training in advanced biomanufacturing and provide educational opportunities in STEM fields. This project supports the national interest by strengthening U.S. leadership in biotechnology and preparing a skilled workforce for the future. This project aims to develop an affinity-based purification platform for CRISPR ribonucleoproteins (RNPs). RNPs are complexes of Cas nucleases and guide RNAs (gRNAs) that are essential for CRISPR. The core challenge addressed by this effort is the scalable, high-purity isolation of active CRISPR RNPs from cell lysates. The project will develop "Sterically-Modulated Affinity by RNA Trigger" (SMART) peptide ligands. These ligands will overcome current manufacturing limitations by removing both process- and product-related impurities, particularly Cas proteins mis-complexed with host RNAs. SMART peptides tethered to a solid support will selectively capture apoCas (inactive Cas protein) from lysates. The pure, active RNPs will then be released by adding gRNA. The project will (1) elucidate the molecular and kinetic mechanisms governing Cas9 capture and gRNA-regulated release. This will be done through a combination of experimental and computational methods; (2) extend the SMART ligand discovery platform to other high-value CRISPR nucleases, including Cas12a and Cas13b; and (3) integrate these findings into a robust and optimized chromatographic purification process. This project will deliver the first stimuli-responsive affinity purification technology for CRISPR-Cas systems. This will accelerate the widespread adoption of CRISPR tools. Additional benefits will come from training a modern STEM workforce through targeted educational programs, and fostering technology transfer through partnerships with industry and regulatory agencies. 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.