Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging

Abstract

The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery but remains a major technical challenge. Here, we describe dynamic protein quantitation (DyProQ) which is effective across microscopy and multiwell platforms. Using collagen as a test protein, CRISPR-Cas9-mediated introduction of nluc (encoding NanoLuciferase, NLuc) into theCol1a2locus enabled simplification and miniaturisation of procollagen-I (PC-I) quantitation. We robustly assessed extracellular, intracellular, and subcellular PC-I levels, by correlating to known concentrations of recombinant NLuc in the presence of substrate. Loss of collagen causes tissue degeneration whereas excess collagen results in fibrosis (often with poor-outcome) and is evident in aggressive cancers; however, treatment options are extremely limited. Using collagen-DyProQ, we screened a library of 1,971 FDA-approved compounds and identified 10 candidates for repurposing in the treatment of fibrotic and 7 for degenerative diseases.