Identity of protease-based biomarkers for viability and cytotoxicity revealed by CRISPR knockouts

A wide variety of assay chemistries are routinely employed to determine cell health within an in vitro test population. Each method relies on the consistent and robust measurement of specific biological surrogates for cell viability or cytotoxicity. Unfortunately, the precise cellular origin or identity of many of these biomarkers remain poorly characterized or unknown, and thus subject to a host of undetermined biological and chemical interferences. This work details efforts to pinpoint the enzymatic sources of a set of proteolytic activity profiles previously discovered in a phenotypic activity screen and measured in a multiplexed viability ("live cell") and cytotoxicity ("dead cell") assay. First, Clustered Regularly Interspaced Short Palindromic Repeats gene editing (CRISPR) was utilized to knockout (KO) genes encoding candidate enzymes in a human cell background to create clones for identity testing. Next, clones demonstrating discrete reduction of either the live or dead cell signals were further characterized by Western blot analysis for presence of immunogenic protein and by Sanger Sequencing of the targeted edit site. The KO data directed the sourcing of potent and selective inhibitors for orthogonal activity studies of the proteases in a parental population. Last, the utility of the multiplexed assay reagent was further explored in both non-human and human primary cell lines to characterize the universality of the application. Collectively, the positive identification of Cathepsin C (CatC) and Tripeptidyl peptidase II (TPP II) informs assay users about potential modulators of activity leading to possible interferences. Last, the work provides new information about assay performance in previously untested cell types.