Molecular recording of cellular protein kinase activity with chemical labeling

Protein kinases control most cellular processes and aberrant kinase activity is involved in numerous diseases. To investigate the link between specific kinase activities and cellular phenotypes in heterogeneous cell populations and in vivo, we introduce molecular recorders of kinase activities for later analysis. Based on split-HaloTag and a phosphorylation-dependent molecular switch, our recorders become rapidly labeled in the presence of a specific kinase activity and a fluorescent HaloTag substrate. The kinase activity in a given cell controls the degree of fluorescent labeling whereas the recording window is set by the presence of the fluorescent substrate. We have designed specific recorders for four protein kinases, including protein kinase A. We apply our protein kinase A recorder to sort heterogeneous cell populations for subsequent transcriptome analysis, in genome-wide CRISPR screens to discover regulators of PKA activity, and to track neuromodulation in freely moving mice. To investigate genetic changes crucial PKA activity regulation. A CRISPR mediated genetic screen was carried out on RKO colorectal cancer cell line using the Heidelberg CRISPR sgRNA library sub-library A. The pool of edited RKO-KinprolaPKA cells were expanded, labeled with CPY-CA dye and flourescently sorted according to the CPY-CA/EGFP signal ratio into 3 population. Important regulator of PKA identified from the sgRNA representation in each of the 3 samples.