Fig. 3: Kinprola enables identification of PKA signaling regulators through CRISPR-knockout screening.
From: Molecular recording of cellular protein kinase activity with chemical labeling
a, A schematic of KinprolaPKA recording in RKO cells for pooled CRISPR-knockout screening. Created with BioRender.com. b, A volcano plot of gene enrichment comparing ‘high’ versus ‘low’ KinprolaPKA-labeled populations. The dashed lines indicate the FDR threshold (0.05). Putative hits and canonical regulators (PRKACA, GNAS and ADCY7) are highlighted. The data are from two independent screens. The statistical significance was calculated by fitting a linear model for each gene, and the resulting P value was corrected for multiple testing using a Benjamini–Hochberg correction. c, GSEA of the top five categories from the ‘high’ versus ‘low’ comparison. d, A dot plot of normalized KinprolaPKA labeling (CPY-CA/EGFP) in cells with individual knockouts of putative regulators. The NT group represents the NT control sgRNAs. e, A validation of putative regulators by ELISA-based PKA activity assay in cell lysates. The PKA activity was normalized to the NT group. The circles and triangles represent two distinct sgRNAs per gene for d and e. The error bars indicate the mean ± standard error of the mean for d and e, and the data are from six (for d) or three (for e) independent experiments with duplicates. The same RKO cell line was used in b–e. The statistical significance was calculated with unpaired two-tailed Welch’s t-test. The P values are shown for d and e. n.s., not significant; UV, ultraviolet.
