Willaume, S.; Benada, J.; Akopyan, K.; Petrosius, V.; Thatte, J. V.; Miller, T. C. R.; Lindqvist, A.; Sorensen, C. S.

Connecting protein post-translational modifications (PTMs) to phenotypic outcomes remains challenging. While phosphoproteomics has catalogued numerous sites, reliable methods for measuring endogenous effects of individual phosphosites on cellular fitness are lacking. Here, we introduce CRISPR-PTM and CRISPR-VEIS as complementary platforms for quantitative, endogenous phosphosite interrogation. CRISPR-PTM is a multiplexed knock-in framework creating defined phosphosite variants with internal allelic markers, enabling precise fitness measurements in pooled populations. CRISPR-VEIS (Visualisation of Edits In Situ) links endogenous edits to single-cell phenotypes via in situ mRNA-genotyping, bypassing clonal isolation. Applied to the essential WEE1-CDK1 pathway, we investigated why canonical CDK1-Y15 phosphorylation alone cannot explain WEE1 loss phenotypes. CRISPR-PTM quantified fitness consequences of CDK1 variants, identifying Y19 as a novel WEE1-dependent inhibitory site. While single Y15 or Y19 substitutions had minimal impact, combined CDK1-Y15F/Y19F editing caused severe fitness defects, phenocopying WEE1 inactivation. CRISPR-VEIS confirmed that acute endogenous editing of both sites correlated with elevated single-cell CDK activity. Together, these methods provide broadly applicable approaches linking endogenous phosphosites to cellular fitness and signaling, revealing critical functional redundancies in essential regulatory networks.