CRISPR-PTM and CRISPR-VEIS: Multiplexed platforms for quantitative functional analysis of endogenous phosphosites

Connecting protein post-translational modifications (PTMs) to phenotypic outcomes is a central challenge. Although phosphoproteomics has richly catalogued specific sites, reliable methods to measure the endogenous effects of individual phosphosites on cellular fitness and signaling are still lacking. Here, we introduce CRISPR-PTM and CRISPR-VEIS as complementary platforms for quantitative, endogenous phosphosite interrogation at both individual and clustered phosphorylation events. CRISPR-PTM is a multiplexed knock-in framework generating defined phosphosite variants with internal allelic markers, enabling precise relative fitness effects in pooled populations. CRISPR-VEIS (Visualisation of Edits In Situ) is an in situ mRNA-genotyping approach that directly links endogenous allelic edits to single-cell phenotypes, addressing needs for subclonal isolation or exogenous reporters. We applied these methods to the WEE1-CDK1 regulatory pathway, where canonical CDK1-Y15 phosphorylation alone cannot explain WEE1 loss or inhibition phenotypes. CRISPR-PTM systematically quantified fitness consequences of CDK1 phosphosite variants and identified Y19 as a previously unrecognized WEE1-dependent inhibitory site. Single non-phosphorylatable substitutions at Y15 or Y19 had minimal impact, but combined CDK1-Y15F/Y19F editing caused pronounced fitness defects, phenocopying WEE1 inactivation and showing epistasis to WEE1 inhibitors. CRISPR-VEIS further demonstrated that acute endogenous editing of both sites correlated with elevated CDK activity at the single-cell level. Together, CRISPR-PTM and CRISPR-VEIS provide broadly applicable approaches for quantitative analysis of PTM function, enabling direct linkage of endogenous phosphosite variation to cellular fitness and signaling phenotypes.