Post-translational modification-centric base editor screens to assess phosphorylation site functionality in high throughput

Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe “signaling-to-transcription network” mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1 which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways. Various phosphosite mutants were introduced into TPR Jurkat cells via in vitro transcription of sgRNA and ABE8e (base editor) complex. To produce purely edited cell populations, single cell clones were isolated from the corresponding bulk edited cell populations by diluting 0.8 cells per well in 96-well plates. Once isolated, these cells were grown until confluence, then individually genotyped through extraction of its gDNA and PCR amplification of the edited genomic region of interest. Once validated, 4-8 single cell clones were then mixed together to avoid clone-specific effects. Transcriptional profiling was completed via single-cell RNA on five selected phosphosite mutants.