Proteome-wide base editor screens to assess phosphorylation site functionality in high-throughput

Abstract

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 the phosphatase 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.

Design and characterization of multiplex gene-edited CAR-T cells resistant to orthogonal immunosuppressive pathways within the solid tumor microenvironment

“…These point mutations are effective at knocking down protein expression by installing premature stop codons within genes or altering conserved intron-exon gene splice junctions resulting in improperly translated proteins, which are eliminated through nonsense mediated decay [102][103][104]. These point mutations may also install in-frame non-synonymouse mutations that alter protein binding pockets, epitopes, phosphorylation sites, or nuclear binding motifs to enact a variety of cellular changes [105][106][107]. In the context of CAR-T cell engineering, BEs could be employed to introduce point mutations that disrupt the function of inhibitory receptors or key elements of immunosuppressive pathways potentially meeting the challenge of addressing multiple pathways upregulated by the immunosuppressive TME.…”