Defining E3 ligase-substrate relationships through multiplex CRISPR screening

Specificity within the ubiquitin-proteasome system (UPS) is primarily achieved through E3 ubiquitin ligases, but for many E3s their substrates - and in particular the molecular features (degrons) that they recognize - remain largely unknown. Current approaches for assigning E3s to their cognate substrates are tedious and low-throughput. Here we developed a multiplex CRISPR screening platform to assign E3 ligases to their cognate substrates at scale. A proof-of-principle multiplex screen successfully performed approximately 100 CRISPR screens in a single experiment, refining known C-degron pathways and identifying a novel pathway through which Cul2FEM1B targets C-terminal proline. Further, by identifying substrates for Cul1FBXO38, Cul2APPBP2, Cul3GAN, Cul3KLHL8, Cul3KLHL9, Cul3KLHL13 and Cul3KLHL15, we demonstrate that the approach is compatible with pools of full-length protein substrates of varying stabilities, and, when combined with site-saturation mutagenesis, can assign E3 ligases to their cognate degron motifs. Thus, multiplex CRISPR screening will accelerate our understanding of how specificity is achieved within the UPS.