Acknowledgements: We thank C. Araneo and his team for FACS. R.T.T. is a Sir Henry Wellcome Postdoctoral Fellow (201387/Z/16/Z) and a Pemberton-Trinity Fellow. E.L.M. is an HHMI Fellow of The Jane Coffin Childs Memorial Fund for Medical Research. I.A.T. is a Damon Runyon-Dale F. Frey Breakthrough Scientist supported (in part) by the Damon Runyon Cancer Research Foundation (DFS-2277-16). I.K. is supported by the European Research Council (ERC-2020-STG 947709), the Israel Science Foundation (2380/21 and 3096/21), an Alon Fellowship and The Applebaum Foundation. This work was supported by an NIH grant AG11085 to S.J.E. S.J.E. is an Investigator with the Howard Hughes Medical Institute.Specificity within the ubiquitin–proteasome system 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 ~100 CRISPR screens in a single experiment, refining known C-degron pathways and identifying an additional pathway through which Cul2FEM1B targets C-terminal proline. Further, by identifying substrates for Cul1FBXO38, Cul2APPBP2, Cul3GAN, Cul3KLHL8, Cul3KLHL9/13 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 ubiquitin–proteasome system
