Defining E3 ligase–substrate relationships through multiplex CRISPR screening
Richard T. Timms, Elijah L. Mena, Yumei Leng, Mamie Z. Li, Iva A. Tchasovnikarova, Itay Koren, Stephen J. Elledge
Abstract
Abstract 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 Cul2 FEM1B targets C-terminal proline. Further, by identifying substrates for Cul1 FBXO38 , Cul2 APPBP2 , Cul3 GAN , Cul3 KLHL8 , Cul3 KLHL9/13 and Cul3 KLHL15 , 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.