Defining E3 Ligase-substrate Relationships Through Multiplex CRISPR Screening

Overview

Journal Nat Cell Biol

Specialty Cell Biology

Date 2023 Sep 22

PMID 37735597

Authors

Richard T Timms

Elijah L Mena

Yumei Leng

Mamie Z Li

Iva A Tchasovnikarova

Itay Koren

Stephen J Elledge

Affiliations

Soon will be listed here.

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 targets C-terminal proline. Further, by identifying substrates for Cul1, Cul2, Cul3, Cul3, Cul3 and Cul3, 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.

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