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Defining E3 ligase-substrate relationships through multiplex CRISPR screening.

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Mena, Elijah L 
Leng, Yumei 
Li, Mamie Z 
Tchasovnikarova, Iva A 


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.


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.


Ubiquitin-Protein Ligases, Proteasome Endopeptidase Complex, Clustered Regularly Interspaced Short Palindromic Repeats, Ubiquitin

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Nat Cell Biol

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Springer Science and Business Media LLC
Wellcome Trust (201387/Z/16/Z)