Fig. 2. A proof-of-principle multiplex CRISPR screen recapitulates known C-degron pathways.

a, Schematic representation of the dual GPS/CRISPR multiplex screening library, in which the GFP-fusion substrates were a pool of peptides enriched for C-terminal degrons targeted by Cul2 or Cul4 E3 ligase complexes, and the CRISPR sgRNA library targeted either Cul2/5 or Cul4 adaptors. b,c, Identification of KLHDC2 substrates bearing C-terminal di-glycine motifs: the multiplex screen results for six example substrates, all of which terminate with two glycine residues (b); the performance of sgRNAs targeting KLHDC2 across all substrates (c). d,e, Cullin adaptors are correctly assigned to their cognate C-terminal degrons. A range of peptide substrates bearing canonical C-degron motifs targeted by Cul2 (d) and Cul4 (e) adaptors were successfully identified. All source numerical data are available in Supplementary Tables 1–6.