Multiplex CRISPR screening to identify E3 ligase substrates

The ubiquitin-proteasome system (UPS) is the primary route through which the cell achieves selective protein degradation. Specificity is thought to be primarily achieved via the E3 ubiquitin ligases, but for many E3s their substrates remain largely unknown. Thus, we have developed a multiplex CRISPR screening platform to assign E3 ligases to their cognate substrates at scale. This approach combines GPS expression screens with loss-of-function CRISPR screening to simultaneously identify the E3 ligases responsible for the instability of hundreds of GFP-tagged substrates in a single experiment. This approach is compatible with both full-length protein substrates and peptide substrates fused to GFP, can accommodate starting pool of substrates of varying stabilities, and, when combined with saturation mutagenesis, can assign E3 ligases to their cognate degron motifs. In total, a typical multiplex screen can be completed in a few months.