Defining E3 ligase-substrate relationships through multiplex CRISPR screening.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Specificity in the ubiquitin-proteasome system relies on E3 ubiquitin ligases, but their substrates and recognition motifs (degrons) are often unknown.
• Existing methods for E3 ligase-substrate assignment are time-consuming and have low throughput.

Purpose of the Study:

• To develop a scalable and high-throughput platform for assigning E3 ubiquitin ligases to their cognate substrates.
• To identify novel substrate recognition pathways and degron motifs for various E3 ligases.

Main Methods:

• Development of a multiplex CRISPR screening platform enabling simultaneous execution of numerous screens.
• Proof-of-principle screen involving ~100 CRISPR screens in a single experiment.
• Integration with site-saturation mutagenesis to identify specific degron motifs.

Main Results:

• Successfully refined known C-degron pathways and identified a new pathway involving Cul2^FEM1B targeting C-terminal proline.
• Identified substrates for multiple E3 ligases, including Cul1^FBXO38, Cul2^APPBP2, Cul3^GAN, Cul3^KLHL8, Cul3^KLHL9/13, and Cul3^KLHL15.
• Demonstrated platform compatibility with diverse protein substrate stabilities and its ability to assign degron motifs.

Conclusions:

• Multiplex CRISPR screening is a powerful tool for large-scale E3 ligase-substrate assignment.
• This approach significantly accelerates the characterization of E3 ligase specificity and degron recognition.
• The findings will advance the understanding of ubiquitin-proteasome system regulation.