Deciphering protein mutation-phenotype linkages from CRISPR-based tiling mutagenesis screens.

Area of Science:

• Computational Biology
• Genomics
• Molecular Biology

Background:

• CRISPR screens map mutations to phenotypes, but linking specific mutations to function is difficult.
• Tiling mutagenesis screens introduce variants across protein sequences for detailed analysis.

Purpose of the Study:

• Introduce ProTiler-Mut, a computational framework for analyzing mutation effects from tiling screens.
• Identify novel mutation categories and functional impacts on protein substructures and interactions.
• Apply the framework to DNA damage response and T cell regulation pathways.

Main Methods:

• Developed ProTiler-Mut for analyzing tiling mutagenesis screen data.
• Applied ProTiler-Mut to base-editing screens of DNA damage response and T cell regulator proteins.
• Defined and validated a separation-of-function (SoF) mutation category.

Main Results:

• ProTiler-Mut analyzes mutation effects at residue, substructure, and protein-protein interaction (PPI) levels.
• Identified a novel separation-of-function (SoF) mutation class, enriched for pathogenic variants.
• Discovered candidate substructures for inferring unscreened mutations and prioritized phenotype-associated PPIs.
• Linked pathogenic gain-of-function mutations to disrupted MAPK1-RSK1 interactions in PD-1 expressing cells.

Conclusions:

• ProTiler-Mut provides a versatile framework for interpreting mutagenesis screens across platforms.
• The SoF mutation category offers new insights into variant pathogenicity.
• ProTiler-Mut aids in understanding mutation impacts on protein function, substructures, and interactions.