Functional dissection of human mitotic genes using CRISPR-Cas9 tiling screens

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• Human protein-coding gene identity is known, but molecular functions and domain architecture require deeper understanding.
• Current methods like homology-based predictions and whole-gene depletion have limitations in identifying precise functional regions.

Purpose of the Study:

• To develop a novel method for de novo identification of functional regions within protein-coding genes.
• To leverage CRISPR-Cas9-induced mutations for high-resolution functional domain mapping.

Main Methods:

• Developed a CRISPR-Cas9-based tiling mutagenesis strategy to induce mutations across protein-coding genes.
• Applied the method to 48 human mitotic genes to identify essential functional regions.
• Validated screen outcomes for specific functional regions, including novel ones.

Main Results:

• Identified hundreds of functional regions essential for cell proliferation across 48 human mitotic genes.
• Discovered experimentally characterized, homology-predicted, and novel functional domains.
• Validated the role of specific regions, such as Mad1 amino acids 387-402, in kinetochore localization and chromosome segregation.

Conclusions:

• CRISPR-Cas9 tiling mutagenesis is an effective de novo method for identifying key functional domains in protein-coding genes.
• This approach facilitates the discovery of separation-of-function mutants and enables comprehensive functional annotation of the human proteome.