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

Abstract

Kinetochores are large protein assemblies that mark the centromere and bind to mitotic spindle microtubules to ensure accurate chromosome segregation. Like most protein-coding genes, the full multifunctional nature of kinetochore factors remains uncharacterized due to the limited experimental tools for unbiased dissection of human protein sequences. Here, we develop a method that leverages CRISPR-Cas9 induced mutations to comprehensively identify key functional regions within protein sequences that are required for cellular outgrowth. Our analysis of 48 human kinetochore genes revealed hundreds of regions required for cell proliferation, corresponding to known and uncharacterized protein domains. We biologically validated 15 of these regions, including amino acids 387-402 of Mad1, which when deleted compromise Mad1 kinetochore localization and chromosome segregation fidelity. Altogether, we demonstrate that CRISPR-Cas9-based tiling mutagenesis enables de novo identification of key functional domains in protein-coding genes, which elucidates separation of function mutants and allows functional annotation across the human proteome.

Zombies Never Die: The Double Life Bub1 Lives in Mitosis

“…How Mad1 interacts with RZZ is not known yet. A recent study identified a region on Mad1 to be involved in interacting with RZZ has shed light on this question (Herman et al, 2021). The mechanism of RZZ-Mad1 facilitating Bub1-Mad1 interaction is also missing.…”