Spatiotemporal proteomic profiling of genome editing-associated microenvironment

Abstract

Genome editing outcomes are influenced by local genomic context, yet the dynamic protein assemblies that form around genome editing sites remain poorly characterized. Here, we developed a Cas9-TurboID proximity labeling platform that combines programmable genomic targeting with rapid and minimally perturbative protein labeling, enabling spatiotemporal mapping of dynamic protein assemblies associated with distinct genome editing processes. Dynamic comparative profiling identified ACTL6A and H2A.Z as candidate regulators of genome editing, and Cas9 fusion with either ACTL6A or H2A.Z enhanced editing efficiency and improved editing outcomes in heterochromatic regions, which are generally refractory to genome editing due to limited DNA accessibility. We further extended the platform to prime editing and identified WRN as a functional regulator. Together, this study provides a strategy for systematically decoding editor-associated protein microenvironments, establishing a framework for understanding genome editing mechanisms and guiding the rational engineering of genome editors.