A CRISPR-based sequence proximity binding protein labelling system for scanning upstream regulatory proteins

Transcriptional regulation involves complex and dynamic protein-DNA interactions, which alter chromatin states and, consequently, regulate gene expression. In plants, current technologies face challenges in efficiently capturing dynamically DNA-binding proteins, especially transcription factors. Here, by leveraging the binding ability of dead Cas9 to specific DNA fragments and the labelling capacity of the TurboID protein for adjacent proteins, we have developed a CRISPR-based sequence proximity binding protein labelling system (CSPL) to detect promoter-binding proteins. Using this approach, we identified both known and novel upstream binding proteins on the PIF4 promoter in Arabidopsis, cabbage and rice. This demonstrates the powerful capabilities and broad potential applications of CSPL for detecting promoter-binding proteins in plants.