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

Area of Science:

• Plant molecular biology
• Genetics
• Biotechnology

Background:

• Transcriptional regulation relies on dynamic protein-DNA interactions influencing gene expression.
• Existing technologies struggle to capture transiently DNA-binding proteins, particularly transcription factors, in plants.

Purpose of the Study:

• To develop a novel CRISPR-based system for detecting promoter-binding proteins in plants.
• To overcome limitations of current methods in capturing dynamic protein-DNA interactions.

Main Methods:

• Developed a CRISPR-based sequence proximity binding protein labelling system (CSPL).
• Leveraged dead Cas9 for specific DNA binding and TurboID for proximity-based protein labeling.
• Applied CSPL to identify proteins binding to the PIF4 promoter.

Main Results:

• Successfully identified both known and novel upstream binding proteins on the PIF4 promoter.
• Demonstrated the efficacy of CSPL in Arabidopsis, cabbage, and rice.
• Validated CSPL's capability to detect promoter-binding proteins.

Conclusions:

• CSPL is a powerful tool for identifying promoter-binding proteins in plants.
• The system has broad potential applications in plant research.
• CSPL advances the study of transcriptional regulation in diverse plant species.