Synthetic CRISPR Networks Driven by Transcription Factors via Structure-Switching DNA Translators

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biochemistry

Background:

• CRISPR-Cas systems are powerful tools in life sciences for gene editing, diagnostics, and biosensing.
• Existing CRISPR applications lack sophisticated regulatory control mechanisms.
• Transcription factors (TFs) are key regulators of gene expression.

Purpose of the Study:

• To develop a novel platform for regulating CRISPR-Cas12a activity using transcription factors.
• To engineer dynamic DNA structures (DNA translators) that respond to TF binding.
• To demonstrate the integration of TF-regulated CRISPR-Cas systems into synthetic biology networks.

Main Methods:

• Engineered DNA translators that undergo conformational changes upon TF binding.
• Utilized TATA-binding protein and Myc-Max as model transcription factors.
• Optimized DNA translators for tunable control and rapid kinetics.
• Integrated the TF-regulated CRISPR-Cas12a system with a fluorogenic RNA aptamer (Mango III).
• Established an artificial communication pathway between CRISPR-Cas12a and CRISPR-Cas13a.

Main Results:

• Demonstrated precise and tunable control over CRISPR-Cas12a trans-cleavage activity via TF binding.
• Achieved rapid response kinetics in the TF-DNA translator system.
• Successfully activated a fluorogenic RNA aptamer (Mango III) using the platform.
• Created a novel artificial communication pathway between Cas12a and Cas13a systems.

Conclusions:

• Transcription factors can effectively regulate CRISPR-Cas systems.
• The developed platform enables novel protein-nucleic acid communication channels.
• This work opens new avenues for sophisticated synthetic biology applications.