Synthetic circuits based on split Cas9 to detect cellular events.

Area of Science:

• Synthetic biology
• Molecular biology
• Biotechnology

Background:

• Synthetic biology utilizes logic gates for controlled biological functions.
• CRISPR technology is widely used but less applied to logic gates beyond gRNA control.
• Split Cas9 offers a novel approach for engineering biological logic gates.

Purpose of the Study:

• To adapt split Cas9 for creating logic gates that sense specific biological events.
• To develop a system for detecting cancer cell origin, EMT, and cell-cell fusion.
• To demonstrate the versatility of split Cas9 as a biosensor and actuator.

Main Methods:

• Engineered split Cas9 protein halves under different promoters.
• Utilized self-assembling inteins for Cas9 reconstitution.
• Integrated a Cas9 reporter (EGxxFP) for fluorescence-based output.

Main Results:

• Developed a logic gate detecting epithelial cancer cell origin via fluorescence.
• Created a logic gate sensing epithelial to mesenchymal transition (EMT) triggered by TWIST1.
• Successfully detected cell-cell fusion using split Cas9 logic gates in induced and natural scenarios.

Conclusions:

• Split Cas9 can be engineered into effective logic gates for sensing biological events.
• The system demonstrates potential for detecting cancer cell states and cell fusion.
• The split Cas9 system offers a simple, integrable platform for biosensing and actuation.