Customizable gene sensing and response without altering endogenous coding sequences

Area of Science:

• Synthetic biology
• Genetic engineering
• Cellular engineering

Background:

• Synthetic biology utilizes genetic circuits to control cellular functions.
• Integrating biosensors into endogenous genes via CRISPR-Cas9 allows gene expression monitoring but can disrupt native regulation.
• A method is needed to embed biosensors without impacting gene coding sequences.

Purpose of the Study:

• To develop a novel method for integrating genetic biosensors into endogenous genes without altering their coding sequences.
• To enable precise control and prediction of biosensor responses using mathematical modeling.
• To engineer a functional cell stress sensor and actuator system in CHO-K1 cells.

Main Methods:

• Engineered a system to insert single-guide RNAs into the terminator regions of endogenous genes, activating downstream circuits.
• Developed a mathematical model to predict and fine-tune sensor dosage responses.
• Constructed a cell stress sensor and actuator in CHO-K1 cells to conditionally activate the antiapoptotic protein BCL-2.

Main Results:

• Successfully integrated genetic biosensors into endogenous genes without modifying coding sequences.
• Demonstrated fine-tuning and prediction of sensor dosage responses using a mathematical model.
• Engineered a functional cell stress sensor and actuator that enhances cell survival under stress by activating BCL-2.

Conclusions:

• The developed platform allows for the integration of genetic biosensors into endogenous genes by targeting terminator regions.
• This approach avoids disruption of native gene coding sequences and allows for predictable biosensor responses.
• The engineered cell stress sensor and actuator system shows potential for biomanufacturing, cell fate control, and cell-based therapeutics.