Multidimensional Control of Cas9 by Evolved RNA Polymerase-Based Biosensors

Area of Science:

• Molecular Biology
• Synthetic Biology
• Gene Editing Technologies

Background:

• Precise control of Cas9 is crucial for minimizing side effects and enabling targeted gene therapies.
• Existing Cas9 systems lack sophisticated temporal and spatial regulation.
• RNA polymerase (RNAP)-based biosensors offer a potential platform for inducible gene regulation.

Purpose of the Study:

• To engineer novel Cas9 controllers using RNAP-based biosensors for precise spatiotemporal gene editing.
• To develop biosensors responsive to small molecules and protein-protein interactions for Cas9 regulation.
• To create an "on-switch/off switch" system for inducible and reversible Cas9-mediated gene knockout.

Main Methods:

• Design and validation of abscisic acid-inducible RNAP biosensors for Cas9 control.
• Optimization of system background using continuous evolution.
• Development of biosensors responsive to protein-protein interactions for targeted knockout.
• Construction of orthogonal RNAP biosensors for integrated signal input and multiple gRNA outputs.
• Implementation of an "on-switch/off switch" controller for inducible and reversible gene knockout.

Main Results:

• Successfully developed and validated RNAP-based biosensors to control Cas9 activity.
• Demonstrated inducible gene knockout using small-molecule inputs and protein-protein interaction-based triggers.
• Engineered an "on-switch/off switch" system with low background and inducible knockout.
• Showcased the integration of multiple input signals to control multiple gRNA outputs with a single Cas9 protein.

Conclusions:

• Engineered RNAP biosensors are effective and deployable control elements for Cas9.
• This approach significantly expands the possibilities for diverse input signals to drive gene editing technologies.
• The developed system offers enhanced precision and safety for future gene therapy applications.