A chemically controlled Cas9 switch enables temporal modulation of diverse effectors

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• CRISPR-Cas9 technology has enabled diverse gene editing tools like base and prime editors.
• Existing methods for inducible Cas9 modulation lack temporal precision and require extensive optimization.
• Precise temporal control is crucial for understanding gene editing kinetics and engineering novel effectors.

Purpose of the Study:

• To develop a versatile, chemically controlled, and rapidly activated CRISPR-Cas9 switch (ciCas9).
• To confer temporal control over various Cas9 effectors, including base and prime editors, and transcriptional activators.
• To enable precise kinetic studies of base editing and investigate factors influencing editing efficiency.

Main Methods:

• Development of a single-component, chemically inducible Cas9 switch (ciCas9).
• Application of ciCas9 to control seven distinct Cas9 effectors: cytidine base editors, adenine base editors, a dual base editor, a prime editor, and a transcriptional activator.
• Analysis of base editing kinetics using temporally controlled ciCas9-effector systems.

Main Results:

• ciCas9 provides rapid activation and temporal control over multiple Cas9 effectors.
• Base editing occurs within hours, with early editing rates predicting final editing outcomes.
• Editing at preferred nucleotides within target sites correlates with increased bystander edits.

Conclusions:

• The ciCas9 switch offers a simple and versatile method for creating chemically controlled Cas9 effectors.
• This technology facilitates precise temporal control for kinetic studies of gene editing.
• ciCas9 advances effector engineering and provides new tools for precise gene modulation.