A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing.

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR/Cas systems offer powerful therapeutic genome editing capabilities.
• Optimal design of CRISPR guides and homology-directed repair (HDR) templates is crucial for effective genome editing.
• Current validation and optimization rely on reporter systems, necessitating improved tools.

Purpose of the Study:

• To introduce BETLE, a novel reporter system for rapid, sensitive, and cell-specific detection of genome editing and HDR.
• To enable validation and optimization of CRISPR/Cas systems and HDR templates.
• To facilitate the development of advanced genome editing tools for therapeutic applications.

Main Methods:

• Developed BETLE, a reporter system encoding multiple reporter proteins in distinct open-reading frames.
• Utilized out-of-frame non-homologous end joining (NHEJ) to express NanoLuc luciferase for sensitive editing analysis or mTagBFP2 for cell localization.
• Integrated a validation site for CRISPR/Cas systems and evaluated BETLE with spCas9, saCas9, and asCas12a using a defective moxGFP reporter.

Main Results:

• BETLE enables fast, sensitive, and cell-specific detection of genome editing and HDR.
• The system allows for low-cost analysis via NanoLuc luciferase and enumeration/localization via mTagBFP2.
• Demonstrated successful editing and HDR validation with various CRISPR/Cas nucleases and reporter configurations.

Conclusions:

• BETLE is a versatile and adaptable tool for rapid detection and optimization of CRISPR/Cas genome editing and HDR.
• The reporter system facilitates efficient validation of CRISPR/Cas nucleases and HDR templates.
• BETLE represents a state-of-the-art technology for advancing in vitro and in vivo genome editing applications.