Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Prolonged Cas9 activity in genome engineering leads to off-target mutations, genotoxicity, and mosaicism.
• Existing methods for controlling Cas9 temporal activity are often cumbersome.

Purpose of the Study:

• To develop a chemogenetic system for rapid and controllable degradation of Cas9.
• To investigate the impact of tunable Cas9 longevity on genome editing outcomes and specificity.

Main Methods:

• Engineered a chemogenetic system to recruit Cas9 to a ubiquitin ligase for proteasomal degradation.
• Demonstrated efficient Cas9 degradation in cells across multiple species.
• Controlled Cas9 lifetime to influence DNA repair pathway choice and editing outcomes.

Main Results:

• Achieved rapid and tunable degradation of Cas9 using the chemogenetic system.
• Demonstrated successful genome editing with biased repair pathway selection and genotypic outcomes.
• Reduced off-target effects by dosably controlling Cas9 activity and specificity.

Conclusions:

• The developed chemogenetic system provides precise temporal control over Cas9 activity.
• This system enhances genome editing precision by mitigating off-target effects and enabling desired repair pathway biasing.