Precise Regulation of Cas9-Mediated Genome Engineering by Anti-CRISPR-Based Inducible CRISPR Controllers

Area of Science:

• Molecular Biology
• Biotechnology
• Gene Editing Technologies

Background:

• CRISPR/Cas9 technology offers powerful genome editing capabilities.
• Off-target cleavage by CRISPR/Cas9 poses risks for therapeutic applications.
• Controlling Cas9 activity is crucial for improving biosafety.

Purpose of the Study:

• To design a tunable CRISPR controller for enhanced specificity and biosafety.
• To engineer a chemically inducible anti-CRISPR protein system.
• To demonstrate dose-dependent control over CRISPR/Cas9 editing.

Main Methods:

• Engineered anti-CRISPR protein AcrIIA4 fused to a ligand-inducible destabilization domain (DHFR(DD)).
• Utilized trimethoprim as an FDA-approved ligand to control AcrIIA4 activity.
• Developed a new inducible promoter system (Acr-OFF) for CRISPR controllers.
• Assessed off-target activity in mammalian cells.

Main Results:

• Achieved dose-dependent control over Cas9 editing and dCas9 induction.
• Significantly reduced off-target activity in mammalian cells.
• Demonstrated trimethoprim-regulated inhibition of Cas9 DNA binding.
• Established a tunable CRISPR controller system with improved specificity.

Conclusions:

• The developed CRISPR controller enhances the specificity and biosafety of CRISPR/Cas9 technology.
• Chemically inducible anti-CRISPR proteins offer precise control over gene editing.
• This system holds promise for safer therapeutic genome editing applications.