CRISPR DNA base editors with reduced RNA off-target and self-editing activities

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• Cytosine base editors (CBEs) and adenine base editors (ABEs) enable precise DNA alterations.
• Previous work identified unintended RNA editing by base editors and developed variants (SECURE-BE3) to mitigate this.
• Base editors can exhibit guide-RNA-independent RNA editing across the transcriptome.

Purpose of the Study:

• To engineer structure-guided SECURE-ABE variants with reduced off-target RNA editing.
• To evaluate the DNA editing activity and size of these novel SECURE-ABE variants.
• To assess RNA editing activity of various CBEs utilizing different cytidine deaminases.

Main Methods:

• Structure-guided engineering of Streptococcus pyogenes Cas9 base editors.
• Assessment of on-target DNA editing and off-target RNA editing activities.
• Comparative analysis of base editors employing APOBEC1, APOBEC3A, A3C, and Target-AID cytidine deaminases.

Main Results:

• Engineered SECURE-ABE variants demonstrated reduced off-target RNA editing while maintaining on-target DNA editing efficiency.
• These engineered variants are among the smallest Streptococcus pyogenes Cas9 base editors reported.
• Human APOBEC3A-based CBEs showed significant RNA editing, while enhanced APOBEC3A, A3C, and Target-AID CBEs exhibited less RNA editing.
• Base editors with RNA off-target activity were found to self-edit their own transcripts, causing coding sequence heterogeneity.

Conclusions:

• Structure-guided engineering successfully reduced off-target RNA editing in ABEs, yielding smaller, efficient base editors.
• Different cytidine deaminases confer varying levels of RNA editing activity to CBEs.
• Self-editing of transcripts by base editors contributes to heterogeneity in base editor populations.