Structure-guided engineering of adenine base editor with minimized RNA off-targeting activity

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• Adenine base editors (ABEs) and cytosine base editors (CBEs) can cause unintended transcriptome-wide RNA off-target edits.
• These off-target edits occur independently of the guide RNA sequence.

Purpose of the Study:

• To develop a strategy for engineering adenine base editors (ABEs) with minimized RNA off-targeting.
• To investigate the role of specific amino acids in ABEs' RNA editing activity.

Main Methods:

• Constructed a reporter system using the E. coli Hokb gene with a tRNA-like motif to detect RNA editing.
• Employed structure-guided principles to design mutations in the TadA component of ABEs.
• Engineered ABEmax variants with deletion of Arginine 153 (R153) in TadA.

Main Results:

• ABEmax efficiently performed A-to-I editing within an E. coli tRNA-like structure.
• Arginine 153 (R153) in TadA was identified as essential for deaminating RNAs with tRNA-like structures.
• Engineered ABE variants (del153/del153* and mini del153) showed significantly reduced RNA off-targeting with comparable DNA on-targeting.
• R153 deletion also reduced RNA off-targeting in ABE8e and ABE8s.

Conclusions:

• A strategy was developed to generate engineered ABEs (eABEs) with minimized RNA off-targeting.
• Targeting R153 in TadA is an effective approach to enhance the specificity of adenine base editors.
• The engineered base editors offer improved precision for gene editing applications.