Library-Assisted Evolution in Eukaryotic Cells Yield Adenine Base Editors with Enhanced Editing Specificity

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Current adenine base editor (ABE) ABE8e shows high efficiency for A-to-G conversion but has limitations.
• ABE8e's broad editing window and significant off-target activity hinder precise therapeutic gene editing.
• There is a need for more specific and accurate base editing tools.

Purpose of the Study:

• To evolve adenine base editor (ABE) variants with enhanced specificity and reduced off-target editing.
• To maintain high editing activity in human cells while narrowing the editing window.
• To develop precise gene editing tools for therapeutic applications.

Main Methods:

• Employed a library-assisted protein evolution approach in eukaryotic cells.
• Generated and screened an expanded set of ABE variants.
• Assessed editing efficiency, editing window, and off-target activity in human cells.

Main Results:

• Identified four evolved ABE variants with efficient editing activity in human cells.
• These variants exhibit a narrower editing window (protospacer position ≈4-7) compared to ABE8e.
• Demonstrated reduced off-target editing events when delivered via plasmid or mRNA.
• Successfully installed disease-suppressing and disease-correcting mutations with minimal bystander editing.

Conclusions:

• Established a novel mutant-library-assisted protein evolution method in eukaryotic cells.
• Generated ABE variants that offer improved specificity and reduced off-target effects for precise genome editing.
• These engineered ABEs are promising tools for specific therapeutic edits in humans.