Journal Article

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

Hsiao, Shenlin, Liao, Jiaoyang, Jiang, Yanhong, Wu, Yuxuan, Zhong, Tao, Chen, Shuanghong, Wang, Qiudao, Yang, Yang, Lai, Yongrong

2024

Overview

The current‐generation adenine base editor (ABE) ABE8e, which has evolved from the prokaryotic evolution system, exhibits high efficiency in mediating A‐to‐G conversion and is presumed to be promising for gene therapy. However, its much wider editing window and substantially higher off‐target editing activity restricted its applications in precise base editing for therapeutic use. This study uses a library‐assisted protein evolution approach using eukaryotic cells to generate ABE variants with improved specificity and reduced off‐target editing while maintaining high activity in human cells. The study generated an expanded set of ABEs with efficient editing activities and chose four evolved variants that offered either similar or modestly higher efficiency within a narrower editing window of protospacer position ≈4–7 compared to that of ABE8e in human cells, which would enable minimized bystander editing. Moreover, these variants resulted in reduced off‐target editing events when delivered as plasmid or mRNA into human cells. Finally, these variants can install both disease‐suppressing mutations and disease‐correcting mutations efficiently with minimal undesired bystander editing making them promising approaches for specific therapeutic edits. In summary, the work establishes a mutant‐library‐assisted protein evolution method in eukaryotic cells and generates alternative ABE variants as efficient tools for precise human genome editing. ABE variants with high editing efficiency and specificity are obtained through the designed library‐assisted evolution of TadA8e in the eukaryotic cell system. The resulting candidate variants, compared to ABE8e, demonstrate narrower editing window and reduced bystander efficiency as well as lower off‐targeting activity.

• Publisher: John Wiley & Sons, Inc,John Wiley and Sons Inc,Wiley
• Subject: Adenine - metabolism / adenine base editor / Amino acids / Conversion / CRISPR-Cas Systems - genetics / Efficiency / Eukaryotic Cells - metabolism / Gene Editing - methods / Gene Library / Genomes / HEK293 Cells / Humans / library‐assisted evolution / Mutagenesis / Mutation / Plasmids / precise gene editing / Proteins