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However, its much wider editing window and substantially higher off\u2010target editing activity restricted its applications in precise base editing for therapeutic use. This study uses a library\u2010assisted\u00a0protein evolution approach using eukaryotic cells to generate ABE variants with improved specificity and reduced off\u2010target editing while maintaining high activity in human cells. The study generated an expanded set of ABEs with efficient editing activities and chose four\u00a0evolved variants that\u00a0offered either similar or modestly higher efficiency within a narrower editing window of protospacer position \u22484\u20137 compared to that of ABE8e in human cells, which would enable minimized bystander editing. Moreover, these variants resulted in reduced off\u2010target editing events when delivered as plasmid or mRNA into human cells. Finally, these variants can install both disease\u2010suppressing mutations and disease\u2010correcting mutations efficiently with minimal undesired bystander editing making them promising approaches for specific therapeutic edits. In summary, the work establishes a mutant\u2010library\u2010assisted protein evolution method in eukaryotic cells and generates alternative ABE variants as efficient tools for precise human genome editing.<\/jats:p>","DOI":"10.1002\/advs.202309004","type":"journal-article","created":{"date-parts":[[2024,6,14]],"date-time":"2024-06-14T09:50:21Z","timestamp":1718358621000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Library\u2010Assisted Evolution in Eukaryotic Cells Yield Adenine Base Editors with Enhanced Editing Specificity"],"prefix":"10.1002","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-5588-9013","authenticated-orcid":false,"given":"Shenlin","family":"Hsiao","sequence":"first","affiliation":[{"name":"Shanghai Frontiers Science Center of Genome Editing and Cell Therapy Shanghai Key Laboratory of Regulatory Biology Institute of Biomedical Sciences and School of Life Sciences East China 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