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. 2026 Feb 20;16:7463. doi: 10.1038/s41598-026-40642-z

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© The Author(s) 2026

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Evaluation of ABE-SpRY constructs in target sites of interest in mouse N2a cells. (a) Design of sgRNAs for optimal adenine base editing. Target adenines (bolded in red) for each locus are positioned within the optimal editing windows of ABE8e and ABE9 deaminases with the corresponding PAM sites shown. (b) The Cas9 variant SpRY can target non-NGG PAM sites, unlike the wild-type SpCas9. (c) N2a cells were co-transfected with three plasmids: the ABE plasmid, sgRNA plasmid, and GFP plasmid. GFP-positive cells were sorted, and genomic DNA (gDNA) was extracted for subsequent targeted sequencing analysis. (d) Maximum editing efficiency observed for the target adenine with ABE8e- or ABE9-SpRY. The “specified mutation (all alleles)” includes all reads that carry the intended substitution, regardless of bystander edits within the analysed window. (e) A-to-G editing efficiencies of ABE8e and ABE9-SpRY at each adenine position along the protospacer region for the four target sites. The positions of adenines of interest are bolded in magenta. (f) Editing efficiency of the target adenine using ABE8e- or ABE9-SpRY. The “specified mutation without bystanders” reports only clean desired-only (“perfect”) alleles within the analysed window. (g) Product purity was calculated as the percentage of reads carrying the desired substitution that were free of bystander edits within the quantification window: 100 × (desired-only / (desired-only + "desired + bystander")). (h) Frequency of indels at each of the four target sites. Individual data points for three independent biological replicates are shown. Error bars indicate standard deviation (SD); bounds were capped at 0% and 100% where the mean ± SD exceeded the valid range. Parts of the schematics in (b) and (c) were created with BioRender.com.