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. 2022 Mar 29;13:1669. doi: 10.1038/s41467-022-29339-9

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© The Author(s) 2022

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Sequences of PBS and RTT of pegRNAs. Intended single-base edits are in cyan, red (pathogenic mutations), or green (corrected base), additional base substitutions are in blue and protospacer adjacent motifs (PAMs) are in brown. The intended single-base editing frequency, unintended indel frequency and fraction of editing product were induced under PE3 setting. Means ± s.d. are from three independent experiments. b Statistical analysis of normalized single-base editing frequencies, setting the frequencies induced by regular pegRNAs (without additional base substitution) as 1. n = intended single-base editing from three independent experiments in a and Supplementary Figs. 1b and 2b, f. c Heatmaps show the normalized single-base editing efficiencies induced by the pegRNAs with one additional base substitution, setting the ones induced by regular pegRNAs as 1. d Statistical analysis of normalized single-base editing frequencies, setting the frequencies induced by regular pegRNAs as 1. n = intended single-base editing from three independent experiments in c. e Heatmaps show the normalized single-base editing efficiencies induced by the pegRNAs with two additional base substitutions, setting the ones induced by regular pegRNAs as 1. f Statistical analysis of normalized single-base editing frequencies, setting the frequencies induced by regular pegRNAs as 1. n = intended single-base editing from three independent experiments in e. g The strategy to design efficient spegRNAs, depending on the relative positions between the ORF of edited gene and the 3’-end of RTT. h, i Statistical analysis of normalized single-base editing frequencies (h) and unintended indel frequencies (i) induced by the pegRNAs designed according to the rule shown in g, setting the frequencies induced by regular pegRNAs as 1. n = 45 intended single-base editing (h) and unintended indel editing (i) from three independent experiments in Supplementary Fig. 10. The data in c and d are from a and Supplementary Figs. 1b, 2b, e, and 8a. The data in e and f are from a and Supplementary Figs. 1b, 2b, e, and 8b. b, d, f, h, i P value, Wilcoxon one-tailed signed-rank test. The median and interquartile range (IQR) are shown. Source data are provided as a Source Data file.