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. 2022 Sep 8;41(1):96–107. doi: 10.1038/s41587-022-01410-2

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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. 3 — a, Overview of PAM-matched sites used to compare eNme2Cas9 variants to SpRY and SpRY-HF1. b, Summary dot plots showing the activity of eNme2-C-ABE8e compared to SpRY-ABE8e and SpRY-HF1-ABE8e at 14 PAM-matched NCN/N₄CN sites in HEK293T cells. Left-most data represent a summary of all 14 sites, and subsequent columns represent a subdivision into specific PAMs. c, Summary dot plots showing the activity of eNme2-T.1-ABE8e and eNme2-T.2-ABE8e compared to SpRY-ABE8e and SpRY-HF1-ABE8e at eight PAM-matched NTN/N₄TN sites in HEK293T cells. d, Summary dot plots showing the activity of eNme2-C-BE4 compared to SpRY-BE4 and SpRY-HF1-BE4 at eight PAM-matched NCN/N₄CN sites in HEK293T cells. e, Summary dot plots showing the activity of eNme2-C nuclease and eNme2-C.NR nuclease compared to SpRY nuclease and SpRY-HF1 nuclease at eight PAM-matched NCN/N₄CN sites in HEK293T cells. f, Overview of protospacer-matched sites used to compare the DNA specificity of eNme2Cas9 variants against SpRY and SpRY-HF1. g, Heat maps showing off-target adenine base editing activity (brown) or off-target indel formation (dark green) at computationally determined off-targets for two sites in HEK293T cells for eNme2-C-ABE8e and eNme2-C.NR nuclease compared to SpRY and SpRY-HF1 adenine base editor and nuclease variants. The left-most column represents on-target activity. Values represent the average of n = 3 independent biological replicates. h, Percentage of on-target GUIDE-seq reads identified at four protospacer-matched sites for eNme2-C nuclease, eNme2-C.NR nuclease, SpRY nuclease and SpRY-HF1 nuclease. Total reads for the given nuclease are listed above each bar. i, Total putative off-target sites identified by GUIDE-seq for eNme2-C nuclease, eNme2-C.NR nuclease, SpRY nuclease and SpRY-HF1 nuclease at four protospacer-matched sites. For b–e, each point represents the average editing of n = 3 independent biological replicates measured at the maximally edited position within each given genomic site. Mean ± s.e.m. is shown and reflects the average activity and standard error of the pooled genomic site averages.