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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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Extended Data Fig. 6 — (a) Adenine base editing activity of eNme2-T.1-ABE8e and eNme2-T.2-ABE8e at 16 N₃NTN PAM-containing sites in HEK293T cells. Mean±SEM is shown and reflects the average activity and standard error of n = 3 independent biological replicates at the maximally edited position within each genomic site. The six sites that exhibited <1% base editing activity for either variant (line shown) that were excluded in subsequent analyses are italicized. (b) Pooled adenine base editing activity of eNme2-T.1-ABE8e and eNme2-T.2-ABE8e from (a). Left: all 10 sites; right: sites pooled by PAM position 6 identity. Each point represents the average of n = 3 independent biological replicates measured at the maximally edited position within each given genomic site. Mean±SEM is shown and reflects the average activity and standard error of the pooled genomic site averages. (c) Editing window of eNme2-T.1-ABE8e (top) or eNme2-T.2-ABE8e (bottom) reflective of pooled adenine base editing activity at all 23 protospacer positions (PAM counted as positions 24–29) of the 10 sites shown in (a). Each point represents the % A•T-to-G•C conversion observed for an adenine that was present in one of the 10 protospacers, normalized to the highest editing observed within that protospacer. The italicized positions (2 and 18) were not present in any protospacers evaluated. Mean±SEM is shown and reflects the average normalized activity and standard error at all observed adenines at that position. (d) Adenine base editing activity of eNme2-T.1- ABE8e (top) or eNme2-T.2-ABE8e (bottom) as a function of protospacer length at three different genomic sites in HEK293T cells. Each point represents the average of n = 3 independent biological replicates observed for a given protospacer length at one genomic site, normalized to the protospacer length with the highest base editing activity for that site. Mean±SEM is shown and reflects the average normalized activity and standard error of the pooled averages at the observed sites. **, p } 0.01 by individual unpaired, two- tailed Student’s t-tests comparing Nme2-ABE8e to either eNme2-T.1-ABE8e (p = 0.0039) or eNme2-T.2-ABE8e (p = 0.0018).