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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. 2 — a, SAC-PACE modifications increasing selection stringency. Left, original selection scheme; middle, split SAC-PACE selection and right, dual-PAM split SAC-PACE. b, Evolution campaigns toward Nme2Cas9 variants with N₄CN or N₄TN PAM compatibility. c, Summary heat map showing ABE-PPA activity for representative variants across both evolutionary trajectories. Values plotted are raw observed percentage A•T-to-G•C conversion for one replicate of each base editor. d, Mutation overview of the eNme2-C variant, mapped onto the crystal structure of wild-type Nme2Cas9 (Protein Data Bank (PDB) 6JE3), mutated positions are shown in blue. The inset shows the wild-type PAM and PAM-interacting residues (D1028, R1033), with evolved mutations listed. e, Summary dot plots showing the progression of mammalian cell adenine base editing activity at eight N₄CN PAM-containing sites for representative variants from the N₄CN evolution trajectory. f, Mutation overview of the eNme2-T.1 and eNme2-T.2 variants, mapped onto the crystal structure of wild-type Nme2Cas9 (PDB 6JE3). Positions mutated in both variants are shown in yellow. Mutations unique to eNme2-T.1 are shown in light green. Mutations unique to eNme2-T.2 are shown in dark green. The insets show the wild-type PAM and PAM-interacting residues (D1028, R1033), along with new mutations listed. g, Summary dot plots showing the progression of mammalian cell adenine base editing activity at eight N₄TN PAM-containing sites for representative variants from the N₄TN evolution trajectory. For e and g, each point represents the average editing of n = 3 independent biological replicates measured at the maximally edited position within each genomic site. Mean ± s.e.m. is shown and reflects the average activity and standard error of the pooled genomic site averages. NS, P > 0.05; *P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. P values determined by Sidak’s multiple comparisons test following ordinary one-way analysis of variance. For e, eNme2-C-ABE8e versus Nme2-ABE8e (P < 0.0001), eNme2E1-2-ABE8e (P = 0.0003), eNme2E2-12-ABE8e (P = 0.0004) and for g eNme2-T.1-ABE8e versus Nme2-ABE8e (P = 0.0020), eNme2E1-2-ABE8e (P = 0.0020), eNme2E2-12-ABE8e (P = 0.1252), eNme2E3-18-ABE8e (P = 0.0157) or eNme2-T.2-ABE8e versus Nme2-ABE8e (P = 0.0037), eNme2E1-2-ABE8e (P = 0.0037), eNme2E2-12-ABE8e (P = 0.1947) and eNme2E3-18-ABE8e (P = 0.0272).