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. 2023 Jan 13;14:207. doi: 10.1038/s41467-022-35508-7

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© The Author(s) 2023

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 Five sgRNAs targeting the core TGN_7-9WGATAR half E-box/GATA1 binding motif (shown in box) at +58 BCL11A erythroid enhancer with predominant base editing position indicated by arrowhead and PAM shaded. b Base editing by ABE8e complexed with each of five sgRNAs in human CD34⁺ HSPCs. Heat map displays base edit frequency. c, h Percentage of γ-gene expression by RT-qPCR analysis after in vitro erythroid maturation of HSPCs edited by each of (or combined) the indicated sgRNAs complexed with ABE8e. Data are plotted as mean ± sd. n = 3 independent experiments. d Frequency of on-target and three validated sg1620-dependent DNA off-target sites with ABE8e mRNA and RNP delivery. Data were presented as mean ± sd, n = 3 independent experiments. e RNA off-target for mRNA and RNP groups. Data were presented as mean ± sd and analyzed with the unpaired two-tailed Student’s t test. p values have been noted on the corresponding comparisons. n = 3 independent experiments. f Sequences of two sgRNAs targeting promoters of the HBG. BCL11A-binding sites are shaded. The −114/−102 13 bp HPFH deletion is indicated by an empty box. Predominant base editing positions were indicated by green arrowheads. g Editing efficiency by ABE8e complexed with sgHBGsense or sgHBGsite1. i Multiplex base editing by ABE8e-sg1620&sgHBGsense. j Percentage of γ-gene expression determined by RT-qPCR in HSPCs edited by ABE8e-sg1620 or/and sgHBGsense, A3A (N57Q)-BE3-sg1620 and 3 × NLS-SpCas9-sg1617 targeting +58 BCL11A enhancer. Data were presented as mean ± sd. n = 4 independent experiments. k Percentage of γ-globin determined by RP-HPLC analysis after in vitro erythroid maturation of HSPCs edited by the sg1620, sgHBGsense, and sg1620&sgHBGsense complexed with ABE8e. Data are plotted as mean ± sd. n = 4 independent experiments. l γ-globin gene expression by RT-qPCR analysis of erythroid progeny after dose response of RNP electroporation of HSPCs. Data were presented as mean ± sd, n = 3 independent experiments. All statistical significance in the figures were analyzed with the unpaired two-tailed Student’s t test, with p values noted where appropriate.