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. 2023 Aug 30;621(7978):404–414. doi: 10.1038/s41586-023-06496-5

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

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Fig. 3 — a, The editing windows within FLT3, KIT and CD123 sgRNAs after electroporation with different doses of mRNA. Data are mean ± s.d. b, Editing efficiencies of bulk CD34⁺ or FACS-sorted subsets. The gating strategy is reported in Supplementary Fig. 2c. n = 4 biological replicates, n = 3 for KIT. Data are mean ± s.d. Statistical analysis was performed using one-way ANOVA. c, The CD90/CD45RA composition of epitope-edited HSPCs measured by FACS analysis within the CD34⁺CD133⁺ subset. Data are mean ± s.d. The sample size is reported within the bars. d, In vitro 4G8-CAR killing assay of FLT3^N399 edited HSPCs. The fraction of persisting live cells (left) and the viability on the basis of annexin V and 7-AAD staining (right) of CD34⁺CD45RA⁺ cells, 48 h after co-culture with 4G8-CAR or untransduced T cells at different effector:target (E:T) ratios. Data are mean ± s.d. n = 4. Statistical analysis was performed using two-way ANOVA. e, In vitro CSL362-CAR killing assay of CD123^S59 edited HSPCs. The fraction of persisting live CD90⁺ cells (left) and the viability of CD34⁺ cells on the basis of annexin V and 7-AAD staining (right), 48 h after co-culture with CSL362-CAR or untransduced T cells at different E:T ratios. n = 8 on 2 donors. Statistical analysis was performed using two-way ANOVA. f, The fraction of absolute live CD34⁺ cells (relative to the no-antibody control) of KIT^H378 or AAVS1^BE HSPCs after 48 h of culture with Fab-79D monoclonal antibodies. Data are mean ± s.d. n = 6 on 2 donors. Statistical analysis was performed using two-way ANOVA. g, RNA-seq analysis of epitope-edited CD34⁺ HSPCs with or without stimulation. log-scale scatter plot of the mean gene expression values of the AAVS1 control and FLT3-, KIT- and CD123-edited cells, either at the baseline (top) or after stimulation with FLT3L, SCF or IL-3 (bottom). n = 3 biological replicates. Unstim., unstimulated. h, The absolute counts of total CD34⁺ and CD90⁺CD45RA⁻ cells (n = 4; top right) and of myeloid and erythroid colonies (n = 2; bottom right) of edited HSPCs. Left, representative colony-forming-unit (CFU) microphotographs. i, Primary and secondary xenotransplantation of FLT3^N399 or AAVS1^BE HSPCs. Top, human engraftment (hCD45⁺) by flow cytometry analysis in primary recipients. Data are mean ± s.d. Statistical analysis was performed using two-way ANOVA. n = 7 (FLT3^N399) and n = 4 (AAVS1^BE). Bottom, FLT3 editing was measured on peripheral blood, haematopoietic organs (BM and spleen (SP)) or CFUs in primary and secondary transplants. Data are mean ± s.d. Statistical analysis was performed using one-way ANOVA. LC, liquid culture; SP, spleen; W, week.

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