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. 2019 Oct 18;10:4730. doi: 10.1038/s41467-019-12726-0

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© The Author(s) 2019

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 — Functional interrogation of single CRISPR/Cas9-edited hematopoietic stem cells in vitro. a Percentage of CRISPR/Cas9 efficiency as determined by single cell-derived colonies that were positive for homozygous deletion of exon 1 in control OR2W5, positive for exclusive assignment to the GATA1-Long isoform through HDR-mediated mutation of the alternative start site or positive for exclusive assignment to the GATA1-Short isoform through deletion of exon 2 (n = 3 experiments with independent cord blood pools). b Percentage of single cells that grew into a colony in the single cell in vitro differentiation assays (n = 3 experiments with independent cord blood pools, unpaired t test P < 0.05 for % colony efficiency in LT-HSCs GATA1-Long versus ST-HSCs GATA1-Long). c Capillary-based western assay of GATA1 in bulk MEPs that were CRISPR/Cas9 edited with control, GATA1-Long and GATA1-Short gRNAs and cultured under erythro-myeloid conditions for three days (n = 2 experiments, second western assay is shown in Supplementary Fig. 6a). d Lineage output from the in vitro single cell differentiation assay of individual CRISPR/Cas9-edited LT-HSCs, ST-HSCs and MEPs with control, GATA1-Long and GATA1-Short gRNAs. Numbers of single cell colonies with positive genotype are indicated at each condition (E = erythroid, M = myeloid, Meg = megakaryocytic, n = 3 experiments with independent cord blood pools, number of single cells assessed in each condition is indicated in each bar graph, unpaired t test P < 0.01 for E, M, Meg in GATA1-Short versus control and E, M, Meg in GATA1-Short versus GATA1-Long among all three cell types). e Overall percentage of erythroid, myeloid and megakaryocyte containing colonies from single cell in vitro differentiation assays (E = erythroid, M = myeloid, Meg = megakaryocytic, n = 3 experiments with independent cord blood pools, unpaired t test P < 0.005 for % Meg in GATA1-Short versus Control or % Meg in GATA1-Short versus GATA1-Long among all three cell types). Error bars represent standard deviations