Fig. 2: Loss of transcriptional HSCs after MECOM perturbation.

a, Uniform Manifold Approximation and Projection (UMAP) plot and cell type clustering of human HSCs after CRISPR editing. UCB CD34⁺ cells underwent CRISPR editing and were sorted 3 d later for CD34⁺CD45RA^-CD90⁺ HSCs followed by scRNA-seq. Cells were clustered by transcriptional signatures using Celltypist²². CMP, common myeloid progenitor; MEMP, megakaryocyte-erythroid-mast cell progenitor; cMEMP, cycling MEMP; MEP, megakaryocyte-erythroid progenitor; cMPP, cycling multipotent progenitor; Ery, early erythroid progenitor; MK, early megakaryocyte progenitor; Eo/baso, eosinophil/basophil progenitor; Macro, macrophage progenitor; Mast, mast cell progenitor. b, UMAP plot of CD34⁺CD45RA^-CD90⁺ HSCs stratified by CRISPR edits, showing the depletion of HSCs following MECOM perturbation. AAVS1-edited sample highlighted in red (left). MECOM-edited sample highlighted in red (right). Each sample is the combination of two biological replicates. c, Bar graph showing the number of cells in the HSC cluster in AAVS1- and MECOM-edited samples. Mean is plotted and each of two biological replicates is shown. Total number of cells profiled in each group was 19,375 (AAVS1) and 19,821 (MECOM). d, UMAP plot of CD34⁺CD45RA⁻CD90⁺ HSCs following CRISPR editing (AAVS1-edited (left), MECOM-edited (right)), colored according to expression of HSC signature (CD34, HLF and CRHBP). e, Bar graph showing the number of cells expressing the three-gene HSC signature. An HSC signature score >0.5 indicates high expression. Mean is plotted and each of two biological replicates is shown. Total number of cells profiled in each group was 19,375 (AAVS1) and 19,821 (MECOM). f–h, UMAP plots of CD34⁺CD45RA⁻CD90⁺CD133⁺EPCR⁺ITGA3⁺ LT-HSCs following CRISPR editing, indicating enrichment of the HSC signature as determined by scRNA-seq using the 10x Genomics platform (f), overlap of AAVS1-edited and the MECOM-edited cells, sequenced using the 10x Genomics platform (g) and distribution of cells with monoallelic MECOM edits determined by G&T sequencing by SmartSeq2, compared to AAVS1-edited cells and LT-HSCs from f (h).

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