CRISPR-Cas9 screen reveals RUNX1 dependency in ML-DS. (A) Schema of the high-throughput Hsa21 CRISPR-Cas9 in vitro screen. (B) Dot plots showing the log₁₀ fold change and −log₁₀ (P 2-sided) of the Hsa21 sgRNAs in CMK (left), K562 (middle), and CMK + K562 (right; maximum likelihood estimation [MLE] β score) cell lines. Dark green dots represent highly depleting sgRNAs in CMK cells that also deplete in K562s. Light green dots represent positive controls (MYB, MYC, ACTB, SF3B3, RPL9, and POLR2A). Blue dots represent sgRNAs targeting RUNX1. (C) Dot plot showing the number of sgRNA-transduced (control sgRNA [sgCtrl] and sgRUNX1.1-1.5 indicated by shape) CMK and K562 cells (with stable Cas9 expression) after 14 days of culture, normalized to day 0 (n = 2 per sgRNA, 2-tailed unpaired t test). (D) Experimental setup for evaluating RUNX1 knockout in vivo. ML-DS blasts (with stable Cas9 expression) were transduced with sgRUNX1 (E2Crimson⁺ and sgRUNX1.1) or a sgCtrl (E2Crimson⁺) and mixed 1:1 with sgCtrl-transduced blasts (GFP⁺), before transplantation into sublethally irradiated recipient mice. (E) Dot plot showing the ratio of E2Crimson⁺ to green fluorescent protein–positive (GFP⁺) cells after 12 days of culture, normalized to day 0 (left; n = 4, 2-tailed unpaired t test) and in the bone marrow of mice euthanized 6 to 8 weeks after transplantation (right; n = 5, Mann Whitney U test). (F) Schematic representation of the human RUNX1 genomic locus (top) and the 3 main RUNX1 transcript isoforms (bottom; not to scale). Functional exons encoding the DNA-binding domain (Runt; red) and transactivation domain (TAD; blue) are indicated. Bar graph showing RUNX1 isoform distribution (Oxford Nanopore sequencing) in polyA⁺-enriched RNA samples from TAM and ML-DS samples. (G) Expression of RUNX1A and RUNX1C isoforms normalized to the expression of β2-microglobulin (B2M) (left graph; 2-way analysis of variance [ANOVA]) and RUNX1A to RUNX1C ratios (right graph; 1-way ANOVA) in CD34⁺ HSPCs, erythrocytes, megakaryocytes, granulocytes, and monocytes isolated from healthy donors, as well as in leukemic blasts from patients with TAM/ML-DS. gDNA, genomic DNA; NGS, next-generation sequencing; PCR, polymerase chain reaction.