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. 2024 Oct 29;14:25940. doi: 10.1038/s41598-024-77314-9

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© The Author(s) 2024, corrected publication 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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-nc-nd/4.0/.

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Fig. 1 — A genome-scale CRISPRi screen reveals gene knockdowns that confer sensitivity or resistance to 5-aza-2’-deoxycytidine (decitabine). (a) The chemical structure of decitabine. (b) Schematic of a genome-scale CRISPRi screen performed in HL-60 cells. (c) Volcano plot of gene-level rho (ρ) phenotypes and Mann–Whitney p-values. Negative rho values represent increased sensitivity to decitabine after knockdown, and positive rho values represent increased resistance. (d–e) Validation of top screen hits. HL-60i cells were transduced with a control sgRNA (black) or an active sgRNA (red or blue) and treated with DMSO or decitabine, and the proportion of sgRNA + cells in the decitabine condition relative to DMSO was observed over time. Data are shown as means ± SD, two sgRNAs per gene and two replicates per sgRNA. (f) Scatter plot showing the correlation between screen rho phenotype and validation phenotype (day 14–15 post-infection) for each validated sgRNA. (g) A cleaved caspase 3/7 assay shows the fraction of apoptotic HL-60 cells at day 5 following treatment with DMSO or decitabine ± RG3039. Data are shown as means ± SD for three replicates. (h) A cell cycle assay shows the fraction of HL-60 cells at different phases of the cell cycle at day 5 following treatment with DMSO or decitabine ± RG3039. Data are shown as means ± SD for three replicates. (i) Normalized counts for genes in GO:1,903,557 (positive regulation of tumor necrosis factor superfamily cytokine production) upregulated upon decitabine and RG3039 treatment.