CRISPR-Cas9–mediated DNT cytotoxicity screen reveals gene targets for DNT therapy in AML. (A) Pie chart illustrates the composition of the epidrug sgRNA library that contains ∼1000 genes. “Other” refers to m⁶A-related genes (eg, METTL3, METTL14, ALKBH5, YTHDF1). (B) Experimental design of the in vitro CRISPR screen with AML3 cells. An epigenetic drug library with ∼12 500 sgRNAs was cloned into lentiviral constructs and Cas9-expressing AML3 cells were transduced and selected with the library at an MOI ∼ 0.3. Ten days postinfection, transduced AML3 cells were cocultured with DNTs for 2 hours, which resulted in 40% to 50% Annexin V staining of target cells. Annexin V⁺ and Annexin V⁻ AML cells were subsequently sorted using FACS. Two independent experiments were performed, and samples were collected for PCR amplification followed by NGS. The data were analyzed by MAGeCK.