MLL, Hox genes, and their partners are required for the survival of Npm1^cA-driven AML cells. (A) Schematic depicting the derivation and liquid culture of Rosa26-EF1a-Cas9 expressing AML cell lines. CRISPR-Cas9–based assessment of individual genes aberrantly expressed in Npm1^cA/+;Nras^G12D/+ and Npm1^cA;Flt3^ITD mice. CAS9 activity of these mouse AML cell lines was validated, as described previously (supplemental Figure 7A).¹¹  Individual Rosa26-EF1a-Cas9–expressing cell lines were derived from 2 mice (ID I and II) of each genotype. In vitro competitive assays were performed over a 23-day period using AML cell lines transduced with lentivirus expressing gRNAs for the indicated gene, and the BFP-positive fraction compared with the nontransduced population. Results were normalized to day 3 for each gRNA. Results from AML cell lines transduced with guide RNAs targeting Hoxa-related (B) and non-Hoxa–related (C) genes. gRNA sequences were selected from a previously published library¹¹  and are detailed in supplemental Table 15. Guides against the pan-essential Npm1 gene are used as a control. Ctrl, control; ID, identity of mouse of origin.