A CRISPR screen identifies Spi1 as a vulnerability of Prdm16s-induced AML. (A) A schematic of the targeted CRISPR dropout screen against 770 candidate genes identified by RNA-seq. (B) Top L-MEP essential genes identified by CRISPR dropout screening included 2 transcription factors Elf1 and Spi1. Top 5 genes with significant robust ranking aggregation (RRA) scores are highlighted. (C) Immunoblotting to detect PU.1 expression after CRISPR editing. Cells deleted of the Rosa26 locus (R26sg) were compared with cells deleted of Spi1 with different sgRNA (sg1-7). (D) In vitro validation of a candidate gene Spi1 by a colony assay (n = 4). (E) Megakaryocyte formation by normal MEPs (left) and L-MEPs with (right) or without (middle) Spi1 deletion shown by Wright-Giemsa staining (n = 4). L-MEPs were deleted of either control Rosa26 or Spi1 and plated. (F) Representative flow cytometric histograms showing that Spi1-deleted L-MEPs form cells with decreased Mac-1 expression (left) and increased CD41 expression (right) compared with control Rosa26-edited cells (n = 4). (G) Spi1 deletion reduced GFP⁺ AML cells in the blood and ameliorated anemia and thrombocytopenia of L-MEP recipients (n = 7). Rosa26- or Spi1-deleted L-MEPs or vector-transduced normal MEPs were transplanted and blood was analyzed by flow cytometry or a hematology analyzer. (H) Spi1 deletion prolonged the survival of recipient mice of L-MEPs (n = 10). Rosa26- or Spi1-deleted L-MEPs were transplanted as in panel G. All data represent mean ± standard deviation. *P < .05, **P < .01, ***P < .001 by Student t test, except for comparison of survival curves, in which significance was assessed by log-rank test. gRNA, guide RNA; PLT, platelet; RBC, red blood cell; WT, wild type.