Genome-wide CRISPR rensensitization screens in FGF2 and FL-derived Early and Late Gilteritinib Resistant MOLM14 cultures using Y. Kosuke library

Early and late gilteritinib resistant MOLM14 cells were transduced with Cas9-blasticidin lentiviral vector to generate Cas9+-expressing cells. These cells were then infected with the Yusa CRISPR library consisting of an average of 5 single-guide RNAs (sgRNAs) per gene for 18,010 genes. After transduction, cells were selected with puromycin for 5 days to ensure stable guide integration and subjected to treatment with 100 nM of gilteritinib or vehicle (DMSO). Early gilteritinib screen cultures were maintained in 10 ng/mL of FGF2 or FL, respectively. DNA was harvested from 0-21-day cultures to amplify library of sgRNA barcodes for deep sequencing analyses. Deep sequencing of early and late resistant screens was performed on the NovaSeq and HiSeq 2500, respectively (Illumina Inc.). Genes that were significantly depleted from gilteritinib-treated cultures relative to controls were deemed essential for the survival of gilteritinib resistant cells. Performed comparisons of sgRNA read counts between DMSO and gilteritinib treated cells at corresponding time points. Genes that were significantly depleted from gilteritinib-treated cultures relative to controls were deemed essential for the survival of gilteritinib resistant cells.