In Vivo pooled CRISPRko screens in PDA Cell Lines

We tested the essentiality of PDA state-specific Master Regulators in vivo by transducing a sgRNA library targeting 93 regulatory proteins into Cas9 expressing KP4 and HPAFII human cell lines. Cells were then injected into immunocompromised mice for genomic sequencing. Overall design: To confirm the essentiality of state-specific MRs in vivo, we transduced a sgRNA library targeting 32 GLS/Lineage and 61 MOS/Morphogenic MRs into Cas9 expressing morphogenic KP4-and lineage HPAFII-cell lines, as representatives of the MOS and GLS state, respectively. All these genes were targeted with 5 sgRNAs / gene. For assay controls, we added 6 core-essential genes as strong positive controls, 100 negative intergenic controls, and 5 non-targeting controls. After puromycin selection, cells were injected into seven immunocompromised mice for each cell line and tumor formation was monitored for 29 days, followed by genomic sequencing