CRISPR screening for cancer dependency gene and genetic modifiers to doxorubicin

The Mouse CRISPR Knockout Pooled Library (Brie, lentiCRISPRv2) was obtained from Addgene (Addgene#73632), which includes 1000 control gRNAs and 78,637 gRNAs targeting 19,674 genes. The plasmid library was amplified and validated in the Center for Advanced Genome Engineering at St. Jude Children’s Research Hospital as described in the Broad GPP protocol (https://portals.broadinstitute.org/gpp/public/resources/protocols) except Endura^TM DUOs (Lucigen) electrocompetent cells were used for the transformation step. The workflow of this whole genome genetic screen is illustrated in Fig. 6a. We used NEJF1, NEJF6, and NEJF-10 cells; three mouse hepatoblastoma cell lines established in our laboratory by culturing dissociated liver mass cells from the ABC-Myc models. The cells were transduced with mouse CRISPR Knockout pooled library (Brie) which contains 78, 637 unique sgRNA sequences targeting 19,674 human genes (4 sgRNAs per gene, and 1000 non-targeting controls) at a low MOI (~0.3) to ensure effective barcoding of individual cells. Cells were replenished with fresh DMEM medium containing 2 μg/mL puromycin (Millipore Sigma) for 36 h. After puromycin selection, cells were washed to eliminate dead cell debris and maintained in complete DMEM medium, and 32 × 10⁶ cells were collected for genomic DNA extraction to ensure over 400× coverage of Brie library. For genetic mapping, the transduced cells were cultured for 5 days for CRISPR editing to generate a mutant cell pool, which was then treated with vehicle (DMSO) and doxorubicin (IC20 ~ 5 nM, 14 days: IC90 ~ 30 nM 21 days), these concentrations were selected from colony formation assay that mimics similar experimental setup for actual experiment (IC20 ~ 11.84 nM, IC90 ~ 35.83 nM, for 4 days), since doxorubicin has a very narrow therapeutic window at the nM level. During the experiment, at least 32 × 10⁶ cells were collected for genomic DNA extraction to ensure over 400× coverage of Brie library. The total genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen) and quantified with a Nanodrop instrument. The sgRNA sequences were amplified using PCR method using NEB Q5 polymerase (New England Biolabs). PCR products were purified by AMPure XP SPRI beads (Beckman Coulter) and quantified by a Qubit dsDNA HS assay (Thermo Fisher Scientific). A total of 16 million reads were sequenced using an Illumina HiSeq sequencer, and the sequencing data were analyzed using MAGeCK-VISPR software. NGS sequencing was performed in the Hartwell Center Genome Sequencing Facility at St. Jude Children’s Research Hospital. Single-end, 100-cycle sequencing was performed on a NovaSeq 6000 (Illumina). Validation to check gRNA presence and representation was performed using calc_auc_v1.1.py (https://github.com/mhegde/) and count_spacers.py. Network analysis performed using STRING program (https://string-db.org/).