CRISPR Knockout Pooled Library Screening for HCC CSCs

Human CRISPR Knockout Pooled Library (GeCKO V2, 1000000049) was purchased from Addgene. The libraries were amplified using Endura cells (catalog 60242, Lucigen). The workflow of CRISPR/Cas9 pooled screen is shown in Figure 1A. PLC cells were infected with lentiviral particles packaging Cas9 protein at an MOI less than 0.7. The blasticidin-selected Cas9-expressing PLC cells were infected with pooled lentiviral CRISPR library at an MOI of 0.3 (1,000× coverage) to ensure single-copy sgRNA integration in each cell. A pool of knockout cells was created after 7 days of selection with 2.5 μg/mL puromycin. CD24⁺CD133⁺ HCC CSCs and CD24^–CD133^– non-HCC CSCs were sorted by FACS. Genomic DNA of cells were extracted using Quick-DNA Microprep Plus Kit (catalog D4074, Zymo Research) according to the manufacturer’s instructions. Sequencing libraries were constructed and sequenced by GENEWIZ company. The sequencing data were analyzed by MAGeCKFlute (49 (link)).