Fig. 1: Schematic and results of functional screening by sgRNA library.

A Structure of Two vector lentiviralGeCKO system. B The best lenvatinib concentration for treatment. Using different lenvatinib concentrations to treat Huh7 cells, after 21 days, the number of surviving cells in 1000 nM group was the closest to that after zero days. C Schematic diagram illustrates the workflow of genome-wide CRISPR/Cas9 knockout library screening. Human genome-wide CRISPR/Cas9 knockout library (GeCKO v2A) was packed into lentiviral particle and transduced into Cas9-overexpressing Huh7 cells at low multiplicity of infection (MOI = 0.5). The sgRNA transduced cells were selected by puromycin for 7 days. The cells were then divided into vehicle and lenvatinib groups for culture. After 21 days, lenvatinib-resistant cells were enriched (Day 21), and then cells of vehicle group (Day 0) and lenvatinib group (Day 21) were collected for genomic DNA extraction and high-throughput sequencing. D GO analysis showed that the candidate 1261 genes are involved in cell proliferation. The red dots indicated the top 5 enriched BP (biological process) of GO analysis. The blue dots indicated the top 5 enriched CC (cellular component). E KEGG analysis showed that the candidate 1261 genes are involved MAPK/ERK and PI3K/AKT signaling pathways. Only showed the top5 signaling pathways. F Diagram of the mechanism of lenvatinib in cancer. Lenvatinib exerts its anticancer effect by inhibiting PI3K/AKT and MEK/ERK signaling pathways through target receptor tyrosine kinase. G The potential 14 genes induced lenvatinib resistance according to bioinformatics analysis and gene function.