Our study used CRISPR-library screening to systematically identify NF1 and DUSP9 as two critical drivers for lenvatinib resistance in HCC, and further clarified the mechanisms by which NF1 loss reactivates the PI3K/AKT and MAPK/ERK signaling pathways, while DUSP9 loss activates the MAPK/ERK signaling pathways, leading to lenvatinib resistance in HCC. Here, MAPK1 is linked to hepatocellular carcinoma.