In this study, we performed a genome-scale CRISPR knockout screening on CD24+CD133+ liver CSCs for determinants of HCC stemness and found that the most enriched were the genes associated with H3K4 methylation, chromatin remodeling, cell cycle, WNT, and noncanonical NF-κB pathway, especially the genes regulating H3K4 methylation, including the H3K4 methyltransferases KMT2A, KMT2D, and SETD1A. Here, SETD1A is linked to hepatocellular carcinoma.