A genome-scale CRISPR-Cas9 knockout screening method was developed to identify genes essential for cell viability and resistance to vemurafenib in melanoma cells. The study used a lentiviral vector (lentiCRISPR) to deliver Cas9 and guide RNAs (sgRNAs) to target 18,080 genes with 64,751 unique guide sequences. This approach enabled both negative and positive selection screens. In negative selection, the depletion of sgRNAs targeting essential survival genes was analyzed, revealing that most depleted sgRNAs targeted essential genes such as ribosomal structural constituents. In positive selection, the screen identified genes whose loss conferred resistance to vemurafenib, a BRAF inhibitor. The highest-ranking candidates included previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. These findings suggest that loss of these genes contributes to vemurafenib resistance. The study compared the efficacy of CRISPR-Cas9 screening with RNAi-based screening. The CRISPR-Cas9 approach showed higher consistency and efficiency in gene knockout, with a high rate of hit confirmation. The RNAi Gene Enrichment Ranking (RIGER) algorithm was used to rank screening hits based on consistent enrichment among multiple sgRNAs targeting the same gene. The results demonstrated that CRISPR-Cas9 screening provides a more reliable and efficient method for genome-scale functional screening. The study also validated the effectiveness of the CRISPR-Cas9 approach by showing that sgRNAs targeting NF2 resulted in high allele modification and reduced protein expression, leading to increased resistance to vemurafenib. The findings highlight the potential of CRISPR-Cas9 technology for systematic perturbation of gene function and its application in functional genomics.A genome-scale CRISPR-Cas9 knockout screening method was developed to identify genes essential for cell viability and resistance to vemurafenib in melanoma cells. The study used a lentiviral vector (lentiCRISPR) to deliver Cas9 and guide RNAs (sgRNAs) to target 18,080 genes with 64,751 unique guide sequences. This approach enabled both negative and positive selection screens. In negative selection, the depletion of sgRNAs targeting essential survival genes was analyzed, revealing that most depleted sgRNAs targeted essential genes such as ribosomal structural constituents. In positive selection, the screen identified genes whose loss conferred resistance to vemurafenib, a BRAF inhibitor. The highest-ranking candidates included previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. These findings suggest that loss of these genes contributes to vemurafenib resistance. The study compared the efficacy of CRISPR-Cas9 screening with RNAi-based screening. The CRISPR-Cas9 approach showed higher consistency and efficiency in gene knockout, with a high rate of hit confirmation. The RNAi Gene Enrichment Ranking (RIGER) algorithm was used to rank screening hits based on consistent enrichment among multiple sgRNAs targeting the same gene. The results demonstrated that CRISPR-Cas9 screening provides a more reliable and efficient method for genome-scale functional screening. The study also validated the effectiveness of the CRISPR-Cas9 approach by showing that sgRNAs targeting NF2 resulted in high allele modification and reduced protein expression, leading to increased resistance to vemurafenib. The findings highlight the potential of CRISPR-Cas9 technology for systematic perturbation of gene function and its application in functional genomics.