The authors present a genome-scale CRISPR-Cas9 knockout (GeCKO) screening method to interrogate gene function in human cells. They designed a lentiviral vector (lentiCRISPR) to deliver Cas9, a single guide RNA (sgRNA), and a selection marker, enabling efficient gene knockout. The GeCKO library targets 18,080 genes with 64,751 unique sgRNAs. The authors used this library to identify essential genes for cell viability in cancer and pluripotent stem cells, as well as genes involved in resistance to the BRAF inhibitor vemurafenib in melanoma. The screen identified previously validated genes like NF1 and MED12, as well as novel hits such as NF2, CUL3, TADA2B, and TADA1. The study demonstrates the high efficacy and consistency of the GeCKO method, showing that it can effectively identify genes involved in drug resistance and other biological processes.The authors present a genome-scale CRISPR-Cas9 knockout (GeCKO) screening method to interrogate gene function in human cells. They designed a lentiviral vector (lentiCRISPR) to deliver Cas9, a single guide RNA (sgRNA), and a selection marker, enabling efficient gene knockout. The GeCKO library targets 18,080 genes with 64,751 unique sgRNAs. The authors used this library to identify essential genes for cell viability in cancer and pluripotent stem cells, as well as genes involved in resistance to the BRAF inhibitor vemurafenib in melanoma. The screen identified previously validated genes like NF1 and MED12, as well as novel hits such as NF2, CUL3, TADA2B, and TADA1. The study demonstrates the high efficacy and consistency of the GeCKO method, showing that it can effectively identify genes involved in drug resistance and other biological processes.