The authors improved CRISPR screening vectors and genome-wide libraries for more efficient and effective screening. Previously, they used a Genome-scale CRISPR Knock-Out (GeCKO) library to identify mutations conferring vemurafenib resistance in melanoma cells. However, initial lentiviral delivery systems had low viral titer or required pre-existing Cas9-expressing cell lines, limiting screening applications. To improve lentiviral packaging and guide sequence selection, they developed a new vector, lentiCRISPRv2, which increased viral titer by ~10-fold over lentiCRISPRv1. They also created a two-vector system, delivering Cas9 and sgRNA via separate vectors with distinct selection markers, increasing viral titer by ~100-fold. Both single and dual-vector systems efficiently knocked out EGFP in human cells. Additionally, they designed and synthesized new human and mouse GeCKOv2 sgRNA libraries with several improvements: 6 sgRNAs per gene, targeting all genes uniformly; improved off-target score calculation; sgRNAs to inactivate miRNAs; and targeting ~1000 additional genes. Both libraries are divided into two sub-libraries, each containing 3 sgRNAs per gene and 1000 non-targeting controls. Screens can be performed by combining both sub-libraries for higher coverage or using individual sub-libraries when cell numbers are limited. The human and mouse libraries have been cloned into lentiCRISPRv2 and lentiGuide-Puro and deep sequenced to ensure uniform representation. These new vectors and libraries improve GeCKO reagents for diverse screening applications. Reagents are available to the academic community through Addgene and the Zhang lab website.The authors improved CRISPR screening vectors and genome-wide libraries for more efficient and effective screening. Previously, they used a Genome-scale CRISPR Knock-Out (GeCKO) library to identify mutations conferring vemurafenib resistance in melanoma cells. However, initial lentiviral delivery systems had low viral titer or required pre-existing Cas9-expressing cell lines, limiting screening applications. To improve lentiviral packaging and guide sequence selection, they developed a new vector, lentiCRISPRv2, which increased viral titer by ~10-fold over lentiCRISPRv1. They also created a two-vector system, delivering Cas9 and sgRNA via separate vectors with distinct selection markers, increasing viral titer by ~100-fold. Both single and dual-vector systems efficiently knocked out EGFP in human cells. Additionally, they designed and synthesized new human and mouse GeCKOv2 sgRNA libraries with several improvements: 6 sgRNAs per gene, targeting all genes uniformly; improved off-target score calculation; sgRNAs to inactivate miRNAs; and targeting ~1000 additional genes. Both libraries are divided into two sub-libraries, each containing 3 sgRNAs per gene and 1000 non-targeting controls. Screens can be performed by combining both sub-libraries for higher coverage or using individual sub-libraries when cell numbers are limited. The human and mouse libraries have been cloned into lentiCRISPRv2 and lentiGuide-Puro and deep sequenced to ensure uniform representation. These new vectors and libraries improve GeCKO reagents for diverse screening applications. Reagents are available to the academic community through Addgene and the Zhang lab website.