The authors, Neville E. Sanjana, Ophir Shalem, and Feng Zhang, from the Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, and the Department of Brain and Cognitive Sciences at MIT, have improved the lentiviral packaging and guide sequences in their original Genome-scale CRISPR Knock-Out (GeCKO) library. They developed a new vector, lentiCRISPRv2, which increased the functional viral titer by ~10-fold compared to the previous version (lentiCRISPRv1). Additionally, they created a two-vector system (lentiCas9-Blast and lentiGuide-Puro) that further increased the viral titer by ~100-fold. These improvements enable more efficient knock-out of genes in human cells. The authors also designed and synthesized new human and mouse GeCKOv2 sgRNA libraries, which include 6 sgRNAs per gene distributed over 3-4 exons, improved off-target score calculations, and additional sgRNAs targeting miRNA pre-mRNA hairpin structures. These libraries are divided into two sub-libraries, each containing 3 sgRNAs per gene and 1000 non-targeting control sgRNAs. The libraries are available to the academic community through Addgene and computational tools via the Zhang lab website. These advancements enhance the capabilities of CRISPR screening for diverse biological models and applications.The authors, Neville E. Sanjana, Ophir Shalem, and Feng Zhang, from the Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, and the Department of Brain and Cognitive Sciences at MIT, have improved the lentiviral packaging and guide sequences in their original Genome-scale CRISPR Knock-Out (GeCKO) library. They developed a new vector, lentiCRISPRv2, which increased the functional viral titer by ~10-fold compared to the previous version (lentiCRISPRv1). Additionally, they created a two-vector system (lentiCas9-Blast and lentiGuide-Puro) that further increased the viral titer by ~100-fold. These improvements enable more efficient knock-out of genes in human cells. The authors also designed and synthesized new human and mouse GeCKOv2 sgRNA libraries, which include 6 sgRNAs per gene distributed over 3-4 exons, improved off-target score calculations, and additional sgRNAs targeting miRNA pre-mRNA hairpin structures. These libraries are divided into two sub-libraries, each containing 3 sgRNAs per gene and 1000 non-targeting control sgRNAs. The libraries are available to the academic community through Addgene and computational tools via the Zhang lab website. These advancements enhance the capabilities of CRISPR screening for diverse biological models and applications.