The authors developed and optimized sgRNA design rules to improve the activity and specificity of CRISPR-Cas9-based genetic screens. They created human and mouse genome-wide libraries, Avana and Asiago, respectively, and performed positive and negative selection screens. The Avana library showed improved performance in identifying genes associated with vemurafenib resistance and essential cellular processes. The authors also developed a new scoring system, the Cutting Frequency Determination (CFD) score, to predict off-target effects of sgRNAs. This score outperformed existing metrics and was used to create optimized libraries, Brunello and Brie, which are designed to maximize on-target activity and minimize off-target effects. These optimized libraries are expected to enhance the efficiency and accuracy of genetic screens and genome engineering.The authors developed and optimized sgRNA design rules to improve the activity and specificity of CRISPR-Cas9-based genetic screens. They created human and mouse genome-wide libraries, Avana and Asiago, respectively, and performed positive and negative selection screens. The Avana library showed improved performance in identifying genes associated with vemurafenib resistance and essential cellular processes. The authors also developed a new scoring system, the Cutting Frequency Determination (CFD) score, to predict off-target effects of sgRNAs. This score outperformed existing metrics and was used to create optimized libraries, Brunello and Brie, which are designed to maximize on-target activity and minimize off-target effects. These optimized libraries are expected to enhance the efficiency and accuracy of genetic screens and genome engineering.