CRISPR/Cas9 technology is a powerful tool for genome engineering, but the variable activity of different single guide RNAs (sgRNAs) remains a significant limitation. The authors analyzed the molecular features influencing sgRNA stability, activity, and loading into Cas9 in vivo. They found that guanine enrichment and adenine depletion increase sgRNA stability and activity, while loading, nucleosome positioning, and Cas9 off-target binding are not major determinants. Truncated and 5′ mismatch-containing sgRNAs were identified as efficient alternatives to canonical sgRNAs. Based on these findings, they developed a predictive sgRNA-scoring algorithm (CRISPRscan.org) that effectively captures sequence features affecting Cas9/sgRNA activity in vivo. Additionally, they demonstrated that targeting Cas9 to the germ line using a Cas9-nanos-3′-UTR fusion can generate maternal-zygotic mutants, increase viability, and reduce somatic mutations. These results provide insights into the determinants of Cas9 activity and a framework for identifying highly efficient sgRNAs for genome targeting in vivo.CRISPR/Cas9 technology is a powerful tool for genome engineering, but the variable activity of different single guide RNAs (sgRNAs) remains a significant limitation. The authors analyzed the molecular features influencing sgRNA stability, activity, and loading into Cas9 in vivo. They found that guanine enrichment and adenine depletion increase sgRNA stability and activity, while loading, nucleosome positioning, and Cas9 off-target binding are not major determinants. Truncated and 5′ mismatch-containing sgRNAs were identified as efficient alternatives to canonical sgRNAs. Based on these findings, they developed a predictive sgRNA-scoring algorithm (CRISPRscan.org) that effectively captures sequence features affecting Cas9/sgRNA activity in vivo. Additionally, they demonstrated that targeting Cas9 to the germ line using a Cas9-nanos-3′-UTR fusion can generate maternal-zygotic mutants, increase viability, and reduce somatic mutations. These results provide insights into the determinants of Cas9 activity and a framework for identifying highly efficient sgRNAs for genome targeting in vivo.