CRISPRscan is a tool designed to identify highly efficient single guide RNAs (sgRNAs) for CRISPR/Cas9 targeting in vivo. The study analyzed the molecular features influencing sgRNA stability, activity, and loading into Cas9. It 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, a predictive sgRNA-scoring algorithm (CRISPRscan.org) was developed to capture sequence features affecting Cas9/sgRNA activity in vivo. The study also showed 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 influencing Cas9 activity and a framework for identifying efficient sgRNAs for genome targeting in vivo. The study analyzed the activity of over 1000 sgRNAs targeting 128 genes in zebrafish, revealing that efficient sgRNAs have biased sequence composition affecting CRISPR/Cas9 stability and activity. Alternative sgRNA formulations were evaluated, showing that shorter sgRNAs and those with 5' mismatches were more efficient. The study also demonstrated that targeting Cas9 to the germ line can generate maternal-zygotic mutants, improving the efficiency and applicability of CRISPR/Cas9-mediated mutagenesis. The findings provide a valuable resource for predicting the most efficient sgRNAs and facilitate direct functional screenings in vivo.CRISPRscan is a tool designed to identify highly efficient single guide RNAs (sgRNAs) for CRISPR/Cas9 targeting in vivo. The study analyzed the molecular features influencing sgRNA stability, activity, and loading into Cas9. It 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, a predictive sgRNA-scoring algorithm (CRISPRscan.org) was developed to capture sequence features affecting Cas9/sgRNA activity in vivo. The study also showed 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 influencing Cas9 activity and a framework for identifying efficient sgRNAs for genome targeting in vivo. The study analyzed the activity of over 1000 sgRNAs targeting 128 genes in zebrafish, revealing that efficient sgRNAs have biased sequence composition affecting CRISPR/Cas9 stability and activity. Alternative sgRNA formulations were evaluated, showing that shorter sgRNAs and those with 5' mismatches were more efficient. The study also demonstrated that targeting Cas9 to the germ line can generate maternal-zygotic mutants, improving the efficiency and applicability of CRISPR/Cas9-mediated mutagenesis. The findings provide a valuable resource for predicting the most efficient sgRNAs and facilitate direct functional screenings in vivo.