Fig. 1.

Genome editing in zebrafish using a CRISPR/Cas9 system. The system consists of two components, a dual NLS-tagged zebrafish codon-optimized Cas9 protein and a single crRNA:tracrRNA chimeric gRNA, comprising a 20-bp target sequence (dark red) complementary to the genomic target adjacent to a PAM site of NGG. Both components are first generated as RNAs by in vitro transcription from the SP6 or T3 (for Cas9) or T7 (for gRNA) promoter. The mix of gRNA and Cas9 RNA was then injected into one-cell–stage embryos to induce RNA-guided targeted DNA double-stranded breaks mediated by the Cas9 enzyme. Arrowheads denote putative cleavage sites.