Despite diverse transfection approaches, zygote micro-injection remains the most widely used method to deliver the CRISPR/Cas9 system into most model organisms, including zebrafish and non-human primates (Jao et al., 2013; Niu et al., 2014). In zebrafish, injection of Cas9 mRNA and single guide RNA (sgRNA) is routine, and sgRNAs are usually produced via in vitro transcription driven by T7 promoter, which prefers 5′-GG as the initial nucleotide of transcripts, constraining the selection of target sites (Sanjana et al., 2014). Recently, several RNA processing mechanisms were employed to generate sgRNAs from cleavable multiplexed transcripts, which not only expand the available target sites but also empower the spatiotemporal regulation of sgRNA expression via injecting vectors carrying RNA polymerase II promoters (Nissim et al., 2014; Xu et al., 2017). Among those mechanisms, RNA endonuclease Csy4 simply requests a 28-nucleotide RNA sequence (‘28’) for recognition. However, a severe teratogenic effect was previously observed after injecting csy4 mRNA into zebrafish zygotes, prohibiting the further application of Csy4 in manipulating vertebrate embryos (Qin et al., 2015). Here we integrated csy4 with the CRISPR/Cas9 system into Tol2-flanked vectors (Kwan et al., 2007), and used conditional and tissue-specific promoters to regulate the expression of all components in vivo. Zebrafish zygotes injected by the cys4-expressing vectors could develop into viable chimeric embryos and larvae with a high frequency of integration, allowing for inducible gene editing without causing malformation.