CRISPR-based Zebrafish Mutant Generation

The CRISPR/Cas9 system was applied to generate the zebrafish mutant lines according to the protocols reported previously (Lau et al., 2016 (link)). The targets were designed with ZIFIT Targeter (http://zifit.partners.org/zifit). Oligonucleotides synthesized were annealed and inserted into the pDR274 vector. The sgRNAs and Cas9 RNA were prepared using MEGAscript T7 and mMESSAGE mMACHINE SP6 kits (Life Technologies, Carlsbad, CA, USA), respectively. The mixture (4.6 nl) of sgRNA (20 ng/μl) and Cas9 mRNA (200 ng/μl) was co-injected into one-cell-stage embryos with a Nanoject system (Drummond, Broomall, PA, USA) to generate the F0 mutant fish. F0 fish were raised and those carrying mutations were outcrossed with WT fish to obtain F1 fish. The oligonucleotides used in this study are listed in Table S1.