Producing gRNA and Cas9 mRNA for Genome Editing

For producing gRNAs for the fads2 genes, the respective pT7-gRNA plasmids were digested with BamHI-HF^TM (NEB) and purified using the DNA Clean and Concentrator^TM-5 (ZYMO RESEARCH). The gRNAs were synthesized using the MEGAscript T7 kit (Ambion). Synthesized gRNAs were purified using the mirVana and miRNA Isolation kit (Ambion). The gRNA for slc45a2 was prepared as previously described^{33 (link)}. For making the Cas9 nuclease mRNA, the pTST3-nCas9n vector, codon optimized for zebrafish (Addgene ID# 46757)^{32 (link)} was digested with XbaI (NEB) and gel-purified using Wizard® SV Gel and PCR clean-up system (Promega). Cas9 mRNA was in vitro transcribed using the mMessage mMachine T3 kit (Ambion) and purified using RNeasy Mini Kit Spin column (Qiagen). The integrity of synthesized gRNAs and Cas9 mRNA was checked using the RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer (Agilent Technologies).