Dr274 vector

The online tool CCTop (Stemmer et al., 2015 (link)) was used to design sgRNAs. sgRNAs were generated as previously described by Hwang et al., 2013 (link). Oligonucleotides were annealed and ligated into DR274 vector (Addgene), which was previously linearized using BsaI-HF (New England Biolabs). Template for in vivo transcription of sgRNA was amplified by PCR. In vivo transcription was performed using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) and RNA was purified using the RNeasy Mini kit (Qiagen). Quantity and quality of sgRNA were analyzed using a NanoDrop and agarose gel electrophoresis. sgRNA cleaving activity was confirmed by an in vivo assay.

Cas9 mRNA was in vitro transcribed from pXT7 using the mMessage mMachine kit (ThermoFisher) as previously described with some modifications [42 (link)]. CRISPR/Cas9 target site in exon 3 of nrg1 were identified using ZiFit software [43 (link)] and zebrafish genomic sequence, build GRCz9 [44 (link)]. Single stranded oligonucleotides corresponding to the targeting sequence were annealed and cloned into the DR274 vector (Addgene) [45 (link)], then transcribed in vitro with T7 MaxiScript kit (ThermoFisher). Cas9 plasmid was generously provided by Dr. Jing-Wei Xiong. Embryos were injected at the one cell stage with 1–2 nl of a mixture containing 1200 ng Cas9, 50–75 ng gRNA, 10 mM MgCl, and 0.01% phenol red. gRNA targeting efficiency was determined by High Resolution Melt Analysis (HRMA) [46 (link)] as described below using primers flanking the target site. F1 offspring from F0 founders that carry favorable mutations, determined by DNA sequencing of the target site, were raised to adulthood. Heterozygous F1 fish were interbred to produce homozygous wild type, homozygous mutant, and heterozygous mutant offspring.