CRISPR-Cas9 Editing of il-34 and csf3r Genes in Zebrafish

The target genes were il-34 (NCBI accession: NM_001128701.1; Gene ID: 560193) and csf3r (NCBI accession: NM_001113377.1; Gene ID: 100134935). Co-injection of Cas9 mRNA and guide RNAs (gRNAs) was conducted in wild-type 1-cell stage zebrafish embryos. Cas9 mRNA was transcribed from XbaI linearalized pT3TS-nCas9n plasmid (Addgene #46757) using mMessage mMachine T3 Kit (Ambion) according to the manufacturer’s instructions. CRISPR targets for gRNA designs were identified using CHOPCHOP (http://chopchop.cbu.uib.no) (Gagnon et al., 2014 (link)). Gene-specific oligonucleotides using T7 promoter were used to make gRNAs as previously described (Gagnon et al., 2014 (link)). gRNA target sequences and genotyping primers are provided in Supplementary file 1. In vitro transcription of gRNAs from assembled oligonucleotides was conducted using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB). To ensure high mutagenesis rate and large deletion mutations, three gRNAs were simultaneously injected with Cas9 mRNA for each gene. Injected clutches of embryos were validated to contain CRISPR mediated mutagenesis by a T7 endonuclease assay. Mutations were analyzed by TOPO TA cloning followed by Sanger sequencing.