Genetic Editing of amhr2b Using CRISPR/Cas9 in Ayu

The sgRNA for amhr2bY was designed using CCTOP [111 (link)]. Forward 5’-TAGGAGTAAGGCTCGGACCAGT-3’ and reverse 5’-AAACACTGGTCCGAGCCTTACT-3’ oligo nucleotide for target sequences of amhr2bY were annealed and cloned into the BsaI site of the pDR274 vector (Addgene, Watertown, MA. catalog number: #42250). The sgRNA was transcribed using DraI-digested gRNA expression vectors as templates using the ScriptMAX Thermo T7 Transcription kit (TOYOBO). Then, sgRNA was treated DNase I and purified by RNeasy Mini Kit (Qiagen). The 150 ng of Cas9 Nuclease protein NLS (Nippon Gene, Tokyo, Japan) and 25 ng of sgRNA were microinjected into fertilized ayu one-cell eggs obtained from the cultured strain derived from the Ota River population using a microinjector BJ-110 (BEX, Tokyo, Japan). The Body of amhr2bY-targeted G0 ayu were fixed in Bouin’s solution and analyzed histology of developing gonads. The head and tail of amhr2bY-targeted G0 ayu were used to extract genomic DNA. Mutations of target site for CRISPR/Cas9 were identified by genomic PCR using the primer set amhr2bY-11F and amhr2bY-12R for amhr2bY and amhr2a-11F and amhr2a-12R for amhr2a (S12 Table). The PCR products were cloned into a pGEM-T easy vector (Promega) and analyzed by Sanger sequencing to identify individual editing patterns. At least 8 clones per individual were examined.