For phf21aa mutant (phf21aaaik4) construction, wild-type AB embryos were co-injected with 3 nl of a mixture containing guide RNA targeting exon 6 (ENSDART00000173629.2) (∼160 ng/µl) along with mRNA encoding nuclear-localized Cas9 (∼160 ng/µl). Nuclear-localized Cas9 mRNA was synthesized from pCS2-nCas9n (Addgene), linearized with NotI-HF (New England Biolabs), column purified (Zyppy Plasmid Miniprep kit; Zymo Research), and mRNA was synthesized (mMessenge mMachine SP6 kit; Thermo Fisher). Mutagenesis efficiency was detected in groups of F0 embryos (5 pooled × 3 replicates) using T7 endonuclease (New England Biolabs) digest of PCR fragments flanking the gRNA target site (PCR product = 995 base-pairs (bp), digestion products are approximately 720 and 275 bp). DNA from potential individual mutants was Sanger sequenced to establish a line with a 7 bp deletion at the gRNA target site in exon 6 ( phf21aaaik4). This mutation results in a frameshift mutation producing extensive missense and a premature stop codon (GenBank accession numbers: wild type MW438986, mutant MW438985). Genotyping of subsequent individual phf21aaaik4 fish utilized primers flanking the InDel site. PCR amplification results in a 641 bp product for wild-type DNA and a 634 bp product for mutant DNA. PCR products are run on a 2.5% agarose gel to resolve bands (wild type = 641 bp, mutant = 634 bp, heterozygotes = 641 + 634 bp bands).

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