Generation of zebrafish mutant lines using Cas9/gRNA system

Mutant pinhead and admp lines were generated using the CRISPR-Cas9 system as previously described (39). Two guide RNAs (gRNAs) were designed to target the sequences 5′-GGAGGTTGTGTGCTCGTG-3′ and 5′-GGTCGGAGGCGATCAGG-3′ within the first exon of the pinhead and admp loci, respectively. The Cas9 mRNA and gRNA were synthesized in vitro and coinjected into one-cell stage wild-type embryos. For screening of the mutant alleles, the genomic regions surrounding gRNA-targeted sequences were amplified by PCR with the following primers: for pinhead mutants, 5′-CATGTGGATTAAACACAAAGGC-3′ (forward) and 5′-GAAATACTGTAAATGGATTGAACGT-3 (reverse); for admp mutants, 5′-TCAGGATCTCCTCGAGGACCACC-3′ (forward) and 5′-TTATCTTACATTTGTCGAAGAAG-3′ (reverse). The amplified DNA fragment was purified for enzymatic digestion with T7 endonuclease I (M0302, New England BioLabs) or subjected to Sanger sequencing.

Confirmed founders were crossed to wild-type animals to raise F1 carriers for each mutant. Homozygous mutants were obtained by incrossing F1 fish carrying mutated genomic DNA. adΔ11 homozygous mutant embryos were injected with Cas9 mRNA and pinhead gRNA to generate germline mutants for pinhead in an adΔ11 mutant background. F1 animals were obtained by crossing the founders and adΔ11 homozygous mutants. Homozygous phΔ49;adΔ11 double mutants were obtained from the offspring of the phΔ49−/−;adΔ11−/− adults.

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