Two sgRNAs targeting igfbp5a exon 1 and two sgRNAs targeting igfbp5b exon 1 were designed using the online tool ZiFiT Targeter V4.2 ( Their sequences are as follows: igfbp5a-sgRNA-1, 5′-GGTGCGGAACGGGTGTCAGGTGG-3′ (sense strand, +114 to +136); igfbp5a-sgRNA-2, 5′-GTTCCGCACCGGAGGACACATGG-3′ (anti-sense strand, +123 to +101); igfbp5b-sgRNA-1, 5′-GGTGGGCTGTCAGCTAGTGAAGG-3′ (sense strand, +114 to +136); and igfbp5b-sgRNA-2, 5′-GTGCGAGCCGTGCGATCAGAAGG-3′ (sense strand, +66 to +88). These sgRNAs were synthesized by in vitro transcription following a published method (52). sgRNAs (30 ng/μl) were mixed with Cas9 protein (700 ng/μl) or mRNA (250 ng/μl) and coinjected into Tg(igfbp5a:GFP) or wild-type embryos at the one-cell stage. The injected embryos were raised in E3 solution until 72 hpf and transferred to either the regular or low-[Ca2+] solution. NaR cells were quantified at 120 hpf as previously reported (4). To generate the igfbp5b mutant line, F0 fish were screened using T7E1 assays with the following primers: igfbp5b-gt-F, 5′-AACCCCAACCTGTGTTTTCA-3′; igfbp5b-gt-R, 5′-GATACAAACCACCGCACCCA-3′. igfbp5b homozygous mutants were obtained following the cross strategy described above.

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