TALEN target sites were designed following published criteria (50) and are shown in fig. S1A. TALEN constructs were assembled using a Golden Gate cloning strategy following a published protocol (51). The assembled plasmids were determined by Stu I and Afl II digestion and confirmed by DNA sequencing.

TALEN-capped mRNA was synthesized by in vitro transcription using T3 polymerase (Ambion mMessage mMachine kit) and injected into embryos at the one-cell stage. After confirming indels using T7E1 assays in a subset of pooled F0 embryos, the remaining F0 embryos were raised to adulthood and crossed with wild-type fish. F1 fish were raised, and caudal fin was severed and genotyped as described below. Positive F1 fish were subjected to DNA sequencing. The F1 adult fish were intercrossed, and the progeny were raised and genotyped as described below. To obtain the viable homozygous fish, progeny from the F1 intercrosses were raised in the 5PPT high [Ca2+] solution in the first month and then transferred into the fish system.

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