Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behaviour in Pacific bluefin tuna (Thunnus orientalis)

Toshiya Yamaguchi,Yoshiaki Iwasaki ,Yukinori Shimada,Satoshi Soma,Aiko Matsuura,Gén Kobashi ,Tetsuji Masaoka,Kentaro Higuchi,Yukinori Kazeto,Yuichi Ozaki,Ryoji Goto ,Takanori Kobayashi,Takashi Yamamoto,Yoshitaka Sakakura,Motoshige Yasuike,Takahiro Matsubara,Shukei Masuma,Tetsushi Sakuma,Issei Nishiki,Yoshiaki Ina,Yoji Nakamura,Atushi Fujiwara

doi:10.1038/s41598-019-50418-3

Abstract

In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance. Here, we report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. A pair of Platinum TALENs targeting exons 2 and 43 of the PBT ryr1b gene induced deletions in each TALEN target site of the injected embryos with extremely high efficiency. In addition, ryr1b expression was significantly decreased in the mutated G0 larvae at 7 days after hatching (DAH). A touch-evoked escape behaviour assay revealed that the ryr1b-mutated PBT larvae swam away much less efficiently in response to mechanosensory stimulation at 7 DAH than did the wild-type larvae. Our results demonstrate that genome editing technologies are effective tools for determining the functional characterization of genes in a comparatively short period, and create avenues for facilitating genetic studies and breeding of bluefin tuna species.

Highlights

In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance
We report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum transcriptional activator-like effector nucleases (TALENs) caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae
These results suggest that the Platinum TALEN system is an effective genome editing tool for breeding new varieties with valuable traits in bluefin tuna

Summary

TALENs causes slow swimming

Received: 18 February 2019 Accepted: 5 September 2019 Published online: 25 September 2019 behaviour in Pacific bluefin tuna (Thunnus orientalis). We report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. We successfully produced Pacific bluefin tuna (PBT, Thunnus orientalis) larvae exhibiting slow swimming in response to tactile stimuli due to mutations of the ryr1b gene induced by Platinum TALENs. The slow swimming characteristics are a useful trait for the prevention of wall collisions in bluefin tuna aquaculture. Sequence analysis of PCR amplicons covering the targeted sites in the embryos injected with TALENs at various concentrations indicated the induction of a spectrum of indel mutation, consistent with what is expected from non-homologous end-joining (Supplementary Figs S3 and S4)

Fast Slow

Discussion

Mutant a

Materials and Methods

Author Contributions

Findings

Additional Information