F0 FOUNDER PRODUCTION OF AVTR-V1A2 KNOCKOUT STRAIN IN CHUB MACKEREL USING GENOME EDITING TECHNIQUE

Hirofumi Ohga*, Koki Shibata, Satoshi Kai, Ryo Sakanoue,
Hajime Kitano, Kohei Ohta, Naoki Nagano, Keishi Sakaguchi, Satoru Kuhara,
Kosuke Tashiro, Sangwan Kim, Tetsushi Sakuma, Takashi Yamamoto, Koichiro Gen, Atsushi Fujiwara, Yukinori Kazeto, Takanori Kobayashi, Michiya Matsuyama

Fisheries Research Institute of Karatsu, Faculty of Agriculture,
Kyushu University, Karatsu 847-0132, Saga, Japan
hohga@agr.kyushu-u.ac.jp

The chub mackerel (Scomber japonicus) is one of the most commercially important fish worldwide. In recent years, we standardized the full life cycle aquaculture of this species due to high consumer demand. However, high depletion during juvenile period stemming from cannibalistic behavior is still a serious problem. Here, we report the F0 founder production of arginine-vasotocin receptor (AVTR-V1a2) knockout strain by using TALENs. In the same study, reports showed that AVTR-V1a2 is involved in aggressiveness of teleost fish. The final vision of this project is to produce "manageable fish" for aquaculture efficiency.

Partial avtr-v1a2 sequence was obtained from genome and TALENs were designed to break S-S bridge. In preliminary trial, 100 ng/uL concentration of TALEN mRNA pairs were microinjected into fertilized eggs. In heteroduplex mobility assay (HMA) analysis, all individuals showed mutation. Moreover, random sequence analysis on PCR products from their genome showed 43.9% mutated cell rate. On May and June 2017, TALENs were microinjected into 1,296 fertilized eggs, 37 of which survived to adulthood. In embryo and juvenile of F0 founder, mutation of avtr-v1a2 was confirmed in over 80% individuals.

This work was supported by Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), "Technologies for creating next-generation agriculture, forestry and fisheries" (funding agency: Bio-oriented Technology Research Advancement Institution, NARO).