Production of a breed of red sea bream Pagrus major with an increase of skeletal muscle mass and reduced body length by genome editing with CRISPR/Cas9

Kishimoto, Kenji; Washio, Youhei; Yoshiura, Yasutoshi; Toyoda, Atsushi; Ueno, Takahiro; Fukuyama, Hidenao; Kato, Keitaro; Kinoshita, Masato

doi:10.1016/j.aquaculture.2018.05.055

Cited by 108 publications

(79 citation statements)

References 31 publications

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“…However, previous reports indicated that there were few or no off-target alternations in pre-selected candidate sites harbouring 2- or fewer-bp mismatches in the 12-bp of the targeting sequence followed by NGG protospacer adjacent motif in mice, Drosophila , C . elegans , and fish 33,34 . Meanwhile, because the DNA-binding domain in each TALEN has a sufficiently long binding sequence that exhibits high DNA-binding specificity, it is likely that there are few off-target effects caused by mutagenesis of potential off-target sites of the TALENs 17 .…”

Section: Discussionmentioning

confidence: 99%

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

Higuchi

Kazeto

Ozaki

et al. 2019

Sci Rep

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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.

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Section: Discussionmentioning

confidence: 99%

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

Higuchi

Kazeto

Ozaki

et al. 2019

Sci Rep

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show abstract

“…However, frame-shift mutation rates were not apparently correlated with the phenotypes in the present study. No clear correlation between frame-shift mutation rates and the severity of the phenotypes was also reported in red sea bream (Pagrus major) 52 . Given that the target area was located towards the 3′ end of piwil2 (21st exon out of a total of 24 exons), the frame-shift mutation may not make an apparent difference in the phenotype in this case.…”

Section: Discussionmentioning

confidence: 93%

Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system

Jin

Liao

Migaud

et al. 2020

Sci Rep

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the application of genome engineering techniques to understand the mechanisms that regulate germ cell development opens promising new avenues to develop methods to control sexual maturation and mitigate associated detrimental effects in fish. In this study, the functional role of piwil2 in primordial germ cells (PGCs) was investigated in Nile tilapia using CRISPR/Cas9 and the resultant genotypes were further explored. piwil2 is a gonad-specific and maternally deposited gene in Nile tilapia eggs which is known to play a role in repression of transposon elements and is therefore thought to be important for maintaining germline cell fate. A functional domain of piwil2, PIWI domain, was targeted by injecting Cas9 mRNA and sgRNAs into Nile tilapia embryos at 1 cell stage. Results showed 54% of injected mutant larvae had no or less putative PGCs compared to control fish, suggesting an essential role of piwil2 in survival of PGCs. The genotypic features of the different phenotypic groups were explored by next generation sequencing (NGS) and other mutant screening methods including T7 endonuclease 1 (T7E1), CRISPR/Cas-derived RNA-guided engineered nuclease (RGEN), high resolution melt curve analysis (HRMA) and fragment analysis. Linking phenotypes to genotypes in F0 was hindered by the complex mosacism and wide indel spectrum revealed by NGS and fragment analysis. This study strongly suggests the functional importance of piwil2 in PGCs survival. Further studies should focus on reducing mosaicism when using CRISPR/Cas9 system to facilitate direct functional analysis in F0. Nile tilapia (Oreochromis niloticus) is one of the fastest growing farmed finfish species with > 120% increase in production volume over the last decade, such that global production has exceeded 4.1 million tonnes, worth USD 7.6 billion in 2017 1 making it the second largest (by volume) farmed finfish globally. While Nile tilapia is native to Northern Africa and Israel, it is now farmed widely out with its native range in many countries, contributing significantly to global food security particularly for poor rural communities 2. A significant hurdle that limited production potential of the species is precocious maturation where individuals direct energy towards sexual maturation to the detriment of somatic growth, which also results in the overproduction of unmarketable fry 3. This challenge has largely been overcome with the production of all male stocks which result in a more efficient production of tilapia with increased harvest weight 4. While this has had a significant beneficial impact on production of the species, contributing to its rapid expansion globally, the farming of reproductively competent animals has also resulted in widespread environmental impacts. Nile tilapia is considered to be an established invasive species in Asia 5 as well as Australia and North and South America, with reported impacts on native species and ecosystems 6. Therefore, there remains a need to develop methodologies to induce sterility and reduce production losses a...

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“…Teleost generally possess two types of MSTN genes (1 and 2 or a and b). The MSTN gene of red sea bream used in this study was identified as MSTNa (Kishimoto et al., 2018). We searched chromosome location of the GH and MSTNa / MSTN1 genes in the genome of selected fish species and found that the genes are located on different chromosomes: GH on ch.22 and MSTN1 on ch.8 in Takifugu rubripes and GH on ch.3 and MSTNa on ch.22 in Danio rerio .…”

Section: Discussionmentioning

confidence: 99%

Polymorphisms and haplotypes of the myostatin gene associated with growth in juvenile red sea breamPagrus major

Sawayama

2020

Aquac Res

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Polymorphisms of the myostatin gene (MSTN) have been studied in vertebrates including several aquaculture species, revealing their role in growth. We attempted to identify polymorphisms in MSTN associated with growth traits of juvenile farmed red sea bream Pagrus major, an important cultured fish species in Japan. Polymorphisms in the coding region of MSTN were screened, and six polymorphisms were found among three exons: a deletion in exon 1, a single nucleotide polymorphism (SNP) in exon 2 and four SNPs in exon 3. The deletion in exon 1 eliminated a codon (glutamine) from the region comprising the polyglutamine structure, but all SNPs were synonymous. We analysed the polymorphisms for association with growth phenotype in large and small P. major specimens obtained from commercial production at 50 days post hatching. Two SNPs located in exon 3 (c.+846T>C and c.+1140T>C) were significantly more frequent in the large group, while no SNPs were significantly associated with the small phenotype. Haplotypes were reconstructed using genotype information of the two phenotype groups, and nine haplotypes (Hap_1 to Hap_9) were successfully reconstructed. Hap_6 and Hap_7 were significantly more often observed in small and large groups respectively. Analysis of diplotypes (haplotype combinations) of individual specimens revealed 19 diplotypes. The Hap_7/Hap_7 diplotype was found in 46.7% of large phenotype specimens, significantly more frequently than in the small group (13.5%). These results will be useful for marker‐assisted selection of red sea bream to improve production with respect to growth.

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Production of a breed of red sea bream Pagrus major with an increase of skeletal muscle mass and reduced body length by genome editing with CRISPR/Cas9

Cited by 108 publications

References 31 publications

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

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

Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system

Polymorphisms and haplotypes of the myostatin gene associated with growth in juvenile red sea breamPagrus major

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