An effective microinjection method for genome editing of marine aquaculture fish: tiger pufferfish Takifugu rubripes and red sea bream Pagrus major

Kishimoto, Kenji; Washio, Youhei; Murakami, Yu; Katayama, Takashi; Kuroyanagi, Miwa; Kato, Keitaro; Yoshiura, Yasutoshi; Kinoshita, Masato

doi:10.1007/s12562-018-1277-3

Cited by 17 publications

(13 citation statements)

References 15 publications

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“…Both before and during injecting, pots containing egg clutches were broken apart into multiple shards (~2.0x4.0 cm) using a hammer and chisel. The shards were then placed in a petri dish and partially submerged in Yamamoto’s ringer’s solution [ 47 ] (see Supporting Information S3 File ) to alleviate osmotic stress associated with injection [ 10 ]. Eggs were viewed under a dissection microscope (3.5x magnification) and microinjected directly into the animal pole ( Fig 1A and 1B ) at a 45° angle with a pulled borosilicate glass pipette (Harvard Apparatus: 1.0x0.58x100 mm) fitted on a pneumatic injector unit (Narishige IM- 400) and micromanipulator (Marzhauser MM3301R).…”

Section: Methodsmentioning

confidence: 99%

“…The injection of sgRNA fused with Cas9 protein has proven to be an effective tool for precise genome editing at target gene sequences in the cell lines of numerous species including many teleost fishes such as zebrafish ( Danio rerio ) [ 2 ], Nile tilapia ( Oreochromis niloticus ) [ 3 , 4 ], medaka ( Oryzias latipes ) [ 5 ], Atlantic salmon ( Salmo salar ) [ 6 ], killifish (spp.) [ 7 , 8 ], pufferfish ( Takifugu rubribes ) [ 9 , 10 ], and red sea bream ( Pagrus major ) [ 11 ]. However, the CRISPR/Cas9 system has yet to be applied to coral reef fishes, a highly diverse assemblage of species with a unique life history and biological adaptations suited for survival in their reef environment (e.g., a pelagic larval stage, demersal spawning, and parental behaviour) [ 12 – 14 ] but make them incompatible with standard CRISPR protocols used on most teleosts.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, a well-designed protocol for the efficient delivery of sgRNA/Cas9 to completely knockout gene function is needed. To achieve this, careful species-specific considerations must be made for sgRNA design, dose toxicity, construct delivery parameters (i.e., air pressure, needle dimensions), and egg/embryo- care both during microinjection and incubation [ 10 ]. Inherent challenges specific to gene-editing anemonefishes and many other demersal spawning reef fishes include the injection and/or care of their substrate-attaching eggs [ 36 ] and pelagic larval stage [ 37 ].…”

Section: Introductionmentioning

confidence: 99%

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CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants

et al. 2021

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Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of zygotes was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 73.3% of injected embryos. Further analysis of the subcloned mutant gene sequences combined with amplicon shotgun sequencing revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in F0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful starting point to further explore the potential application of CRISPR/Cas9 in A. ocellaris, as a platform for studying gene function in anemonefishes and other reef fishes.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants

et al. 2021

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“…6,28 In over 20 aquaculture species, CRISPR/Cas9 system has been used in studies on gene functions and breeding (Table 1). These species include Nile tilapia (O. niloticus), 23,79 Atlantic salmon (Salmo salar), 26,80,81 Atlantic killifish (Fundulus heteroclitus), 25 oriental prawn (Exopalaemon carinicauda), 82 Labeo rohita, a farmed carp (known as rohu), 83 common carp (Cyprinus carpio), 76,84 The Burton's mouth-brooder (Astatotilapia burtoni), 61 Chinese tongue sole (Cynoglossus semilaevis), 85 Red sea bream (Pagrus major), [86][87][88] channel catfish (Ictalurus punctatus), [89][90][91] grass carp (Ctenopharyngodon idella), 92 sterlet (Acipenser ruthenus), 93 olive flounder (Paralichthys olivaceus), 27,94,95 Pacific oyster (Crassostrea gigas), 96 blunt snout bream (Megalobrama amblycephala), 97 white prawn (Exopalaemon carinicauda), 82 fighting fish (Betta 98 and other species. 12 Studies on genome editing in aquaculture species confirmed that the CRISPR/Cas system is highly efficient, has lower off-target tendencies, and is able to generate long fragment deletions.…”

Section: S Tatus Of G Enome Ed Iting In Aq Uacu Ltu Rementioning

confidence: 99%

Genome editing and its applications in genetic improvement in aquaculture

Yang

Tay

et al. 2021

Reviews in Aquaculture

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In the aquaculture industry, selective breeding has played an important role in increasing aquaculture production significantly. To meet the global growing demand for high quality proteins, it is essential to apply novel technologies to accelerate breeding to facilitate the increase in aquaculture production. Gene and genome editing technologies, including ZFNs, TALLENS and Crispr/Cas9, are promising tools to speed up genetic improvement. Among all the genome editing approaches, the CRISPR/Cas9 system is faster, cheaper and precise in editing genes/genomes. Therefore, the application of CRISPR/Cas9 technology in the editing of genomes in aquaculture species is emerging rapidly. It has been applied to precisely edit genes to identify gene functions and generate the preferred traits in over 20 aquaculture species. This review summarises the genome editing technologies and their applications in the rapid improvement of economic traits in the aquaculture industry. Challenges and future directions of genome editing are also discussed.

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“…Further application of CRISPR/Cas in other fish species of commercial importance (for foods and ornamental value) include editing of disease resistance genes in grass carp (Ctenopharyngdon idella ) (Ma et al 2018 ), farmed carp (Chakrapani et al 2016 ), and channel catfish (Elaswad and Dunham 2017 ; Elaswad et al 2018a , b ). Editing of growth-related genes has been conducted in common carp (Zhong et al 2016 ), channel catfish (Khalil et al 2017 ), tiger puffer fish ( Takifugu rubripes ), red sea bream ( Pagrus major ) (Kishimoto et al 2018 , 2019 ) and in olive flounder ( Paralichthys olivaceus ) (eg. Kim et al 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Sustainable use of CRISPR/Cas in fish aquaculture: the biosafety perspective

et al. 2021

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Aquaculture is becoming the primary source of seafood for human diets, and farmed fish aquaculture is one of its fastest growing sectors. The industry currently faces several challenges including infectious and parasitic diseases, reduced viability, fertility reduction, slow growth, escapee fish and environmental pollution. The commercialization of the growth-enhanced AquAdvantage salmon and the CRISPR/Cas9-developed tilapia (Oreochromis niloticus) proffers genetic engineering and genome editing tools, e.g. CRISPR/Cas, as potential solutions to these challenges. Future traits being developed in different fish species include disease resistance, sterility, and enhanced growth. Despite these notable advances, off-target effect and non-clarification of trait-related genes among other technical challenges hinder full realization of CRISPR/Cas potentials in fish breeding. In addition, current regulatory and risk assessment frameworks are not fit-for purpose regarding the challenges of CRISPR/Cas notwithstanding that public and regulatory acceptance are key to commercialization of products of the new technology. In this study, we discuss how CRISPR/Cas can be used to overcome some of these limitations focusing on diseases and environmental release in farmed fish aquaculture. We further present technical limitations, regulatory and risk assessment challenges of the use of CRISPR/Cas, and proffer research strategies that will provide much-needed data for regulatory decisions, risk assessments, increased public awareness and sustainable applications of CRISPR/Cas in fish aquaculture with emphasis on Atlantic salmon (Salmo salar) breeding.

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An effective microinjection method for genome editing of marine aquaculture fish: tiger pufferfish Takifugu rubripes and red sea bream Pagrus major

Cited by 17 publications

References 15 publications

CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants

CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants

Genome editing and its applications in genetic improvement in aquaculture

Sustainable use of CRISPR/Cas in fish aquaculture: the biosafety perspective

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