CRISPR/Cas9-Mediated Transgenesis of the Masu Salmon (Oncorhynchus masou) elovl2 Gene Improves n-3 Fatty Acid Content in Channel Catfish (Ictalurus punctatus)

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play a very important role in human health. Channel catfish ( Ictalurus punctatus ) is one of the leading freshwater aquaculture species in the USA, but has low...

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Published in:Marine biotechnology (New York, N.Y.) N.Y.), 2022-06, Vol.24 (3), p.513-523
Main Authors: Xing, De, Su, Baofeng, Li, Shangjia, Bangs, Max, Creamer, David, Coogan, Michael, Wang, Jinhai, Simora, Rhoda, Ma, Xiaoli, Hettiarachchi, Darshika, Alston, Veronica, Wang, Wenwen, Johnson, Andrew, Lu, Cuiyu, Hasin, Tasnuba, Qin, Zhenkui, Dunham, Rex
Format: Article
Language:English
Subjects:
Actin
Aquaculture
Biomedical and Life Sciences
Biosynthesis
Catfish
CRISPR
Deformation effects
Design optimization
Docosahexaenoic acid
Eicosapentaenoic acid
Engineering
Fatty acids
Fish
Freshwater
Freshwater & Marine Ecology
Freshwater aquaculture
Freshwater fishes
Gene editing
Gene expression
gRNA
Ictalurus punctatus
Inland water environment
Life Sciences
Microbiology
Morphometry
Muscles
Oncorhynchus masou
Original Article
Plasmids
Polyunsaturated fatty acids
Salmon
Tissue
Transgenic fish
Zoology
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cited_by cdi_FETCH-LOGICAL-c282t-1aa1be8f921f334bab7aa86763e918d6a2d42fd5dacdbd52aafd1e8f8960e79c3
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container_title Marine biotechnology (New York, N.Y.)
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creator Xing, De
Su, Baofeng
Li, Shangjia
Bangs, Max
Creamer, David
Coogan, Michael
Wang, Jinhai
Simora, Rhoda
Ma, Xiaoli
Hettiarachchi, Darshika
Alston, Veronica
Wang, Wenwen
Johnson, Andrew
Lu, Cuiyu
Hasin, Tasnuba
Qin, Zhenkui
Dunham, Rex
description Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play a very important role in human health. Channel catfish ( Ictalurus punctatus ) is one of the leading freshwater aquaculture species in the USA, but has low levels of EPA and DHA compared to some fish such as salmon. To improve EPA and DHA content, a modification of the n-3 PUFA biosynthetic pathway was achieved through the insertion of an elovl2 transgene isolated from masu salmon ( Oncorhynchus masou ) driven by a carp β-actin promoter using a two-hit by gRNA and two oligos with a targeting plasmid (2H2OP) CRISPR/Cas9 approach. Integration rate of the transgene was high (37.5%) and detected in twelve different tissues of P 1 transgenic fish with tissue-specific gene expression. Liver and muscle had relative high gene expression (13.4- and 9.2-fold change, respectively). Fatty acid analysis showed DHA content in the muscle from transgenic fish was 1.62-fold higher than in non-transgenic fish ( P  
doi_str_mv 10.1007/s10126-022-10110-6
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Channel catfish ( Ictalurus punctatus ) is one of the leading freshwater aquaculture species in the USA, but has low levels of EPA and DHA compared to some fish such as salmon. To improve EPA and DHA content, a modification of the n-3 PUFA biosynthetic pathway was achieved through the insertion of an elovl2 transgene isolated from masu salmon ( Oncorhynchus masou ) driven by a carp β-actin promoter using a two-hit by gRNA and two oligos with a targeting plasmid (2H2OP) CRISPR/Cas9 approach. Integration rate of the transgene was high (37.5%) and detected in twelve different tissues of P 1 transgenic fish with tissue-specific gene expression. Liver and muscle had relative high gene expression (13.4- and 9.2-fold change, respectively). Fatty acid analysis showed DHA content in the muscle from transgenic fish was 1.62-fold higher than in non-transgenic fish ( P  &lt; 0.05). Additionally, total n-3 PUFAs and omega-6 polyunsaturated fatty acids (n-6 PUFAs) increased to 1.41-fold and 1.50-fold, respectively, suggesting the β-actin- elovl2 transgene improved biosynthesis of PUFAs in channel catfish as a whole. The n-9 fatty acid level decreased in the transgenic fish compared to the control. Morphometric analysis showed that there were significant differences between injected fish with sgRNAs (including positive and negative fish) and sham-injected controls ( P  &lt; 0.001). Potential off-target effects are likely the major factor responsible for morphological deformities. 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Su, Baofeng ; Li, Shangjia ; Bangs, Max ; Creamer, David ; Coogan, Michael ; Wang, Jinhai ; Simora, Rhoda ; Ma, Xiaoli ; Hettiarachchi, Darshika ; Alston, Veronica ; Wang, Wenwen ; Johnson, Andrew ; Lu, Cuiyu ; Hasin, Tasnuba ; Qin, Zhenkui ; Dunham, Rex</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c282t-1aa1be8f921f334bab7aa86763e918d6a2d42fd5dacdbd52aafd1e8f8960e79c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>Actin</topic><topic>Aquaculture</topic><topic>Biomedical and Life Sciences</topic><topic>Biosynthesis</topic><topic>Catfish</topic><topic>CRISPR</topic><topic>Deformation effects</topic><topic>Design optimization</topic><topic>Docosahexaenoic acid</topic><topic>Eicosapentaenoic acid</topic><topic>Engineering</topic><topic>Fatty acids</topic><topic>Fish</topic><topic>Freshwater</topic><topic>Freshwater &amp; Marine Ecology</topic><topic>Freshwater aquaculture</topic><topic>Freshwater fishes</topic><topic>Gene editing</topic><topic>Gene expression</topic><topic>gRNA</topic><topic>Ictalurus punctatus</topic><topic>Inland water environment</topic><topic>Life Sciences</topic><topic>Microbiology</topic><topic>Morphometry</topic><topic>Muscles</topic><topic>Oncorhynchus masou</topic><topic>Original Article</topic><topic>Plasmids</topic><topic>Polyunsaturated fatty acids</topic><topic>Salmon</topic><topic>Tissue</topic><topic>Transgenic fish</topic><topic>Zoology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Xing, De</creatorcontrib><creatorcontrib>Su, Baofeng</creatorcontrib><creatorcontrib>Li, Shangjia</creatorcontrib><creatorcontrib>Bangs, Max</creatorcontrib><creatorcontrib>Creamer, David</creatorcontrib><creatorcontrib>Coogan, Michael</creatorcontrib><creatorcontrib>Wang, Jinhai</creatorcontrib><creatorcontrib>Simora, Rhoda</creatorcontrib><creatorcontrib>Ma, Xiaoli</creatorcontrib><creatorcontrib>Hettiarachchi, Darshika</creatorcontrib><creatorcontrib>Alston, Veronica</creatorcontrib><creatorcontrib>Wang, Wenwen</creatorcontrib><creatorcontrib>Johnson, Andrew</creatorcontrib><creatorcontrib>Lu, Cuiyu</creatorcontrib><creatorcontrib>Hasin, Tasnuba</creatorcontrib><creatorcontrib>Qin, Zhenkui</creatorcontrib><creatorcontrib>Dunham, Rex</creatorcontrib><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Oceanic Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>ABI商业信息数据库</collection><collection>ABI/INFORM Global (PDF only)</collection><collection>Health &amp; 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Channel catfish ( Ictalurus punctatus ) is one of the leading freshwater aquaculture species in the USA, but has low levels of EPA and DHA compared to some fish such as salmon. To improve EPA and DHA content, a modification of the n-3 PUFA biosynthetic pathway was achieved through the insertion of an elovl2 transgene isolated from masu salmon ( Oncorhynchus masou ) driven by a carp β-actin promoter using a two-hit by gRNA and two oligos with a targeting plasmid (2H2OP) CRISPR/Cas9 approach. Integration rate of the transgene was high (37.5%) and detected in twelve different tissues of P 1 transgenic fish with tissue-specific gene expression. Liver and muscle had relative high gene expression (13.4- and 9.2-fold change, respectively). Fatty acid analysis showed DHA content in the muscle from transgenic fish was 1.62-fold higher than in non-transgenic fish ( P  &lt; 0.05). Additionally, total n-3 PUFAs and omega-6 polyunsaturated fatty acids (n-6 PUFAs) increased to 1.41-fold and 1.50-fold, respectively, suggesting the β-actin- elovl2 transgene improved biosynthesis of PUFAs in channel catfish as a whole. The n-9 fatty acid level decreased in the transgenic fish compared to the control. Morphometric analysis showed that there were significant differences between injected fish with sgRNAs (including positive and negative fish) and sham-injected controls ( P  &lt; 0.001). Potential off-target effects are likely the major factor responsible for morphological deformities. Optimization of sgRNA design to maximize activity and reduce off-target effects of CRISPR/Cas9 should be examined in future transgenic research, but this research shows a promising first step in the improvement of n-3 PUFAs in channel catfish.</abstract><cop>New York</cop><pub>Springer US</pub><doi>10.1007/s10126-022-10110-6</doi><tpages>11</tpages><orcidid>https://orcid.org/0000-0002-6149-6017</orcidid></addata></record>
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identifier ISSN: 1436-2228
ispartof Marine biotechnology (New York, N.Y.), 2022-06, Vol.24 (3), p.513-523
issn 1436-2228
1436-2236
language eng
recordid cdi_proquest_miscellaneous_2649998455
source ABI/INFORM Global (ProQuest); Springer Nature
subjects Actin
Aquaculture
Biomedical and Life Sciences
Biosynthesis
Catfish
CRISPR
Deformation effects
Design optimization
Docosahexaenoic acid
Eicosapentaenoic acid
Engineering
Fatty acids
Fish
Freshwater
Freshwater & Marine Ecology
Freshwater aquaculture
Freshwater fishes
Gene editing
Gene expression
gRNA
Ictalurus punctatus
Inland water environment
Life Sciences
Microbiology
Morphometry
Muscles
Oncorhynchus masou
Original Article
Plasmids
Polyunsaturated fatty acids
Salmon
Tissue
Transgenic fish
Zoology
title CRISPR/Cas9-Mediated Transgenesis of the Masu Salmon (Oncorhynchus masou) elovl2 Gene Improves n-3 Fatty Acid Content in Channel Catfish (Ictalurus punctatus)
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