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

Xing, De; Su, Baofeng; Li, Shangjia; Bangs, Max R.; Creamer, David; Coogan, Michael; Wang, Jinhai; Simora, Rhoda Mae C.; Ma, Xiaoli; Hettiarachchi, Darshika; Alston, Veronica; Wang, Wenwen; Johnson, Andrew; Lu, Cuiyu; Hasin, Tasnuba; Qin, Zhenkui; Dunham, Rex A.

doi:10.1007/s10126-022-10110-6

Cited by 18 publications

(10 citation statements)

References 59 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Docosahexaenoic acid (DHA), an omega-3 fatty acid, is essential for the development of human eye and nerve tissues (Judge et al, 2007), and the DHA biosynthesis pathway requires the elongase gene Elovl2 to function (Gregory and James, 2014). Recent studies showed that transgenic fish carrying elongase-like or Elovl2 gene exhibited higher DHA content compared to non-edited individuals (Alimuddin et al 2008; Xing et al, 2022). Thus, compared with knocking out reproductive-related genes to achieve sterilization, DHA-enrich fish are usually created by knocking in elongase-like genes.…”

Section: Discussionmentioning

confidence: 99%

Harnessing antimicrobial peptide genes to expedite disease-resistant enhancement in aquaculture: Transgenesis and genome editing

Wang

Cheng

2023

Preprint

Self Cite

View full text Add to dashboard Cite

Numerous studies have demonstrated that genome editing and transgenesis by integrating vector-engineered antimicrobial peptide genes (AMGs) is effective to modulate the fish's innate immune system. To generalize the knowledge of AMG application in aquaculture, here we recruited 540 data entries from a pool of empirical studies, which included 18 peer-reviewed publications and spanning 12 diseases. We systematically re-processed and re-analyzed these data by harnessing a cross-disease meta-analysis. On aggregate, AMG-genetic engineering aimed at enhancing disease resistance was shown to decrease the number of colony-forming units of bacteria, improve lysozyme activity, increase the post-infection survival rates, and alter the expression of AMGs and immune-related genes in aquatic animals. Furthermore, the AMG-pathogen combating activity was triggered within two hours after infection and lasted 48 hours, and the overexpression of AMGs was dominant in the spleen and skin, followed by the kidney and liver during this period. Typically, regardless of the type of AMGs, the synergistic expression of AMGs with IL, IKβ, TGFβ, C3b and TLR in AMG-integrated fish contributed to activating inflammatory/immune responses against pathogens. In addition, innovative CRISPR/Cas9-mediated systems enabling the site-directed knock-in of foreign genes at multiple loci were presented and prospected for disease-resistant enhancement in combination with other favorable fish-producing traits, including fast-growing, sterility, and enriched fatty acid. Altogether, our findings indicated that AMGs as transgenes have substantial potential to modulate the fish's innate immune system and accelerate disease-resistant enhancement combined with genetic engineering.

show abstract

Section: Discussionmentioning

confidence: 99%

Harnessing antimicrobial peptide genes to expedite disease-resistant enhancement in aquaculture: Transgenesis and genome editing

Wang

Cheng

2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…We observed that masu salmon elovl2 transgene was transferred into channel catfish, but the other two transgenes were not detectable in strategies 1 and 2, suggesting that the masu salmon elovl2 gene was more amenable to gene transfer than the rabbitfish desaturase genes. Previous studies demonstrated successes of HDRmediated and ssODN-mediated end joining KI of single transgenes, 21,32,33 but we failed to generate any transgenic fish simultaneously carrying three transgenes using strategy 1 or strategy 2 in our previous study 34 and this study. The efficiency of KI via HDR is low due to the constraint of the competing NHEJ pathway.…”

Section: ■ Discussionmentioning

confidence: 99%

A New Strategy for Increasing Knock-in Efficiency: Multiple Elongase and Desaturase Transgenes Knock-in by Targeting Long Repeated Sequences

Xing

Shang

et al. 2022

ACS Synth. Biol.

Self Cite

View full text Add to dashboard Cite

CRISPR/Cas9-mediated knock-in (KI) has a wide application in gene therapy, gene function study, and transgenic breeding programs. Unlike gene therapy, which requires accurate KI to correct gene mutation, transgenic breeding programs can accept robust KI as long as integration does not interrupt normal gene functions and result in any negative pleiotropic effects. High KI efficiency is required to reduce the breeding cost and shorten the breeding period, especially in transferring multiple foreign genes to a single individual. To elevate the KI efficacy and achieve multiple gene KIs simultaneously, we introduced a new strategy that enables transgene integration into numerous sites of the genome by targeting long repeated sequences (LRSs). Using this simple strategy, for the first time we successfully generated transgenic fish carrying the masu salmon (Oncorhynchus masou) elovl2 gene and rabbitfish (Siganus canaliculatus) Δ4 fad and Δ6 fad genes, and achieved robust target KI of elovl2 and Δ6 fad genes at multiple sites of LRS1 and LRS3, respectively, in the initial generation. This demonstrated that donor plasmid homology arms, which were nearly identical but not completely the same as the genome sequence, still led to on-target KI. Although the target KI efficiencies at LRS1, LRS2, and LRS3 sites were still relatively low in the current study, it is very promising that 100% KI efficiency in the future could be realized and perfected by selection of better LRSs and optimization of sgRNAs.

show abstract

“…Beyond the successful achievement of one desired trait, it is possible to use CRISPR/Cas9 to simultaneously improve multiple characteristics based on empirical data and theoretical foundations, 89–92 which means that we could alter other traits through the construction of different vectors while we focus on enhancement of disease resistance. Publications have already shown that single or multiple transgenes can be integrated into the fish genome by single sgRNA‐mediated genome editing (ssGE) or multi‐sgRNA‐mediated multiple genome editing assisted using plasmid donors 92,93 . Wang et al 34 demonstrated that the cathelicidin transgene can be knocked into the LH locus of channel catfish by double‐stranded DNA‐mediated ssGE.…”

Section: Future Perspectivesmentioning

confidence: 99%

“…Publications have already shown that single or multiple transgenes can be integrated into the fish genome by single sgRNA-mediated genome editing (ssGE) or multi-sgRNA-mediated multiple genome editing assisted using plasmid donors. 92,93 Wang et al 34 demonstrated that the cathelicidin transgene can be knocked into the LH locus of channel catfish by double-stranded DNA-mediated ssGE. In addition, a double-cut plasmid containing a foreign AMG transgene as a donor is beneficial to improve integration rates using CRISPR/Cas9-mediated ssGE.…”

Section: Transgenic Amgs and Fish Immune Systemmentioning

confidence: 99%

Enhancing aquaculture disease resistance: Antimicrobial peptides and gene editing

Wang

Cheng

2023

Reviews in Aquaculture

View full text Add to dashboard Cite

Previous studies have demonstrated that CRISPR/Cas9‐mediated genome editing and transgenesis by integrating vector‐engineered antimicrobial peptide genes (AMGs) are effective to modulate the fish's innate immune system. To generalise the knowledge of AMG application in aquaculture, we recruited 544 data entries from a pool of empirical studies, which included 23 studies (two unpublished from our team) spanning 12 diseases. We systematically re‐processed and re‐analysed these data by harnessing a cross‐disease meta‐analysis. On aggregate, AMG‐genetic engineering aimed at enhancing disease resistance was shown to decrease the number of colony‐forming units of bacteria from fish tissues, increase post‐infection survival rates, and alter the expression of AMGs and immune‐related genes. Further, AMG pathogen‐combating activity was triggered within 2 h after infection and lasted 48 h. The overexpression of AMGs was highest in the spleen and skin, followed by the kidney and liver during this period. Typically, regardless of the type of AMGs, the synergistic expression of AMGs with IL‐10, IKβ, TGFβ, C3b and TLR genes in AMG‐integrated fish contributed to activating inflammatory/immune responses against pathogens. Our findings revealed that the efficiency of transgenic AMGs against pathogens was fish‐, pathogen‐ and AMG‐specific. Compared to bacteria, transgenic AMGs were less inhibitory to viruses and parasites. In addition, innovative CRISPR/Cas9‐based transgenesis enabling the site‐directed knock‐in of foreign genes at multiple loci was assessed for the enhancement of disease resistance in combination with other favourable fish‐production traits, including fast growth, sterility and enriched fatty‐acid content. Altogether, our findings indicated that AMGs as transgenes have substantial promise to modulate the fish's innate immune system and enhance disease resistance.

show abstract

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

Cited by 18 publications

References 59 publications

Harnessing antimicrobial peptide genes to expedite disease-resistant enhancement in aquaculture: Transgenesis and genome editing

Harnessing antimicrobial peptide genes to expedite disease-resistant enhancement in aquaculture: Transgenesis and genome editing

A New Strategy for Increasing Knock-in Efficiency: Multiple Elongase and Desaturase Transgenes Knock-in by Targeting Long Repeated Sequences

Enhancing aquaculture disease resistance: Antimicrobial peptides and gene editing

Contact Info

Product

Resources

About