CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome

Abstract CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model...

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Autores principales:	Rhoda Mae C. Simora, De Xing, Max R. Bangs, Wenwen Wang, Xiaoli Ma, Baofeng Su, Mohd G. Q. Khan, Zhenkui Qin, Cuiyu Lu, Veronica Alston, Darshika Hettiarachchi, Andrew Johnson, Shangjia Li, Michael Coogan, Jeremy Gurbatow, Jeffery S. Terhune, Xu Wang, Rex A. Dunham
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/cdedef54f68c49f1baf0d0bd4a6c8ded
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spelling	oai:doaj.org-article:cdedef54f68c49f1baf0d0bd4a6c8ded2021-12-02T12:03:15ZCRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome10.1038/s41598-020-79409-52045-2322https://doaj.org/article/cdedef54f68c49f1baf0d0bd4a6c8ded2020-12-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-79409-5https://doaj.org/toc/2045-2322Abstract CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.Rhoda Mae C. SimoraDe XingMax R. BangsWenwen WangXiaoli MaBaofeng SuMohd G. Q. KhanZhenkui QinCuiyu LuVeronica AlstonDarshika HettiarachchiAndrew JohnsonShangjia LiMichael CooganJeremy GurbatowJeffery S. TerhuneXu WangRex A. DunhamNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-14 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Rhoda Mae C. Simora De Xing Max R. Bangs Wenwen Wang Xiaoli Ma Baofeng Su Mohd G. Q. Khan Zhenkui Qin Cuiyu Lu Veronica Alston Darshika Hettiarachchi Andrew Johnson Shangjia Li Michael Coogan Jeremy Gurbatow Jeffery S. Terhune Xu Wang Rex A. Dunham CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome
description	Abstract CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.
format	article
author	Rhoda Mae C. Simora De Xing Max R. Bangs Wenwen Wang Xiaoli Ma Baofeng Su Mohd G. Q. Khan Zhenkui Qin Cuiyu Lu Veronica Alston Darshika Hettiarachchi Andrew Johnson Shangjia Li Michael Coogan Jeremy Gurbatow Jeffery S. Terhune Xu Wang Rex A. Dunham
author_facet	Rhoda Mae C. Simora De Xing Max R. Bangs Wenwen Wang Xiaoli Ma Baofeng Su Mohd G. Q. Khan Zhenkui Qin Cuiyu Lu Veronica Alston Darshika Hettiarachchi Andrew Johnson Shangjia Li Michael Coogan Jeremy Gurbatow Jeffery S. Terhune Xu Wang Rex A. Dunham
author_sort	Rhoda Mae C. Simora
title	CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome
title_short	CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome
title_full	CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome
title_fullStr	CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome
title_full_unstemmed	CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome
title_sort	crispr/cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/cdedef54f68c49f1baf0d0bd4a6c8ded
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CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome

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