An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

Abstract CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribon...

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Autores principales:	Jens Hamar, Dietmar Kültz
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f4dc1460df1e45f59466f0141dfc9410
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spelling	oai:doaj.org-article:f4dc1460df1e45f59466f0141dfc94102021-12-02T14:26:13ZAn efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters10.1038/s41598-021-87068-32045-2322https://doaj.org/article/f4dc1460df1e45f59466f0141dfc94102021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-87068-3https://doaj.org/toc/2045-2322Abstract CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.Jens HamarDietmar KültzNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-17 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jens Hamar Dietmar Kültz An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters
description	Abstract CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.
format	article
author	Jens Hamar Dietmar Kültz
author_facet	Jens Hamar Dietmar Kültz
author_sort	Jens Hamar
title	An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters
title_short	An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters
title_full	An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters
title_fullStr	An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters
title_full_unstemmed	An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters
title_sort	efficient vector-based crispr/cas9 system in an oreochromis mossambicus cell line using endogenous promoters
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/f4dc1460df1e45f59466f0141dfc9410
work_keys_str_mv	AT jenshamar anefficientvectorbasedcrisprcas9systeminanoreochromismossambicuscelllineusingendogenouspromoters AT dietmarkultz anefficientvectorbasedcrisprcas9systeminanoreochromismossambicuscelllineusingendogenouspromoters AT jenshamar efficientvectorbasedcrisprcas9systeminanoreochromismossambicuscelllineusingendogenouspromoters AT dietmarkultz efficientvectorbasedcrisprcas9systeminanoreochromismossambicuscelllineusingendogenouspromoters
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An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

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