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Cells 2019, 8(1), 75; https://doi.org/10.3390/cells8010075

Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines

1
FAVET-INBIOGEN, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Avda. Santa Rosa, 11735 Santiago, Chile
2
Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, 7520245 Santiago, Chile
3
Departamento Biomédico, Facultad de Ciencias de la Salud; Instituto Antofagasta, Universidad de Antofagasta, Avenida Angamos 601, 1240000 Antofagasta, Chile
*
Author to whom correspondence should be addressed.
Received: 30 November 2018 / Revised: 14 January 2019 / Accepted: 16 January 2019 / Published: 21 January 2019
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Abstract

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely used in animals as an efficient genome editing tool. In fish cells, the technique has been difficult to implement due to the lack of proper vectors that use active promoters to drive the expression of both small guide RNA (sgRNA) and the S. pyogenes Cas9 (spCas9) protein within a single expression platform. Until now, fish cells have been modified using co-transfection of the mRNA of both the sgRNA and the spCas9. In the present study, we describe the optimization of a new vector for the expression of a CRISPR/Cas9 system, designed to edit the genome of fish cell lines, that combines a gene reporter (mCherry), sgRNA, and spCas9 in a single vector, facilitating the study of the efficiency of piscine and non-piscine promoters. A cassette containing the zebrafish U6 RNA III polymerase (U6ZF) promoter was used for the expression of the sgRNA. The new plasmid displayed the expression of spCas9, mCherry, and sgRNA in CHSE/F fish cells. The results demonstrate the functionality of the mammalian promoter and the U6ZF promoter in fish cell lines. This is the first approach aimed at developing a unified genome editing system in fish cells using bicistronic vectors, thus creating a powerful biotechnological platform to study gene function. View Full-Text
Keywords: CRISPR/Cas9; U6 promoter; fish cells CRISPR/Cas9; U6 promoter; fish cells
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Escobar-Aguirre, S.; Arancibia, D.; Escorza, A.; Bravo, C.; Andrés, M.E.; Zamorano, P.; Martínez, V. Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines. Cells 2019, 8, 75.

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