Next Article in Journal
NUP214 in Leukemia: It’s More than Transport
Previous Article in Journal
Application of Prostate Cancer Models for Preclinical Study: Advantages and Limitations of Cell Lines, Patient-Derived Xenografts, and Three-Dimensional Culture of Patient-Derived Cells
Article Menu
Issue 1 (January) cover image

Export Article

Open AccessCommunication
Cells 2019, 8(1), 75;

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

FAVET-INBIOGEN, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Avda. Santa Rosa, 11735 Santiago, Chile
Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, 7520245 Santiago, Chile
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
Full-Text   |   PDF [1864 KB, uploaded 24 January 2019]   |  


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

Graphical abstract

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).

Supplementary materials


Share & Cite This Article

MDPI and ACS Style

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.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics



[Return to top]
Cells EISSN 2073-4409 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top