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.
Highlights
A new gene-editing system with high targeting efficiency and low cell toxicity, known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9(Cas9), has been widely applied across species [1]
The present study reveals a novel and unified method to express a CRISPR/Cas9 system in the fish cell line CHSE/F
The vector LcU6ZF was adapted from mammalians, to express small guide RNA (sgRNA) and the Cas9 nuclease in fish cells
Summary
A new gene-editing system with high targeting efficiency and low cell toxicity, known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9(Cas9), has been widely applied across species [1]. There are few reports describing the editing of fish cell lines by the CRISPR/cas system because co-expression and joint delivery are challenging due to low transfection efficiency [12]. In medaka cell lines genome editing using the CRISPR/Cas system by ribonucleoprotein (RNP) complex was carried out using electroporation [13]. This methodology has been difficult to implement in practice due to the lack of fish-specific promoters within a single vector that can drive the co-expression of sgRNAs and the Cas protein, and limitations of high throughput delivery into individual cells
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