Abstract
ABSTRACTGene editing with CRISPR/Cas9 is a powerful tool to study the function of target genes. Although this technology has demonstrated wide efficiency in many species, including fertilized zebrafish and medaka fish embryos when microinjected, its application to achieve efficient gene editing in cultured fish cells have met some difficulty. Here, we report an efficient and reliable approach to edit genes in cultured medaka (Oryzias latipes) fish cells using pre-formed gRNA-Cas9 ribonucleoprotein (RNP) complex. Both medaka fish haploid and diploid cells were transfected with the RNP complex by electroporation. Efficient gene editing was demonstrated by polymerase chain reaction (PCR) amplification of the target gene from genomic DNA and heteroduplex mobility assay carried out with polyacrylamide gel electrophoresis (PAGE). The heteroduplex bands caused by RNP cleavage and non-homologous end joining could be readily detected by PAGE. DNA sequencing confirmed that these heteroduplex bands contains the mutated target gene sequence. The average gene editing efficiency in haploid cells reached 50%, enabling us to generate a clonal cell line with ntrk3b gene mutation for further study. This RNP transfection method also works efficiently in diploid medaka cells, with the highest mutation efficiency of 61.5%. The specificity of this synthetic RNP CRISPR/Cas9 approach was verified by candidate off-target gene sequencing. Our result indicated that transfection of pre-formed gRNA-Cas9 RNP into fish cells is efficient and reliable to edit target genes in cultured medaka fish cells. This method will be very useful for gene function studies using cultured fish cells.
Highlights
The CRISPR/Cas9 gene editing technology was originally derived from the bacterial adaptive immune system, using a guide RNA activated Cas9 nuclease to cleave double-stranded DNA targets (Horvath and Barrangou, 2010)
RNP-mediated CRISPR/Cas9 gene editing in cultured medaka cells using a reporter plasmid pCut After failed attempts to achieve gene editing using expression plasmids or single guide RNA (sgRNA), we tried the electroporation of the Cas9: tracrRNA:crRNA RNP complex method in cultured medaka fish cells
The crRNA targeting either exon 1 or exon 2 of these genes were designed according to the CCTop - CRISPR/Cas9 target online predictor (Stemmer et al, 2015)
Summary
The CRISPR/Cas9 gene editing technology was originally derived from the bacterial adaptive immune system, using a guide RNA activated Cas9 nuclease to cleave double-stranded DNA targets (Horvath and Barrangou, 2010). We report a simple and efficient CRISPR/Cas9 gene editing method for cultured medaka fish cells by electroporation of pre-formed gRNA/Cas9 ribonucleoprotein (RNP) complex. RESULTS AND DISCUSSION RNP-mediated CRISPR/Cas9 gene editing in cultured medaka cells using a reporter plasmid pCut After failed attempts to achieve gene editing using expression plasmids or sgRNA, we tried the electroporation of the Cas9: tracrRNA:crRNA RNP complex method in cultured medaka fish cells.
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