Please use this identifier to cite or link to this item: https://doi.org/10.1242/bio.035170
Title: Efficient genome editing using CRISPR/Cas9 ribonucleoprotein approach in cultured Medaka fish cells
Authors: Liu, Q 
Yuan, Y 
Zhu, F 
Hong, Y 
Ge, R 
Keywords: cas9 protein
cellular apoptosis susceptibility protein
CRISPR associated protein
DNA
guide RNA
heteroduplex
ntrk3b protein
protein tyrosine kinase
ribonucleoprotein
unclassified drug
animal cell
Article
cell assay
cell culture
clonal cell line
controlled study
CRISPR-CAS9 system
diploidy
DNA end joining repair
DNA sequence
electroporation
gene editing
gene expression
gene function
gene mutation
gene targeting
genetic transfection
haploidy
in vitro study
nonhuman
Oryzias latipes
polyacrylamide gel electrophoresis
polymerase chain reaction
protein synthesis
Issue Date: 2018
Citation: Liu, Q, Yuan, Y, Zhu, F, Hong, Y, Ge, R (2018). Efficient genome editing using CRISPR/Cas9 ribonucleoprotein approach in cultured Medaka fish cells. Biology Open 7 (8) : bio035170. ScholarBank@NUS Repository. https://doi.org/10.1242/bio.035170
Abstract: Gene 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. © 2018. Published by The Company of Biologists Ltd
Source Title: Biology Open
URI: https://scholarbank.nus.edu.sg/handle/10635/175074
ISSN: 20466390
DOI: 10.1242/bio.035170
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