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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 |
Appears in Collections: | Elements Staff Publications |
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