Applications of plasmid CRISPR/Cas9 system in gene editing of Opsariichthys bidens spermatogonial stem cell line

【Objective】To test the gene editing effects of a plasmid CRISPR/Cas9 gene editing system in Opsariichthys bidens spermatogonial stem cells（ObSSCs）and zebrafish embryos，which could provide technical support for conducting the transplantation of gene-edited SSCs and in turn，promote the rapid development of gene-edited breeding technology in farmed fish.【Method】The gRNA targeting the red fluorescence protein fusion gene（RFP）was incorporated into the integrated plasmid pCas9-zU6sgRNA（a guide RNA with scaffold sequence，driven by the U6 promoter from zebrafish）and the reporter plasmid pCVpf-gRNA（guide RNA）for in vitro and in vivo gene editing，followed by transfection of ObSSCs and microinjection of zebrafish embryos. The gene editing effects of the plasmid CRISPR/Cas9 system in ObSSCs and zebrafish embryos were detected by fluorescence microscopy and PCR.【Result】The integrated plasmid pCas9-zU6sgRNA and the reporter plasmid pCVpf-gRNA were co-transfected into ObSSCs and ObSSCs：pCVpr. Green fluorescence signals were observed in ObSSCs. A clear decrease in the red fluorescence signal was observed in cells expressing green fluorescence protein（GFP）. No green fluorescence signal was observed in the non-transfected ObSSCs. As the dose of integrated plasmid pCas9-zU6sgRNA increased from 340 ng to 410 ng，the gene editing efficiency improved from 0.10% to 0.63%. The editing efficiency of the plasmid CRISPR/Cas9 system in genomes and exogenous plasmids was similar. To further test the editing efficiency of the plasmid CRISPR/Cas9 system in vivo，the integrated plasmid pCas9-zU6sgRNA and the reporter plasmid pCVpf-gRNA were co-injected into zebrafish embryos. Green fluorescence signals were observed after 24 h，while no green fluorescence signal was observed in the blank and negative control groups of zebrafish embryos. The gene editing effect in ObSSCs and zebrafish was detected by PCR. GFP fragments repaired were detected in all experimental groups but not in the blank control group. Compared to ObSSCs，the gene editing efficiency of the integrated plasmid pCas9-zU6sgRNA in zebrafish was significantly higher（100% vs 0.63%）（P<0.05）. 【Conclusion】The gene editing efficiency of the plasmid CRISPR/Cas9 system，consisting of the integrating plasmid pCas9-zU6sgRNA and the reporter plasmid pCVpf-gRNA，can be visually assessed in ObSSCs，which is up to 100% in zebrafish embryos of the same Cyprinidae family of fish. Consequently，the plasmid CRISPR/Cas9 system can be used to screen sgRNAs in Opsariichthys bidens and zebrafish，and provide a new way for the creation of new varieties of farmed fish.