【摘要】 【目的】检测质粒CRISPR/Cas9系统在马口鱼精原干细胞（ObSSCs）和斑马鱼胚胎中的基因编辑效果，为开展基因编辑的SSCs移植提供技术支撑，进而推动养殖鱼类基因编辑育种工作的快速发展。【方法】将靶向红色荧光蛋白（RFP）的gRNA整合至可用于体内外基因编辑的整合质粒pCas9-zU6sgRNA（带有支架序列的向导RNA，由来自斑马鱼的U6启动子驱动）和报告质粒pCVpf-gRNA（向导RNA）中，然后分别转染ObSSCs和显微注射斑马鱼胚胎，通过荧光显微镜观察和PCR检测质粒CRISPR/Cas9系统在ObSSCs及斑马鱼胚胎中的基因编辑效果。【结果】以整合质粒pCas9-zU6sgRNA与报告质粒pCVpf-gRNA共转染正常ObSSCs及pCVpr质粒转染ObSSCs，在ObSSCs中能观察到绿色荧光，且在表达绿色荧光蛋白（GFP）的ObSSCs中观察到红色荧光信号明显减弱，而不转染质粒的ObSSCs未观察到绿色荧光信号；随着整合质粒pCas9-zU6sgRNA转染剂量由340 ng增加到410 ng，其基因编辑效率由0.10%增加到0.63%；此外，质粒CRISPR/Cas9系统在基因组中的编辑效率与在外源质粒中的编辑效率基本一致。为进一步检测质粒CRISPR/Cas9系统在体内的编辑效率，以整合质粒pCas9-zU6sgRNA与报告质粒pCVpf-gRNA共注射斑马鱼胚胎，24h后能观察到绿色荧光信号，而空白对照组和阴性对照组斑马鱼胚胎均未观察到绿色荧光信号。对ObSSCs和斑马鱼的基因编辑效果进行PCR验证，发现试验组均能检测到修复的GFP片段，而空白对照组未检测到修复的GFP片段。此外，整合质粒pCas9-zU6sgRNA在斑马鱼胚胎中的基因编辑效率显著高于ObSSCs(100%vs 0.63%)(P<0.05)。【结论】由整合质粒p Cas9-zU6sgRNA与报告质粒pCVpf-gRNA构成的质粒CRISPR/Cas9系统能在ObSSCs中直观评估基因编辑效率，且质粒CRISPR/Cas9系统在同为鲤科鱼类斑马鱼胚胎中的基因编辑效率高达100%。因此，质粒CRISPR/Cas9系统可用于马口鱼和斑马鱼的sgRNA筛选，为创制养殖鱼类新品种（系）提供新思路。更多还原

【Abstract】 【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 g RNA 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.更多还原