基于CRISPR/Cas9技术制备NCAPG基因敲除牛成纤维细胞系

doi:10.13560/j.cnki.biotech.bull.1985.2025-0792

摘要/Abstract

摘要：

目的利用CRISPR/Cas9技术构建NCAPG基因敲除的牛胎儿成纤维细胞模型，并探究NCAPG基因缺失对细胞活性的影响。方法在牛NCAPG基因转录本第6外显子区域设计特异性sgRNA，将其与SpCas9蛋白孵育形成核糖核蛋白（RNP）复合物，通过电转染技术将RNP复合物导入牛胎儿成纤维细胞。随后采用ClonePlus™技术进行单克隆分离培养，经PCR扩增和测序验证后成功获得NCAPG基因敲除的阳性单克隆细胞株。结果 ICE软件分析表明，sgRNA的敲除效率达到49%；TA克隆测序结果证实，所获得的单克隆细胞均为杂合子，其靶位点存在5 bp的缺失突变；Western blot检测显示，与野生型细胞相比，敲除细胞株中NCAPG蛋白表达水平极显著降低（P<0.01）；CCK-8细胞活性检测结果表明，NCAPG基因敲除显著抑制了细胞增殖能力（P<0.01），表现为细胞生长速率明显减缓。结论利用CRISPR/Cas9技术成功构建了牛胎儿成纤维细胞NCAPG基因高效敲除方法，并证实NCAPG基因缺失会显著抑制细胞活性，为深入研究牛NCAPG基因的功能及其分子机制提供了重要的实验材料。

关键词: 牛胎儿成纤维细胞, NCAPG基因敲除, CRISPR/Cas9系统, 细胞活性

Abstract:

Objective This study employed CRISPR/Cas9 technology to establish a bovine fetal fibroblast cell model with knockout of the NCAPG gene and to investigate the impact of NCAPG deficiency on cell viability. Method A specific sgRNA targeting exon 6 of the bovine NCAPG transcript was designed. The sgRNA was complexed with SpCas9 protein to form a ribonucleoprotein (RNP) complex, which was subsequently introduced into bovine fetal fibroblasts with electroporation. Following transfection, single-cell clones were isolated and expanded using the ClonePlus™ technique. Putative knockout monoclonal cell lines were identified by PCR amplification and sequencing. Result ICE software analysis indicated a knockout efficiency of sgRNA reached 49%. TA cloning and sequencing confirmed that the monoclonal cells were heterozygous knockouts, carrying a 5 bp deletion at the target site. Western blot analysis revealed a highly significant reduction in NCAPG protein expression in the knockout cells compared to wild-type controls (P<0.01). CCK-8 cell viability assays revealed that NCAPG knockout significantly inhibited cell proliferation (P<0.01), as evidenced by a markedly reduced growth rate. Conclusion An efficient CRISPR/Cas9-mediated NCAPG knockout method was successfully established in bovine fetal fibroblasts, and NCAPG deficiency was found to significantly suppress cell viability. This work provides crucial experimental resources for further investigation into the function and molecular mechanisms of the bovine NCAPG gene.

Key words: bovine fetal fibroblasts, NCAPG gene knockout, CRISPR/Cas9 technology, cell activity

王婷婷, 何孟雅, 盛家顺, 高晨, 蔡含芳, 付彤, 孙宇, 高腾云, 张天留. 基于CRISPR/Cas9技术制备NCAPG基因敲除牛成纤维细胞系[J]. 生物技术通报, 2026, 42(2): 317-324.

WANG Ting-ting, HE Meng-ya, SHENG Jia-shun, GAO Chen, CAI Han-fang, FU Tong, SUN Yu, GAO Teng-yun, ZHANG Tian-liu. Preparation of NCAPG Knockout Bovine Fibroblast Cell Lines Using CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2026, 42(2): 317-324.

图/表 6

图1 电转染成纤维细胞（400×）

Fig. 1 Electroporation transfection of fibroblast (400×)

图2 ClonePlusTM技术单克隆培养与筛选

Fig. 2 ClonePlusTM technology for monoclonal culture and screening

图3 ICE软件分析sgRNA的编辑效率A：sgRNA在不同单克隆中的切割效率；B：Sanger序列视图，分别代表已编辑和野生型序列；sgRNA靶序列用黑色下划线标注；PAM序列以红色虚线标出；垂直虚线指示预测的Cas9切割位点；C：序列差异度分析，显示了编辑样本（绿色）和对照样本（橙色）的测序轨迹的不一致性；D：Indel类型统计：展示了插入和缺失（Insert/Indel）大小及其相对频率

Fig. 3 Analysis of sgRNA editing efficiency using ICE softwareA: The cutting efficiency of sgRNA in different monoclonal cell lines. B: Sanger sequence view, respectively representing the edited and wild-type sequences. The sgRNA target sequence is annotated with a black underline; the PAM sequence is marked with a red dashed line; and a vertical dashed line indicates the predicted Cas9 cleavage site. C: Trace discrepancy analysis, showing the misalignment between the sequencing traces of the edited sample (green) and the control sample (orange). D: Indel type statistics: Displays the size of insertions and deletions (Insert/Deletion) and their relative frequency

图4 敲除NCAPG基因型的鉴定A：NCAPG基因敲除靶位点PCR扩增产物琼脂凝胶电泳检测；M：DL10000 DNA marker；1：NCAPG基因敲除靶位点PCR扩增结果；WT：野生型对照组；H2O：阴性对照；B：阳性单克隆细胞峰图；C：TA克隆测序比对结果

Fig. 4 Identification of NCAPG knockout genotypesA: Agarose gel electrophoresis of PCR amplified products of NCAPG gene knockout target sites. M: DL10000 DNA marker. 1: PCR amplified results of NCAPG gene knockout target sites. WT: Wild type control. H2O: Negative control. B: Sequencing peak map of positive cell clones. C: Sequencing alignment results after TA cloning

图5 Western blotting检测NCAPG蛋白表达A：野生型和基因敲除的NCAPG蛋白条带图；B：NCAPG蛋白水平的定量分析；NCAPG+/+：野生型成纤维细胞；NCAPG+/-：基因敲除的成纤维细胞；GAPDH为内参。**P<0.01，下同

Fig. 5 Expression of NCAPG protein detected using Western blottingA: Western blot analysis of NCAPG expression in wild-type and knockout cells; B: Quantitative densitometric analysis of NCAPG protein levels. NCAPG+/+: Wild type fibroblasts. NCAPG+/-: Fibroblasts with gene knockout; GAPDH was used as a loading control. *P<0.01, the same below

图6 CCK8检测结果

Fig. 6 Results of CCK-8 assay

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