Preparation of NCAPG Knockout Bovine Fibroblast Cell Lines Using CRISPR/Cas9 Technology

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

Abstract

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

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.

Figures/Tables 6

Fig. 1 Electroporation transfection of fibroblast (400×)

Fig. 2 ClonePlusTM technology for monoclonal culture and screening

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

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

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

Fig. 6 Results of CCK-8 assay

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