兔成纤维细胞 Tet-On 3G 系统诱导 SpCas9 的表达

Expression of SpCas9 induced by Tet-On 3G system in rabbit fibroblasts cells

摘要

CRISPR/Cas9系统中SpCas9的持续表达易导致脱靶效应与细胞毒性,这一问题限制了其在体内基因编辑中的应用。为克服该局限,成功在兔成纤维细胞中建立了Tet-On 3G诱导型CRISPR/Cas9基因编辑系统。通过设计靶向兔H11安全港基因座的特异性sgRNA,并利用CRISPR/Cas9介导的同源重组修复,将Tre3G-SpCas9-P2A-EGFP-pA表达框精确整合至H11位点。结合PiggyBac转座子实现rtTA稳定表达,经多西环素诱导后,可有效启动SpCas9及EGFP报告基因表达,并显著降低背景泄露。研究结果在兔成纤维细胞中验证了Tet-On 3G系统的可行性,为构建可诱导基因编辑的兔模型提供技术基础。

Abstract

The continuous expression of SpCas9 in the CRISPR/Cas9 system is prone to cause off-target effects and cytotoxicity, thereby limiting its applicability in vivo. To address this limitation, this study successfully established a Tet-On 3G inducible CRISPR/Cas9 gene editing system in rabbit fibroblasts. A specific sgRNA targeting the rabbit H11 safe harbor locus was designed, and the Tre3G-SpCas9-P2A-EGFP-pA expression cassette was precisely integrated into the H11 site via CRISPR/Cas9-mediated homologous recombination repair. Combined with the PiggyBac for stable rtTA expression, doxycycline induction effectively activated SpCas9 and EGFP reporter gene expression, significantly reducing background leakage. This study validated the feasibility of the Tet-On 3G system in rabbit fibroblasts, providing a technical foundation for constructing inducible gene-edited rabbit models.

关键词

Tet-On 3G / CRISPR/Cas9 / H11 / 转基因 / 兔成纤维细胞

Key words

Tet-On 3G / CRISPR/Cas9 / H11 / transgenic / rabbit fibroblast cells

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基金资助

国家自然科学基金项目(32070540)