兔成纤维细胞 Tet-On 3G 系统诱导 SpCas9 的表达

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。

关键词

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

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基金资助

国家自然科学基金项目(32070540)