为了解决CRISPR-Cas9的局限性,包括脱靶效应和商业用途的高额许可费,Cas-CLOVER,由两个引导RNA激活的二聚体基因编辑工具,是最近开发的。这项研究的重点是通过靶向信号转导和转录激活因子1(STAT1)基因座,在用于重组腺相关病毒(rAAV)生产的HEK-293细胞中实施和评估Cas-CLOVER,这对于细胞生长调节至关重要,并可能影响rAAV的产量。Cas-CLOVER在基因编辑方面表现出令人印象深刻的效率,达到90%以上的淘汰赛(KO)成功率。对13个选定的HEK-293STAT1KO亚克隆进行广泛的分析表征,以评估其基因组稳定性。对于保持细胞的完整性和功能至关重要。此外,rAAV9生产力,Rep蛋白图谱,和效力,其中,被评估。克隆显示衣壳和载体基因组滴度的显着变化,衣壳滴度降低15%至98%,载体基因组滴度从16%至55%。有趣的是,Cas-CLOVER介导的STAT1KO大细胞群显示出更好的全衣壳与空衣壳比例。我们的研究还建立了一个全面的分析工作流程来检测和评估这种创新工具产生的基因KO,为未来精确基因编辑技术的研究提供了坚实的基础。
In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.