CRISPR/Cas9系统在通过基因疗法治疗人类遗传疾病方面显示出巨大的潜力。然而，人们对该系统的安全性存在担忧，特别是与无引导 Cas9 的使用有关。先前的研究表明，无引导的Cas9可以在体外诱导基因组不稳定。然而，与无引导Cas9相关的体内安全风险尚未得到评估，这对于临床环境中基因治疗的发展是必要的。在本研究中，我们使用多西环素诱导的 Cas9 表达猪来评估无引导 Cas9 的体内安全风险。我们的研究结果表明，无引导Cas9的表达可以在体内诱导基因组损伤和转录组变化。基因组损伤和转录组变化的严重程度与Cas9蛋白的表达水平相关。此外，Cas9在猪体内的长时间表达导致异常表型，包括体重显着下降，这可能归因于基因组损伤引起的营养吸收和代谢功能障碍。此外，我们观察到长期表达Cas9的猪的全基因组和肿瘤驱动基因突变增加，增加了肿瘤发生的风险。我们对猪体内无引导 Cas9 的体内评估强调了在临床基因治疗中实施通过 CRISPR/Cas9 系统进行基因组编辑时考虑和监测单独使用 Cas9 的有害影响的必要性。这项研究强调了进一步研究和实施安全措施的重要性，以确保 CRISPR/Cas9 系统在临床实践中成功和安全的应用。© 2024。作者。

The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy. However, there are concerns about the safety of this system, specifically related to the use of guide-free Cas9. Previous studies have shown that guide-free Cas9 can induce genomic instability in vitro. However, the in vivo safety risks associated with guide-free Cas9 have not been evaluated, which is necessary for the development of gene therapy in clinical settings. In this study, we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo. Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo. The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein. Moreover, prolonged expression of Cas9 in pigs led to abnormal phenotypes, including a significant decrease in body weight, which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction. Furthermore, we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression, raising the risk of tumor occurrence. Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy. This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRISPR/Cas9 system in clinical practice.© 2024. The Author(s).