美国斯坦福大学Hiromitsu Nakauchi等研究人员合作发现，使用Cas9和AAV修复模板的基因组工程会产生频繁的病毒载体串联插入。相关论文于2024年4月8日在线发表在《自然—生物技术》杂志上。

据研究人员介绍，CRISPR-Cas9与腺相关病毒血清型6（AAV6）配对是产生靶向基因敲除的最有效工具之一。

研究人员报告了这一系统可能导致病毒载体基因组，在目标位点频繁出现难以检测的串联插入。这种错误会导致不良和不可靠的表型，与精准基因组工程的目标背道而驰。无论基因座、载体浓度、细胞系或细胞类型，包括人类多能干细胞和造血干细胞，都会出现串联基因敲入。虽然在一半以上的编辑细胞中发现了这些高含量的错误，但普通的分析方法无法轻易检测到它们。

研究人员介绍了检测和彻底鉴定串联病毒载体插入的策略，并强调了掩盖其普遍性的分析陷阱。然后，研究人员介绍了通过在转导后切割载体基因组，来防止串联插入的策略。这种方法与现有的基因编辑管线兼容，可实现更安全、更可靠、更可重复的稳健基因敲除。

附：英文原文

Title: Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors

Author: Suchy, Fabian P., Karigane, Daiki, Nakauchi, Yusuke, Higuchi, Maimi, Zhang, Jinyu, Pekrun, Katja, Hsu, Ian, Fan, Amy C., Nishimura, Toshinobu, Charlesworth, Carsten T., Bhadury, Joydeep, Nishimura, Toshiya, Wilkinson, Adam C., Kay, Mark A., Majeti, Ravindra, Nakauchi, Hiromitsu

Issue&Volume: 2024-04-08

Abstract: CRISPR–Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering. The concatemeric knockins occurred regardless of locus, vector concentration, cell line or cell type, including human pluripotent and hematopoietic stem cells. Although these highly abundant errors were found in more than half of the edited cells, they could not be readily detected by common analytical methods. We describe strategies to detect and thoroughly characterize the concatemeric viral vector insertions, and we highlight analytical pitfalls that mask their prevalence. We then describe strategies to prevent the concatemeric inserts by cutting the vector genome after transduction. This approach is compatible with established gene editing pipelines, enabling robust genetic knockins that are safer, more reliable and more reproducible.

DOI: 10.1038/s41587-024-02171-w

Source: https://www.nature.com/articles/s41587-024-02171-w