Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities.
Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities.
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DOI：
10.1016/j.omtn.2022.07.026
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发表时间：
2022-09-13
期刊：
影响因子：
--
通讯作者：
Lin, Chao-Po
中科院分区：
文献类型：
--
作者：
Zhang, Zhenwu;Tao, Wanyu;Huang, Shisheng;Sun, Wenjun;Wang, Yue;Jiang, Wen;Huang, Xingxu;Lin, Chao-Po
使用CRISPR技术对多能干细胞（PSCs）进行基因组编辑在治疗应用方面具有巨大潜力。然而，据报道，Cas9介导的切割可能导致DNA的大片段缺失或重排，并且经过编辑的PSCs的筛选可能会获得p53突变。腺嘌呤碱基编辑器（ABEs）不会引入DNA双链断裂，因此被提议作为规避这些问题的替代方法，但其脱靶效应仍然限制了它们的应用。在此，我们测试了不同的脱靶减少方法组合，以在不影响其靶向编辑效率的情况下进一步降低ABEs的脱靶效应。我们随后选择了最佳编辑器CE - 8e - dV，它包含V106W替换、R153缺失和Cas嵌入策略，以建立一个表达四环素诱导型CE - 8e - dV的单细胞来源的人类胚胎干细胞（hESC）系。通过进行RNA和全基因组测序，我们证明了CE - 8e - dV的表达在hESCs中几乎没有产生任何DNA或RNA脱靶效应。我们的结果为ABEs在PSCs中的安全性提供了严格的证据，并表明CE - 8e - dV可能适用于相关的治疗策略，例如体外工程干细胞的产生和体内基因治疗。
CE - 8e - dV是一种包含V106W替换、R153缺失和Cas嵌入策略的腺嘌呤碱基编辑器（ABE），它在人类胚胎干细胞（hESCs）中被证明具有强大的碱基编辑效率，同时几乎不产生任何DNA或RNA脱靶效应，使其适用于体外和体内的相关治疗策略。
Genome editing in pluripotent stem cells (PSCs) using CRISPR technology holds great promise for therapeutic applications. Yet, it has been reported that Cas9-mediated cleavage could cause large deletions or rearrangements of DNA, and the selection of edited PSCs could acquire p53 mutations. Adenine base editors (ABEs) do not introduce DNA double-strand breaks and thus have been proposed as alternatives to circumvent those problems, but their off-target effects still limit their applications. Here, we tested different combinations of off-target reduction methods to further diminish off-target effects of ABEs without compromising their on-target editing efficiencies. We subsequently chose the best editor, CE-8e-dV, which contains V106W substitution, R153 deletion, and Cas-embedding strategy, to establish a single-cell-derived human embryonic stem cell (hESC) line expressing tetracycline-inducible CE-8e-dV. By performing RNA and whole-genome sequencing, we demonstrated that the expression of CE-8e-dV did not produce nearly any DNA or RNA off-target effects in hESCs. Our results provide stringent proof of the safety of ABEs in PSCs and suggest that CE-8e-dV could be suitable for related therapeutic strategies, such as generation of engineered stem cells in vitro and gene therapy in vivo. CE-8e-dV, an adenine base editor (ABE) containing V106W substitution, R153 deletion, and Cas-embedding strategy, was shown to possess robust base-editing efficiency while not producing almost any DNA or RNA off-target effects in human embryonic stem cells (hESCs), making it suitable for related therapeutic strategies in vitro and in vivo.
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