CRISPR–Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1–6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe ‘verification of in vivo off-targets’ (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR–Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR–Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.A strategy developed to define off-target effects of gene-editing nucleases in whole organisms is validated and leveraged to show that CRISPR–Cas9 nucleases can be used effectively in vivo without inducing detectable off-target mutations.

中文翻译：

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没有可检测的全基因组脱靶突变的体内 CRISPR 编辑

CRISPR-Cas 基因组编辑核酸酶在开发人类治疗应用方面具有重大前景 1-6，但识别不需要的脱靶突变对于临床转化很重要 7。迄今为止，尚未描述一种能够可靠地识别体内脱靶的经过充分验证的方法，这意味着目前尚不清楚这些突变是否发生以及发生的频率。在这里，我们描述了“体内脱靶验证”（VIVO），这是一种高度敏感的策略，可以在体内可靠地识别 CRISPR-Cas 核酸酶的全基因组脱靶效应。我们使用 VIVO 和故意设计为混杂的引导 RNA 来表明 CRISPR-Cas 核酸酶可以在体内诱导小鼠肝脏中的大量脱靶突变。更重要的是，我们还使用 VIVO 来证明，适当设计的引导 RNA 可以在小鼠肝脏中指导有效的体内编辑，而没有可检测到的脱靶突变。VIVO 提供了定义和量化基因编辑核酸酶在整个生物体中的脱靶效应的通用策略，从而提供了一个蓝图，以促进使用体内基因编辑的治疗策略的开发。开发用于定义脱靶效应的策略对整个生物体中基因编辑核酸酶的研究进行了验证和利用，以表明 CRISPR-Cas9 核酸酶可以在体内有效使用，而不会诱导可检测的脱靶突变。