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Fanconi Anemia Gene Editing by the CRISPR/Cas9 System

机译：通过CRISPR / Cas9系统编辑Fanconi贫血基因

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Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder.
机译：具有设计者核酸酶的基因组工程是一个快速发展的领域，非常需要能够原位校正人类基因突变的能力。我们采用了范可尼贫血患者的成纤维细胞作为模型，以测试成簇的规则间隔的短回文重复序列/ Cas9核酸酶系统介导基因校正的能力。我们显示，Cas9核酸酶和切口酶均导致基因校正，但是切口酶由于其优先介导同源性导向修复的能力，导致校正克隆分离物的频率更高。为了评估脱靶效应，我们使用了预测软件平台来鉴定同源性的基因内序列，以及使用线性扩增介导的PCR进行全基因组筛选。我们没有观察到脱靶活性，并显示了不具有低序列复杂性功能的RNA指导的核酸内切酶候选位点。总的来说，我们提供了Fanconi贫血（一种DNA修复缺陷型人类疾病）中精确基因组编辑的原理证明。

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