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Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep

机译：在Cas9介导的多重编辑绵羊三基因组中SNV和插入缺失的发生率低

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The simplicity of the CRISPR/Cas9 system has enabled its widespread applications in generating animal models, functional genomic screening and in treating genetic and infectious diseases. However, unintended mutations produced by off-target CRISPR/Cas9 nuclease activity may lead to negative consequences. Especially, a very recent study found that gene editing can introduce hundreds of unintended mutations into the genome, and have attracted wide attention. To address the off-target concerns, urgent characterization of the CRISPR/Cas9-mediated off-target mutagenesis is highly anticipated. Here we took advantage of our previously generated gene-edited sheep and performed family trio-based whole genome sequencing which is capable of discriminating variants in the edited progenies that are inherited, naturally generated, or induced by genetic modification. Three family trios were re-sequenced at a high average depth of genomic coverage (~?25.8×). After developing a pipeline to comprehensively analyze the sequence data for de novo single nucleotide variants, indels and structural variations from the genome; we only found a single unintended event in the form of a 2.4?kb inversion induced by site-specific double-strand breaks between two sgRNA targeting sites at the MSTN locus with a low incidence. We provide the first report on the fidelity of CRISPR-based modification for sheep genomes targeted simultaneously for gene breaks at three coding sequence locations. The trio-based sequencing approach revealed almost negligible off-target modifications, providing timely evidences of the safe application of genome editing in vivo with CRISPR/Cas9.
机译：CRISPR / Cas9系统的简单性使其能够广泛应用于生成动物模型，功能基因组筛选以及治疗遗传和传染病。然而，脱靶的CRISPR / Cas9核酸酶活性产生的意外突变可能导致负面后果。特别是，最近的一项研究发现，基因编辑可以将数百个意料之外的突变引入基因组，并引起了广泛关注。为了解决脱靶问题，迫切需要对CRISPR / Cas9介导的脱靶诱变进行紧急表征。在这里，我们利用了先前生成的基因编辑的绵羊，并进行了基于家族三重基因的全基因组测序，该序列能够区分编辑后代中遗传，自然生成或通过遗传修饰诱导的变体。三个家庭三重奏在基因组覆盖率较高的平均深度（〜25.8倍）处重新排序。在开发出一条管道以全面分析从基因组中从头开始的单核苷酸变异，插入缺失和结构变异的序列数据之后;我们只发现了一个意外事件，它是由MSTN基因座上两个sgRNA靶向位点之间的位点特异性双链断裂引起的，以2.4？kb倒置的形式发生的，发生率低。我们提供有关基于CRISPR修饰的绵羊基因组保真度的第一份报告，该基因组同时针对三个编码序列位置处的基因断裂。基于三重序列的测序方法揭示了几乎可以忽略的脱靶修饰，为使用CRISPR / Cas9进行体内基因组编辑的安全应用提供了及时的证据。

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