College of Animal Science and Technology, Yangling, 712100, China.
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, China.
BMC Genomics. 2018 May 25;19(1):397. doi: 10.1186/s12864-018-4712-z.
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.
CRISPR/Cas9 系统的简单性使其在生成动物模型、功能基因组筛选和治疗遗传和传染病方面得到了广泛的应用。然而，脱靶 CRISPR/Cas9 核酸酶活性产生的非预期突变可能会导致负面后果。特别是，最近的一项研究发现，基因编辑可以将数百个非预期突变引入基因组，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特征描述。在这里，我们利用之前生成的基因编辑绵羊进行了基于家族三人组的全基因组测序，该方法能够区分编辑后代中遗传、自然产生或遗传修饰诱导的变体。对三个家族三人组进行了平均深度约为 25.8×的高基因组覆盖重测序。在开发了一种全面分析来自基因组的新单核苷酸变异、插入缺失和结构变异的序列数据的流水线后；我们仅在 MSTN 基因座的两个 sgRNA 靶向位点之间的特异性双链断裂诱导下发现了一个 2.4 kb 的倒位，发生率较低，这是一种非预期事件。
我们首次报道了同时针对三个编码序列位置的基因断裂的绵羊基因组的基于 CRISPR 的修饰的保真度。基于三人组的测序方法揭示了几乎可以忽略不计的脱靶修饰，为 CRISPR/Cas9 体内基因组编辑的安全应用提供了及时的证据。
