Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep.
Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep.
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通过 CRISPR/Cas9 进行多重基因编辑，在绵羊中表现出理想的肌肉肥大，且没有可检测到的脱靶效应
DOI：
10.1038/srep32271
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发表时间：
2016-08-26
影响因子：
4.6
通讯作者：
Chen Y
中科院分区：
文献类型：
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作者：
Wang X;Niu Y;Zhou J;Yu H;Kou Q;Lei A;Zhao X;Yan H;Cai B;Shen Q;Zhou S;Zhu H;Zhou G;Niu W;Hua J;Jiang Y;Huang X;Ma B;Chen Y
CRISPR/Cas9系统为基因位点的基因组工程提供了一种灵活的方法。在这里，我们通过将Cas9 mRNA和针对三个基因（MSTN、ASIP和BCO2）的指导RNA共同注射到单细胞阶段胚胎中，成功地在绵羊中实现了精确的基因靶向。我们通过克隆和测序仔细检查了 sgRNA:Cas9 介导的注射胚胎、体细胞组织以及性腺的靶向效应。在生成的羔羊中，这三个基因的靶向效率在 27-33% 范围内，同时靶向这三个基因的效率为 5.6%，这表明受精卵显微注射是生成基因修饰羊的有效方法。有趣的是，我们观察到 MSTN 基因的破坏导致了所需的肌肉肥大，其特征是肌纤维增大，从而提供了第一个详细的证据，支持基因修饰在遗传和形态水平上发生。此外，在显微注射前对成纤维细胞进行了 sgRNA 脱靶效应的预筛选，以确保不存在可检测到的来自创始动物的脱靶突变。我们的研究结果表明，CRISPR/Cas9 方法可以通过同时靶向负责经济重要性状的多个基因，作为牲畜改良的强大工具。
The CRISPR/Cas9 system provides a flexible approach for genome engineering of genetic loci. Here, we successfully achieved precise gene targeting in sheep by co-injecting one-cell-stage embryos with Cas9 mRNA and RNA guides targeting three genes (MSTN,ASIPandBCO2). We carefully examined the sgRNAs:Cas9-mediated targeting effects in injected embryos, somatic tissues, as well as gonads via cloning and sequencing. The targeting efficiencies in these three genes were within the range of 27–33% in generated lambs and that of simultaneously targeting the three genes was 5.6%, which demonstrated that micro-injection of zygotes is an efficient approach for generating gene-modified sheep. Interestingly, we observed that disruption of theMSTNgene resulted in the desired muscle hypertrophy that is characterized by enlarged myofibers, thereby providing the first detailed evidence supporting that gene modifications had occurred at both the genetic and morphological levels. In addition, prescreening for the off-target effect of sgRNAs was performed on fibroblasts before microinjection, to ensure that no detectable off-target mutations from founder animals existed. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by simultaneously targeting multiple genes that are responsible for economically significant traits.