Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects.
Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects.
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DOI：
10.1186/s13059-016-1144-4
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发表时间：
2017-02-01
期刊：
影响因子：
12.3
通讯作者：
Zhang Y
中科院分区：
文献类型：
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作者：
Gao Y;Wu H;Wang Y;Liu X;Chen L;Li Q;Cui C;Liu X;Zhang J;Zhang Y
CRISPR-CAS9系统是在各种物种中广泛使用的转基因动物生产平台，尽管它的非靶标效应应该得到解决。该工具在模型动物中的几个应用已被提出，但仍不足以用于转基因牲畜的生产。在这里，我们报道了首次应用单个Cas9镍酶(Cas9n)来诱导牛的基因插入到选定的基因座上。我们用染色质免疫沉淀测序技术(CHIP-SEQ)鉴定了牛胎儿成纤维细胞(BFF)中催化失活的Cas9(DCas9)蛋白的主要结合部位。随后，我们证明了单一的Cas9n诱导的单链断裂可以刺激自然耐药相关巨噬细胞蛋白-1(NRAMP1)基因的插入，减少了但仍然相当大的脱靶效应。通过体细胞核移植，我们最终获得了对结核病抗性增强的转基因牛。我们的研究成果有助于CRISPR-CAS9农业应用系统的开发。本文的在线版本(doi：10.1186/s13059-0161144-4)包含补充材料，授权用户可以使用。
The CRISPR-Cas9 system is a widely utilized platform for transgenic animal production in various species, although its off-target effects should be addressed. Several applications of this tool have been proposed in model animals but remain insufficient for transgenic livestock production. Here, we report the first application of single Cas9 nickase (Cas9n) to induce gene insertion at a selected locus in cattle. We identify the main binding sites of a catalytically inactive Cas9 (dCas9) protein in bovine fetal fibroblast cells (BFFs) with chromatin immunoprecipitation sequencing (ChIP-seq). Subsequently, we demonstrate that a single Cas9n-induced single-strand break can stimulate the insertion of the natural resistance-associated macrophage protein-1 (NRAMP1) gene with reduced, but still considerable, off-target effects. Through somatic cell nuclear transfer, we finally obtain transgenic cattle with increased resistance to tuberculosis. Our results contribute to the development of CRISPR-Cas9 system for agriculture applications. The online version of this article (doi:10.1186/s13059-016-1144-4) contains supplementary material, which is available to authorized users.