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利用CRISPR/Cas9系统对海胆胚胎进行基因组编辑。

Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.

作者信息

Lin Che-Yi, Su Yi-Hsien

机构信息

Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan.

Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan.

出版信息

Dev Biol. 2016 Jan 15;409(2):420-8. doi: 10.1016/j.ydbio.2015.11.018. Epub 2015 Nov 26.

Abstract

Sea urchin embryos are a useful model system for investigating early developmental processes and the underlying gene regulatory networks. Most functional studies using sea urchin embryos rely on antisense morpholino oligonucleotides to knockdown gene functions. However, major concerns related to this technique include off-target effects, variations in morpholino efficiency, and potential morpholino toxicity; furthermore, such problems are difficult to discern. Recent advances in genome editing technologies have introduced the prospect of not only generating sequence-specific knockouts, but also providing genome-engineering applications. Two genome editing tools, zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs), have been utilized in sea urchin embryos, but the resulting efficiencies are far from satisfactory. The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system serves as an easy and efficient method with which to edit the genomes of several established and emerging model organisms in the field of developmental biology. Here, we apply the CRISPR/Cas9 system to the sea urchin embryo. We designed six guide RNAs (gRNAs) against the well-studied nodal gene and discovered that five of the gRNAs induced the expected phenotype in 60-80% of the injected embryos. In addition, we developed a simple method for isolating genomic DNA from individual embryos, enabling phenotype to be precisely linked to genotype, and revealed that the mutation rates were 67-100% among the sequenced clones. Of the two potential off-target sites we examined, no off-target effects were observed. The detailed procedures described herein promise to accelerate the usage of CRISPR/Cas9 system for genome editing in sea urchin embryos.

摘要

海胆胚胎是研究早期发育过程及潜在基因调控网络的有用模型系统。大多数利用海胆胚胎进行的功能研究依赖反义吗啉代寡核苷酸来敲低基因功能。然而,与该技术相关的主要问题包括脱靶效应、吗啉代效率的差异以及潜在的吗啉代毒性;此外,这些问题难以辨别。基因组编辑技术的最新进展不仅带来了产生序列特异性敲除的前景,还提供了基因组工程应用。两种基因组编辑工具,锌指核酸酶(ZFN)和转录激活样效应核酸酶(TALENs),已被用于海胆胚胎,但所获效率远不尽人意。CRISPR(成簇规律间隔短回文重复序列)-Cas9(CRISPR相关核酸酶9)系统是一种简便高效的方法,可用于编辑发育生物学领域中几种已确立和新兴的模式生物的基因组。在此,我们将CRISPR/Cas9系统应用于海胆胚胎。我们针对研究充分的节点基因设计了六种引导RNA(gRNA),发现其中五种gRNA在60%-80%的注射胚胎中诱导出预期表型。此外,我们开发了一种从单个胚胎中分离基因组DNA的简单方法,使表型能够精确地与基因型相关联,并揭示测序克隆中的突变率为67%-100%。在我们检测的两个潜在脱靶位点中,未观察到脱靶效应。本文所述的详细程序有望加速CRISPR/Cas9系统在海胆胚胎基因组编辑中的应用。

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