Efficient In Vivo Genome Editing Using RNA-Guided Nucleases
Efficient In Vivo Genome Editing Using RNA-Guided Nucleases
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发表时间：
2013
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通讯作者：
Woong Y. Hwang;Yanfang Fu;D. Reyon;Morgan L. Maeder;S. Tsai;Jeffry D. Sander;R. Peterson;J.-R. Joanna Yeh-J.
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作者：
Woong Y. Hwang;Yanfang Fu;D. Reyon;Morgan L. Maeder;S. Tsai;Jeffry D. Sander;R. Peterson;J.-R. Joanna Yeh-J.
规则间隔短回文重复序列（CRISPR）/CRISPR相关（Cas）系统已经在细菌和古细菌中进化为沉默病毒和质粒的外源核酸的防御机制。最近的工作表明，细菌II型CRISPR系统可以适应于产生能够在体外指导Cas9核酸酶的位点特异性DNA切割的指导RNA（gRNA）。在这里，我们表明，该系统可以在体内诱导斑马鱼胚胎中的靶向遗传修饰，其效率与使用ZFN和TALENs获得的相同基因的效率相当。RNA引导的核酸酶在测试的11个不同位点中的9个位点上稳健地实现了基因组编辑，其中包括TALEN先前未能诱导改变的两个位点。这些结果表明，可编程CRISPR/Cas系统提供了一种简单、快速和高度可扩展的方法来改变体内基因，为在广泛的生物体中使用RNA引导的核酸酶进行基因组编辑打开了大门。细菌和古细菌已经进化出一种优雅的适应性防御机制，该机制使用成簇的规则间隔短回文重复序列（CRISPR）以及CRISPR相关（Cas）蛋白，以提供对入侵病毒和质粒的获得性抗性1 -3。II型CRISPR/Cas系统依赖于摄取外来DNA片段用户可以查看，打印，复制，下载和文本和数据挖掘这些文件中的内容，用于学术研究的目的，始终受到完整的用途：http://www.nature.com/authors/editorial_policies/license.html#terms通信：jjoung@partners.org或jyeh1@partners. org. †这些作者对这项工作同样做出了贡献利益冲突声明J. K. J.在Transposagen Biopharmaceuticals中拥有经济利益。J.K.J.'的利益由马萨诸塞州总医院和Partners HealthCare根据其利益冲突政策进行审查和管理。HHS公共访问作者手稿Nat Biotechnol。作者手稿;可在PMC 2013年9月1日获得。以最终编辑形式发表：Nat Biotechnol。2013年3月; 31（3）：227-229。doi：10.1038/nbt.2501。A uhor M anscript
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNAguided nucleases for genome editing in a wide range of organisms. Bacteria and archaea have evolved an elegant adaptive defense mechanism which uses clustered regularly interspaced short palindromic repeats (CRISPR), together with CRISPRassociated (Cas) proteins, to provide acquired resistance to invading viruses and plasmids1–3. The type II CRISPR/Cas system relies on uptake of foreign DNA fragments Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence to: jjoung@partners.org or jyeh1@partners.org. †These authors contributed equally to this work Conflict of Interest Statement J.K.J. has a financial interest in Transposagen Biopharmaceuticals. J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. HHS Public Access Author manuscript Nat Biotechnol. Author manuscript; available in PMC 2013 September 01. Published in final edited form as: Nat Biotechnol. 2013 March ; 31(3): 227–229. doi:10.1038/nbt.2501. A uhor M anscript