In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

清脆的 基因组编辑 Cas9 计算生物学 色素性视网膜炎 生物 同源定向修复 基因组工程 基因组 转基因 遗传增强 引导RNA 回文 遗传学 基因 DNA修复 DNA错配修复
作者
Keiichiro Suzuki,Yuji Tsunekawa,Reyna Hernández‐Benítez,Jun Wu,Jie Zhu,Euiseok J. Kim,Fumiyuki Hatanaka,Mako Yamamoto,Toshikazu Araoka,Zhe Li,Masakazu Kurita,Tomoaki Hishida,Mo Li,Emi Aizawa,Shicheng Guo,Song Chen,April Goebl,Rupa Devi Soligalla,Jing Qu,Yan He,Xin Fu,Maryam Jafari,Concepción Rodrı́guez Esteban,W. Travis Berggren,Jerónimo Lajara Blesa,Estrella Núñez‐Delicado,Pedro Guillén,Josep M. Campistol,Fumio Matsuzaki,Guang‐Hui Liu,Pierre J. Magistretti,Kun Zhang,Edward M. Callaway,Kang Zhang,Juan Carlos Izpisúa Belmonte
出处
期刊:Nature [Springer Nature]
卷期号:540 (7631): 144-149 被引量:977
标识
DOI:10.1038/nature20565
摘要

A method for CRISPR-based genome editing that harnesses cellular non-homologous end joining activity to achieve targeted DNA knock-in in non-dividing tissues. A current challenge in genome editing is achieving efficient targeted integration of transgenes in post-mitotic cells. These authors develop a method for CRISPR-based genome editing that harnesses the non-homologous-end-joining double-strand-break repair pathway to achieve targeted knock-in in dividing and non-dividing tissues. Although further development is needed to increase efficacy, the authors show the potential application of this method for targeted knock-in in post-mitotic neurons and other non-dividing tissues, and provide initial exploratory data on its potential application for disease correction in retinal pigment epithelium models. Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient1, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders2. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)3,4 technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.
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