Homology-mediated end joining-based targeted integration using CRISPR/Cas9
Targeted integration of transgenes can be achieved by strategies based on homologous recombination （HR）, mi- crohomology-mediated end joining （MMEJ） or non-homologous end joining （NHEJ）. The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because i...
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| Published in | Cell research Vol. 27; no. 6; pp. 801 - 814 |
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| Main Authors | , , , , , , , , , , , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
London
Nature Publishing Group UK
01.06.2017
Nature Publishing Group |
| Subjects | |
| Online Access | Get full text |
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| Summary: | Targeted integration of transgenes can be achieved by strategies based on homologous recombination （HR）, mi- crohomology-mediated end joining （MMEJ） or non-homologous end joining （NHEJ）. The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining （HMEJ）-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and -800 bp of homology arms, and the targeted genome. We found no significant improve- ment of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblas- toma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock- in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies. |
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| Bibliography: | Targeted integration of transgenes can be achieved by strategies based on homologous recombination （HR）, mi- crohomology-mediated end joining （MMEJ） or non-homologous end joining （NHEJ）. The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining （HMEJ）-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and -800 bp of homology arms, and the targeted genome. We found no significant improve- ment of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblas- toma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock- in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies. Xuan Yaol＇2＇, Xing Wang , Xinde Hu1 , Zhen Liu, Junlai Liu2＇3＇ 4, Haibo Zhou, Xiaowen Shen, Yu Wei~＇5, Zijian Huang1＇2, Wenqin Ying, Yan Wang, Yan-Hong Nie, Chen-Chen Zhang, Sanlan Li, Leping Cheng, Qifang Wang1, Yan Wu6, Pengyu Huang3, Qiang Sun1, Linyu Shi1, Hui Yang~（1Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Ex- cellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China,＂ e College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China; 3School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; 41nstitute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; 5Shanghai University, Shang- hai 200444, China; 6National _Institute of Biological Sciences, Beijing 102206, China） 31-1568 homology-mediated end joining; CRISPR/Cas9; monkey embryos; neurons; knock-in ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 These four authors contributed equally to this work. |
| ISSN: | 1001-0602 1748-7838 1748-7838 |
| DOI: | 10.1038/cr.2017.76 |