Homology-mediated end joining-based targeted integration using CRISPR/Cas9

doi:10.1038/cr.2017.76

	Homology-mediated end joining-based targeted integration using CRISPR/Cas9
	Yao, Xuan 1,2; Wang, Xing 1,2; Hu, Xinde 1; Liu, Zhen 1; Liu, Junlai2,3,4 ; Zhou, Haibo 1; Shen, Xiaowen 1; Wei, Yu 1,5; Huang, Zijian 1,2; Ying, Wenqin 1; Wang, Yan 1; Nie, Yan-Hong 1; Zhang, Chen-Chen 1; Li, Sanlan 1; Cheng, Leping 1; Wang, Qifang 1; Wu, Yan 6; Huang, Pengyu3 ; Sun, Qiang 1; Shi, Linyu 1; Yang, Hui 1
	2017-06
发表期刊	CELL RESEARCH
ISSN	1001-0602
卷号	27 期号:6 页码:801-814
发表状态	已发表
DOI	10.1038/cr.2017.76
摘要	Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-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 similar to 800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knockin 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.
关键词	homology-mediated end joining CRISPR/Cas9 monkey embryos neurons knock-in
收录类别	SCI ; CSCD
语种	英语
资助项目	Shanghai City Committee of science and technology project[16JC1420202] ; Shanghai City Committee of science and technology project[14140900100]
WOS研究方向	Cell Biology
WOS类目	Cell Biology
WOS记录号	WOS:000402545200010
CSCD记录号	CSCD:6016604
出版者	NATURE PUBLISHING GROUP
WOS关键词	KNOCK-IN ; CYNOMOLGUS MONKEY ; GENE ; DNA ; GENERATION ; ZEBRAFISH ; CELLS ; CRISPR-CAS9 ; CLEAVAGE ; TALENS
原始文献类型	Article
引用统计
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/1348
专题	生命科学与技术学院生命科学与技术学院_PI研究组_黄鹏羽组生命科学与技术学院_博士生
通讯作者	Sun, Qiang; Shi, Linyu; Yang, Hui
作者单位	1.Chinese Acad Sci, Shanghai Inst Biol Sci, CAS Ctr Excellence Brain Sci & Intelligence Techn, Key Lab Primate Neurobiol,Inst Neurosci,State Key, Shanghai 200031, Peoples R China 2.Univ Chinese Acad Sci, Coll Life Sci, Beijing 100049, Peoples R China 3.ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai 201210, Peoples R China 4.Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Biochem & Cell Biol, Shanghai 200031, Peoples R China 5.Shanghai Univ, Shanghai 200444, Peoples R China 6.Natl Inst Biol Sci, Beijing 102206, Peoples R China
推荐引用方式 GB/T 7714	Yao, Xuan,Wang, Xing,Hu, Xinde,et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9[J]. CELL RESEARCH,2017,27(6):801-814.
APA	Yao, Xuan.,Wang, Xing.,Hu, Xinde.,Liu, Zhen.,Liu, Junlai.,...&Yang, Hui.(2017).Homology-mediated end joining-based targeted integration using CRISPR/Cas9.CELL RESEARCH,27(6),801-814.
MLA	Yao, Xuan,et al."Homology-mediated end joining-based targeted integration using CRISPR/Cas9".CELL RESEARCH 27.6(2017):801-814.