Homology-mediated end joining-based targeted integration using CRISPR/Cas9
Yao, Xuan1,2; Wang, Xing1,2; Hu, Xinde1; Liu, Zhen1; Liu, Junlai2,3,4; Zhou, Haibo1; Shen, Xiaowen1; Wei, Yu1,5; Huang, Zijian1,2; Ying, Wenqin1; Wang, Yan1; Nie, Yan-Hong1; Zhang, Chen-Chen1; Li, Sanlan1; Cheng, Leping1; Wang, Qifang1; Wu, Yan6; Huang, Pengyu3; Sun, Qiang1; Shi, Linyu1; Yang, Hui1
2017-06
Source PublicationCELL RESEARCH
ISSN1001-0602
Volume27Issue:6Pages:801-814
Status已发表
DOI10.1038/cr.2017.76
AbstractTargeted 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.
Keywordhomology-mediated end joining CRISPR/Cas9 monkey embryos neurons knock-in
Indexed BySCI ; CSCD
Language英语
Funding ProjectShanghai City Committee of science and technology project[16JC1420202] ; Shanghai City Committee of science and technology project[14140900100]
WOS Research AreaCell Biology
WOS SubjectCell Biology
WOS IDWOS:000402545200010
CSCD IDCSCD:6016604
PublisherNATURE PUBLISHING GROUP
WOS KeywordKNOCK-IN ; CYNOMOLGUS MONKEY ; GENE ; DNA ; GENERATION ; ZEBRAFISH ; CELLS ; CRISPR-CAS9 ; CLEAVAGE ; TALENS
Original Document TypeArticle
Citation statistics
Cited Times:138[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttps://kms.shanghaitech.edu.cn/handle/2MSLDSTB/1348
Collection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
Corresponding AuthorSun, Qiang; Shi, Linyu; Yang, Hui
Affiliation1.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
Recommended Citation
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.
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