Homology-mediated end joining-based targeted integration using CRISPR/Cas9
| العنوان: | Homology-mediated end joining-based targeted integration using CRISPR/Cas9 |
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| المؤلفون: | Zhen Liu, Haibo Zhou, Chen-Chen Zhang, Leping Cheng, Xiaowen Shen, Hui Yang, Xuan Yao, Zijian Huang, Junlai Liu, Qiang Sun, Qifang Wang, Sanlan Li, Yan Wang, Yanhong Nie, Yan Wu, Xing Wang, Wenqin Ying, Pengyu Huang, Linyu Shi, Xinde Hu, Yu Wei |
| المصدر: | Cell Research |
| بيانات النشر: | Springer Science and Business Media LLC, 2017. |
| سنة النشر: | 2017 |
| مصطلحات موضوعية: | 0301 basic medicine, DNA End-Joining Repair, monkey embryos, Transgene, neurons, Biology, knock-in, Mice, 03 medical and health sciences, Genome editing, Gene knockin, Animals, Humans, CRISPR, Gene Knock-In Techniques, Guide RNA, CRISPR/Cas9, Molecular Biology, Genetics, HEK 293 cells, Cell Biology, Embryonic stem cell, Cell biology, HEK293 Cells, 030104 developmental biology, Hepatocytes, Original Article, CRISPR-Cas Systems, Genetic Engineering, Homologous recombination, homology-mediated end joining, RNA, Guide, Kinetoplastida |
| الوصف: | 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 ∼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 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. |
| تدمد: | 1748-7838 1001-0602 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a3216153871e9b06c44ec9037a02e87c https://doi.org/10.1038/cr.2017.76 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....a3216153871e9b06c44ec9037a02e87c |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 17487838 10010602 |
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