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

Cell Research
Xuan YaoHui Yang

Abstract

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...Continue Reading

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Citations

Dec 23, 2017·Nucleic Acids Research·Xin HanTangliang Li
Aug 23, 2017·Cell Biology and Toxicology·Tetsushi Sakuma, Takashi Yamamoto
Dec 1, 2017·Proceedings of the National Academy of Sciences of the United States of America·Alexandre PaixGeraldine Seydoux
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May 16, 2019·FEMS Microbiology Letters·Belén Adiego-PérezJohn van der Oost
Jan 13, 2018·Cell Research·Xuan YaoHui Yang
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Feb 23, 2020·Biotechnology and Bioengineering·Sung Wook Shin, Jae Seong Lee
Sep 22, 2018·Applied Microbiology and Biotechnology·Tian-Qiong ShiXiao-Jun Ji
Dec 11, 2019·Proceedings of the National Academy of Sciences of the United States of America·Yang ZhangChunliang Li
Aug 26, 2020·Scientific Reports·Riki IshibashiFumiko Toyoshima
Jul 14, 2018·Chromosoma·Yutaka Yamamoto, Susan A Gerbi
Mar 6, 2019·Cell Death & Disease·Ruiqi WangJin Xu
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Methods Mentioned

BETA
gene knock-in
transfection
fluorescence-activated cell sorting
genotyping
PCR
transgenic
in vitro transcription
dissection

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