LION: a simple and rapid method to achieve CRISPR gene editing

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

DOI

  • Xi Xiang
  • Lidan Luo, Department of Liver Disease, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, 518033, China.
  • ,
  • Michał Nodzyński, Department of General Biochemistry, Jagiellonian University, ul. Gronostajowa 7, 30-387, Kraków, Poland.
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  • Conghui Li, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Peng Han, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Hongwei Dou, BGI-Shenzhen, 518083 Shenzhen, China; China National GeneBank-Shenzhen, BGI-Shenzhen, 518083 Shenzhen, China.
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  • Trine Skov Petersen
  • Xue Liang, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Xiaoguang Pan, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
  • ,
  • Kunli Qu, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China.
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  • Ling Yang, BGI-Shenzhen, 518083 Shenzhen, China; China National GeneBank-Shenzhen, BGI-Shenzhen, 518083 Shenzhen, China.
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  • Yonghui Dang, Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China., College of Forensics and Medicine, Xi'an Jiaotong University Health Science Centre, Xi'an, 710049, China.
  • ,
  • Xin Liu, China National GeneBank, BGI-Shenzhen, Shenzhen 518120, China.
  • ,
  • Lars Bolund
  • Xiuqing Zhang, BGI Education Center, University of Chinese Academy of Sciences, Shenzhen 518083, China., Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China., BGI-Shenzhen, 518083 Shenzhen, China; China National GeneBank-Shenzhen, BGI-Shenzhen, 518083 Shenzhen, China., Department of General Biochemistry, Jagiellonian University, ul. Gronostajowa 7, 30-387, Kraków, Poland.
  • ,
  • Guangdong Tong, Department of Liver Disease, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, 518033, China.
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  • Yufeng Xing, Department of Liver Disease, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, 518033, China. yufeng000729@163.com.
  • ,
  • Yonglun Luo
  • Lin Lin

The RNA-guided CRISPR-Cas9 technology has paved the way for rapid and cost-effective gene editing. However, there is still a great need for effective methods for rapid generation and validation of CRISPR/Cas9 gRNAs. Previously, we have demonstrated that highly efficient generation of multiplexed CRISPR guide RNA (gRNA) expression array can be achieved with Golden Gate Assembly (GGA). Here, we present an optimized and rapid method for generation and validation in less than 1 day of CRISPR gene targeting vectors. The method (LION) is based on ligation of double-stranded gRNA oligos into CRISPR vectors with GGA followed by nucleic acid purification. Using a dual-fluorescent reporter vector (C-Check), T7E1 assay, TIDE assay and a traffic light reporter assay, we proved that the LION-based generation of CRISPR vectors are functionally active, and equivalent to CRISPR plasmids generated by traditional methods. We also tested the activity of LION CRISPR vectors in different human cell types. The LION method presented here advances the rapid functional validation and application of CRISPR system for gene editing and simplified the CRISPR gene-editing procedures.

OriginalsprogEngelsk
TidsskriftCellular and Molecular Life Sciences
Vol/bind76
Nummer13
Sider (fra-til)2633-2645
Antal sider13
ISSN1420-682X
DOI
StatusUdgivet - jul. 2019

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