Nuclear Techniques ›› 2018, Vol. 41 ›› Issue (8): 80502-080502.doi: 10.11889/j.0253-3219.2018.hjs.41.080502

• NUCLEAR PHYSICS, INTERDISCIPLINARY RESEARCH • Previous Articles     Next Articles

Electroporation conditions for CRISPR/Cas9-mediated genome editing

XIA Kai1,3, HAN Ling2, KONG Huating1, YAN Qinglong1,3, ZHANG Yu1, ZHU Ying1   

  1. 1. Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Jiading Campus, Shanghai 201800, China;
    2. Department of Molten Salt Chemistry and Engineering, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Jiading Campus, Shanghai 201800, China;
    3. University of Chinese Academy of Sciences, Beijing 100049, China
  • Received:2018-03-28 Revised:2018-04-09 Online:2018-08-10 Published:2018-08-15
  • Supported by:
    Supported by the National Key R&D Program of China (No.2016YFA0400902), the Open Large Infrastructure Research of Chinese Academy of Sciences, National Natural Science Foundation of China (No.11675251, No.21390414), Instrument Developing Project of Chinese Academy of Sciences, the Youth Innovation Promotion Association of Chinese Academy of Sciences (No.2016236) and the China Postdoctoral Science Foundation (No.2016M601679, No.2018M632189)

Abstract: [Background] Direct cellular delivery of CRISPR/Cas9 complexes is of great significance for genome editing, gene expression regulation, and DNA/RNA imaging. Electroporation is an important approach for the delivery of Cas9 ribonucleoproteins (RNP).[Purpose] This study aims to screen the optimal electroporation condition for CRISPR/Cas9-mediated genome editing.[Methods] SDS PAGE, Western bolt, Agarose gel electrophoresis and T7E1 cleavage assay were used to investigate the CRISPR/Cas9-mediated gene editing efficiency under various electroporation conditions. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and Caspase-3 activity determination methods were used to investigate the biocompatibility of various electroporation conditions.[Results] When the voltage is 1 300 V, pulse number is 1, and pulse width is 30 ms, we get the optimal genome editing efficiency with low cellular toxicity.[Conclusion] Our work provides useful information of using CRISPR/Cas9 complexes for further biomedical applications.

Key words: Cas9 ribonucleoproteins, Electroporation condition, Direct delivery, Gene editing efficiency, Biocompatibility

CLC Number: 

  • TL99