Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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A qPCR Method to Assay Endonuclease Activity of Cas9-sgRNA Ribonucleoprotein Complexes

  • Minh Tri Nguyen (Brain Korea 21 Center for Bio-Health Industry, Division of Animal, Horticultural, and Food Science, Chungbuk National University) ;
  • Seul-Ah Kim (Brain Korea 21 Center for Bio-Health Industry, Division of Animal, Horticultural, and Food Science, Chungbuk National University) ;
  • Ya-Yun Cheng (Brain Korea 21 Center for Bio-Health Industry, Division of Animal, Horticultural, and Food Science, Chungbuk National University) ;
  • Sung Hoon Hong (Brain Korea 21 Center for Bio-Health Industry, Division of Animal, Horticultural, and Food Science, Chungbuk National University) ;
  • Yong-Su Jin (Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign) ;
  • Nam Soo Han (Brain Korea 21 Center for Bio-Health Industry, Division of Animal, Horticultural, and Food Science, Chungbuk National University)
  • Received : 2023.05.10
  • Accepted : 2023.06.06
  • Published : 2023.09.28
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Abstract

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/㎍ RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

Keywords

Acknowledgement

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2021R1I1A3049738).

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