Expression, purification and enzymatic characterization of recombinant Streptococcus pyogenes Cas9 nuclease

BAO Ling-Na1 WU Yong1,2 LIN Jun1 DONG Yuan-Zhen1,2 HUANG Zong-Qing2

(1.Shanghai Institute of Pharmaceutical Industry, China Institute of Pharmaceutical Industry, Shanghai, China 201203)
(2.Shanghai Domray Biotechnology Co., Ltd., Shanghai, China 201203)

【Abstract】[Background] Cas9 nuclease is a site-specific RNA-guided endonuclease that can form a stable ribonucleoprotein complex with single-guide RNA (sgRNA), which recognizes and cleaves target DNA molecules. Due to its high flexibility and efficiency, Cas9 is the most widely used gene-editing tool in both basic research and clinical treatment. [Objective] To provide a theoretical basis for the rational development and utilization of Cas9 nuclease. [Methods] The wild type Cas9 nuclease from Streptococcus pyogenes was expressed in the Escherichia coli system. The expressed enzyme was then purified by ammonium sulfate precipitation and Ni2+-affinity chromatography. Finally, the purified Cas9 nuclease was characterized for its thermal stability, pH stability, and the influence of metal ions. [Results] The results showed that the wet weight of the bacteria was 191.0 g/L after high-density fermentation. The specific activity was 641.29 U/mg and the bacteria was purified up to 16.02 folds with a recovery of 46.40% after purification. Cas9 nuclease retained over 65% of its initial activity after incubation at 25–42 °C for 2 h, but it was completely deactivated after treatment at 45 °C for 15 min. The nuclease was stable between pH 6.0 and 10.0 with residual nuclease activity more than 68%, and especially the highest stability at pH 9.0. Mg2+ at concentration of 0.5–20.0 mmol/Lactivated the nuclease, and 10.0 mmol/L Mg2+ increased the nuclease activity by 23%. Besides, this nuclease was inhibited by some metal ions such as Ba2+, Co2+, Ca2+, Mn2+, Cu2+, Fe2+and Zn2+, wherein Cu2+ and Fe2+ completely inhibited Cas9 nuclease at a concentration of 0.5 mmol/L. [Conclusion] Cas9 nuclease from Streptococcus pyogenes was heterologously expressed and purified with high purity and high activity. The purified Cas9 nuclease was characterized, serving as a reference for further promotion and application of this nuclease in CRISPR/Cas9 technology.

【Keywords】 Streptococcus pyogenes Cas9 nuclease; Purification; Ammonium sulfate precipitation; Affinity chromatography; Enzymatic characteristics;


【Funds】 National Key Research and Development Program of China (2017YFD0501404)

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    [1] Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea [J]. Science, 2010, 327 (5962): 167–170

    [2] Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea [J]. Nature, 2012, 482 (7385): 331–338

    [3] Kim H, Kim JS. A guide to genome engineering with programmable nucleases [J]. Nature Reviews Genetics, 2014, 15 (5): 321–334

    [4] Jinek M, Jiang FG, Taylor DW, et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation [J]. Science, 2014, 343 (6176): 1247997

    [5] Chen HF, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease [J]. The Journal of Biological Chemistry, 2014, 289 (19): 13284–13294

    [6] Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity [J]. Science, 2012, 337 (6096): 816–821

    [7] Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria [J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109 (39): E2579–E2586

    [8] Ma CX, Ha K, Kim MS, et al. The anaphase promoting complex promotes NHEJ repair through stabilizing Ku80 at DNA damage sites [J]. Cell Cycle, 2018, 17 (9): 1138–1145

    [9] Zhang XH, Tee LY, Wang XG, et al. Off-target effects in CRISPR/Cas9-mediated genome engineering [J]. Molecular Therapy—Nucleic Acids, 2015, 4: e264

    [10] Sezonov G, Joseleau-Petit D, D’Ari R. Escherichia coli physiology in luria-bertani broth [J]. Journal of Bacteriology, 2007, 189 (23): 8746–8749

    [11] Gao YL, Yang SW, Fan KQ, et al. Development of SDS-PAGE in protein [J]. Liaoning Chemical Industry, 2007, 36 (7): 460–463 (in Chinese)

    [12] Zhang XJ, Abell C. The determination of saturation percentages of ammonium [J]. Journal of Guizhou University of Technology (Natural Science Edition), 2000, 29 (6): 25–29 (in Chinese)

    [13] Li HL, Peng SM, Li L, et al. Studies on four conventional methods for protein determination [J]. Chinese Journal of Biochemical Pharmaceutics, 2008, 29 (4): 277–278, 282 (in Chinese)

    [14] Ng SR, Rideout III WM, Akama-Garren EH, et al.CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer [J]. Proceedings of the National Academy of Sciences of the United States of America, 2020, 117 (1): 513–521

    [15] Xu L, Wang J, Liu YL, et al. CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia [J]. The New England Journal of Medicine, 2019, 381 (13): 1240–1247

    [16] Jubair L, Fallaha S, Mc Millan NAJ. Systemic delivery of CRISPR/Cas9 targeting HPV oncogenes is effective at eliminating established tumors [J]. Molecular Therapy, 2019, 27 (12): 2091–2099

    [17] Harrington LB, Paez-Espino D, Staahl BT, et al. A thermostable Cas9 with increased lifetime in human plasma [J]. Nature Communications, 2017, 8: 1424

This Article


CN: 11-1996/Q

Vol 47, No. 07, Pages 2003-2011

July 2020


Article Outline



  • 1 Material
  • 2 Method
  • 3 Results and analysis
  • 4 Discussion and conclusion
  • References