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

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 times with a recovery of 46.40% after purification. Cas9 nuclease retained over 65% of its initial activity after incubation between 25 and 42 °C for 2 h, but it was completely deactivated after treatment at 45 °C for 15 min. The enzyme was stable between pH 6.0 and 10.0 with residual enzyme activity more than 68%, and especially the highest stability at pH 9.0. Mg2+ activated the enzyme between 0.5 and 20.0 mmol/L, and 10.0 mmol/L Mg2+ increased the enzyme activity by 23%. Besides, this enzyme was inhibited by some metal ions such as Ba2+, Co2+, Ca2+, Mn2+, Cu2+, Fe2+ and Zn2+, wherein Cu2+ and Fe2+ completly 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 were characterized, serving as reference for further promotion and application of this enzyme in CRISPR/Cas9 technology.