The Development of Recombinant hSpCas9 Production System in E. coli

Author:

Number of pages: 36-43
Year-Number: 2021-2

Abstract

The development of CRISPR/Cas9 system has revolutionized and accelerated the era of programmable nuclease mediated gene editing technologies. The CRISPR/Cas9 system consists of a Cas9 nuclease and a synthetic chimeric RNA known as guide RNA (gRNA). Cas9 endonuclease cleaves target DNA at a sequence-specific site with the help of designed sgRNA. The utilization of Cas9 nuclease protein as an important molecular tool in gene editing makes a great emphasis on the production of recombinant Cas9 protein. Due to Cas9 protein’s large size and its complexity, recombinant Cas9 protein production can be challenged. Here, we subcloned a sequence encoding human codon-optimized Streptococcus pyogenes Cas9 (hSpCas9), and recombinant protein production of hSpCas9 was evaluated in different E. coli strains at different induction settings. The expression of recombinant hSpCas9 protein was determined within BL21(DE3) host strain at 25 °C induction temperature for four hours and following overnight induction. The purification of HisTagged recombinant hSpCas9 protein was performed by affinity chromatography. These findings imply that recombinant hSpCas9 protein could be produced on a large scale for potential utilization in the biotechnology market. The production of recombinant hSpCas9 nuclease protein could provide a significant tool for the advancement in the ribonucleoprotein complex utilization in CRISPR/Cas9 technology as well as the improvement of pharmaceutical-based approaches to modulate Cas9 nuclease protein activity for precise gene editing.

Keywords

Abstract

The development of CRISPR/Cas9 system has revolutionized and accelerated the era of programmable nuclease mediated gene editing technologies. The CRISPR/Cas9 system consists of a Cas9 nuclease and a synthetic chimeric RNA known as guide RNA (gRNA). Cas9 endonuclease cleaves target DNA at a sequence-specific site with the help of designed sgRNA. The utilization of Cas9 nuclease protein as an important molecular tool in gene editing makes a great emphasis on the production of recombinant Cas9 protein. Due to Cas9 protein’s large size and its complexity, recombinant Cas9 protein production can be challenged. Here, we subcloned a sequence encoding human codon-optimized Streptococcus pyogenes Cas9 (hSpCas9), and recombinant protein production of hSpCas9 was evaluated in different E. coli strains at different induction settings. The expression of recombinant hSpCas9 protein was determined within BL21(DE3) host strain at 25 °C induction temperature for four hours and following overnight induction. The purification of HisTagged recombinant hSpCas9 protein was performed by affinity chromatography. These findings imply that recombinant hSpCas9 protein could be produced on a large scale for potential utilization in the biotechnology market. The production of recombinant hSpCas9 nuclease protein could provide a significant tool for the advancement in the ribonucleoprotein complex utilization in CRISPR/Cas9 technology as well as the improvement of pharmaceutical-based approaches to modulate Cas9 nuclease protein activity for precise gene editing.

Keywords