Ferdowsi University of Mashhad
Abstract: (452 Views)
Genome editing based on CRISPR/Cas9 technology is rapidly expanding as a powerful tool for crop breeding. One of the advantages of this technology genome editing without introducing foreign DNA into plant genome.to achieve this, Cas9 and guide RNA need delivered to plant cell in form of protein-RNA complex (RNP). Hence the production of Cas9 enzyme in laboratory is the first step of CRISPR based on RNP method. Here, we evaluated the expression and purification of recombinant Cas9 protein in E. coli. In this study Cas9 gene was cloned into pBADM30 vector and transferred to the E. coli CodonPlus strain. The result of colony PCR and double digest confirmed the presence of Cas9 gene into pBAD vector. Cas9 expression was induced by applying 0.2% L-Arabinose and then isolated from the total soluble protein. Cas9 protein was also purified from cell crude extract using Nickel affinity chromatography. The purification was analyzed by SDS-PAGE and show the presence of a 180 kDa band in the fraction collected during the elution step. The Cas9 protein in E. coli was successfully expressed and purified. According to the obtained results, it can be said that after measuring the catalytic activity of Cas9 enzyme, this enzyme can be used together with guide RNA and fabrication of RNP complex and its transfer to the plant by protoplast or electroporation method in further research.
Article number: 9
Type of Study:
Applicable |
Subject:
Subject 01 Received: 2022/01/4 | Accepted: 2023/01/18 | Published: 2023/04/22