Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran
Department of Tissue Engineering and Applied Cell Sciences, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran
Department of Medical Genetics, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
The clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) used for genome editing. The usage of CRISPR-Cas9 in gene editing is faced with certain limitations including off-target mutation, decreased homologous recombination (HR) repair, and immune system responses. It seems that if Cas9 expressed in an inducible manner, off-target mutations may decrease. P53 decreases the activity of the HR pathway in the cell cycle, so, the decrease in P53 level may increase the activity of this pathway. Based on this topic, for the first time, we designed ''px601-Turbo GFP-TRE-shRNA P53'' as a CRISPR-based vector. The use of this vector can simultaneously induce expression of Cas9 and shutdown transiently P53 under an inducible promoter and an inducing agent. Therefore, shutdown transiently P53 may be leading to reduced off-targets and increased accuracy of genome editing. In the human gastric cancer MKN45 cell line, the P53 gene expresses at a normal level. Also, CD44 in this cell line has overexpression and is a gastric cancer stem cell marker. To evaluate this hypothesis, CD44 will be targeted for a specific sequence change (editing) by the px601-Turbo GFP-TRE-shRNA P53 vector. Accordingly, after cloning and virus preparation, MKN45 cell lines will be transduced in the presence of the appropriate doxycycline (DOX) dosage. Ultimately, to evaluate the vector efficiency, DNA extraction and whole-genome sequencing (WGS) will be done and compared with the transduced MKN45 cells without an inducible prompter and DOX as control. Also, the Sanger sequencing for the target gene must be done. This temporary inducible expression may appear to increase the efficiency of the CD44 gene editing and reduce off-targets.