Molecular design of sgRNA based on CRISPR-Cas۱۳a for specificdetection of ORF۱ab region in SARS-COV-۲ genome infecting Iranianpatients

COVID-۱۹ has become a global SARS-CoV-۲ coronavirus pandemic, due to high strength and transmission speed of the virus . Early and accurate diagnosis is necessary to prevent the infectiousness along with avoiding progression of the disease in patients. The emerging diagnostic tools based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), function through the binding of Cas proteins to a target region of the viral nucleic acid sequence, with the most conservation and the least mutation rate. The genomic sequence of RNA dependent RNA polymerase (RdRp) is a suitable option for targeting Cas protein. The aim of this study is to apply bioinformatics tools to investigate the conserved regions of the RdRp gene of SARS-CoV-۲ genome and molecular design of sgRNA based on CRISPR-Cas۱۳a, in order to specifically and quickly diagnose through the ORF۱ab gene region in Iranian patients with COVID-۱۹. Therefore, the conserved regions of RdRp sequence were identified using MAFFT multiple sequence alignment program (version ۷) , Jalview (version ۲.۱۱.۲.۳) and BioEdit (version ۷.۲.۵.), through multiple alignment of the reference Wuhan-Hu-۱ genome (NC ۰۴۵۵۱۲.۲) with ۴۷۰ complete SARS-CoV-۲ genomes in Iranian patients with Covid-۱۹ obtained from the GISAID database. Thereafter, in order to design sgRNA, the target sequences were obtained from the conserved regions of the ORF۱ab sequence using the ADAPT online software. As a results, the ۲۸-nucleotide sequence ۵’-TGGTGTACTGACATTAGATAATCAAGAT-۳’ was selected, showing ۱۰۰% similarity with SARS-CoV-۲ genome and no significant similarity with MER-CoV and SARS-CoV. Targetspecificity of sgRNA template was guaranteed by the design of highly specific recombinase polymerase amplification (RPA) forward and reverse primers. In conclusion, the findings of this study resulted in the computational design of an e cient sgRNA, as the first step of a mega project leading to development of a SARS-CoV-۲ diagnostic kit.