Palindromic target site identification in SARS-CoV-2, MERS-CoV and SARS-CoV-1 by adopting CRISPR-Cas technique.

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated Cas protein (CRISPR-Cas) has turned out to be a very important tool for the rapid detection of viruses. This can be used for the identification of the target site in a virus by identifying a 3–6 nt length Protospacer Adjacent Motif (PAM) adjacent to the potential target site, thus motivating us to adopt CRISPR-Cas technique to identify SARS-CoV-2 as well as other members of Coronaviridae family. In this regard, we have developed a fast and effective method using k-mer technique in order to identify the PAM by scanning the whole genome of the respective virus. Subsequently, palindromic sequences adjacent to the PAM locations are identified as the potential target sites. Palindromes are considered in this work as they are known to identify viruses. Once all the palindrome-PAM combinations are identified, PAMs specific for the RNA-guided DNA Cas9/Cas12 endonuclease are identified to bind and cut the target sites. In this regard, PAMs such as 5’-TGG-3’ and 5’-TTTA-3’ in NSP3 and Exon for SARS-CoV-2, 5’-GGG-3’ and 5’-TGG-3’ in Exon and NSP2 for MERS-CoV and 5’-AGG-3’ and 5’-TTTG-3’ in Helicase and NSP3 respectively for SARS-CoV-1 are identified corresponding to SpCas9 and FnCas12a endonucleases. Finally, to recognise the target sites of Coronaviridae family as cleaved by SpCas9 and FnCas12a, complements of the palindromic target regions are designed as primers or guide RNA (gRNA). Therefore, such complementary gRNAs along with respective Cas proteins can be considered in assays for the identification of SARS-CoV-2, MERS-CoV and SARS-CoV-1.