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

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.

1H, 13C, 15N and 31P chemical shift assignment for stem-loop 4 from the 5'-UTR of SARS-CoV-2.

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5'-untranslated region (5'-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1H, 13C, 15N and 31P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy.

Using All-Atom Potentials to Refine RNA Structure Predictions of SARS-CoV-2 Stem Loops.

A considerable amount of rapid-paced research is underway to combat the SARS-CoV-2 pandemic. In this work, we assess the 3D structure of the 5' untranslated region of its RNA, in the hopes that stable secondary structures can be targeted, interrupted, or otherwise measured. To this end, we have combined molecular dynamics simulations with previous Nuclear Magnetic Resonance measurements for stem loop 2 of SARS-CoV-1 to refine 3D structure predictions of that stem loop. We find that relatively short sampling times allow for loop rearrangement from predicted structures determined in absence of water or ions, to structures better aligned with experimental data. We then use molecular dynamics to predict the refined structure of the transcription regulatory leader sequence (TRS-L) region which includes stem loop 3, and show that arrangement of the loop around exchangeable monovalent potassium can interpret the conformational equilibrium determined by in-cell dimethyl sulfate (DMS) data.