Devaraju, N. | bioRxiv Science

bioRxiv2022

Correlating the differences in the receptor binding domain of SARS-CoV-2 spike variants on their interactions with human ACE2 receptor

Spike protein of SARS-CoV-2 variants play critical role in the infection and transmission through its interaction with hACE2 receptor. Prior findings using molecular docking and biomolecular studies reported varied findings on the difference in the interactions among the spike variants with hACE2 receptor. Hence, it is a prerequisite to understand these interactions in a more precise manner. To this end, firstly, we performed ELISA with trimeric spike proteins of Wild (Wuhan Hu-1), Delta, C.1.2 and Omicron variants. Further, to study the interactions in a more specific manner by mimicking the natural infection, we developed hACE2 receptor expressing HEK-293T cell line and evaluated binding efficiencies of the variants and competitive binding of spike variants with D614G spike pseudotyped virus. In lines with the existing findings, we observed that Omicron had higher binding efficiency compared to Delta in both ELISA and Cellular models. Intriguingly, we found that cellular models could differentiate the subtle differences between the closely related C.1.2 and Delta in their binding to hACE2. From the analysis in receptor binding domain (RBD) revealed that a single common modification, N501Y, present in both Omicron and C.1.2 is driving the enhanced spike binding to the receptor and showed two-fold superior competitive binding than Delta. Our study using cellular model provides a precise method to evaluate the binding interactions between spike sub-lineages to hACE2 receptors and signifies the role of single common modification N501Y in RBD towards imparting superior binding efficiencies. Our approach would be instrumental in understanding the disease progression and developing therapeutics. Author SummarySpike proteins of evolving SARS-CoV2 variants demonstrated their signature binding to hACE2 receptor, in turn contributed to driving the infection and transmission. Prior studies to scale the binding efficiencies between the spike variant and the receptor had consensus in distinct variants, but discrepancies in the closely related ones. To this end, we compared spike variants-receptor interactions with ELISA, from cells expressing hACE2 receptor. Intriguingly, we found that cellular models could differentiate the subtle differences between the closely related C.1.2 and Delta in their binding to hACE2. More importantly, competitive binding studies in presence of pseudovirus, demonstrated that a single common modification, N501Y, present in both Omicron and C.1.2 showed two fold superior competitive binding than Delta. Collectively, our study suggests a precise approach to evaluate the binding interactions between spike sub-lineages to hACE2 receptor. This would be instrumental in understanding the disease progression and developing therapeutics.

pharmacology and toxicology↗

bioRxiv2022

Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors

{beta}-thalassemia and HbE result from mutations in the {beta}-globin locus that impedes the production of functional {beta}-hemoglobin and represents one of the most common genetic disorders worldwide. Recent advances in genome engineering have opened up new therapeutic opportunities to directly correct these pathogenic mutations using base editors that install transition mutations (A>G and C>T) in the target region with minimal generation of indels. Herein, for the first time, we demonstrate the usage of base editor in the correction of point mutations spanning multiple regions of the HBB gene, including promoter, intron and exon. To this end, we have engineered human erythroid cells harbouring the diverse HBB mutations, thus eliminating the requirement of patient CD34+ HSPCs with desired mutations for the primary screening by base editors. We further performed precise creation and correction of individual HBB point mutations in human erythroid cells using base editors, which were effectively corrected in the HBB-engineered erythroid model. Intriguingly, most bystander effects produced by the base editor at the target site were reported to exhibit normal hemoglobin variants. Overall, our study provides the proof-of-concept for the precise, efficient and scarless creation and correction of various pathogenic mutations at the coding and non-coding regions of HBB gene in human erythroid cells using base editors and establishes a novel therapeutic platform for the treatment of {beta}-thalassemia/HbE patients. This study can be further explored in correcting the other monogenic disorders caused due to single base substitutions.

genomics↗