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The COVID-19 crisis, incited by the zoonotic SARS-CoV-2 virus, has quickly escalated into a catastrophic public health issue and a grave threat to humankind owing to the advent of mutant viruses. Multiple pharmaceutical therapies or biologics envision stopping the virus from spreading further; however, WHO has voiced concerns about the variants of concern (VoCs) inability to respond. Nanobodies are a new class of antibody mimics with binding affinity and specificity similar to classical mAbs, as well as the privileges of a small molecular weight, ease of entry into solid tissues, and binding cryptic epitopes of the antigen. Herein, we investigated multiple putative anti-SARS-CoV-2 nanobodies targeting the Receptor binding domain of the WHO-listed SARS-CoV-2 variants of concern using a comprehensive computational immunoinformatics methodology. With affinity maturation via alanine scanning mutagenesis, we remodeled an ultrapotent nanobody with substantial breadth and potency, exhibiting pico-molar binding affinities against all the VoCs. An antiviral peptide with specificity for ACE-2 receptors was affixed to make it multispecific and discourage viral entry. Collectively, we constructed a broad-spectrum therapeutic biparatopic nanobody-peptide conjugate (NPC) extending coverage to SARS-CoV-2 VoCs RBDs. We PEGylated the biparatopic construct with 20kD maleimide-terminated PEG (MAL-(PEG)n-OMe) to improve its clinical efficacy limiting rapid renal clearance, and performed in silico cloning to facilitate future experimental studies. Our findings suggest that combining biparatopic nanobody conjugate with standard treatment may be a promising bivariate tool for combating viral entry during COVID-19 illness. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s11030-022-10570-x.

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CRISPR-CAS9, a bacterial defensive genome editing mechanism against phages, introduced by Jennifer Doudna and Emmanuelle Charpentier as a system that can be programmed and reprogrammed in 2012, is a modern revolutionary bioscience tool with applications far beyond the present-day scientic boundaries. With its accelerating advancements in elds ranging from agriculture, horticulture, genetic disorders, biomedicines, live-imaging of genes, epigenetic editing, CRISPR technology has already altered the biological perspective in a great deal. In this review paper, an overview of the CRISPR/CAS9 technology and its applications in agricultural and bio-medicinal eld of studies based on previously available works of literature, has been shortly summarized.