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Footnote 1: [[https://www.mdpi.com/journal/applsci](https://www.mdpi.com/journal/applsci)]([https://www.mdpi.com/journal/applsci](https://www.mdpi.com/journal/applsci))  
Footnote 2: email: {[[https://www.mdpi.com/journals/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdfpdf/pdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdf](https://www.mdpi.com/journals/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdfpdf/pdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdf)]([https://www.mdpi.com/journals/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdfpdf/pdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdf](https://www.mdpi.com/journals/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdf/pdfpdf/pdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdfpdf/pdf))efficient infection of lung cells, thereby decreasing the incidence of severe pneumonia [2]. Recent scientific revelations have underscored the significance of the Omicron subvariant BA.5 as a pivotal progression within this viral landscape. Unlike preceding Omicron subvariants, Omicron subvariant BA.5 demonstrates efficient lung cell entry caused by specific spike protein mutations, which enhance viral cell fusion and replication within lung cells [3]. This variant was then used for bivalent vaccine development by Pfizer-BioNTech and Moderna. This study utilized nanobodies (Nbs), recombinantly produced single antigen-binding domains derived from the heavy-chain only antibodies (HCab) present in animals of the Camelidae family, as potent tools targeting the RBD's functional intricacies. Nbs offer higher stability and solubility, and more efficient target-binding, than conventional monoclonal antibodies (mAbs) due to their diminutive size, which confers superior tissue penetration capabilities essential in impeding viral intrusion, as well as greater accessibility to difficult-to-reach tissue areas [4; 5]. Several studies have demonstrated the potentials of Nbs for COVID-19 treatment by blocking RBD-ACE2 interaction [6; 7; 8; 9]. The functional ligand-binding identification by Tat-based recognition of associating proteins (FLI-TRAP) technique is an in vivo technique that is useful for the selection of binding proteins. It has previously been utilized for both identification and enhancement of antibody fragments (single-chain variable fragment, scFv, and single-domain antibody, sdAb, also known as Nb) specific to various targets [10; 11; 12; 13]. Our study focused on the directed evolution of Nbs that specifically target the RBD of the Omicron subvariant BA.5, using the FLI-TRAP technique as an in vivo selection method [10]. Our goal was to create Nbs that could bind the SARS-CoV-2 Omicron subvariant BA.5 by redirecting the specificity of the existing broad-spectrum Nb, the 1.29 Nb, capable of effectively binding several SARS-CoV-2 variants of concern (VCCs) [9]. To achieve this, we employed error-prone PCR to generate Nb mutation libraries and FLI-TRAP to isolate Nbs specific to the RBD of the SARS-CoV-2 spike protein of the Omicron subvariant BA.5. After two rounds of evolution, Nbs C11 and K9 emerged as potential candidates, displaying notable binding abilities, particularly toward the RBD of Omicron subvariant BA.5 as well as other Omicron subvariants tested, including XBB.1.5 and XBB.1.16. The combined use of error-prone PCR and the FLI-TRAP technique has shown the potential to redirect the specificity of the currently available Nb toward targets with mutations. Our tool offers a rapid and simple evolution method facilitating the creation of tailored treatments for various diseases other than viral infections.