## 2 Results and Discussion  
**Selection of the target RBD variant and the prototype Nb. ** The Omicron variant has displaced other variants, with its rising prevalence and continued propensity for mutation [14]. In addition, the amino acid sequences of the RBD of Omicron subvariants BA.4/5 were used to design the most updated bivalent booster vaccines, BNT162b2 BA.4/5 bivalent mRNA vaccine (Pfizer-BioNTech) and SPIKEVAX Bivalent Original/Omicron BA.4-5 (Moderna). Unfortunately, the accumulated additional spike mutations may reduce the effectiveness of the vaccine, as a study found that the BA.5 bivalent booster did not provide strong neutralization against some of the Omicron sublineages, including the BA.2-derived BA.2.75.2 and the BA.5-derived BQ.1.1 and XBB.1 [15]. From a therapeutic standpoint, the search for broad-spectrum antibodies continues. Our literature review revealed that the 1.29 Nb exhibits comprehensive binding to RBD variants of SARS-CoV-2, including the VOCs Wuhan (WA1), Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). Nonetheless, its efficacy in binding to the Omicron BA.1 variant is notably diminished compared to WA1 [9]. This poses a challenge for the development of the 1.29 Nb against the new Omicron variants. To address this challenge, we utilized the 1.29 Nb as a prototype Nb to generate a library of diverse Nbs using an error-prone PCR technique, and the library was selected against the RBD of the BA.5 variant using the FLI-TRAP technique. **Redirection of a broad-spectrum anti-RBD 1.29 Nb to the Omicron subvariant BA.5. ** Figure 1 illustrates the overall selection process used for identification of the 1.29 Nb mutants that could bind to the RBD of BA.5 variant. For FLI-TRAP screening, we first constructed RBD fused to mature TEM-1 \(\beta\)-lactamase (Bla) protein in the pDD18 plasmid [10], and the parental 1.29 Nb was then cloned with an N-terminal Tat signal peptide in the same plasmid (Figure S1). The mutant 1.29 Nb library gene created by an error-prone PCR technique utilizing Mutazyme II DNA polymerase from a GeneMorph II mutagenesis kit was subcloned by replacing the parental 1.29 Nb. For the first round of mutation, the library size and error rate were 9.8 \(\times\) 10\({}^{5}\) members and 1-2 mutations per gene, respectively. For the second round of mutation, the library size and error rate were determined to be 4.2 \(\times\) 10\({}^{5}\) members and 1-4 mutations per gene. respectivel. Since we wanted to identify mutant Nbs that could bind to the RBD of the BA.5 variant better than the parental 1.29 Nb, this original 1.29 Nb sequence was used to establish the library selection conditions by spot plating (Figure 2A). During this first round of mutation, 51 hits were obtained at 100 \(\mu\)g/mL Carb and 10\({}^{-4}\) cell dilution. Figure 2B shows the results of the spot plating, which was performed to compare the cell resistance of Carb among 15 randomly selected clones and the parental 1.29 Nb. The sequencing of 13 of 15 colonies was successful and revealed that 10 of them had the same amino acid sequence as the 1.29 Nb. Of the three unique sequences, i.e., C3, C4, and C11, that we obtained, two contained stop codons (C3 and C4) hence were false positives. Therefore, C11 was the only unique Nb with a sequence with two nucleotide mutations resulting in R38C and V64E mutations (Figure S2). It should be noted that the cell resistance of the colonies that contained the same sequence as the 1.29 Nb could be due to natural mutations of the bacterial cells that created resistance to the antibiotic that was unrelated to the FLI-TRAP selection system [16]. Figure 1: **The FLI-TRAP selection system. ** Schematic representation of the overall process used in this study. IM: inner membrane; TatABC: Twin-arginine translocation (Tat) ABC complex; TEM-1 Bla: Temoniera \(\beta\)-lactamase; Nb: nanobody; Carb: carbenicillin; RBD: receptor binding domain; FLI-TRAP: Tat-based recognition of associating proteins.