## 3 Results
### Generation of VNA CDR3-Targeted Mutagenesis Library  
The design of a randomized CDR3 loop of a VNA antibody library is adapted from the work of [PERSON] et al. [35], with the change of the phage display system to a yeast surface display system. Six CDR3 sizes (12, 13, 16, 18, or 20 and 23 amino acids) of the HEL-5A7 VNAR protein sequence were randomized by a splice-overlap polymerase chain reaction, while two atypical cysteines remained unchanged. The library DNA cassettes were mixed with linearized pNACP-GFP and electroporated into EBY100 yeast cells to generate a semi-synthetic VNAR antibody library by in vivo homologous recombination.  
The library was induced and subjected to three rounds of negative selection against streptavidin-coated magnetic beads, resulting in a yield of 6.4 \(\times\) 10\({}^{9}\) transformants. To calculate the diversity of the library, we extracted plasmids from yeast cells and performed next-generation sequencing (NGS). The NGS results are exhibited in Table 2. After trimming and merging the original sequence, 249,437 sequences were used for subsequent analysis. There were 10,092 (4%) scaffold gene (HEL-5A7) sequences, and 118,791 sequences (47.6%) were in line with the expected six lengths of the CDR3 open reading frame. Overall, 76,780 sequences (30.08%) were unique. Within the unique sequences, the percentage of the VNAR  
Figure 2: Determination of surface-displayed HEL5A7 binding against lysozyme. (**A**) HEL5A7-displaying yeasts were incubated with various concentrations of lysozyme. The detection of bound yeast was employed by rabbit polyclonal anti-lysozyme antibody and HRP-conjugated goat anti-rabbit IgG via cell-based ELISA. Yeasts harboring the pNACP-GFP plasmid were the negative control group. Data are mean \(\pm\) SE (\(n=3\)). \(n\)**s**” indicates not significant (\(p>0.05\)), whereas the asterisk indicates significant difference (\(p<0.05\)) between each group (\(t\)-test) in the presence of different amounts of lysozyme. (**B**) HEL5A7-displaying yeasts were incubated with various concentrations of biotinylated lysozyme. The bound yeast was detected by PE-conjugated streptavidin, and the fluorescence intensity of GFP and PE were measured by fluorescent plate reader. Data are mean \(\pm\) SE (\(n=3\)). Means with different letters among groups are significantly different (\(p<0.05\)) by [PERSON]’s multiple range test. (**C**) The biotinylated lysozyme-bound yeasts were incubated with PE-conjugated streptavidin, and the percentage changes of GFP\({}^{+}\)PE\({}^{-}\) and GFP\({}^{+}\)PE\({}^{+}\) cells were analyzed by flow cytometry while at least 3.5 \(\times\) 10\({}^{5}\) yeast cells per sample were analyzed (\(n=1\)). The HEL5A7-displaying yeasts without biotinylated lysozyme were the untreated control group.  
sequences with 12, 13, 16, 18, 20, or 23 residues of CDR3 were 36.5, 22, 22, 16.7, 13.3, 7.7, or 3.6%, respectively (Figure 3). It seems that the frequency of the individual set of unique sequences is CDR3 length-dependent. The proportion of unique sequences can be used to deduce that the diversity of the semi-synthetic VNAR antibody library is 1.97 \(\times\) 10\({}^{9}\).  
To ensure the NNK site-saturation mutagenesis was successfully covering all kinds of amino acids in the mutation sites, the analysis of the amino acid composition in the library was performed. As in Figure 4, the amino acid percentage on each loop length of the randomized CDR3 sequence in the library is shown in a heatmap. The amino acids distribution is similar between different lengths of randomized CDR3 sequences. The most abundant amino acids are arginine (12.20%), proline (11.90%), serine (8.80%), alanine (8.70%), threonine (8.60%), and leucine (8.00%), while others are ranged from 1.4% to 6.1%.