###### Abstract
Although the VNAR affinity is moderate when isolated from non-immune antibody libraries, it has still shown a broad spectrum of specificity against antigens [21; 22; 23]. The moderate-to-low affinity can be improved by increasing the library diversity [24] and in vitro affinity maturation, such as the target-site mutation or saturation mutation of CDR1 [25; 26; 27]. Another aspect of improving the affinity is to increase the diversity of the non-immune antibody library. The naive antibody libraries are antibody libraries constructed from B cells of nonimmunized sharks. The VNAR gene sequence is isolated from a large portion of white blood cells and then cloned into display platforms for screening, requiring a large quantity of animal tissues and potentially causing some ethical issues. In contrast, the starting material for building a semi-synthetic VNAR antibody library only needs the unrearranged IgNARgenes from B cells or the VNAR antibody gene scaffold. And at least one hypervariable loop of VNAR was genetically randomized to build the diversity of a semi-synthetic library. For example, [PERSON] et al. used the striped shark VNAR as the gene scaffold and randomized the CDR3 region by saturation mutation to construct the first semi-synthetic phage VNAR library. They have successfully sorted out the VNARs against AMA-1, Tom70, or Gingipain K protease and proved that these recombinantly expressed VNARs can bind with its antigen [27; 28; 29; 18]. In addition to the CDR3 region, the VNAR contains another three hypervariable loops, designated as CDR1, hypervariable loop 2 (HV2), and hypervariable loop 4 (HV4), respectively. The HV2 region of the VNAR gene can also be randomized to generate a new antigen-binding site for screening bispecific VNAR antibodies. [PERSON] et al. employed the anti-epithelial cell adhesion molecule (EpCAM) VNAR gene as the scaffold and mutated its HV2 region to construct a semi-synthetic yeast VNAR library. Thereafter, the bispecific VNARs against EpCAM and with a cluster of differentiation of 3\(\epsilon\) (CD3\(\epsilon\)) or human Fcy were screened and isolated [30]. During the screening process to identify high-affinity binders from the yeast VNAR library, a peptide tag must be fused to VNARs to aid the analysis of the display level of the VNAR library on the surface of the yeast cell. Further, in cases where no direct fluorophore-labeled antibodies against the peptide tags are available, a tag-specific primary antibody and a fluorophore-conjugated secondary antibody might be needed for fluorescence-activated cell sorting (FACS). The complexity of the analysis and possible batch-to-batch differences of antibodies may pose reproducibility challenges to the screening process. Therefore, an improved yeast surface display platform for screening nanobody immune libraries is designed to overcome these hurdles [31]. The nanobody library fused to the N-terminus end of Aga2p and an orthogonal acyl carrier protein (ACP) linked to the C-terminus end of Aga2p. After Sfp synthase attaches fluorophore-labeled CoA derivatives to the ACP tag, the display level of the Nanobody on the yeast cell can be monitored by fluorometry. Although this Nanobody-Aga2p-ACP fusion avoided the possible steric hindrance by a peptide tag and can be easily stained in a one-step reaction, specific reagents and extra steps are still needed in the staining process. A more straightforward strategy is employed in this study, where the GFP protein replaces the ACP tag and leads to the skipping of staining [32]. Additionally, to speed up the VNAR discovery, a work flow was taken advantage of using the animal-free semi-synthetic yeast VNAR library and the efficient selection of antigen-specific VNAR by sequential magnetic activated cell sorting (MACS) and FACS. Eventually, a comprehensive survey of the candidate binders was done by next-generation sequencing.