Construction of Synthetic Nanobody Library in Mammalian Cells by dsDNA‐Based Strategies**

Abstract A nanobody is an antibody fragment consisting of a single monomeric variable antigen‐binding domain. Mammalian cells are ideal platforms for identifying nanobodies targeting hard‐to‐display transmembrane proteins and nanobodies that function as modulators of cellular phenotypes. However, the introduction of a high‐diversity nanobody library into mammalian cells is challenging. We have developed two novel methods for constructing a nanobody library in mammalian cells. Complementarity‐determining region (CDR) random sequences were first incorporated into upstream and downstream dsDNAs by PCR. In the first method, named dsDNA‐HR, upstream and downstream dsDNAs containing an identical overlapping sequence were co‐transfected into cultured mammalian cells for intracellular homologous recombination that resulted in the formation of an intact nanobody library expression cassette. In the second method, named in vitro ligation, we generated full‐length nanobody expression dsDNAs via ligation of restriction digested upstream and downstream dsDNAs. The obtained full‐length dsDNAs were transfected into mammalian cells for nanobody library expression. Using both methods, we generated over a million unique nanobody sequences, as revealed by high‐throughput sequencing. Single‐cell sequencing was employed to resolve the diversity of the dsDNA‐HR nanobody library. We also identified a small molecule, Nocodazole, which could enhance the efficacy of dsDNA‐HR.