Despite the significant advances of antibodies as therapeutic agents, there is 
still much room for improvement concerning the discovery of these 
macromolecules. Here, we present a new synthetic cell-based strategy that takes 
advantage of eukaryotic cell biology to produce highly diverse antibody 
libraries and, simultaneously, link them to a high-throughput selection 
mechanism, replicating B cell diversification mechanisms. The interference of 
site-specific recognition by CRISPR/Cas9 with error-prone DNA repair mechanisms 
was explored for the generation of diversity, in a cell population containing a 
gene for a light chain antibody fragment. We achieved up to 93% of cells 
containing a mutated antibody gene after diversification mechanisms, 
specifically inside one of the antigen-binding sites. This targeted variability 
strategy was then integrated into an intracellular selection mechanism. By 
fusing the antibody with a KDEL retention signal, the interaction of antibodies 
and native membrane antigens occurs inside the endoplasmic reticulum during the 
process of protein secretion, enabling the detection of high-quality leads for 
expression and affinity by flow cytometry. We successfully obtained antibody 
lead candidates against CD3 as proof of concept. In summary, we developed a 
novel antibody discovery platform against native antigens by endoplasmic 
synthetic library generation using CRISPR/Cas9, which will contribute to a 
faster discovery of new biotherapeutic molecules, reducing the time-to-market.