TY - JOUR T1 - High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis JF - bioRxiv DO - 10.1101/285015 SP - 285015 AU - Derek M Mason AU - Cédric R Weber AU - Cristina Parola AU - Simon M Meng AU - Victor Greiff AU - William J Kelton AU - Sai T Reddy Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/03/19/285015.abstract N2 - Antibody engineering is performed to improve therapeutic properties by directed evolution, usually by high-throughput screening of phage or yeast display libraries. Engineering antibodies in mammalian cells offers advantages associated with expression in their final therapeutic format (full-length glycosylated IgG), however, the inability to express large and diverse libraries severely limits their potential throughput. To address this limitation, we have developed homology-directed mutagenesis (HDM), a novel method which extends the concept of CRISPR/Cas9-mediated homology-directed repair (HDR). HDM leverages oligonucleotides with degenerate codons to generate site-directed mutagenesis libraries in mammalian cells. By improving HDM efficiency (>35-fold) and combining mammalian display screening with next-generation sequencing (NGS), we validated this approach can be used for key applications in antibody engineering at high-throughput: rational library construction, novel variant discovery, affinity maturation, and deep mutational scanning (DMS). We anticipate that HDM will be a valuable tool for engineering and optimizing antibodies in mammalian cells, and eventually enable directed evolution of other complex proteins and cellular therapeutics. ER -