High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis

Nucleic Acids Res. 2018 Aug 21;46(14):7436-7449. doi: 10.1093/nar/gky550.

Abstract

Antibody engineering is often performed to improve therapeutic properties by directed evolution, usually by high-throughput screening of phage or yeast display libraries. Engineering antibodies in mammalian cells offer 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 HDR to a robust efficiency of 15-35% and combining mammalian display screening with next-generation sequencing, 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies / genetics
  • Antibodies / immunology*
  • Antibodies / metabolism
  • Antibody Affinity / genetics
  • Antibody Affinity / immunology
  • Base Sequence
  • CRISPR-Cas Systems*
  • Cell Line
  • DNA Breaks, Double-Stranded
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Hybridomas
  • Mutagenesis, Site-Directed*
  • Oligonucleotides / genetics
  • Oligonucleotides / metabolism
  • Protein Engineering / methods*
  • Recombinational DNA Repair

Substances

  • Antibodies
  • Oligonucleotides