Faculty, Staff and Student Publications
Publication Date
12-11-2023
Journal
Nucleic Acids Research
Abstract
Mammalian cells carrying defined genetic variations have shown great potentials in both fundamental research and therapeutic development. However, their full use was limited by lack of a robust method to construct large monoclonal high-quality combinatorial libraries. This study developed cell cycle arrested recombinase-mediated cassette exchange (aRMCE), able to provide monoclonality, precise genomic integration and uniform transgene expression. Via optimized nocodazole-mediated mitotic arrest, 20% target gene replacement efficiency was achieved without antibiotic selection, and the improved aRMCE efficiency was applicable to a variety of tested cell clones, transgene targets and transfection methods. As a demonstration of this versatile method, we performed directed evolution of fragment crystallizable (Fc), for which error-prone libraries of over 107 variants were constructed and displayed as IgG on surface of CHO cells. Diversities of constructed libraries were validated by deep sequencing, and panels of novel Fc mutants were identified showing improved binding towards specific Fc gamma receptors and enhanced effector functions. Due to its large cargo capacity and compatibility with different mutagenesis approaches, we expect this mammalian cell platform technology has broad applications for directed evolution, multiplex genetic assays, cell line development and stem cell engineering.
Keywords
Cricetinae, Animals, Recombinases, Cricetulus, CHO Cells, Transfection, Cell Cycle
DOI
10.1093/nar/gkad1001
PMID
37941133
PMCID
PMC10711435
PubMedCentral® Posted Date
11-6-2023
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes