[1S06a] 15. Advanced Cell and Cell Culture Engineering for Biopharmaceuticals and Functional Food - Part 2

2:00 PM - 2:30 PM JST(5:00 AM - 5:30 AM UTC)

[1ACCE-06-KL] RMCE-mediated genome-wide CRISPR screening: Emerging strategies for engineering high-performance mammalian producers

○Gyun Min Lee¹ (1. KAIST (Korea))

Keywords:

CRISPR screening,RMCE,mammalian cells,therapeutic protein

Current mammalian cell engineering strategies to enhance therapeutic protein yield, quality, and manufacturing efficiency largely focus on manipulating known regulators of apoptosis, autophagy, glycosylation, and endoplasmic reticulum stress. However, critical production traits—including stress tolerance, secretion capacity, and glycan processing—are polygenic, epigenetically regulated, and highly context dependent. As a result, knowledge-driven engineering often yields clone-, product-, and process-specific improvements with limited generalizability. These complex phenotypes arise from interconnected regulatory networks that remain incompletely characterized, rendering rational target selection labor-intensive and unpredictable.The advent of CRISPR/Cas9 technology has enabled high-throughput functional genomics approaches to systematically uncover genes associated with desirable production phenotypes. In particular, pooled CRISPR-based screens have revealed unexpected regulators that would likely escape conventional hypothesis-driven strategies. Most pooled screens rely on lentiviral delivery of guide RNAs (gRNAs); however, lentiviral systems present challenges related to biosafety requirements, technical accessibility, and variable gRNA copy number integration despite careful control of multiplicity of infection.Recombinase-mediated cassette exchange (RMCE) provides a non-viral alternative that enables site-specific integration of single sgRNA cassettes into engineered landing pads at defined safe harbor loci. This approach ensures uniform single-copy insertion, minimizes positional effects, and improves signal-to-noise ratios compared to lentiviral delivery. Recent RMCE-based genome-wide CRISPR knockout and activation screens have identified novel regulators of industrially relevant stress responses in CHO and HEK293 cell platforms.In this presentation, I will discuss key design considerations for RMCE-mediated genome-wide CRISPR screening strategies aimed at improving therapeutic protein production in mammalian cells, with particular emphasis on CHO and HEK293 systems and on the strategic selection of phenotype-relevant screening readouts.