[P002] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P01-142] Sorting-free enrichment of biallelic two-copy recombinase-mediated cassette exchange (RMCE) integrants in GS-KO CHO cells via SiMPl-GS

○JaeJun Lee¹, Chansik Yoon², Yujin Kim³, Gyun Min Lee^1,3 (1. Department of Biological Sciences, KAIST, Daejeon, Republic of Korea (Korea), 2. Center for Cellular Therapies and Cancer Immunology, The University of Texas Southwestern Medical Center, Dallas, TX (USA), 3. Graduate School of Engineering Biology, KAIST, Daejeon, Republic of Korea (Korea))

Keywords:

Site-specific integration (SSI),Recombinase-mediated cassette exchange (RMCE),GS-KO CHO cells,SiMPl-GS (split-intein GS),Biallelic two-copy integration

Site-specific integration (SSI) using recombinase-mediated cassette exchange (RMCE) provides a rational route to stable and predictable expression in CHO cell line development. However, single-copy SSI often falls short of practical manufacturing targets, motivating multicopy SSI strategies. Achieving controlled multicopy integration while maintaining genotypic clarity and minimizing random integration remains challenging, and it can increase engineering and selection complexity. To address this, we established a biallelic SSI/RMCE platform in glutamine synthetase knockout (GS-KO) CHO cells by targeting both alleles of the well-characterized T2 hotspot (chromosome 3, C6orf62 locus) with an mCherry landing pad and performing cassette exchange using Bxb1 recombinase. In this design, mCherry serves as an exchange indicator, enabling efficient estimation of RMCE performance through mCherry dropout while providing an orthogonal readout to genotyping. The platform supports direct interpretation of two-copy exchange outcomes at the single-clone level and is compatible with production cassettes. For sorting-free enrichment of true exchange products, we implemented SiMPl-GS, an AND-gate synthetic selection module based on split-intein-mediated reconstitution of GS. To optimize this system for the SSI setting, we re-evaluated GS split-site design and selected a new split between residues 72 and 73 based on superior recovery in transient screening. By coupling GS reconstitution to survival under glutamine-free selection, this design provides stringent enrichment and may further favor outgrowth of cells with higher functional GS activity. Using an etanercept production cassette as a model insert, conventional full-length GS selection produced 15.7% mCherry loss, whereas SiMPl-GS selection produced 99.3% mCherry loss. Junction PCR genotyping confirmed biallelic (two-copy) cassette exchange in 8/55 clones for full-GS and 51/54 clones for SiMPl-GS. SiMPl-GS therefore enables efficient recovery of correctly exchanged two-copy integrants without flow sorting. Importantly, antibiotic resistance markers are not required for maintenance of the final producer cell line, supporting a streamlined GS-based selection workflow. Building on this two-copy SSI framework, we designed monoclonal antibody RMCE donor architectures compatible with biallelic exchange to enable efficient LC/HC co-expression and straightforward interpretation from exchange to production.