Evaluation of Prime Editing Efficiency in Human Immortalized MSC-TERT Cells with Osteogenic Potential for Modeling <i>FGFR2</i> -Linked Craniosynostosis

Craniosynostosis is a rare congenital bone condition where skull sutures fuse prematurely and is linked to mutations in over 60 genes. Generating mutation-specific in vitro models allows investigation of craniosynostosis-associated mutations without the need for patient-derived material or transgenic gene expression. Here, we developed a human in vitro disease model with the CRISPR-Cas9 prime editing variant, using an immortalized TERT-immortalized mesenchymal bone marrow-derived stem (MSC-TERT) cell line with osteogenic potential. MSC-TERT cells showed a higher resistance to prime editing, compared with HEK293FT cells. Addition of dnMLH1 and epegRNAs resulted in higher editing efficiencies in HEK293FT cells, but not in MSC-TERT cells. Prime editing efficiency varied between targeted loci and was found to be more efficient in nonadherent cells compared with adherent cells. Prime editing continued over 4 days in an isolated nonadherent HEK293FT culture. Our results present a foundation on the use of prime editing to establish FGFR2 mutation-specific in vitro models and their application in MSC-TERT cells.