在具有成骨潜力的人永生MSC-TERT细胞中评估主要编辑效率，以模拟FGFR2连锁的颅神经病

摘要(中文译文)

颅缝早闭是一种罕见的先天性骨骼疾病，表现为颅骨骨缝过早融合，与超过60个基因的突变相关。构建突变特异性模型可在无需患者来源材料或转基因基因表达的情况下研究颅缝早闭相关突变。本研究利用具有成骨潜能的永生化TERT永生化间充质骨髓来源干细胞（MSC-TERT）细胞系，采用CRISPR-Cas9先导编辑（prime editing）变体构建了人类疾病模型。与HEK293FT细胞相比，MSC-TERT细胞对先导编辑表现出更高的抵抗性。加入dnMLH1和epegRNA后，HEK293FT细胞的编辑效率提高，但MSC-TERT细胞未见提高。先导编辑效率因靶位点而异，且非贴壁细胞较贴壁细胞更为高效。在分离的非贴壁HEK293FT培养物中，先导编辑持续超过4天。本研究结果为利用先导编辑建立突变特异性模型及其在MSC-TERT细胞中的应用奠定了基础。

Abstract

Craniosynostosis is a rare congenital bone condition where skull sutures fuse prematurely and is linked to mutations in over 60 genes. Generating mutation-specific models allows investigation of craniosynostosis-associated mutations without the need for patient-derived material or transgenic gene expression. Here, we developed a human 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 mutation-specific models and their application in MSC-TERT cells.

如何引用