Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations

Cells

Safaa M. Naes¹  Asilah Ahmad Tajudin²  Amir Syahir Amir Hamzah²  Asmaa Y. Abuhamad²  Ling Sheen³  Siti Nur Hasanah Mohd Yusuf³  Nurul Nadia Mohamad Zamberi³  Saiful Effendi Syafruddin³  M. Aiman Mohtar³ 

[1] Department of Biochemistry & Molecular Medicine, Faculty of Medicine, University Teknologi MARA, Cawangan Selangor, Kampus Sungai Buloh, Sungai Buloh 47000, Selangor, Malaysia;Nanobiotechnology Research Group, Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, Serdang 43400, Selangor, Malaysia;UKM Medical Molecular Biology Institute, University Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, Kuala Lumpur 56000, Malaysia;

关键词: prime editing;    CRISPR;    TP53;    missense mutation;    tumor suppressor;    breast cancer;   

DOI  :  10.3390/cells11101612

来源: DOAJ

【 摘 要 】

Breast cancer is the leading cause of cancer-related deaths in women. The aggressive breast cancer subtype is commonly linked to the genetic alterations in the TP53 tumor suppressor gene, predominantly the missense mutations. Robust experimental models are needed to gain better insights into these mutations’ molecular properties and implications in tumorigenesis. The generation of such models harboring the alterations is feasible with the CRISPR-based gene editing technology. Moreover, the development of new CRISPR applications, particularly DNA base and prime editing, has considerably improved the precision and versatility of gene editing. Here, we employed the prime editing tool to revert a TP53 missense C > T mutation (L194F) in a T47D luminal A breast cancer cell line. In parallel, this prime editing tool was also utilized to introduce the L194F mutation in HEK293T cells. To assess the prime editing efficiency in both cell lines, we first performed Sanger sequencing in the prime-edited cells pool and single cell-derived clones. However, the Sanger sequencing approach did not detect any base substitution in these cell lines. Next, by employing the more sensitive amplicon target sequencing, we managed to identify the expected substitution in these T47D and HEK293T cells, albeit the editing efficiency was low. In light of these findings, we discussed the technical aspects and provided suggestions for improve the prime editing workflow and efficiency for future experiments.

【 授权许可】

Unknown