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

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 m...

Full description

Bibliographic Details
Published in:Cells
Main Author: Abuhamad A.Y.; Mohamad Zamberi N.N.; Sheen L.; Naes S.M.; Mohd Yusuf S.N.H.; Ahmad Tajudin A.; Mohtar M.A.; Amir Hamzah A.S.; Syafruddin S.E.
Format: Article
Language:English
Published: MDPI 2022
Online Access:https://www.scopus.com/inward/record.uri?eid=2-s2.0-85129759011&doi=10.3390%2fcells11101612&partnerID=40&md5=68c9b4c1a942d499b9f16d99718a8d01
id 2-s2.0-85129759011
spelling 2-s2.0-85129759011
Abuhamad A.Y.; Mohamad Zamberi N.N.; Sheen L.; Naes S.M.; Mohd Yusuf S.N.H.; Ahmad Tajudin A.; Mohtar M.A.; Amir Hamzah A.S.; Syafruddin S.E.
Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations
2022
Cells
11
10
10.3390/cells11101612
https://www.scopus.com/inward/record.uri?eid=2-s2.0-85129759011&doi=10.3390%2fcells11101612&partnerID=40&md5=68c9b4c1a942d499b9f16d99718a8d01
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. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
MDPI
20734409
English
Article
All Open Access; Gold Open Access
author Abuhamad A.Y.; Mohamad Zamberi N.N.; Sheen L.; Naes S.M.; Mohd Yusuf S.N.H.; Ahmad Tajudin A.; Mohtar M.A.; Amir Hamzah A.S.; Syafruddin S.E.
spellingShingle Abuhamad A.Y.; Mohamad Zamberi N.N.; Sheen L.; Naes S.M.; Mohd Yusuf S.N.H.; Ahmad Tajudin A.; Mohtar M.A.; Amir Hamzah A.S.; Syafruddin S.E.
Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations
author_facet Abuhamad A.Y.; Mohamad Zamberi N.N.; Sheen L.; Naes S.M.; Mohd Yusuf S.N.H.; Ahmad Tajudin A.; Mohtar M.A.; Amir Hamzah A.S.; Syafruddin S.E.
author_sort Abuhamad A.Y.; Mohamad Zamberi N.N.; Sheen L.; Naes S.M.; Mohd Yusuf S.N.H.; Ahmad Tajudin A.; Mohtar M.A.; Amir Hamzah A.S.; Syafruddin S.E.
title Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations
title_short Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations
title_full Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations
title_fullStr Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations
title_full_unstemmed Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations
title_sort Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations
publishDate 2022
container_title Cells
container_volume 11
container_issue 10
doi_str_mv 10.3390/cells11101612
url https://www.scopus.com/inward/record.uri?eid=2-s2.0-85129759011&doi=10.3390%2fcells11101612&partnerID=40&md5=68c9b4c1a942d499b9f16d99718a8d01
description 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. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
publisher MDPI
issn 20734409
language English
format Article
accesstype All Open Access; Gold Open Access
record_format scopus
collection Scopus
_version_ 1809678025159081984