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Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor
Base editing is an efficient and precise gene-editing technique, by which a single base can be changed without introducing double-strand breaks, and it is currently widely used in studies of various species. In this study, we used hA3A-BE3-Y130F to simultaneously introduce premature stop codons (TAG...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9140995/ https://www.ncbi.nlm.nih.gov/pubmed/35627220 http://dx.doi.org/10.3390/genes13050835 |
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author | Jiang, Haoyun Jing, Qiqi Yang, Qiang Qiao, Chuanmin Liao, Yaya Liu, Weiwei Xing, Yuyun |
author_facet | Jiang, Haoyun Jing, Qiqi Yang, Qiang Qiao, Chuanmin Liao, Yaya Liu, Weiwei Xing, Yuyun |
author_sort | Jiang, Haoyun |
collection | PubMed |
description | Base editing is an efficient and precise gene-editing technique, by which a single base can be changed without introducing double-strand breaks, and it is currently widely used in studies of various species. In this study, we used hA3A-BE3-Y130F to simultaneously introduce premature stop codons (TAG, TGA, and TAA) into three tumor suppressor genes, TP53, PTEN, and APC, in large white porcine fetal fibroblasts (PFFs). Among the isolated 290 single-cell colonies, 232 (80%) had premature stop codons in all the three genes. C−to−T conversion was found in 98.6%, 92.8%, and 87.2% of these cell colonies for TP53, PTEN, and APC, respectively. High frequencies of bystander C−to−T edits were observed within the editing window (positions 3–8), and there were nine (3.01%) clones with the designed simultaneous three-gene C−to−T conversion without bystander conversion. C−to−T conversion outside the editing window was found in 9.0%, 14.1%, and 26.2% of the 290 cell colonies for TP53, PTEN, and APC, respectively. Low-frequency C−to−G or C−to−A transversion occurred in APC. The mRNA levels of the three genes showed significant declines in triple-gene-mutant (Tri-Mut) cells as expected. No PTEN and a significantly lower (p < 0.05) APC protein expression were detected in Tri-Mut cells. Interestingly, the premature stop codon introduced into the TP53 gene did not eliminate the expression of its full-length protein in the Tri-Mut cells, suggesting that stop codon read-through occurred. Tri-Mut cells showed a significantly higher (p < 0.05) proliferation rate than WT cells. Furthermore, we identified 1418 differentially expressed genes (DEGs) between the Tri-Mut and WT groups, which were mainly involved in functions such as tumor progression, cell cycle, and DNA repair. This study indicates that hA3A-BE3-Y130F can be a powerful tool to create diverse knockout cell models without double-strand breaks (DSBs), with further possibilities to produce porcine models with various purposes. |
format | Online Article Text |
id | pubmed-9140995 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-91409952022-05-28 Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor Jiang, Haoyun Jing, Qiqi Yang, Qiang Qiao, Chuanmin Liao, Yaya Liu, Weiwei Xing, Yuyun Genes (Basel) Article Base editing is an efficient and precise gene-editing technique, by which a single base can be changed without introducing double-strand breaks, and it is currently widely used in studies of various species. In this study, we used hA3A-BE3-Y130F to simultaneously introduce premature stop codons (TAG, TGA, and TAA) into three tumor suppressor genes, TP53, PTEN, and APC, in large white porcine fetal fibroblasts (PFFs). Among the isolated 290 single-cell colonies, 232 (80%) had premature stop codons in all the three genes. C−to−T conversion was found in 98.6%, 92.8%, and 87.2% of these cell colonies for TP53, PTEN, and APC, respectively. High frequencies of bystander C−to−T edits were observed within the editing window (positions 3–8), and there were nine (3.01%) clones with the designed simultaneous three-gene C−to−T conversion without bystander conversion. C−to−T conversion outside the editing window was found in 9.0%, 14.1%, and 26.2% of the 290 cell colonies for TP53, PTEN, and APC, respectively. Low-frequency C−to−G or C−to−A transversion occurred in APC. The mRNA levels of the three genes showed significant declines in triple-gene-mutant (Tri-Mut) cells as expected. No PTEN and a significantly lower (p < 0.05) APC protein expression were detected in Tri-Mut cells. Interestingly, the premature stop codon introduced into the TP53 gene did not eliminate the expression of its full-length protein in the Tri-Mut cells, suggesting that stop codon read-through occurred. Tri-Mut cells showed a significantly higher (p < 0.05) proliferation rate than WT cells. Furthermore, we identified 1418 differentially expressed genes (DEGs) between the Tri-Mut and WT groups, which were mainly involved in functions such as tumor progression, cell cycle, and DNA repair. This study indicates that hA3A-BE3-Y130F can be a powerful tool to create diverse knockout cell models without double-strand breaks (DSBs), with further possibilities to produce porcine models with various purposes. MDPI 2022-05-07 /pmc/articles/PMC9140995/ /pubmed/35627220 http://dx.doi.org/10.3390/genes13050835 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Jiang, Haoyun Jing, Qiqi Yang, Qiang Qiao, Chuanmin Liao, Yaya Liu, Weiwei Xing, Yuyun Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor |
title | Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor |
title_full | Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor |
title_fullStr | Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor |
title_full_unstemmed | Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor |
title_short | Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor |
title_sort | efficient simultaneous introduction of premature stop codons in three tumor suppressor genes in pffs via a cytosine base editor |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9140995/ https://www.ncbi.nlm.nih.gov/pubmed/35627220 http://dx.doi.org/10.3390/genes13050835 |
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