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Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology

BACKGROUND: Hemophilia B is a rare inherited genetic bleeding disorder caused by a deficiency or lack of coagulation factor IX, the gene for which (F9) is located on the X chromosome. Hemophilia B is currently incurable and the standard treatment is coagulation factor replacement therapy. Although g...

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Autores principales: Ma, Yanchun, Sun, Wenwen, Zhao, Lidong, Yao, Mingze, Wu, Changxin, Su, Pengfei, Yang, Linhua, Wang, Gang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9327398/
https://www.ncbi.nlm.nih.gov/pubmed/35883203
http://dx.doi.org/10.1186/s13287-022-03036-2
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author Ma, Yanchun
Sun, Wenwen
Zhao, Lidong
Yao, Mingze
Wu, Changxin
Su, Pengfei
Yang, Linhua
Wang, Gang
author_facet Ma, Yanchun
Sun, Wenwen
Zhao, Lidong
Yao, Mingze
Wu, Changxin
Su, Pengfei
Yang, Linhua
Wang, Gang
author_sort Ma, Yanchun
collection PubMed
description BACKGROUND: Hemophilia B is a rare inherited genetic bleeding disorder caused by a deficiency or lack of coagulation factor IX, the gene for which (F9) is located on the X chromosome. Hemophilia B is currently incurable and the standard treatment is coagulation factor replacement therapy. Although gene therapy has the potential to cure hemophilia, significant barriers are still needed to be overcome, e.g., off-target effects and immunoreactivity, so new approaches must be explored. Nonsense mutations account for 8% of all the hemophilia B mutation types and can result in the development of coagulation factor inhibitors. In this study, CRISPR/Cas9 technology was used to construct a mouse embryonic stem cell model with a hemophilia B nonsense mutation (F9 c.223C > T) in humans to investigate the pathogenesis and treatment of nonsense mutations in hemophilia B. METHODS: First, a donor plasmid with a mutation (F9 c.223 C > T) and sgRNAs were constructed. Second, both the donor plasmid and the px330-sgRNA were electroporated into mouse embryonic stem cell, and the mutant cells were then screened using puromycin and red fluorescence. Third, the mutant cell lines were tested for pluripotency and the ability to differentiate into three layers. Finally, the effect of mutation on gene function was studied in the differentiation system. RESULTS: The mutant vector and effective sgRNA were constructed, and the mutant cell line was screened. This mutant cell line exhibited pluripotency and the ability to differentiate into three layers. This point mutation affects F9 expression at both the RNA and protein levels in the differentiation system. CONCLUSIONS: The mutant cell line obtained in the current study had a single-base mutation rather than a base deletion or insertion in the exon, which is more similar to clinical cases. In addition, the mutant has the characteristics of mouse embryonic stem cells, and this point mutation affects F9 gene transcription and translation, which can be used as a disease model for studying the pathogenesis and treatment of hemophilia at the stem cell level. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13287-022-03036-2.
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spelling pubmed-93273982022-07-28 Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology Ma, Yanchun Sun, Wenwen Zhao, Lidong Yao, Mingze Wu, Changxin Su, Pengfei Yang, Linhua Wang, Gang Stem Cell Res Ther Research BACKGROUND: Hemophilia B is a rare inherited genetic bleeding disorder caused by a deficiency or lack of coagulation factor IX, the gene for which (F9) is located on the X chromosome. Hemophilia B is currently incurable and the standard treatment is coagulation factor replacement therapy. Although gene therapy has the potential to cure hemophilia, significant barriers are still needed to be overcome, e.g., off-target effects and immunoreactivity, so new approaches must be explored. Nonsense mutations account for 8% of all the hemophilia B mutation types and can result in the development of coagulation factor inhibitors. In this study, CRISPR/Cas9 technology was used to construct a mouse embryonic stem cell model with a hemophilia B nonsense mutation (F9 c.223C > T) in humans to investigate the pathogenesis and treatment of nonsense mutations in hemophilia B. METHODS: First, a donor plasmid with a mutation (F9 c.223 C > T) and sgRNAs were constructed. Second, both the donor plasmid and the px330-sgRNA were electroporated into mouse embryonic stem cell, and the mutant cells were then screened using puromycin and red fluorescence. Third, the mutant cell lines were tested for pluripotency and the ability to differentiate into three layers. Finally, the effect of mutation on gene function was studied in the differentiation system. RESULTS: The mutant vector and effective sgRNA were constructed, and the mutant cell line was screened. This mutant cell line exhibited pluripotency and the ability to differentiate into three layers. This point mutation affects F9 expression at both the RNA and protein levels in the differentiation system. CONCLUSIONS: The mutant cell line obtained in the current study had a single-base mutation rather than a base deletion or insertion in the exon, which is more similar to clinical cases. In addition, the mutant has the characteristics of mouse embryonic stem cells, and this point mutation affects F9 gene transcription and translation, which can be used as a disease model for studying the pathogenesis and treatment of hemophilia at the stem cell level. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13287-022-03036-2. BioMed Central 2022-07-26 /pmc/articles/PMC9327398/ /pubmed/35883203 http://dx.doi.org/10.1186/s13287-022-03036-2 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Ma, Yanchun
Sun, Wenwen
Zhao, Lidong
Yao, Mingze
Wu, Changxin
Su, Pengfei
Yang, Linhua
Wang, Gang
Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology
title Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology
title_full Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology
title_fullStr Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology
title_full_unstemmed Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology
title_short Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology
title_sort generation of an mesc model with a human hemophilia b nonsense mutation via crispr/cas9 technology
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9327398/
https://www.ncbi.nlm.nih.gov/pubmed/35883203
http://dx.doi.org/10.1186/s13287-022-03036-2
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