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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 (<jats:italic>F9 c.223C<\/jats:italic>\u2009&gt;\u2009<jats:italic>T<\/jats:italic>) in humans to investigate the pathogenesis and treatment of nonsense mutations in hemophilia B.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>First, a donor plasmid with a mutation (<jats:italic>F9 c.223 C<\/jats:italic>\u2009&gt;\u2009<jats:italic>T<\/jats:italic>) 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.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>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.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>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 <jats:italic>F9<\/jats:italic> 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.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s13287-022-03036-2","type":"journal-article","created":{"date-parts":[[2022,7,27]],"date-time":"2022-07-27T13:48:54Z","timestamp":1658929734000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR\/Cas9 technology"],"prefix":"10.1186","volume":"13","author":[{"given":"Yanchun","family":"Ma","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenwen","family":"Sun","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lidong","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mingze","family":"Yao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Changxin","family":"Wu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pengfei","family":"Su","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Linhua","family":"Yang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2717-4575","authenticated-orcid":false,"given":"Gang","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2022,7,26]]},"reference":[{"key":"3036_CR1","doi-asserted-by":"publisher","first-page":"118969","DOI":"10.1016\/j.lfs.2020.118969","volume":"267","author":"A Ghaemi","year":"2021","unstructured":"Ghaemi A, Bagheri E, Abnous K, Taghdisi SM, Ramezani M, Alibolandi M. 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