Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing
Introduction: Hemophilia A (HA) is the most common genetic bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). As the second predominant pathogenic mutation in hemophilia A severe patients, F8 Intron one inversion (Inv1) completely splits the F8 gene into t...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-03-01
|
| Series: | Frontiers in Genetics |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fgene.2023.1115831/full |
| _version_ | 1811157898200350720 |
|---|---|
| author | Zhiqing Hu Yong Wu Rou Xiao Junya Zhao Yan Chen Lingqian Wu Miaojin Zhou Desheng Liang |
| author_facet | Zhiqing Hu Yong Wu Rou Xiao Junya Zhao Yan Chen Lingqian Wu Miaojin Zhou Desheng Liang |
| author_sort | Zhiqing Hu |
| collection | DOAJ |
| description | Introduction: Hemophilia A (HA) is the most common genetic bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). As the second predominant pathogenic mutation in hemophilia A severe patients, F8 Intron one inversion (Inv1) completely splits the F8 gene into two parts and disrupts the F8 transcription, resulting in no FVIII protein production. The part which contains exon 2-exon 26 covers 98% of F8 coding region.Methods: We hypothesized that in situ genetic manipulation of F8 to add a promoter and exon one before the exon two could restore the F8 expression. The donor plasmid included human alpha 1-antitrypsin (hAAT) promoter, exon one and splicing donor site (SD) based on homology-mediated end joining (HMEJ) strategy was targeted addition in hemophilia A patient-derived induced pluripotent stem cell (HA-iPSCs) using CRISPR/Cas9. The iPSCs were differentiated into hepatocyte-like cells (HPLCs).Results: The hAAT promoter and exon one were targeted addition in HA-iPSCs with a high efficiency of 10.19% via HMEJ. The FVIII expression, secretion, and activity were detected in HPLCs derived from gene-targeted iPSCs.Discussion: Thus, we firstly rescued the 140 kb reversion mutation by gene addition of a 975 bp fragment in the HA-iPSCs with Inv1 mutation, providing a promising gene correction strategy for genetic disease with large sequence variants. |
| first_indexed | 2024-04-10T05:14:09Z |
| format | Article |
| id | doaj.art-c661de3703ae4fd282b5bbe3040bb26d |
| institution | Directory Open Access Journal |
| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-04-10T05:14:09Z |
| publishDate | 2023-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genetics |
| spelling | doaj.art-c661de3703ae4fd282b5bbe3040bb26d2023-03-09T04:57:39ZengFrontiers Media S.A.Frontiers in Genetics1664-80212023-03-011410.3389/fgene.2023.11158311115831Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editingZhiqing Hu0Yong Wu1Rou Xiao2Junya Zhao3Yan Chen4Lingqian Wu5Miaojin Zhou6Desheng Liang7Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, ChinaShenzhen Baoan Women’s and Children’s Hospital, Jinan University, Shenzhen, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha, ChinaIntroduction: Hemophilia A (HA) is the most common genetic bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). As the second predominant pathogenic mutation in hemophilia A severe patients, F8 Intron one inversion (Inv1) completely splits the F8 gene into two parts and disrupts the F8 transcription, resulting in no FVIII protein production. The part which contains exon 2-exon 26 covers 98% of F8 coding region.Methods: We hypothesized that in situ genetic manipulation of F8 to add a promoter and exon one before the exon two could restore the F8 expression. The donor plasmid included human alpha 1-antitrypsin (hAAT) promoter, exon one and splicing donor site (SD) based on homology-mediated end joining (HMEJ) strategy was targeted addition in hemophilia A patient-derived induced pluripotent stem cell (HA-iPSCs) using CRISPR/Cas9. The iPSCs were differentiated into hepatocyte-like cells (HPLCs).Results: The hAAT promoter and exon one were targeted addition in HA-iPSCs with a high efficiency of 10.19% via HMEJ. The FVIII expression, secretion, and activity were detected in HPLCs derived from gene-targeted iPSCs.Discussion: Thus, we firstly rescued the 140 kb reversion mutation by gene addition of a 975 bp fragment in the HA-iPSCs with Inv1 mutation, providing a promising gene correction strategy for genetic disease with large sequence variants.https://www.frontiersin.org/articles/10.3389/fgene.2023.1115831/fullhemophilia AF8 intron 1 inversionin situ gene additionhomology-mediated end joininghepatocyte-like cells |
| spellingShingle | Zhiqing Hu Yong Wu Rou Xiao Junya Zhao Yan Chen Lingqian Wu Miaojin Zhou Desheng Liang Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing Frontiers in Genetics hemophilia A F8 intron 1 inversion in situ gene addition homology-mediated end joining hepatocyte-like cells |
| title | Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing |
| title_full | Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing |
| title_fullStr | Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing |
| title_full_unstemmed | Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing |
| title_short | Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing |
| title_sort | correction of f8 intron 1 inversion in hemophilia a patient specific ipscs by crispr cas9 mediated gene editing |
| topic | hemophilia A F8 intron 1 inversion in situ gene addition homology-mediated end joining hepatocyte-like cells |
| url | https://www.frontiersin.org/articles/10.3389/fgene.2023.1115831/full |
| work_keys_str_mv | AT zhiqinghu correctionoff8intron1inversioninhemophiliaapatientspecificipscsbycrisprcas9mediatedgeneediting AT yongwu correctionoff8intron1inversioninhemophiliaapatientspecificipscsbycrisprcas9mediatedgeneediting AT rouxiao correctionoff8intron1inversioninhemophiliaapatientspecificipscsbycrisprcas9mediatedgeneediting AT junyazhao correctionoff8intron1inversioninhemophiliaapatientspecificipscsbycrisprcas9mediatedgeneediting AT yanchen correctionoff8intron1inversioninhemophiliaapatientspecificipscsbycrisprcas9mediatedgeneediting AT lingqianwu correctionoff8intron1inversioninhemophiliaapatientspecificipscsbycrisprcas9mediatedgeneediting AT miaojinzhou correctionoff8intron1inversioninhemophiliaapatientspecificipscsbycrisprcas9mediatedgeneediting AT deshengliang correctionoff8intron1inversioninhemophiliaapatientspecificipscsbycrisprcas9mediatedgeneediting |