Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs
Hemophilia B (HB) is an X-linked recessive bleeding disorder, caused by F9 gene deficiency. Gene therapy combined with the CRISPR/Cas9 technology offers a potential cure for hemophilia B. Now the Cas9 nickase (Cas9n) shows a great advantage in reducing off-target effect compared with wild-type Cas9....
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2018-10-01
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| Series: | International Journal of Molecular Sciences |
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| author | Yanchi Wang Junya Zhao Nannan Duan Wei Liu Yuxuan Zhang Miaojin Zhou Zhiqing Hu Mai Feng Xionghao Liu Lingqian Wu Zhuo Li Desheng Liang |
| author_facet | Yanchi Wang Junya Zhao Nannan Duan Wei Liu Yuxuan Zhang Miaojin Zhou Zhiqing Hu Mai Feng Xionghao Liu Lingqian Wu Zhuo Li Desheng Liang |
| author_sort | Yanchi Wang |
| collection | DOAJ |
| description | Hemophilia B (HB) is an X-linked recessive bleeding disorder, caused by F9 gene deficiency. Gene therapy combined with the CRISPR/Cas9 technology offers a potential cure for hemophilia B. Now the Cas9 nickase (Cas9n) shows a great advantage in reducing off-target effect compared with wild-type Cas9. In this study, we found that in the multicopy ribosomal DNA (rDNA) locus, the homology directed recombination (HDR) efficiency induced by sgRNA-Cas9n was much higher than sgRNA-Cas9, meanwhile without off-target in six predicted sites. After co-transfection into mESCs with sgRNA-Cas9n and a non-viral rDNA targeting vector pMrnF9, harboring the homology donor template and the human F9 expression cassette, a recombination efficiency of 66.7% was achieved and all targeted clones were confirmed to be site-specific integration of F9 in the rDNA locus by PCR and southern blotting. Targeted mESCs retained the main pluripotent properties and were then differentiated into hepatic progenitor like cells (HPLCs) and mature hepatocytes, which were characterized by hepatic markers and functional assays. Importantly, the differentiated cells could transcribe exogenous F9 and secrete coagulation factor IX (FIX) proteins, suggesting active transcription and stable inheritance of transgenes in the rDNA locus. After intrasplenical transplantation in severe combined immune deficiency (SCID) mice, targeted HPLCs could survive and migrate from spleen to liver, resulting in secretion of exogenous FIX into blood. In summary, we demonstrate an efficient and site-specific gene targeting strategy in rDNA locus for stem cell-based gene therapy for hemophilia B. |
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| language | English |
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| publishDate | 2018-10-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-05aea0edeb194fbfa659e5cef3a7075d2022-12-22T03:11:05ZengMDPI AGInternational Journal of Molecular Sciences1422-00672018-10-011910303510.3390/ijms19103035ijms19103035Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCsYanchi Wang0Junya Zhao1Nannan Duan2Wei Liu3Yuxuan Zhang4Miaojin Zhou5Zhiqing Hu6Mai Feng7Xionghao Liu8Lingqian Wu9Zhuo Li10Desheng Liang11Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha 410000, ChinaHemophilia B (HB) is an X-linked recessive bleeding disorder, caused by F9 gene deficiency. Gene therapy combined with the CRISPR/Cas9 technology offers a potential cure for hemophilia B. Now the Cas9 nickase (Cas9n) shows a great advantage in reducing off-target effect compared with wild-type Cas9. In this study, we found that in the multicopy ribosomal DNA (rDNA) locus, the homology directed recombination (HDR) efficiency induced by sgRNA-Cas9n was much higher than sgRNA-Cas9, meanwhile without off-target in six predicted sites. After co-transfection into mESCs with sgRNA-Cas9n and a non-viral rDNA targeting vector pMrnF9, harboring the homology donor template and the human F9 expression cassette, a recombination efficiency of 66.7% was achieved and all targeted clones were confirmed to be site-specific integration of F9 in the rDNA locus by PCR and southern blotting. Targeted mESCs retained the main pluripotent properties and were then differentiated into hepatic progenitor like cells (HPLCs) and mature hepatocytes, which were characterized by hepatic markers and functional assays. Importantly, the differentiated cells could transcribe exogenous F9 and secrete coagulation factor IX (FIX) proteins, suggesting active transcription and stable inheritance of transgenes in the rDNA locus. After intrasplenical transplantation in severe combined immune deficiency (SCID) mice, targeted HPLCs could survive and migrate from spleen to liver, resulting in secretion of exogenous FIX into blood. In summary, we demonstrate an efficient and site-specific gene targeting strategy in rDNA locus for stem cell-based gene therapy for hemophilia B.http://www.mdpi.com/1422-0067/19/10/3035CRISPR/Cas9 nickasegene targetingHemophilia Bribosomal DNAgene therapymESCshepatic progenitor like cellsintrasplenic transplantation |
| spellingShingle | Yanchi Wang Junya Zhao Nannan Duan Wei Liu Yuxuan Zhang Miaojin Zhou Zhiqing Hu Mai Feng Xionghao Liu Lingqian Wu Zhuo Li Desheng Liang Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs International Journal of Molecular Sciences CRISPR/Cas9 nickase gene targeting Hemophilia B ribosomal DNA gene therapy mESCs hepatic progenitor like cells intrasplenic transplantation |
| title | Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs |
| title_full | Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs |
| title_fullStr | Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs |
| title_full_unstemmed | Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs |
| title_short | Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs |
| title_sort | paired crispr cas9 nickases mediate efficient site specific integration of f9 into rdna locus of mouse escs |
| topic | CRISPR/Cas9 nickase gene targeting Hemophilia B ribosomal DNA gene therapy mESCs hepatic progenitor like cells intrasplenic transplantation |
| url | http://www.mdpi.com/1422-0067/19/10/3035 |
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