Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs.
Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs.
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配对的 CRISPR/Cas9 切口酶介导 F9 高效位点特异性整合到小鼠 ESC 的 rDNA 基因座中。
DOI：
10.3390/ijms19103035
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发表时间：
2018-10-05
影响因子：
5.6
通讯作者：
Liang D
中科院分区：
文献类型：
--
作者：
Wang Y;Zhao J;Duan N;Liu W;Zhang Y;Zhou M;Hu Z;Feng M;Liu X;Wu L;Li Z;Liang D
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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影响因子：
7
作者：
Cho SW;Kim S;Kim Y;Kweon J;Kim HS;Bae S;Kim JS
通讯作者：
Kim JS
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
12.4
作者：
Lisowski, Leszek;Lau, Ashley;Kay, Mark A.
通讯作者：
Kay, Mark A.
靶向人类间充质干细胞 rDNA 位点的非病毒基因
影响因子：
--
作者：
Hu Y;Liu X;Long P;Xiao D;Cun J;Li Z;Xue J;Wu Y;Luo S;Wu L;Liang D
通讯作者：
Liang D
影响因子：
4
作者：
Lu Xiao-Jie;Xue Hui-Ying;Ji Li-Juan
通讯作者：
Ji Li-Juan