Ectopic expression of factor VIII in MSCs and hepatocytes derived from rDNA targeted hESCs

Sun, Qiuhong; Liu, Xionghao; Wu, Yong; Niu, Wenbin; Long, Panpan; Liu, Jing; Lei, Ming; Hu, Youjin; Wu, Lingqian; Li, Zhuo

doi:10.1016/j.cca.2018.08.007

Cited by 2 publications

(1 citation statement)

References 42 publications

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“…After co-transfection into mESCs with paired sgRNA-Cas9n and pMrnF9, in which pMrnF9 serves as the donor template, 16 out 24 mESCs clones were verified to be rDNA site-specific targeting. The relative targeting efficiency reached about 66.7%, demonstrating that with the assistance of sgRNA-Cas9n in the rDNA region, HDR-mediated gene targeting could be achieved at a high level even in stem cells, compared with our previous studies [ 27 ]. Except the main function of sgRNA-Cas9n, the special rDNA properties and the promoter trap strategy should also contribute to promote the relative targeting efficiency.…”

Section: Discussionmentioning

confidence: 60%

Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs

Wang

Zhao

Duan

et al. 2018

IJMS

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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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Section: Discussionmentioning

confidence: 60%

Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs

Wang

Zhao

Duan

et al. 2018

IJMS

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Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci

Zhang,

Van Treeck,

Horton

et al. 2024

Nat Biotechnol

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Current approaches for inserting autonomous transgenes into the genome, such as CRISPR–Cas9 or virus-based strategies, have limitations including low efficiency and high risk of untargeted genome mutagenesis. Here, we describe precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro transcribed RNAs: messenger RNA encoding avian R2 retroelement-protein and template RNA encoding a transgene of length validated up to 4 kb. The R2 protein coordinately recognizes the target site, nicks one strand at a precise location and primes complementary DNA synthesis for stable transgene insertion. With a cultured human primary cell line, over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. PRINT advantages include no extragenomic DNA, limiting risk of deleterious mutagenesis and innate immune responses, and the relatively low cost, rapid production and scalability of RNA-only delivery.

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Ectopic expression of factor VIII in MSCs and hepatocytes derived from rDNA targeted hESCs

Cited by 2 publications

References 42 publications

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

Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci

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