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

Yanchi Wang,Desheng Liang,Lingqian Wu,Zhiqing Hu,Mai Feng,Nannan Duan,Zhuo Li,Yuxuan Zhang,Miaojin Zhou,Junya Zhao,Xionghao Liu,Wei Liu

doi:10.3390/ijms19103035

Yanchi Wang, Desheng Liang + Show 10 more

Open Access

https://doi.org/10.3390/ijms19103035

Copy DOI

Abstract

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.

Highlights

Hemophilia B is an X-linked congenital bleeding disorder resulting from a deficiency of the functional coagulation factor IX (FIX), encoded by the F9 gene
To facilitate the integration of exogenous genes into the mouse rDNA region via CRISPR/Cas9 nickase (Cas9n), which was a gift from Feng Zhang (Addgene plasmid #42335), we tried to design and screened the most effective sgRNAs around the targeted site—the 5646 locus of musculus ribosomal DNA complete
Among these four sgRNAs, sg1-Cas9n paired with sg6-Cas9n and sg2-Cas9n paired with sg5-Cas9n would function together to induce double strand break (DSB) with cohesive ends

Summary

Introduction

Hemophilia B is an X-linked congenital bleeding disorder resulting from a deficiency of the functional coagulation factor IX (FIX), encoded by the F9 gene. Since the size of the F9 gene is small and a slight increase of FIX levels will modify the bleeding diathesis, hemophilia B is relatively ideal for gene therapy. Based on viral vectors, especially adeno-associated virus vectors (AAV), several clinical trials have been conducted in small cohorts of hemophilia B patients. On the basis of gene targeting into stem cells via non-viral vectors, would be an attractive choice for gene therapy. This approach allows for the selection and removal of potential hazard mutagenesis and benefits from its less immunotoxicity. Specificity, gene expression duration, and safety led to an increased number of non-viral vector products entering clinical trials. One of the key points of this strategy in future clinical applications is to explore a safe genome site for integration of exogenous genes via gene targeting

Methods

Results

Conclusion