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Targeted genome engineering based on CRISPR/Cas9 system to enhance FVIII expression in vitro.

Authors :
Zhao, Lidong
Fang, Shuai
Ma, Yanchun
Ren, Juan
Hao, Lixia
Wang, Lei
Yang, Jia
Lu, Xiaomei
Yang, Linhua
Wang, Gang
Source :
Gene. Feb2024, Vol. 896, pN.PAG-N.PAG. 1p.
Publication Year :
2024

Abstract

• The p18T-BDD-F8-V3 variant has high FVIII activity. • Targeted knock-in of BDD-hF8 cDNA at the mROSA26 locus was achieved by HDR or NHEJ, which significantly increased FVIII expression. • Gene editing was successfully achieved in vitro using CRISPR/Cas9. Hemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will make it possible to alter the expression of the F8 gene in hemophiliacs, while achieving a potential cure for the disease. Initially, we identified high-activity variants of FVIII and constructed donor plasmids using enzymatic digestion and ligation techniques. Subsequently, the donor plasmids were co-transfected with sgRNA-Cas9 protein into mouse Neuro-2a cells, followed by flow cytometry-based cell sorting and puromycin selection. Finally, BDD-hF8 targeted to knock-in the mROSA26 genomic locus was identified and validated for FVIII expression. We identified the p18T-BDD-F8-V3 variant with high FVIII activity and detected the strongest pX458-mROSA26-int1-sgRNA1 targeted cleavage ability and no cleavage events were found at potential off-target sites. Targeted knock-in of BDD-hF8 cDNA at the mROSA26 locus was achieved based on both HDR/NHEJ gene repair approaches, and high level and stable FVIII expression was obtained, successfully realizing gene editing in vitro. Knock-in of exogenous genes based on the CRISPR/Cas9 system targeting genomic loci is promising for the research and treatment of a variety of single-gene diseases. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
03781119
Volume :
896
Database :
Academic Search Index
Journal :
Gene
Publication Type :
Academic Journal
Accession number :
174665675
Full Text :
https://doi.org/10.1016/j.gene.2023.148038