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Updated: Jul 9, 2025
Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
Published on: November 3, 2020
Targeted genome engineering based on CRISPR/Cas9 system to enhance FVIII expression in vitro
Lidong Zhao1, Shuai Fang2, Yanchun Ma3
1Department of Hematology, The Second Hospital of Shanxi Medical University, Taiyuan, China; Department of Hematology, Linfen Central Hospital, Linfen, Shanxi, China.
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
