Gene
Department of Hematology, The Second Hospital of Shanxi Medical University, Taiyuan, China. Electronic address:
Published: February 2024
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.
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http://dx.doi.org/10.1016/j.gene.2023.148038 | DOI Listing |
Cancer Immunol Res
January 2025
Baylor College of Medicine, Houston, TX, United States.
Natural killer T cells (NKTs) are a promising platform for cancer immunotherapy, but few genes involved in regulation of NKT therapeutic activity have been identified. To find regulators of NKT functional fitness, we developed a CRISPR/Cas9-based mutagenesis screen that employs a guide RNA (gRNA) library targeting 1,118 immune-related genes. Unmodified NKTs and NKTs expressing a GD2-specific chimeric antigen receptor (GD2.
View Article and Find Full Text PDFMol Biol Evol
January 2025
Key Laboratory of Genetic Evolution & Animal Models, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China.
Our recent investigations on the function of Drosophila CG11700 and CG32744 (Ubi-p5E) genes using CRISPR/Cas9 deletion technology could not repeat or confirm the results on CG11700 shown in our previous study which was based on P-element excision assay (Zhan et al. 2012). Here by CRISPR/Cas9 editing, we generated mutants of CG32744 with the whole gene body fully deleted from the genome, and truncated mutants of CG11700 with N-terminal 103 aa deleted out of its total 301 aa peptide sequence.
View Article and Find Full Text PDFElife
January 2025
Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
Heritable fragile bone disorders (FBDs), ranging from multifactorial to rare monogenic conditions, are characterized by an elevated fracture risk. Validating causative genes and understanding their mechanisms remain challenging. We assessed a semi-high throughput zebrafish screening platform for rapid in vivo functional testing of candidate FBD genes.
View Article and Find Full Text PDFMol Ther
January 2025
Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, 53715, USA; Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, 53715, USA. Electronic address:
Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood.
View Article and Find Full Text PDFLife Sci
January 2025
Institute of Toxicology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany. Electronic address:
The protein deacetylase HDAC6 has been controversially linked to cancer cell proliferation and viral propagation. We analyzed whether a pharmacological depletion of HDAC6 with a recent proteolysis-targeting chimera (PROTAC) kills tumor cells. We show that low micromolar doses of the cereblon-based PROTAC TH170, but not its inactive analog TH170E, induce proteasomal degradation of HDAC6.
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