HuH-7 is a type of human liver cell line cancer cell line, originally taken from a liver tumor in a 56-year-old Japanese male in 1982. HuH-7 is an Adult hepatocellular carcinoma cell line, which has been used extensively in hepatitis C and dengue virus research, sometimes by using CRISPR/Cas9 technologies to create CRISPR knockout cells. These years, HuH-7 cells are primarily grown in the laboratory for research purposes, especially in research that involves hepatitis C knockout. The introduction of the Huh7 cell line permitted the screening of drug candidates against laboratory-cultured hepatitis C virus and permitted the development of new drugs against hepatitis C.
HuH-7 is an immortal cell line composed of epithelial-like and tumorigenic cells. The majority of HuH-7 cells are highly heterogeneous, with a chromosome number between 55 to 63. The cells are adherent to the surface of flasks or plates. According to previous studies, it is assumed that HuH-7 cells can produce alpha-fetoprotein, pancreatin alpha antibody, plasma ceruloplasmin, fibrinogen, fibronectin, etc. Therefore gene-editing knockout cells HuH-7 is helpful when it comes to research that involves these kinds of proteins.
Application of gene-editing in HuH-7 cell line:
1. CRISPR/Cas9 Genetic Modification of CYP3A5 *3 in HuH-7 Human Hepatocyte Cell Line
The HuH-7 cell line was derived from a hepatic carcinoma that can convert the substrate MDZ in cell culture to its metabolite products; however, HuH-7 cells are not very efficient at MDZ metabolism because they are homozygous for the slow metabolizing CYP3A5 *3 allele. Thus, there is a need to develop a gene knockout and point mutation liver cell line that mimics the rapid drug metabolism associated with the CYP3A5 *1 genotype in cell culture. Researchers hypothesized that by genetically modifying the HuH-7 cell line to the more metabolically active CYP3A5 *1/*1 or *1/*3 genotypes, the cells would have increased MDZ and Tac metabolic activity. To test the hypothesis, they used CRISPR/Cas9 bioengineering to develop and characterize new cell lines and then phenotypically evaluate the genotypes’ effects on MDZ and Tac metabolism. These newly engineered cells can be used as a parental cell line in future studies to assess the association of additional genetic variants with drug metabolism and the metabolism of other drugs.
2. Coexpression of HBV-specific gRNAs and Cas9 suppressed the production of HBV proteins in vitro
To examine whether the clustered regularly interspaced short palindromic repeats CRISPR/Cas9 system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in HuH-7 cells transfected with an HBV-expression vector, which created an overexpression HuH-7 cell line. Among the eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, researchers further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in a reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.
3. Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication
The destruction of HBV-expressing vector was examined in HuH-7 cells co-transfected with dual-gRNAs and HBV-expressing vector using PCR and sequencing method, and the destruction of cccDNA was examined in HepAD38 cells using KCl precipitation, PSAD digestion, rolling circle amplification and quantitative PCR combined method. The cytotoxicity of these gRNAs was assessed by a mitochondrial tetrazolium assay. The results suggested that the CRISPR/Cas9 system could efficiently destroy HBV expressing templates (genotypes A-D) without apparent cytotoxicity. It may be a potential approach for eradication of persistent HBV cccDNA in chronic HBV infection patients.
4. The CRISPR/Cas9 genome editing methodology as a weapon against human viruses
Curing chronic HBV infection will require the specific eradication of the persistent HBV cccDNA from infected cells. Scientists designed eight gRNAs against HBV and showed that the CRISPR/Cas9 system significantly reduced the production of HBV core and HBsAg proteins in the HuH-7 cells transfected with an HBV-expression vector. Further, this system could cleave intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo in a mouse model resulting in a reduction in serum HBsAg level. Thus, the CRISPR/Cas9 system could disrupt the HBV templates both in vitro and in vivo and may have the potential in eradicating persistent HBV infection.
Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.
Make genome editing easier is the goal of Ubigene. We developed CRISPR-U™ (based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, and CRISPR-U™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. With CRISPR-U™, Ubigene has successfully edit genes on more than 100 cell lines.
Ubigene developed CRISPR-B™ which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Easily achieve microbial gene knockout (KO), point mutation (PM), and knockin (KI).
Ubigene has more than 400 types of primary cells, including epithelial cells, endothelial cells, smooth muscle cells, and fibroblasts from different species, such as human, rat, and mouse. We can provide a validation report for each primary cell. Our primary cells have been widely used in many research institutes and pharmaceutical enterprises.
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