HuH-7 knockout cell line | 10x Efficiency

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HuH-7 knockout cell line | 10x Efficiency

Definition of HuH-7 cell:

 

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 cell. 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 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 knockou 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 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 a overexpression HuH-7 cell line. Among 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 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 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. Lin et al (2014) 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, CRISPR/Cas9 system could disrupt the HBV templates both in vitro and in vivo and may have a potential in eradicating persistent HBV infection. 

 

CRISPR/Cas9 knockout HuH-7 cells strategies

 

 

Workflow of knockout HuH-7 cell line

 

References:

1. C. Hsu, T. Tokiwa, W. Bennett, R.A. Metcalf, J.A. Welsh, T. Sun, C.C. Harris, p53 gene mutation and integrated hepatitis B viral DNA sequences in human liver cancer cell lines, Carcinogenesis, Volume 14, Issue 5, May 1993, Pages 987–992.

2. Masaki Kawamoto, Toshiyuki Yamaji, Kyoko Saito, Kazuhiro Satomura, Toshinori Endo, Masayoshi Fukasawa, Kentaro Hanada, Naoki Osada. Identification of Characteristic Genomic Markers in Human Hepatoma Huh7 and Huh7.5.1-8 Cell Lines. bioRxiv 2020.02.17.953281

3. Casey R. Dorr, Rory P. Remmel, Amutha Muthusamy, James Fisher, Branden S. Moriarity, Kazuto Yasuda, Baolin Wu, Weihua Guan, Erin G. Schuetz, William S. Oetting, Pamala A. Jacobson, and Ajay K. Israni. Drug Metabolism and Disposition August 2017, 45 (8) 957-965

4. The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo. Su-Ru Lin, Hung-Chih Yang, Yi-Ting Kuo, Chun-Jen Liu, Ta-Yu Yang, Ku-Chun Sung, You-Yu Lin, Huang-Yi Wang, Chih-Chiang Wang, Yueh-Chi Shen, Fang-Yi Wu, Jia-Horng Kao, Ding-Shinn Chen, Pei-Jer Chen.

5. Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication. Jie Wang, Zhong-Wei Xu, Shuang Liu, Rui-Yang Zhang, Shan-Long Ding, Xiao-Meng Xie, Lu Long, Xiang-Mei Chen, Hui Zhuang, and Feng-Min Lu.

6. The CRISPR/Cas9 genome editing methodology as a weapon against human viruses. Martyn K. White, Wenhui Hu, and Kamel Khalili*.