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Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

1
Laboratory of Virology, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Foundation, Shinagawa-ku, Tokyo 141-0021, Japan
2
Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
3
Center for Biomedical Research Resources, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan
4
Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan
*
Author to whom correspondence should be addressed.
Academic Editor: Luisa Barzon
Int. J. Mol. Sci. 2021, 22(19), 10570; https://doi.org/10.3390/ijms221910570
Received: 2 September 2021 / Revised: 22 September 2021 / Accepted: 24 September 2021 / Published: 29 September 2021
Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy. View Full-Text
Keywords: CRISPR/Cas9; genome editing; adenovirus vector; multiplex guide RNAs; hepatitis B virus (HBV); hepatocellular carcinoma; gene therapy CRISPR/Cas9; genome editing; adenovirus vector; multiplex guide RNAs; hepatitis B virus (HBV); hepatocellular carcinoma; gene therapy
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MDPI and ACS Style

Kato, Y.; Tabata, H.; Sato, K.; Nakamura, M.; Saito, I.; Nakanishi, T. Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient. Int. J. Mol. Sci. 2021, 22, 10570. https://doi.org/10.3390/ijms221910570

AMA Style

Kato Y, Tabata H, Sato K, Nakamura M, Saito I, Nakanishi T. Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient. International Journal of Molecular Sciences. 2021; 22(19):10570. https://doi.org/10.3390/ijms221910570

Chicago/Turabian Style

Kato, Yuya, Hirotaka Tabata, Kumiko Sato, Mariko Nakamura, Izumu Saito, and Tomoko Nakanishi. 2021. "Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient" International Journal of Molecular Sciences 22, no. 19: 10570. https://doi.org/10.3390/ijms221910570

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