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

• Published in: International journal of molecular sciences 2021-09, Vol.22 (19), p.10570
• Main Authors: Kato, Yuya, Tabata, Hirotaka, Sato, Kumiko, Nakamura, Mariko, Saito, Izumu, Nakanishi, Tomoko
• Format: Article
• Language: English
• Subjects: Adenoviridae - genetics; Adenoviridae - physiology; adenovirus vector; Adenoviruses; Carcinoma, Hepatocellular - genetics; Carcinoma, Hepatocellular - therapy; Carcinoma, Hepatocellular - virology; Cell Line, Tumor; Chromosome deletion; Chromosomes; Cirrhosis; Cloning; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9; Drug resistance; Editing; Expression vectors; Gene deletion; Gene Editing - methods; Genetic Vectors - genetics; Genome editing; Genomes; gRNA; HEK293 Cells; Hep G2 Cells; Hepatitis B; hepatitis B virus (HBV); Hepatitis B virus - genetics; Hepatitis B virus - metabolism; Hepatitis B, Chronic - genetics; Hepatitis B, Chronic - therapy; Hepatitis B, Chronic - virology; Hepatocellular carcinoma; Humans; In vivo methods and tests; Liver cancer; Liver cirrhosis; Liver Neoplasms - genetics; Liver Neoplasms - therapy; Liver Neoplasms - virology; multiplex guide RNAs; Multiplexing; Nucleotide sequence; Nucleotides; Patients; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Trans-Activators - genetics; Trans-Activators - metabolism; Viral Regulatory and Accessory Proteins - genetics; Viral Regulatory and Accessory Proteins - metabolism; Virus Replication - genetics
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms221910570

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.