title: Highly multiplexed CRISPR-Cas9-nuclease and Cas9-nickase vectors for inactivation of hepatitis B virus.
abstract: CRISPR-Cas9-mediated genome-editing technology contributes not only to basic genomic studies but also to clinical studies such as genetic correction and virus inactivation. Hepatitis B virus (HBV) is a major target for potential application of CRISPR-Cas9 in eliminating viral DNA from human cells. However, the high stability of covalently closed circular DNA (cccDNA) makes it difficult to completely clear HBV infection. Here, we report highly multiplexed CRISPR-Cas9-nuclease and Cas9-nickase vector systems that simultaneously target three critical domains of the HBV genome. Co-transfection of an HBV-expressing plasmid and all-in-one CRISPR-Cas9 vectors resulted in significant reduction in viral replicative intermediates and extracellular hepatitis B surface and envelope antigens. In addition, successful fragmentation of the HBV genome was confirmed by DNA sequencing. Despite its high efficacy in suppressing HBV, no apparent off-target mutations were detected by genomic cleavage detection assay and the small number of observed mutations was extremely rare and could only be detected by deep sequencing analysis. Thus, our all-in-one CRISPR-Cas9-nuclease and Cas9-nickase vectors present a model for simultaneous targeting of multiple HBV domains, potentially contributing to a well-designed therapeutic approach for curing HBV patients.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Masaki
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan.|Center for Medical Specialist Graduate Education and Research, Institute of Biomedical & Health Science, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Abe-Chayama
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Mochida
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan.|Laboratory for Digestive Diseases, RIKEN Center for Genomic Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Chayama
mesh_terms: D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D004279:DNA, Viral; D003850:Deoxyribonuclease I / Q000378:metabolism; D004720:Endonucleases; D005822:Genetic Vectors; D016679:Genome, Viral; D056945:Hep G2 Cells; D006515:Hepatitis B virus; D006801:Humans; D038301:Virus Inactivation
chemical_list: D001426:Bacterial Proteins; D004279:DNA, Viral; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D003850:Deoxyribonuclease I