title: CRISPR/Cas9 System as an Agent for Eliminating Polyomavirus JC Infection.
abstract: Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by reactivation of the human polyomavirus JCV gene expression and its replication in oligodendrocytes, the myelin producing cells in the brain. Once a rare disease seen in patients with lymphotproliferative and myeloproliferative disorders, PML has been seen more frequently in HIV-1 positive/AIDS patients as well as patients undergoing immunomodulatory therapy due for autoimmune disorders including multiple sclerosis, rheumatoid arthritis, and others. As of now there is no cure for PML and in most cases disease progression leads to death within two years. Similar to other polyomaviruses, the JCV genome is small circular double stranded DNA that includes coding sequences for the viral early protein, T-antigen, which is critical for directing viral reactivation and lytic infection. Here, we employ a newly developed gene editing strategy, CRISPR/Cas9, to introduce mutations in the viral genome and, by inactivating the gene encoding T-antigen, inhibit viral replication. We first used bioinformatics screening and identified several potential targets within the JCV T-antigen gene that can serve as sites for the creation of guide RNAs (gRNAs) for positioning the Cas9 nuclease on the designated area of the viral genome for editing. Results from a series of integrated genetic and functional studies showed that transient or conditional expression of Cas9 and gRNAs specifically targets the DNA sequences corresponding to the N-terminal region of T-antigen, and by introducing mutation, interferes with expression and function of of the viral protein, hence suppressing viral replication in permissive cells. Results from SURVEYOR assay revealed no off-target effects of the JCV-specific CRISPR/Cas9 editing apparatus. These observations provide the first evidence for the employment of a gene editing strategy as a promising tool for the elimination of the JCV genome and a potential cure for PML.
authors.affiliation: Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, 19122, United States of America.
authors.forename: Hassen S
authors.initials: HS
authors.lastname: Wollebo
authors.affiliation: Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, 19122, United States of America.
authors.forename: Anna
authors.initials: A
authors.lastname: Bellizzi
authors.affiliation: Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, 19122, United States of America.
authors.forename: Rafal
authors.initials: R
authors.lastname: Kaminski
authors.affiliation: Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, 19122, United States of America.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Hu
authors.affiliation: Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, 19122, United States of America.
authors.forename: Martyn K
authors.initials: MK
authors.lastname: White
authors.affiliation: Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, 19122, United States of America.
authors.forename: Kamel
authors.initials: K
authors.lastname: Khalili
mesh_terms: D000957:Antigens, Viral, Tumor / Q000235:genetics; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D018390:Gene Targeting; D015316:Genetic Therapy / Q000379:methods; D016679:Genome, Viral; D006801:Humans; D007577:JC Virus / Q000235:genetics*; D007968:Leukoencephalopathy, Progressive Multifocal / Q000628:therapy / Q000821:virology*; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D014779:Virus Replication
chemical_list: D000957:Antigens, Viral, Tumor; D000094704:RNA, Guide, CRISPR-Cas Systems