title: A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase tag insertion at the 3'end using CRISPR-Cas9 genome editing technique.
abstract: α-synuclein (α-SYN) is a major pathologic contributor to Parkinson's disease (PD). Multiplication of α-SYN encoding gene (SNCA) is correlated with early onset of the disease underlining the significance of its transcriptional regulation. Thus, monitoring endogenous transcription of SNCA is of utmost importance to understand PD pathology. We developed a stable cell line expressing α-SYN endogenously tagged with NanoLuc luciferase reporter using CRISPR/Cas9-mediated genome editing. This allows efficient measurement of transcriptional activity of α-SYN in its native epigenetic landscape which is not achievable using exogenous transfection-based luciferase reporter assays. The NanoLuc activity faithfully monitored the transcriptional regulation of SNCA following treatment with different drugs known to regulate α-SYN expression; while exogenous promoter-reporter assays failed to reproduce the similar outcomes. To our knowledge, this is the first report showing endogenous monitoring of α-SYN transcription, thus making it an efficient drug screening tool that can be used for therapeutic intervention in PD.
authors.affiliation: Division of Neurosciences, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 6900 Lake Nona Blvd, Orlando, FL 32827, USA.
authors.forename: Sambuddha
authors.initials: S
authors.lastname: Basu
authors.affiliation: Division of Neurosciences, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 6900 Lake Nona Blvd, Orlando, FL 32827, USA.
authors.forename: Levi
authors.initials: L
authors.lastname: Adams
authors.affiliation: Division of Neurosciences, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 6900 Lake Nona Blvd, Orlando, FL 32827, USA.
authors.forename: Subhrangshu
authors.initials: S
authors.lastname: Guhathakurta
authors.affiliation: Division of Neurosciences, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 6900 Lake Nona Blvd, Orlando, FL 32827, USA.|Kyunghee University Medical College, Seoul, South Korea.
authors.forename: Yoon-Seong
authors.initials: YS
authors.lastname: Kim
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D000072669:Gene Editing / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D008156:Luciferases / Q000737:chemistry / Q000235:genetics; D010300:Parkinson Disease / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection; D051844:alpha-Synuclein / Q000235:genetics*
chemical_list: C497604:SNCA protein, human; D051844:alpha-Synuclein; D008156:Luciferases