Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson's disease

• Published in: Scientific reports Vol. 13; no. 1; p. 17796
• Main Authors: Sastre, Danuta, Zafar, Faria, Torres, C Alejandra Morato, Piper, Desiree, Kirik, Deniz, Sanders, Laurie H, Qi, L Stanley, Schüle, Birgitt
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 18.10.2023; Nature Publishing Group UK; Nature Portfolio
• Subjects: alpha-Synuclein - genetics; alpha-Synuclein - metabolism; Basic Medicine; CRISPR; CRISPR-Cas Systems; Disease; DNA damage; DNA, Mitochondrial - metabolism; Down-regulation; Gene regulation; Humans; Lewy bodies; Medical and Health Sciences; Medicin och hälsovetenskap; Medicinska och farmaceutiska grundvetenskaper; Methicillin-Resistant Staphylococcus aureus - genetics; Mitochondrial DNA; Movement disorders; Neurodegenerative diseases; Neurosciences; Neurovetenskaper; Nuclease; Oxidative stress; Oxidative Stress - genetics; Parkinson Disease; Parkinson's disease; Patients; Precision medicine; Recovery of function; RNA, Guide, CRISPR-Cas Systems; Staphylococcus aureus - genetics; Stem cells; Stem Cells - metabolism; Synuclein
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-023-45078-3

Parkinson's disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.