Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson’s disease.
Publication Year:
2023
PubMed ID:
37853101
Public Summary:
Parkinson’s disease is a brain disorder that affects movement, balance, and coordination. It is one of the most common diseases of aging, but there are currently no treatments that can stop or slow it down. A protein called alpha-synuclein builds up in the brains of people with Parkinson’s disease and forms clumps known as Lewy bodies. These clumps may damage brain cells and are thought to play an important role in the disease.
In our research, we used a new type of gene-editing tool called CRISPR interference (CRISPRi) to lower the amount of alpha-synuclein made by brain cells. This version of CRISPR does not cut DNA—instead, it safely reduces the activity of a gene. We focused on the SNCA gene, which tells cells to make alpha-synuclein. Using special guide molecules called sgRNAs, we tested different spots in the SNCA gene to find the best way to turn it down.
We tested this system in brain cells grown from stem cells taken from a patient with a rare inherited form of Parkinson’s disease that causes too much alpha-synuclein. After using our CRISPRi tool, the cells made less alpha-synuclein and showed signs of improved health, including less stress and less damage to their DNA.
These results show that it is possible to use CRISPRi to carefully lower alpha-synuclein in brain cells. This could lead to a new and safer treatment for Parkinson’s disease and other related disorders. Our approach also helps researchers learn how much alpha-synuclein can be safely reduced without harming the cells.
Scientific Abstract:
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.
