Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc), leading to dopamine deficiency and motor dysfunction. Clustered regularly interspaced palindromic repeats (CRISPR)-based technologies provide a potential approach for targeted gene modulation, although their effectiveness and safety in cellular models of PD remain to be established. This study investigated the feasibility and transcriptional effects of CRISPR activation (CRISPRa) targeting Dopa decarboxylase (DDC) and GTP cyclohydrolase 1 (GCH1) in human embryonic kidney (HEK293) cells and a 6-hydroxydopamine (6-OHDA)-induced neurotoxicity model using human neuroblastoma (SH-SY5Y) cells. Parkinsonian-like toxicity was induced in SH-SY5Y cells with 6-OHDA prior to CRISPRa transfection. A vector-free CRISPRa system was used to target DDC and GCH1, with single-guide RNAs (sgRNAs) assembled with dCas9-VP64 and MS2-p65-HSF1 (MPH) to form ribonucleoprotein (RNP) complexes and delivered using Lipofectamine 3000. CRISPRa targeting of DDC was associated with increased DDC expression in SH-SY5Y cells relative to untreated controls at 24 h (p = 0.0496) and 48 h (p = 0.0064). However, expression levels did not differ significantly from those observed in the 6-OHDA-treated PD control group. In contrast, GCH1 expression was not significantly altered following CRISPRa. CRISPRa also had no significant effect on cell viability in HEK293 cells. In SH-SY5Y cells, 6-OHDA significantly reduced cell viability compared with untreated controls (p = 0.0129). Similar reductions were observed in DDC-targeted cells at 24 h (p = 0.0026) and 48 h (p = 0.0017), and in GCH1-targeted cells at 24 h (p = 0.0017) and 48 h (p = 0.0008); however, none of the CRISPRa-treated groups differed significantly from the 6-OHDA-treated PD control. These findings indicate that CRISPRa elicited a modest transcriptional response, characterized by upregulation of DDC without detectable modulation of GCH1, and did not exacerbate 6-OHDA-induced cytotoxicity. Further optimisation, together with protein-level, enzymatic activity, and dopamine measurements, is required to determine whether these transcriptional changes translate into functional effects on dopamine biosynthesis.
