CRISPRa-mediated activation of Tfeb in a fibril-induced alpha-synuclein aggregation model

Abstract

Accumulation of misfolded alpha-synuclein (a-syn) is a key hallmark of Parkinson's disease (PD), making it a key target for novel therapeutic strategies. Here, we use a previously described screening assay to select multiple efficient single guide RNA (sgRNA) combinations targeting transcription factor EB (TFEB) for CRISPR-activation (CRISPRa) using a nuclease-deficient Staphylococcus aureus Cas9 coupled to a truncated VP64-p53-Rta (VPR) activator complex. Selected sgRNA are applied in a rat neuroblastoma in-vitro model of fibril-induced a-syn aggregation with the goal of inducing autophagy and reduce protein aggregation. We successfully induce a-syn aggregation in this model and observe a stagnation in phosphorylated a-syn (p-a-syn) compared to non-phosphorylated a-syn across treatment groups. While we achieve robust expression of Tfeb in the exogenous screening assay and at endogenous mRNA level, we did not observe proportional increase in protein expression. Additionally, we do not observe any additive effects of CRISPRa induction combined with media treatments inducing Tfeb nuclear translocation. Taken together, these findings illustrate difficulties in epigenetic induction of Tfeb using CRISPRa and highlights the need for additional studies exploring this application.