Large-scale bidirectional arrayed CRISPR screens identify OXR1 and EMC4 as modifiers of α-synuclein aggregation

In synucleinopathies, including Parkinson’s disease, α-synuclein (α-Syn) misfolds and forms Ser129-phosphorylated aggregates (pSyn). Mitochondrial and lysosomal dysfunction, along with impairments in protein and organelle trafficking, exacerbate α-Syn pathology through poorly defined molecular mechanisms. We used CRISPR-mediated gene activation and ablation to systematically interrogate mitochondrial, trafficking and motility-related genes to identify pSyn regulators in HEK293 cells exposed to α-Syn fibrils. We found that activation of the mitochondrial protein OXR1 increased pSyn by impairing ATP synthesis and altering the mitochondrial membrane potential, whereas ablation of the endoplasmic reticulum (ER)-associated protein EMC4 reduced pSyn by enhancing ER-driven autophagic flux and lysosomal clearance. Assays with distinct strains of α-Syn fibrils revealed that OXR1 was preferentially synergistic with multiple system atrophy (MSA)-associated fibrils, whereas EMC4 broadly reduced pSyn across diverse α-Syn polymorphs. These findings were validated in human iPSC-derived cortical and dopaminergic neurons, with OXR1 preferentially driving somatic aggregation and EMC4 depleting both somatic and neuritic aggregates. This work identifies OXR1 and EMC4 as organelle-specific regulators of α-Syn proteostasis and underscores the involvement of mitochondrial and ER-lysosomal pathways in synucleinopathies. Summary This study investigates transcriptomic alterations resulting from CRISPR-based modulation of OXR1 and EMC4 in a human cellular model of α-Synucleinopathy. HEK293 cells stably overexpressing human α-Synuclein (α-Syn) were subjected to CRISPRa-mediated activation of OXR1 and CRISPR/Cas9 CRISPRo-mediated ablation of EMC4, respectively. Total RNA was isolated and subjected to bulk RNA-sequencing. Differential gene expression analyses were performed to uncover downstream pathways associated with mitochondrial and ER-lysosomal function in the context of α-Syn accumulation, with relevance to Parkinson’s disease. Overall Design Two independent bulk RNA-seq experiments were conducted: CRISPRa: OXR1 activation Cell line: HEK293 cells stably overexpressing human α-Synuclein Perturbation: CRISPR activation of OXR1 using a dCas9-VPR system Control: Non-targeting guide RNA (NTG) Biological replicates: 3 per condition (OXR1 and NTG) CRISPRo: EMC4 ablation Cell line: HEK293 cells stably overexpressing human α-Synuclen Perturbation: CRISPR repression (CRISPRo) of EMC4 using a Cas9 system Control: Non-targeting guide RNA (NTG) Biological replicates: 3 per condition (EMC4 and NTG)