Neurodegenerative diseases (NDD) involve the progressive degeneration of the neurons in various brain regions. The complexity and heterogeneity of these diseases, coupled with a limited understanding of their mechanisms, pose challenges for developing effective therapies. To accelerate drug discovery and to systematically explore the cell biology of these diseases at scale, we developed two CRISPR/Cas9-based high-throughput screening platforms: 1) Using cancer cells and iPSC-derived neurons (iNeurons), we conducted a protein-level enrichment focusing on endogenous alpha-synuclein (αSyn), increased levels of which are implicated in Parkinson’s disease (PD). Clearance of toxic or wild-type proteins has been shown to reverse disease phenotypes in NDD animal models. Using αSyn as a prototype protein, we decided to map the regulation network of the endogenous protein levels using sequential CRISPR-knockout and CRISPR-interference screens in αSyn gene (SNCA) tagged cell lines. We successfully identified enzymes of the N-terminal acetyltransferase B (NatB) complex as the most potent modifiers of endogenous αSyn. N-terminal acetylation proved crucial for αSyn stability, specifically protecting the cytosolic αSyn from rapid degradation by the proteasome in a Ube2W-dependent manner. We also demonstrated that pharmacological inhibition of the NatB regulator, methionyl-aminopeptidase 2 (METAP2), attenuates endogenous αSyn in iNeurons carrying SNCA triplication. 2) By pooled tagging of hundreds of RNA-binding proteins (RBPs), mutations that have been associated with amyotrophic lateral sclerosis, we conducted a high-content screen to systematically profile stress-related alterations in their localization and condensation using microscopy. Using our pooled optical screen, we successfully identified both known and novel stress granule constituents and established an initial deep-learning framework to systematically assess the behavior of RBPs under different cellular states. In summary, our approach using pooled CRISPR screens contributes to an increased understanding of NDD mechanisms and simultaneously suggests potential novel targets for disease intervention.