Randomized CRISPR-Cas Transcriptional Perturbation Screening Reveals Protective Genes against Alpha-Synuclein Toxicity

Abstract

SUMMARY The genome-wide perturbation of transcriptional networks with CRISPR-Cas technology has primarily involved systematic and targeted gene modulation. Here, we developed PRISM (Perturbing Regulatory Interactions by Synthetic Modulators), a screening platform that uses randomized CRISPR-Cas transcription factors (crisprTFs) to globally perturb transcriptional networks. By applying PRISM to a yeast model of Parkinson’s disease (PD), we identified guide RNAs (gRNAs) that modulate transcriptional networks and protect cells from alpha-synuclein (αSyn) toxicity. One gRNA identified in this screen outperformed the most protective suppressors of αSyn toxicity reported previously, highlighting PRISM’s ability to identify modulators of important phenotypes. Gene expression profiling revealed genes differentially modulated by this strong protective gRNA that rescued yeast from αSyn toxicity when overexpressed. Human homologs of top-ranked hits protected against αSyn-induced cell death in a human neuronal PD model. Thus, high-throughput and unbiased perturbation of transcriptional networks via randomized crisprTFs can reveal complex biological phenotypes and effective disease modulators.

Introductory Chapter: Gene Editing Technologies and Applications

“…By applying the Perturbing Regulatory Interactions by Synthetic Modulators (PRISM) to a yeast model of PD, sgRNAs were identified to modulate transcriptional networks and protect cells from α-Syn toxicity [38]. The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes Alzheimer's disease (AD).…”

Yeast genetic interaction screens in the age of CRISPR/Cas

“…Because of this unpredictable variability, several gRNAs must be tested for each target gene (Ryan et al 2014; Shalem et al 2014; Wang et al 2014; Koike-Yusa et al 2014; Zhou et al 2014; Konermann et al 2015; Chen et al 2017; Smith et al 2017; Sadhu et al 2018). It is generally presumed that as more gRNAs are tested in yeast, empirical or mechanistic design rules will improve.…”

Quantitative Characterization of α-Synuclein Aggregation in Living Cells through Automated Microfluidics Feedback Control

“…Biological processes involved in α-synuclein inclusion formation and clearance are well conserved across evolution, hence yeast can be used to elucidate the molecular basis of the human disease and to screen for therapeutic drugs ( Menezes et al., 2015 , Schneider et al., 2018 ). Since its inception ( Outeiro and Lindquist, 2003 ), the yeast PD model with heterologous expression of α-synuclein has been successfully used not only to study molecular mechanisms of the PD but also for high-throughput drug and genetic screenings ( Zabrocki et al., 2008 , Menezes et al., 2015 , Chen et al., 2017 ). In this model, α-synuclein is expressed from the galactose-inducible promoter, and protein inclusions form with ensuing growth defects and cell death ( Outeiro and Lindquist, 2003 , Cooper et al., 2006 , Petroi et al., 2012 ).…”

“…In addition to contributing to the biology of PD, our results have relevance for therapeutic applications because the yeast PD model is extensively used in large-scale screening of therapeutic small molecules and modifier genes ( Cooper et al., 2006 , Chen et al., 2017 ).…”