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

ABSTRACT: 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 (?Syn) toxicity. One gRNA identified in this screen outperformed the most protective suppressors of ?Syn 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 ?Syn toxicity when overexpressed. Human homologs of top-ranked hits protected against ?Syn-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.

SUBMITTER: Chen YC

PROVIDER: S-EPMC5702536 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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

Chen Ying-Chou YC Farzadfard Fahim F Gharaei Nava N Chen William C W WCW Cao Jicong J Lu Timothy K TK

Molecular cell 20171001 1

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 netwo ...[more]

PMID: 28985507

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Project description:Prevention of abnormal misfolding and aggregation of alpha synuclein (syn) protein in vulnerable neurons should be viable therapeutic strategies for reducing pathogenesis in Parkinson's disease. The nonamyloid component (NAC) region of alpha-syn shows strong tendencies to form beta-sheet structures, and deletion of this region has been shown to reduce aggregation and toxicity in vitro and in vivo. The binding of a molecular species to this region may mimic the effects of such deletions. Single-chain variable fragment (scFv) antibodies retain the binding specificity of antibodies and, when genetically manipulated to create high-diversity libraries, allow in vitro selection against peptides. Accordingly, we used a yeast surface display library of an entire naive repertoire of human scFv antibodies to select for binding to a NAC peptide. Candidate scFv antibodies (after transfer to mammalian expression vectors) were screened for viability in a neuronal cell line by transient cotransfection with A53T mutant alpha-syn. This provided a ranking of the protective efficacies of the initial panel of intracellular antibodies (intrabodies). High steady-state expression levels and apparent conformational epitope binding appeared more important than in vitro affinity in these assays. None of the scFv antibodies selected matched the sequences of previously reported anti-alpha-syn scFv antibodies. A stable cell line expressing the most effective intrabody, NAC32, showed highly significant reductions in abnormal aggregation in two separate models. Recently, intrabodies have shown promising antiaggregation and neuroprotective effects against misfolded mutant huntingtin protein. The NAC32 study extends such work significantly by utilizing information about the pathogenic capacity of a specific alpha-syn region to offer a new generation of in vitro-derived antibody fragments, both for further engineering as direct therapeutics and as a tool for rational drug design for Parkinson's disease.

Dataset Information

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

Publications

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

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