随机CRISPR-Cas转录干扰筛选揭示抗α-突触核蛋白毒性的保护基因。
Randomized CRISPR-Cas Transcriptional Perturbation Screening Reveals Protective Genes against Alpha-Synuclein Toxicity.
作者信息
Chen Ying-Chou, Farzadfard Fahim, Gharaei Nava, Chen William C W, Cao Jicong, Lu Timothy K
机构信息
Synthetic Biology Group, MIT Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Synthetic Biology Group, MIT Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; MIT Microbiology Program, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering and Electrical Engineering & Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
出版信息
Mol Cell. 2017 Oct 5;68(1):247-257.e5. doi: 10.1016/j.molcel.2017.09.014.
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.
利用CRISPR-Cas技术对转录网络进行全基因组扰动主要涉及系统性和靶向性基因调控。在此，我们开发了PRISM（通过合成调节剂扰动调控相互作用），这是一种筛选平台，它使用随机的CRISPR-Cas转录因子（crisprTFs）对转录网络进行全局扰动。通过将PRISM应用于帕金森病（PD）的酵母模型，我们鉴定出了可调节转录网络并保护细胞免受α-突触核蛋白（αSyn）毒性影响的向导RNA（gRNAs）。在此筛选中鉴定出的一种gRNA比之前报道的最具保护作用的αSyn毒性抑制剂表现更优，突出了PRISM识别重要表型调节剂的能力。基因表达谱分析揭示了受这种强大的保护性gRNA差异调节的基因，当这些基因过表达时可使酵母免受αSyn毒性影响。排名靠前的命中基因的人类同源物在人类神经元PD模型中可抵御αSyn诱导的细胞死亡。因此，通过随机crisprTFs对转录网络进行高通量和无偏扰动能够揭示复杂的生物学表型和有效的疾病调节剂。
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