Alcohol abuse can have devastating impacts on the brain and behavior and is a big burden on public health. However, the molecular underpinnings of alcohol induced neural cell injury are not fully understood. Recent advances in functional genomics suggest that long non-coding RNAs (lncRNAs) may play critical roles in alcohol- induced neural cell death. The recent work from our laboratory support this hypothesis. Therefore, a comprehensive screening system of lncRNAs related to alcohol-induced neural cell death is of significant benefits for identifying potential therapeutic targets. Genetic editing tools such as Zinc Finger Nuclease (ZFN) and Transcription Activation-Like Element Nuclease (TALEN) have great potential for the functional study of genes or the application of gene therapy through knockout or knockin techniques. A new type of genetic editing tool based on bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR?Associated System [Cas]) has been successfully used in various organisms from bacteria and yeast to mammals. Relative to ZFN and TALEN, CRISPR/Cas is advantageous because it only requires changing the sequence of the guide RNA (gRNA) and it can also be directly delivered into embryos to generate gene-modified organisms. Furthermore, the multiplexing capability of CRISPR/Cas makes it possible to target multiple genes simultaneously. In this study we will use our recently developed dual guide CRISPR/Cas approach to generate an lncRNA knockout (gRNA) library in SH-SY5Y cell model. We will then perform a genome-wide screening for lncRNAs involved in alcohol-induced cell death pathways. Our preliminary data indicated that alcohol increased the expression of a nuclear paraspeckle lncRNA in SH-SY5Y cells. Our previous work have demonstrated that alcohol activates the oxidative stress-apoptotic cell death pathway in a dose dependent manner to reduce neuronal cell viability and increase cellular oxidative stress. We expect that the application of the high throughput screening platform in alcohol-induced neuronal death system will allow for the accurate identification of all other specific lncRNAs as well as their potential roles in mediating alcohol-induced neurodegeneration. The generated lncRNA gRNA library in this study can also be available for the characterization of cell toxicity induced by other neurotoxins or other substance abuse. Thus this research has a potential to reduce the health impacts of alcohol abuse and also has benefits for other substances abuse related public heath burdens.
Recent research findings indicate that long non-coding RNAs (lncRNAs) are a potential source for the identification of new molecular targets involved in alcohol-induced neurodegeneration in alcohol use disorders (AUD). In this project, we propose to build a knockout lncRNA library using our recently developed dual guide CRISPR/Cas9 knockout approach and screen this library in our established alcohol-induced neuronal cell death system. The outcomes of this research will allow the rapid and accurate identification of lncRNAs and their potential mediating roles in alcohol-induced neurodegeneration. It will also provide a valuable tool for further research on lncRNAs involved in AUD.
