CRISPR technology helps discover new targets for the treatment of gradual freezing March 09, 2018 Source: WuXi PharmaTech Amytrophic lateral sclerosis (ALS), commonly known as gradual freezing, is a progressive and fatal neurodegenerative disease. Due to the gradual death of motor neurons, the patient's muscle function is degraded, and simple muscle movements such as brushing, talking, and even breathing are ultimately impossible. Abnormal protein deposition in the brain of patients is one of the important hallmarks of ALS, but scientists are not sure how these protein deposits lead to neuronal death. Recently, researchers at Stanford University used CRISPR/Cas9 gene editing technology to screen genes affecting ALS symptoms. The results not only helped scientists understand the pathogenesis of ALS, but also provided new targets for the treatment of ALS. point. ▲Image source: 123RF In ALS patients, a common cause is the hexanucleotide repeat expansion on the C90RF72 gene, and the mutated gene expresses a dipeptide repeat (DPR) that is very easy to polymerize. protein. This deposition of DPR in cells is thought to be responsible for nerve cell death. The results, published in the Nature Genetics study, used CRISPR/Cas9 gene editing technology to perform genome-wide knockout screens in human cell lines called K562. The CRISPR technology is commonly used to modify gene sequences, but in this study it was used to inactivate specific genes in the human genome in cells. The researchers then added DPR proteins to the culture medium of the cells and tested whether knocking out specific genes in the cells made them more sensitive or resistant to DPR-induced cytotoxicity. ▲Image source: 123RF The researchers first screened all genes in the human genome in human cell lines and found that hundreds of genes have a positive or negative effect on the cytotoxicity of DPR. To test the function of these genes in a cell environment closer to ALS, the researchers further examined approximately 200 genes that have the greatest impact on DPR toxicity in human cells in a mouse neuron cell culture model that mimics ALS. . After two rounds of screening, the researchers found that more than a dozen genes have the greatest impact on the cytotoxicity of DPR. Among these dozens of genes, several genes are knocked out and have a strong protective effect on cells. For example, the gene named RBA7A is an endolysosomal trafficking gene that may play an important role in mediating DPR invasion and transduction between cells. Another gene that has attracted the attention of researchers is Tmx2. When it is knocked out in cells, almost 100% of cells can survive in the presence of DPR, and usually only 10% of cells can survive. "You can imagine that Tmx2 protein may be a good drug target," said Michael Haney, a graduate student at Stanford University, the first author of the article. "If you have a small molecule compound that can suppress the function of Tmx2, then It has the potential to be a treatment for ALS." The Tmx2 protein is a protein in the endoplasmic reticulum of the cell and its function has not been fully clarified. It has been suggested that it may play an important role in the response of the mediator endoplasmic reticulum to stress factors in the environment, especially as it may trigger cell death. In this study, Stanford scholars believe that Tmx2 can regulate other genes that initiate cell death programs. "I think understanding the normal function of the Tmx2 protein in cells is the next step, which will help us discover which functions are affected when toxic proteins kill cells and point out which signal pathways we should continue to examine," Mr. Nicholas Kramer, a co-first author and a graduate student at Stanford University, said. The CRISPR screening technology used in this study can be used to study the signaling pathways of other diseases. The team at the current study is trying to use the same technique to study other neurodegenerative diseases caused by toxic protein deposition, including Huntington's disease, Parkinson's disease, and Alzheimer's disease. Disease). “I think this is a very exciting application for CRISPR screening, and it has only just begun,†said Dr. Michael Bassik, a senior author of the article and a professor of genetics at Stanford University. Reference materials: [1] Potential drug targets for ALS revealed in Stanford-led study using CRISPR [2] CRISPR–Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity Paddy Harrow,Paddy Field Harrow,Blade Harrow,Gear Drive Harrow Taizhou Yingtian Agricultural Machinery Manufacturing Co., Ltd. , https://www.sakuradaagc.com
