CRISPR/Cas9,基因组编辑 z-bo 2020-09-23 403

　　In a new study, the researchers used genetically modified CRISPR/Cas9-a new revolutionary gene editing technology-to directly convert fibroblasts isolated from mouse connective tissue into nerves yuan.

　　In 2006, Professor Shinya Yamanaka from the Institute of Advanced Medical Sciences, Kyoto University, Japan discovered how to return fibroblasts from adult connective tissue to immature stem cells that can differentiate into any cell type. These so-called induced pluripotent stem cells (iPS cells) earned Professor Shinya Yamanaka the Nobel Prize only 6 years later because of their huge potential in research and medicine.

　　Since then, scientists have discovered other ways to transform one type of cell into another. This is mainly achieved by introducing a variety of additional copies of the "master switch" gene-the protein that expresses the entire gene network needed to activate a specific cell type.

　　Now, in this new study, researchers from Duke University in the United States have developed a strategy that eliminates the need to introduce additional gene copies. Instead, they used a genetically modified CRISPR/Cas9 gene programming technology to directly activate the natural copy that already exists in the cell's genome. The relevant research results were published online on August 11, 2016 in the journal Cell Stem Cell, with the title of the paper "Targeted Epigenetic Remodeling of Endogenous Loci by CRISPR/Cas9-Based Transcriptional Activators Directly Converts Fibroblasts to Neuronal Cells".

　　These early research results indicate that compared to the method of permanently adding new genes to the host cell genome, the genetically modified CRISPR/Cas9 method is used to achieve the transformation of mouse embryonic fibroblasts directly into neurons The process is more complete and lasting.

　　These neuronal cells may be used to build neurological disease models, discover new treatment methods and develop personalized therapies, and may carry out cell therapy in the future.

　　The corresponding author of the paper, Director of the Center for Biomolecular and Tissue Engineering and Associate Professor of Biomedical Engineering, Dr. Charles Gersbach, said, “This technology has many applications in scientific research and medicine. For example, how we might treat most people’s neurons? Reacting to drugs creates a rough impression. Obtaining a biopsy of your brain to test your neurons is ethically not allowed. But if we can get skin cells from your arm, convert them into neurons , And then use a combination of multiple drugs to deal with it, we may determine an optimal personalized therapy."

　　The first author of the paper and a graduate student in Gersbach’s laboratory, Joshua Black, said, “The challenge is to efficiently generate stable neurons, and they look very similar to real neurons in your body. This has always been a major obstacle in this field.

　　In the 1950s, British developmental biologist Professor Conrad Waddington proposed that the differentiation of immature stem cells into specific types of adult cells is like rolling from a ridged mountain side to many valleys. When a cell chooses each path along a particular slope, its choice of ultimate destination will become less and less.

　　If you want to change this destination, one way is to push the cell vertically back to the top of the mountain---this is the idea of reprogramming the cell into an iPS cell. Another option is to horizontally let this cell climb up a small mountain peak and directly into another valley.

　　Gersbach said, "If you can specifically activate all neuron genes, then you may not necessarily need to climb up this small mountain."

　　The previous method did this by introducing viruses that carry extra copies of genes, which can produce large amounts of proteins called master transcription factors in cells. These proteins (which exist according to different cell types) bind to thousands of sites on the genome and activate specific gene networks in target cells. However, this method also has some disadvantages.

　　Black said, "It would be desirable not to permanently introduce extra copies of existing genes through a virus but to provide a temporary signal to change the cell type in a steady manner. However, doing this efficiently may require The genetic program of the cell makes a very specific change."

　　In this new study, Black, Gersbach and colleagues used genetically modified CRISPR/Cas9 technology to accurately activate the three genes Brn2, Ascl1, and Myt1l to naturally produce these master transcription factors that control the neuronal gene network. Instead of introducing viruses that carry copies of these extra genes.

　　The researchers first genetically modified the bacterial defense system CRISPR/Cas9 to couple it with a gene activator, so that specific DNA fragments can still be identified, but the target fragments will not be cut, but they will remain intact , While using gene activator to activate the target fragment.

　　In the laboratory, the genetically modified CRISPR/Cas9 system was injected into mouse embryonic fibroblasts. The test results show that once activated by this system, these three neuronal master transcription factor genes strongly activate neuronal genes. This causes these fibroblasts to conduct electrical signals--a typical feature of neurons. And even after removing the gene activator coupled with CRISPR/Cas9, these cells still retain their neuronal properties.

　　Gersbach said, "When viruses carrying genes expressing these three primary transcription factors are introduced into cells, it is possible that these cells may behave like neurons. But if they really become autonomous neurons, then They shouldn't need this kind of external stimulus to persist."

　　These experiments confirmed that the epigenetic program of neurons generated by this genetically modified CRISPR/Cas9 technology on the target gene matches the neuron markers naturally found in mouse brain tissue.

　　Black explained, "Using viruses to introduce additional gene copies to produce these transcription factors in large quantities, but the natural copies of these genes produce very few of these proteins. In contrast, this genetically modified CRISPR/Cas9 method generally They don’t produce so many transcription factors, but they are produced by normal chromatin sites. This is the significant difference that these genes make when they are steadily activated. We activate this epigenetic switch to change cell types, not Forcing them to do this through synthesis."

　　According to Black, the next course of action is to extend this method to human cells, improve the efficiency of this technology, and try to eliminate other epigenetic disorders, so that it may be used to build specific disease models.

　　Gersbach said, “In the future, you can imagine making neurons outside the body and transplanting them into the brain to treat Parkinson’s disease or other neurodegenerative diseases. But even if we can’t get to this point, we can still work in the laboratory. China uses this method to help develop better treatments."