基因组编辑系统CRISPR z-bo 2020-10-21 282

　　To knock out genes in zebrafish embryos, Harvard University developmental biologist Alexander Sie used the genome editing system CRISPR. However, Schell eventually eliminated one or more genes. He and his colleagues devised a new method for labeling and tracking developing animal cells. The results of the study were published online in the journal Science on the 26th. The researchers revealed a surprising discovery of mutations induced using CRISPR. Many tissues and organs of adult zebrafish are composed of a small number of embryonic cells.

　　Some researchers have begun to use this method to study developmental processes. James Brisco, a developmental biologist at the Kerrick Institute in London, calls this method "an innovative application of CRISPR technology." "This technology helps rebuild the "family tree" of all the cells that make up the animal's body. "Some scientists plan to use this method to track the progression of tumors. Some people use CRISPR to record cell history, including the impact of cells on the environment.

　　GESTALT's new technology: Establish a "family tree" of all cells in the animal body

　　Schier and his colleague are geneticist George Church of Harvard University. We used CRISPR's "genome disruption" function. In normal CRISPR editing, a molecule called guide RNA guides the Cas9 enzyme to a specific site in the genome, where it cuts double-stranded DNA. Template DNA can guide cells to repair double-strand breaks, and editing accuracy can reach the level of single nucleotide changes. However, if the scientist does not provide the template strand, the cell will not be able to accurately repair the break, and eventually the gene will form a "scar", which may cause nucleotide loss or insertion. To ensure that

　　"Target zebrafish genes, Schier introduced several different guide RNAs to target multiple sites in the gene." However, the results of some repeated experiments are quite different. The size of the deletion is different, and you will see inserts of different sizes in the scar area of the gene. Geneticists Schier and Jay Shendure of the University of Washington have realized that this genetically disrupted diversity can be further exploited. Schier and Shendure have inserted a set of foreign DNA containing 10 CRISPR targeting sequences into the genome of zebrafish embryos. Next, the Cas9 enzyme and 10 guide RNAs corresponding to the target sequence were injected into the single-cell embryo. As the embryo develops, the CRISPR system in each cell destroys the target DNA multiple times and marks it with a barcode (using unique deletions or insertions). When splitting a cell, the child cells first have the same barcode label and are cut by Cas9 at different positions before making different changes. The first bar code change should occur in the two-unit stage. After about 4 hours, the editing tool will slowly close. At this time, the embryo contains thousands of cells. Then follow the rest of the barcode. The continued proliferation of these cells occurs in adult animals. Four months later, the scientists collected adult zebrafish organs and isolated more than 1,000 different barcodes from approximately 200,000 cells. Cells with similar barcodes are likely to divide and form in later development, so scientists can use computer programs to calculate the family tree of these 200,000 cells. In other words, you can use a pedigree chart to find the source of each cell.

　　One of the most surprising findings is that many tissues in all organs are produced by a small number of cells. In most organs, more than half of the cells share less than seven barcodes. In all organs except the brain, there are 25 different barcodes that make up more than 90% of the cells. Briscoe said: "The number of cells that make up a tissue may be less than I expected." Similarly, in some cancer research, such as the number of precursor cells that produce tumors, how the cells in the tumor are related, and how the spreading cancer cells are related to the original tumor. It also helps to clarify related important issues.

　　Schier said that this technology also has some disadvantages. For example, not all new generation cells can be reliably labeled. However, compared to other methods of tracking cells and their progeny (such as staining methods and dependence on spontaneous mutations), the use of barcode markers generated by CRSIPR for subsequent analysis is more effective and useful. It's simple. Leonard Zorn, Director of the Stem Cell Program at Boston Children’s Hospital, said: “This method of labeling cells is easier than existing methods, so I think cancer biologists will start to consider using this method to study cancer. Of course, please try Click." Another mSCRIBE system: Record the environmental effects experienced by cells

　　Researchers have proposed other ways to turn CRISPR into a type of cellular memory. She said: "Conceptually, this is the most exciting thing. History can be recorded in DNA." The MIT research team is already conducting this research. Last week, Timothy Lu and his colleagues uploaded an article on the preprint website bioRxiv.org. This article describes a system called mSCRIBE (Mammalian Synthetic Cell Recorder Integrated Biological Events). Instead of adding a barcode containing 10 CRSIPR targeting sequences, the researchers inserted a single CRISPR targeting sequence into the cell and modified the site to encode a guide RNA. Ultimately, the system targets itself. The guide RNA guides Cas9 to its source DNA, which destroys the DNA, causes sequence mutations, and finally produces a guide RNA containing the mutation. The mutated guide RNA further guides Cas9 to the modified target sequence, after which the process continues to circulate, during which the DNA sequence and guide RNA also continue to change. Through research

　　CRISPR target sequence changes in thousands of single cells. Researchers estimate that Cas9 needs to run for several rounds to generate a specific sequence. (This process is similar to the number of rounds needed in a communication game) Convert the first phrase "lobster boil" to "loser". In the next experiment, we will combine the activity of Cas9 targeting genes with the activity of intracellular inflammatory pathways. In cells exposed to more inflammatory factor TNFα, more rounds of Cas9 mutations were recorded in the target sequence.

　　Then they tested the CRISPR recording tool in mice, injected modified cells into animals, and injected pro-inflammatory molecules into some cells. Compared with untreated mice, the CRIPR targeting sequence changed significantly in mice injected with these molecules. The author writes that the method can be applied to record physiologically relevant biological signals in a simulated manner in vivo. Lu and his colleagues suggested that this method can also record the stimulation of cancer cells in the tumor microenvironment and track the activity of specific pathways in the cells during the development of the disease. Church said that this method is also very useful for brain research, such as recording the activities of pathways involved in basic memory. "This method can be used to turn a temporary process into a permanent record and present it to all neuronal cells in the brain."