CRISPR,基因组编辑系统 z-bo 2020-09-22 374

　　To knock out a gene in a zebrafish embryo, Alexander Schier, a developmental biologist at Harvard University, used the genome editing system CRISPR. But Schier ended up knocking out more than just one gene. He and his colleagues devised a new method to label and track cells in developing animals. The results of the study were published online on the 26th in the journal Science. The researchers used CRISPR-induced mutations to reveal a surprising discovery: Many tissues and organs in adult zebrafish are formed from only a small part of embryonic cells.

　　Some researchers have begun to try to use this method to explore developmental processes. James Briscoe, a developmental biologist at the Kerrick Institute in London, called this method an "innovative application of CRISPR technology." He said, "This technology can help us reconstruct the'family tree' of all the cells that make up an animal body. "Some scientists plan to use this method to track the evolution of tumors. Some people are developing similar methods to use CRISPR to record the history of cells, such as the environmental impact of cells.

　　GESTALT new technology: constructing the ‘family tree’ of all cells in the animal body

　　Schier and his colleagues used what Harvard University geneticist George Church called CRISPR's "genome disruption" ability. In normal CRISPR editing, a molecule called a guide RNA will accurately guide the Cas9 enzyme to a specific site in the genome, and then cut the double-stranded DNA there. The template DNA can guide the cell to repair double-strand breaks, and the 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" where nucleotide loss or insertion may occur.

　　In order to ensure that the target zebrafish gene is really knocked out, Schier introduced several different guide RNAs to target multiple sites of the gene. But the results of several repeated experiments are very different: the size of the deletion is different, and small and large insertions appear in the genetic scar area. Schier and Jay Shendure, a geneticist at the University of Washington, realized that this genetically disrupted diversity could be further exploited.

　　Schier and Shendure inserted a set of foreign DNA into the zebrafish embryo's genome, which contained 10 different CRISPR targeting sequences. Then they injected the Cas9 enzyme and 10 guide RNAs corresponding to the targeted sequence into single-cell embryos. As the embryo develops, the CRISPR system in each cell destroys the target DNA multiple times, marking it with a barcode (with a unique deletion or insertion). When a cell divides, the daughter cells will initially carry the same barcode label, and then will undergo different changes after being cut at different locations by Cas9. The first change in the barcode should occur in the two-cell stage. The editing tool will slowly become invalid after about 4 hours. At this time, the embryo contains thousands of cells. After that, the remaining barcode will follow. The continuous proliferation of these cells occurs in adult animals.

　　Four months later, the scientists collected organs of adult zebrafish and isolated more than 1,000 different barcodes from about 200,000 cells. Cells with similar barcodes are likely to divide and form late in development, so scientists can use computer programs to calculate the family tree of these 200,000 cells, that is, use a pedigree map to reveal where each cell came from.

　　One of the most surprising findings is that a large number of tissues in all organs are produced by a few cells. In most organs, more than half of the cells share fewer than seven barcodes. In all organs except the brain, 25 different barcodes make up more than 90% of the cells. Briscoe said, "The cell population that makes up a tissue may be less than I expected."

　　For developmental biologists, this new technology called GESTALT (Genome Editing of Synthetic Target Arrays for Lineage Tracing) can help understand how animals are formed from single cells. It can also help us clarify some important questions in cancer research, such as how many precursor cells produce tumors, how the cells in the tumor are related to each other, and how the spreading cancer cells are related to the original tumor.

　　Schier said that this technology also has some shortcomings. For example, it cannot reliably label every new generation of cells. However, compared with other methods of tracking cells and their offspring (such as staining methods or relying on natural mutations), the use of barcode markers generated by CRSIPR for tracking analysis is more effective and easier to use. Leonard Zon, director of the stem cell project at Boston Children’s Hospital, said, “I think cancer biologists will start to consider using this method to study cancer, because this method of labeling cells will be simpler than our existing methods. Of course we want to Give it a try."

　　Another mSCRIBE system: recording the environmental impact experienced by cells

　　Researchers have proposed other ways to turn CRISPR into a type of cellular memory. Schier said, "I think this is the most exciting thing conceptually. You can record history in DNA." A research team from the Massachusetts Institute of Technology has already conducted this research. Last week, Timothy Lu and his colleagues uploaded an article to the preprinted website bioRxiv.org describing a system called mSCRIBE (mammalian Synthetic Cellular Recorder Integrating 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. Eventually the system targets itself: the guide RNA guides Cas9 to its source DNA, Cas9 destroys the DNA, causing mutations in the sequence, and finally generates a guide RNA containing mutations. The mutated guide RNA further guides Cas9 to the changed target sequence, and then the process continues to cycle, during which the DNA sequence and guide RNA also continue to change.

　　By studying how the CRISPR target sequence changes in thousands of single cells, the researchers estimated that Cas9 needs to run several rounds to generate a specific sequence (this process is like a game of communication, how many rounds are needed to change the initial sequence) The phrase "lobster boil" is converted to "losing team"). In the next experiment, they coupled the activity of Cas9 targeted genes with the activity of an inflammatory pathway in the cell. In cells exposed to more inflammatory factor TNFα, more rounds of Cas9 mutations will be recorded on the targeted sequence.

　　Then they tested the CRISPR recording tool in mice, injected modified cells into animals, and injected inflammation-inducing molecules into some cells. In mice injected with these molecules, the CRIPR targeting sequence changed more than in untreated mice. The author writes that this method can be applied in vivo to record physiologically relevant biological signals in a simulated manner.

　　Lu and his colleagues suggested that this method can also record the stimuli of cancer cells in the tumor microenvironment, or track the activity of specific pathways in the cells during the course of disease. Church said this method is also very valuable in brain research, such as recording the activity of pathways involved in basic memory. He said, "Using this method, you can turn a transient process into a permanent record and present it in every neuron cell of the brain."