CRISPR markers
Organoids are mini–organs that can be grown in the laboratory. They grow from a small fragment of tissue of any organ. The ability to genetically modify these organoids is very useful for studying biological processes and modeling diseases. However, until now, obtaining genetically modified organoids has been a problem due to the lack of suitable genetic engineering methods.
CRISPR-HOT
A few years ago, researchers discovered that the CRISPR/Cas9 tool, which is a molecular scissors, can precisely cut a DNA strand in a certain place. This technology has advanced genetic engineering.
DNA damage activates one of two different repair mechanisms in cells that can be used by researchers to force cells to capture a new piece of DNA. It was believed that one of these methods – non–homologous connection of the ends - often gives errors and therefore was almost never used to insert new DNA fragments. However, some earlier work on mice has shown that new DNA fragments can be inserted by non-homologous joining of the ends.
Researchers from the Hubrecht Institute, the Netherlands, decided to test the method on human organoids. They found that inserting any DNA fragment into human organoids by non-homologous joining of the ends was more efficient and reliable than the method that has been used so far. They called their new method CRISPR-HOT.
Ultrastructural determination of human liver cells. When the keratin protein forming the cytoskeleton is stained, small structural details of the skeleton (blue) in the cells of the human liver ducts become visible. Source hereafter: Benedetta Artegiani and Delilah Hendriks, Hubrecht Institute.
Colored cells
Using CRISPR-HOT, the researchers inserted fluorescent tags into the DNA of human organoids in such a way that they were attached to the genes of interest. At first they worked with enteroendocrine cells of the intestine. These cells produce hormones that regulate processes such as maintaining glucose levels, food intake and gastric emptying. They are extremely rare, so they are difficult to study. However, with the CRISPR-HOT tool, researchers easily "painted" enteroendocrine cells in different colors, identified and analyzed them.
The researchers also tagged organoids derived from liver duct cells. Using CRISPR-HOT, they visualized the protein keratin, which forms the skeleton of cells. This made it possible to examine keratin in detail and with high resolution, they revealed the organization of the protein in an ultrastructural way and found out that keratins are expressed differently during cell specialization or differentiation.
The researchers suggest that CRISPR-HOT may be useful for studying the life and differentiation of cells.
Abnormal cell division in the liver
There are many hepatocytes in the liver, which contain two or more times more DNA than ordinary cells. How these cells are formed and whether they are able to divide due to an abnormal amount of DNA is unknown. The cells of the elderly contain more of these pathological hepatocytes, but whether they are associated with diseases such as cancer was unclear. The researchers used CRISPR-HOT to label specific components of cell division in hepatocyte organoids and studied the process of cell division. Normal hepatocytes divided in a very orderly manner, always forming two daughter cells. But several departments were found in which pathological hepatocytes were formed from normal hepatocytes. In addition, the researchers studied the effect of a mutation in the TP53 gene, often found in liver cancer, on abnormal cell division in hepatocytes. Turning off TP53 led to a much more frequent division of pathological hepatocytes. Perhaps the TP53 mutation is one of the mechanisms contributing to the development of liver cancer.
Visualization of human liver cell division. Healthy (left) organoids show organized division (arrow), while organoids in which the TP53 gene is turned off (right) show chaotic cell divisions (arrows).
Researchers believe that CRISPR-HOT is applicable to many types of human organoids to visualize any gene or cell type, as well as to study many issues related to the development of diseases.
Article by B.Artegiani et al. Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing is published in the journal Nature Cell Biology.
Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on Hubrecht Institute materials: CRISPR-HOT: a new tool to "color" specific genes and cells.