Nat Methods:the development of the CRISPR-assisted technology to detect living cells in a RNA-binding…

Nat Methods:the development of the CRISPR-assisted technology to detect living cells in a RNA-binding…

2020 7 November 27 hearing /Bio ValleyBIOON/ –despite the current scientists are still not completely understood RNA molecule diversity, but they believe that with these RNA molecules The Binding of RNA-binding proteins or with various body diseases is directly related to, recently, a study published in the International JournalNature Methodsentitled“The CRISPR-assisted detection of RNA–protein interactions in living cells”of the study, from the City University of Hong Kong and other Agency scientists have developed a called CARPID novel detection method, which can identify viable cells in the special RNAs the binding protein, such new method or can be applied to multiple types of cell research, such as from the identification of cancerbiomarkersto detect the treatment of various viral diseases of potential drug targets, etc.

Molecular Biology the Central dogma DNA transcription become RNA, and RNA is ultimately translated into protein, but in fact, only about 2% of the RNAs can encode proteins, while the remaining 98%is called non-coding RNAs（ncRNA）RNA molecule is due to its mysterious function is treated as“dark matter.” In recent years, scientists have begun research efforts aimed at unraveling the RNA of the real function, especially long-chainnon coding RNAs lncRNA, the length is only 200 more nucleotides, the lncRNA is widely accepted to be involved in the regulation of gene expression of important cellular components, while it is also the researchers are most interested in the study of the RNA of one.

Image Source:Nature Methods (2020) doi: 10.1038/s41592-020-0866-0

although the lncRNA do not produce proteins, but it can with the protein-protein interactions, and the interaction between the be able to determine lncRNA function, therefore, to identify the binding proteins for the understanding of lncRNA functions are essential, however the current lot of ways there are some limitations, for example it will produce some false positive signals, and the inability of living cells to operate, etc.

in order to overcome current method limitations, researchers have developed a new method, which can be used to target RNA to the role of the advanced gene editing technology-CRISPR/dCasRx system with close-Biotin labeling technique combined to identify living cells in protein-protein interactions; the researchers, this method will be named CARPID（CRISPR-assisted RNA-protein interaction detection technology, CRISPR-Assisted RNA-Protein Interaction Detection, which is able to sensitively detect any length or concentration of the RNAs binding protein, while most other methods can only be used for long-chainnon coding RNAs for testing.

This named CARPID the new method consists of two parts, i.e., the navigation and the adjacent Biotin-labeled, the first researchers to use CRISPR/CasRx system to perform the navigation so as to include the name BASU markup tools, including CARPID component can be close to the target RNA, BASU is an engineered Biotin ligase, which ligase is capable of Biotin, which is a strong binding force vitamins with The Binding of the target to the RNA protein binding, these proteins will in this way come closer to be the mark of the targeted RNA molecule. When marked, the researchers will use the named streptavidin Biotin-binding protein to identify the BASU of the labeled protein, followed by researchers can be clearly observed in these binding proteins anymore.

in order to detect CARPID technology-specific, the researchers of the three different lncRNAs（DANCR, the XIST and MALAT1 were tested, the experimental results show that the binding protein and not too much overlap, which is described, CARPID for different lengths and expression levels of lncRNAs with high specificity and applicability. The investigator Yan said, CARPID technology can achieve high specificity, because CRISPR navigation is very accurate, we can even get very precise information, i.e., the protein, The Binding of RNA to the specific site. In addition, CARPID does not affect targeting to the cell physiological conditions, and cells will in the normal gene expression level and continue to survive, in this the help of new technology, if researchers in different time of checkpointing to the same RNA target, then they will be able to get dynamic results. In addition, in theproteomicsthe help of Technology, researchers can also be found and confirmed previously in mammalian cells has not been elucidated the characteristics of the two lncRNAs binding protein properties.

researchers believe that CARPID has a wide range of applications, including detection of viral RNA binding protein, for example, SARS-CoV-2 is a cause COVID-19 RNA virus, once the virus enters the host cells, researchers will be able to apply CARPID technology to detect the virus used for the life cycle of the recruitment to the special cell protein, if removed, the binding protein while, researchers will be able to inhibit the replication of the virus, the relevant information or can help researchers identify potential antiviral targets.

in addition, many lncRNAs can also be used as a cancer of thediagnosisbiomarkers, as compared to normal cells, in cancer cells the level is very high, CARPID technology can also be used to detect cancer cells in these lncRNAs binding protein, which perhaps can help clarify the underlying carcinogenic mechanisms and later used to develop cancerdiagnosistechnology and novel therapies of potential protein targets. Finally, the researchers indicated that they spent one year time to develop CARPID technology, most of the experiments are in City University of Hong Kong carried out, the next step they will also or continue tostem cellsand DANCR in-depth research, DANCR is a can be used as atumorpromote sub-lncRNA in. （生物谷Bioon.com

reference:

【1】CRISPR-assisted novel method detects RNA-binding proteins in living cells

【2】Yi, W., Li, J., Zhu, X. et al. CRISPR-assisted detection of RNA–protein interactions in living cells. Nat Methods 17, 685-688 (2020). doi: 10.1038/s41592-020-0866-0

Published at Sun, 26 Jul 2020 16:12:39 +0000