Nat Methods: The development of crispR-assisted new technologies to detect RNA binding proteins in living cells is expected to help research in human diseases in the future.

!--:page title"---- Although scientists do not yet fully understand the diversity of RNA molecules, they believe that RNA binding proteins bound to these RNA molecules are directly related to the development of a variety of body diseases, a recent report published in the international journal Nature Methods, "CRISPR-R-CrispR"-- In the study, scientists from the City University of Hong Kong and other institutions developed a new detection method called CARPID that identifies binding proteins in live cells with special RNAs, a new method that can be applied to multiple types of cell research, from identifying cancer biomarkers to detecting potential drug targets for treating multiple viruses. The central rule of
molecular biology is that DNA is transcribed into RNA, and RNA eventually translates into proteins, but in reality only about 2% of RNAs can encode proteins, while the remaining 98% of RNA molecules called non-coding RNAs (ncRNAs) are considered "dark matter" because of their mysterious function.
in recent years, scientists have been working to uncover the true function of RNA, especially long-chain non-coding RNAs (lncRNA, with just over 200 nucleotides) that are widely accepted as important cellular components that can be used to regulate gene expression, and which researchers are most interested in studying.
picture source: Nature Methods (2020) doi:10.1038/s41592-020-0866-0 Although lNCRNA does not produce proteins, it can interact with proteins, and their interactions can determine the function of lNCRNA, so identifying binding proteins is essential to understand the function of lNCRNA, although many current methods have certain limitations, such as some positive signals, and can not produce some of the wrong signals.
To overcome the limitations of current methods, researchers have developed a new method that can be used in RNA-targeted advanced gene editing techniques - CRISPR/dCasRx systems combined with close biotin marker technology to identify protein-protein interactions in living cells; ID (CRISPR-assisted RNA-protein interaction detection technology, CRISPR-Assisted RNA-Protein Interaction Detection), which sensitively detects the binding proteins of RNAs of any length or concentration, while most other methods can only be used to detect long-chain non-coding RNAs.
the new method, called CARPID, consists of two parts, navigation and proximity biomarkers, first using the CRISPR/CasRx system to navigate the CARPID components, including a marker tool called BASU, to be able to approach the target RNA, an engineered biotin joining enzyme that binds biotin (a strongly binding vitamin) to a protein that binds to the target RNA.
when tagged, the researchers used a biotin binding protein called streptomycin to identify the proteins that were labeled by BASU, and then the researchers were able to observe the binding proteins clearly.
to detect the specificity of CARPID technology, the researchers tested three different lncRNAs (DANCR, XIST, and MALAT1), and the results showed that the binding proteins did not overlap much, which indicates that CARPID has a high degree of specificity and applicability to lncRNAs of different lengths and expression levels. 'CarpID technology enables high specificity because CRISPR navigates so precisely that we can even get very precise information about the specific location of the RNA that the protein binds to,' said Yan, a
researcher.
, CARPID does not affect the physiology of the target cell, and the cells will continue to survive at normal levels of gene expression, and with the help of this new technique, if researchers examine the same RNA targets at different points in time, they can get dynamic results.
, with the help of proteomics, the researchers were able to identify and confirm the properties of two lncRNAs binding proteins that had not previously been articulated in mammalian cells.
researchers believe that CARPID has a wide range of applications, including the ability to detect viral RNA binding proteins, for example, SARS-CoV-2 is a COVID-19-trigger RNA virus, once the virus into the host cell, researchers can use CARPID technology to detect the virus used for life cycle recruitment of special cell proteins, if rejected binding proteins, the researchers can inhibit the virus replication information, or help researchers identify potential antiviral drug targets.
, many lncRNAs can also be used as biomarkers for cancer diagnosis, because they are very high in cancer cells compared to normal cells, and CARPID technology can also be used to detect these lncRNAs binding proteins in cancer cells, which may help to clarify potential carcinogenic mechanisms and potential protein targets for later use to develop cancer diagnostic techniques and new therapies.
the final researchers say they spent a year developing carpID technology, most of which were conducted at the City University of Hong Kong, and that they will continue to study stem cells and DANCR, a type of lncRNA that acts as a tumor promoter.
() References: !--/ewebeditor: page- and !--webeditor: page title""--1CrispR-assisted novel-cells RNA-binding proteins in living cells 2 Yi, W., Li, J., Zhu, X. et al CRISPR-assisted Nat Methods 17, 685-688 (2020). doi: 10.1038/s41592-020-0866-0 !--/ewebeditor.com.