Although scientists have not yet fully understood the diversity of RNA molecules, they believe that the RNA binding proteins binding to these RNA molecules may be directly related to the occurrence of a variety of diseases. Recently, research led by biomedical scientists from City University of Hong Kong (CityU) has developed CRISPR-assisted RNA-protein interaction detection method (CARPID), which leverages CRISPR-CasRx-based RNA targeting and proximity labeling to identify binding proteins of specific long non-coding RNAs (lncRNAs) in the native cellular context. This new method may be applied to multiple types of cell research, such as the identification of cancer biomarkers and the detection of potential drug targets for the treatment of various viral diseases. This study, published in the international journal Nature Methods, is entitled "CRISPR assisted detection of RNA-protein interactions in living cells."
The central principle of molecular biology is that DNA is transcribed into RNA, and RNA is eventually translated into protein. But in fact, only about 2% of RNAs can encode proteins, while the remaining 98% of RNA molecules known as non coding RNAs (ncRNAs) are regarded as "dark matter" because of their mysterious functions. In recent years, scientists have begun to work hard to reveal the real functions of RNA, especially long non coding RNAs (lncRNAs, defined as non-coding RNA of more than 200 nucleotides in length)). lncRNAs are widely accepted as an important cell component that can regulate gene expression, and it is also one of the most interesting RNAs. The interplay with RNA-binding proteins (RBPs) dictates the function and fate of RNA. Despite their importance, notable technical limitations exist in elucidating lncRNA-protein interactions in living cells.
To circumvent the limitations of existing methods and to detect RBPs in living cells, researchers developed a method termed CARPID. Inspired by a strategy that utilized CRISPR-dead Cas9 (dCas9) to navigate biotin ligase to specific genomic loci, they used a nuclease-activity-free form of the compact RNA-targeting type VI-D CRISPR single-effector system dCasRx for specific lncRNA targeting. This dCasRx effector protein was capable of processing guide RNA (gRNA) arrays to two or more component gRNAs without cleaving targeted RNA transcripts. CARPID can sensitively detect binding proteins of RNAs in any lengths or concentrations whereas most other methods can only be applied to very long non-coding RNAs.
Figure 1. Scheme of the CARPID workflow.
The CARPID is composed of two parts: navigation and proximity biotin-labeling. First, the team employed the CRISPR/CasRx system to navigate so that the CARPID components, including a 'labeling tool' called BASU, can approach the targeted RNA. Researchers fused the dCasRx with the engineered biotin ligase BASU, followed by a self-cleaving T2A peptide and an enhanced green fluorescent protein (eGFP) to monitor their expression in living cells.
To test the specificity of CARPID, the team applied it on three different lncRNAs, namely DANCR, XIST, and MALAT1. Comparison of the CARPID results among the three lncRNAs with partially shared subcellular distribution displayed virtually no overlap, demonstrating the high specificity and applicability of the CARPID method for lncRNAs of different lengths and expression levels in various subcellular localizations.
Figure 2. Comparison of CARPID results among different lncRNAs.
"CARPID can achieve high specificity, because CRISPR navigation is very accurate. We can even get very accurate information, that is, the specific location of RNA binding protein," explained Dr. Yan. In addition, researchers did not observe significantly altered gene expression in cells overexpressing BASU-dCasRx and gRNAs, confirming that CARPID did not interfere with the physiology of transfected cells. With the help of CARPID technology, if researchers detect the same RNA target at different time points, they can get dynamic results.
The researchers believe that CARPID has a wide range of applications, including detection of viral RNA binding proteins. For example, SARS-CoV-2 is an RNA virus that causes COVID-19. Once the virus enters the host cell, researchers can use CARPID technology to detect the specific cellular proteins that the virus recruits for its life cycle. If the binding protein is removed, the researchers can inhibit the replication of the virus, and the relevant information may help researchers identify potential antiviral drug targets.
Besides, many lncRNAs can also be used as diagnostic biomarkers for cancer, because their levels in cancer cells are very high compared with normal cells. CARPID can also be used to detect these lncRNAs binding proteins in cancer cells, which may help find tumorigenic mechanisms and potential protein targets for cancer diagnosis or treatment. However, CARPID has room for improvement, for example, biotin-label-based pulldown remains unavoidable. Some technologies, such as XRNAX21, PTex23 and OOPS22, utilize physicochemical properties to allow isolation and avoid affinity capture. But these methods do not allow recognition of individual RNA-protein pairs. Thus, CARPID and these methods can be complementary in investigating RBP-lncRNA interactions.