Creative Biolabs is pushing a unique service referred to the Magic™ CARRY two-hybrid system which is a CRISPR-based yeast two-hybrid assay for RNA-protein interactions.

CRISPR/Cas9

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a family of DNA sequences in bacteria which contains fractions of DNA from viruses which have attacked the bacterium. That sequences are used by the bacterium to detect and destroy DNA of similar viruses and constitute the basis of a technique known as CRISPR/Cas9 (CRISPR-associated nuclease 9) system. It is a prokaryotic immune system that changes genes within organisms effectively and specifically.

The simple version of the CRISPR/Cas9 has been modified to edit varieties of genomes by delivering the Cas9 nuclease complexed with a synthetic guide RNA into cells. Thus, the cell's genome will be cut at a designated location, allowing existing genes to be removed or new genes added. First introduced into mammalian cells in 2013, CRISPR/Cas9 genome editing tool creates some advantageous aspects, such as simple-to-design, easy-to-use, and multiplexing (capable of editing multiple genes concurrently) and is adapted from microbial immune defense system. The core components of CRISPR/Cas9 are a nuclease Cas9 comprising two catalytic active domains RuvC and HNH, together with a single guide RNA (sgRNA) from CRISPR RNA (crRNA) and transacting CRISPR RNA. On the presence of a protospacer-adjacent motif (PAM) on the opposite strand, sgRNA directs Cas9 to the target site via base-pairing which results in Cas9-generated site-specific DNA double-strand breaks (DSBs) then repaired by homologous directed repair (HDR) if the homologous sequences are available. Otherwise, DSBs will be repaired by non-homologous end-joining (NHEJ). HDR decides the precise gene correction or replacement while NHEJ is error prone and may induce small insert or delete mutations. In addition, Cas9 is able to be reprogrammed into nickase (nCas9) through inactivating either RuvC or HNH, or into deactivated Cas9 (dCas9) through inactivating both of them.

Fig.1 Mechanisms of CRISPR/Cas9-mediated genome editing and epigenome modulation.

The CARRY Two-Hybrid Systems

It is commonly acknowledged that RNA-protein interactions are integral to the function of RNA in probably every cellular process. As for functional RNA that finally acts protein-independently, such as peptide-bond formation by ribosomal RNA and mRNA splicing by spliceosomal RNA, these transcripts also require associated proteins for their accurate folding, processing, modification, and localization. However, there is still a very limited set of biotechniques available for detecting proteins that bind to a specific RNA.

According to the current report, Creative Biolabs has performed a novel technique, CARRY two-hybrid system, to identify binding partners for a given RNA. The full name of this method is CRISPR-assisted RNA/RBP yeast (CARRY) two-hybrid system, which combines the technology of CRISPR with the highly effective yeast two-hybrid (Y2H) protein-protein interaction assay for the purpose of investigating RNA-protein interactions. In this CRISPR-based yeast two-hybrid system, an RNA of interest is targeted to the promoters of normal Y2H reporter genes by fusing it to the CRISPR guide RNA in a strain expressing catalytically dCas9. If the promoter linked RNA binds to a protein fused to Gal4 transcriptional activation domain (GAD), the reporter genes are then transcribed just as/like in the standard protein-protein Y2H assay. In conclusion, the new CARRY two-hybrid method provides an easily operable, much-needed instrument to confirm proteins that bind to a particular RNA

Mechanisms of CARRY Two-Hybrid Systems

Similarly to the original Y2H, CARRY two-hybrid scheme interrogates binding between two biological macromolecules by means of tethering one to the promoter of a reporter gene and fusing the other one to a transcriptional activation domain. The reporter gene will be expressed when there is binding occurred on two biomolecules. Unlike the Y2H method, instead of tethering a bait protein to the promoter by fusing it to a DNA-binding domain, CARRY two-hybrid system is using an RNA of interest tethered. Here, RNA tethering is achieved relying on the Streptococcus pyogenes CRISP machinery. While the CRISPR/Cas9 system has generally been co-opted in order to make targeted cuts in DNA, nuclease dCas9 can target an RNA or protein of interest to a specific genomic locus by fusing it to the CRISPR sgRNA or to Cas9, respectively. As we can see in the below diagram, CARRY two-hybrid assay utilizes the former of these two strategies to target the desired RNA to the shared sequences at the promoters of the Y2H reporter genes, HIS3 and LacZ. The reporter genes are subsequently activated if a protein which has been fused to the GAD binds to the promoter-tethered RNA.

Fig.2 The CARRY two-hybrid RNA-protein interaction system.

Creative Biolabs provides the Magic™ CARRY two-hybrid services by combining CRISPR/dCas9 RNP-mediated targeting of RNA to a specific DNA sequence with the yeast two-hybrid protein-protein interaction assay. It is a specific and sufficiently sensitive approach to detect RNA-protein interactions with near-micromolar dissociation constants. Please feel free to contact us for more information and a detailed quote.

Other optional protein-nucleic acid interaction (PNI) assay services:

• Yeast One-Hybrid (Y1H) Service
• Yeast Three-Hybrid (Y3H) Service

Published Data

• Using the CARRY Two-Hybrid Assay to Assess the Binding of CEH Mutant RNAs to TERT

Fig. 3 TERT-binding to telomerase RNA is impaired by a core-enclosing helix shorter than 4 bp. (Melissa A. Mefford, 2020)

Telomerase ribonucleoprotein (RNP) can solve the problem of chromosome terminal replication to prevent cell senescence in yeast, humans, and most other eukaryotes. The telomerase RNP core enzyme consists of a specific RNA subunit and telomerase reverse transcriptase (TERT). Here, the researchers have shown that a 4 bp core-enclosing helix (CEH) is necessary for telomerase activity in vitro and the maintenance of yeast telomeres in vivo. Through the CARRY two-hybrid assay based on CRISPR/nuclease-deactivated Cas9 (dCas9), they evaluated the binding of CEH mutant RNA to TERT and found that 4 bp CEH RNA binds to TERT, but not to the shorter CEH construct, which is consistent with telomerase activity and in vivo complementarity. It is concluded that CEH is essential in yeast telomerase RNA because it needs to bind TERT to form a core RNP enzyme.

FAQ

1. What is the CRISPR-assisted RNA/RBP yeast (CARRY) two-hybrid system and how does it work?

   The CARRY two-hybrid system is a specialized tool designed to study RNA-protein interactions within a yeast model. This innovative system leverages the CRISPR technology to target and manipulate RNA molecules, thereby facilitating the identification and characterization of interactions between RNA and RNA-binding proteins (RBPs). In the CARRY system, the CRISPR complex is engineered to include a catalytically inactive Cas protein (dCas) that is fused to an RNA-binding protein of interest. This fusion protein is directed to a specific RNA sequence within the yeast, where it can interact with other RBPs tagged with a reporter molecule. The presence of interaction between the RNA and RBP results in a detectable signal, typically through the activation of a reporter gene, which provides insights into the binding specificity and dynamics of RNA-protein interactions.

2. What are the advantages of using the CARRY two-hybrid system over traditional methods for studying RNA-protein interactions?

   Firstly, the specificity and versatility of CARRY two-hybrid technology allow for precise targeting of RNA molecules, which can be challenging with other methods. This precision enables the study of interactions under more physiologically relevant conditions. Secondly, the system can be used to investigate a wide range of RNA sequences and RBPs in a high-throughput manner, providing a more comprehensive analysis of RNA-protein interactions. Lastly, the use of a live yeast model permits the examination of these interactions in the context of a living cell, accounting for the effects of cellular environment on RNA and protein behavior, which is often overlooked in in vitro systems.

3. What types of RNA-protein interactions can be studied using the CARRY two-hybrid system?

   The CARRY two-hybrid system is capable of studying a wide variety of RNA-protein interactions, including those involving mRNA, non-coding RNA, and even viral RNA segments. This system is particularly useful for identifying and characterizing interactions with RNA-binding proteins (RBPs) that play critical roles in RNA splicing, stability, localization, and translation. It can be employed to investigate interactions under different environmental conditions or in the presence of various modifications to the RNA, thereby providing insights into how these factors influence RNA-protein binding dynamics.

4. How does the CARRY two-hybrid system contribute to biomedical research and drug development?

   The CARRY two-hybrid system has significant implications for biomedical research and the development of therapeutic strategies targeting RNA-protein interactions. By elucidating the mechanisms of these interactions, researchers can identify potential targets for drugs aimed at diseases where RNA processing or function is disrupted, such as cancer, neurodegenerative disorders, and viral infections. For instance, understanding how specific proteins interact with RNA to regulate gene expression can lead to the development of novel RNA-based therapeutics or small molecules that modulate these interactions. Additionally, the system's ability to screen numerous interactions quickly makes it an invaluable tool for identifying new drug targets and understanding disease mechanisms at a molecular level.

5. How does the CARRY two-hybrid system improve the specificity of RNA-protein interaction studies?

   The CARRY two-hybrid system enhances specificity in RNA-protein interaction studies by utilizing the CRISPR/Cas9 technology, which allows for targeted binding of the dCas (dead Cas9) protein to a specific RNA sequence using a guide RNA (gRNA). This specificity is crucial because it minimizes off-target effects common in other RNA-protein interaction assays, such as RNA immunoprecipitation, which can capture non-specific bindings. In the CARRY two-hybrid system, the dCas protein is fused to a RNA-binding protein (RBP) of interest, and this complex is guided to the RNA sequence targeted by the gRNA. The precision of CRISPR/Cas9 ensures that only the interactions between the targeted RNA and the protein of interest are measured, providing clearer, more accurate results about specific binding events.

6. Can the CARRY two-hybrid system be used in organisms other than yeast?

   While the CARRY two-hybrid system is initially developed and optimized for use in yeast, its underlying principles and components can potentially be adapted for use in other organisms, including mammalian cells. This adaptation would involve optimizing the delivery of CRISPR components and ensuring the functionality of the system in different cellular environments. The flexibility of CRISPR two-hybrid technology and the universal nature of RNA-protein interactions make it feasible to expand the use of the CARRY two-hybrid system beyond yeast, which could open new avenues for studying cellular processes and disease mechanisms in more complex biological models. This adaptation is particularly valuable for medical research where human cell models provide more relevant insights into human diseases.

7. What are the potential future developments for the CARRY two-hybrid system?

   It could include expanding its application to a broader range of biological models, such as mammalian cells, to study disease-relevant RNA-protein interactions more directly. There is also potential for integrating next-generation sequencing technologies with the CARRY two-hybrid system to enable high-throughput analysis of RNA-protein interactions, which would accelerate the discovery of novel interactions and their functional implications. Additionally, advancements might focus on refining the system's components, such as enhancing the efficiency of CRISPR delivery and the stability of the RNA-protein complexes, to improve both the sensitivity and the scalability of the system. These enhancements could significantly broaden the utility of the CARRY two-hybrid system in both basic research and therapeutic development.

8. What specific modifications can be studied using the CARRY two-hybrid system in RNA-protein interactions?

   The CARRY two-hybrid system allows researchers to study how various RNA modifications affect RNA-protein interactions. Modifications such as methylation, pseudouridylation, and others can significantly alter RNA structure and function, impacting how proteins recognize and interact with RNA. By using CRISPR/Cas technology to direct RBPs to specifically modified RNAs within the yeast model, the CARRY two-hybrid system enables the investigation of these interactions in a controlled setting. This capability is particularly valuable in understanding processes such as RNA editing, splicing regulation, and the molecular mechanisms underpinning diseases linked to RNA dysregulation.

9. How can the CARRY two-hybrid system aid in the validation of therapeutic targets involving RNA-protein interactions?

   By enabling precise manipulation and observation of these interactions within a living cell, researchers can evaluate the effects of disrupting or enhancing specific interactions. This approach is crucial for drug development, as it allows for the assessment of potential targets by simulating the inhibition or modification of these interactions in a model organism. For instance, in diseases where aberrant RNA-protein interactions are implicated, such as certain cancers or genetic disorders, the CARRY two-hybrid system can help in identifying and confirming the therapeutic potential of targeting these interactions before moving to more complex systems or clinical trials.

10. How does the CARRY two-hybrid system handle multiple RNA targets simultaneously?

    The CARRY two-hybrid system is capable of handling multiple RNA targets simultaneously by utilizing different guide RNAs (gRNAs) that direct the CRISPR/Cas9 complex to different RNA sequences within the same yeast cell. This multiplexing capability allows for the simultaneous study of various RNA-protein interactions, facilitating complex interaction networks and pathway analyses within a single experimental setup. Researchers can thus investigate the effects of multiple RNA-binding proteins on different RNA targets, or how a single RNA-binding protein interacts with various RNAs, providing comprehensive insights into the regulatory roles of these molecules.

11. What are the limitations of the CARRY two-hybrid system, and how can they be addressed?

    One of the main limitations is the potential for off-target effects, where the CRISPR/Cas9 system might bind to unintended RNA sequences, leading to false positives. Additionally, the efficiency of CRISPR/Cas9 delivery and the expression of fusion proteins can vary, affecting the system's reliability. Addressing these issues involves refining the specificity of guide RNAs and optimizing the yeast expression system to ensure stable and effective delivery of all components. Further research and development may also focus on improving the fidelity of CRISPR/Cas9 components to minimize off-target interactions and enhance the system's overall precision and usability.

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