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 (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.