Method for purifying target RNA

Abstract

This invention provides a method for purifying target RNA. [Solution] A target RNA purification carrier comprising a CRISPR-dCas (dead Cas) protein capable of forming a complex with guide RNA and target RNA and lacking nuclease activity, supported on a solid phase carrier, wherein the guide RNA and target RNA are reacted to the carrier to form a complex containing the target RNA, the guide RNA, and the CRISPR-dCas protein, and A method for purifying target RNA, comprising the step of eluting the target RNA and a target RNA binding factor selected from the group consisting of genomic DNA, protein, and non-target RNA from the complex.

Description

[Technical Field]

[0001] The present invention relates to a method for purifying target RNA. [Background Art]

[0002] It is said that there are approximately 340,000 human RNAs in vivo, of which only about 300 RNAs have been studied, and the functions of approximately 99% of human RNAs remain unelucidated. On the other hand, the number of human proteins is approximately 21,000, of which about 70% have been studied.

[0003] The delay in elucidating the functions of human RNA is because RNA is a gene that is transiently expressed in a cell-specific manner, and it is difficult to label RNA via antibody recognition, biotin, or other methods, which makes it difficult to purify RNA having a specific nucleotide sequence under arbitrary conditions. [Summary of the Invention] [Problem to be Solved by the Invention]

[0004] An object of the present invention is to provide a method for purifying target RNA as a targeting technique capable of evaluating the functions of RNA molecules.

[0005]

[0006] [Means for Solving the Problem]

[0007] The present invention provides a method for purifying target RNA, comprising: allowing guide RNA and target RNA to act on a target RNA purification carrier, wherein said carrier is obtained by supporting a CRISPR-dCas (dead Cas) protein on a solid-phase carrier, said CRISPR-dCas protein being capable of forming a complex with guide RNA and target RNA and having no nuclease activity, to form a complex containing said target RNA, said guide RNA and said CRISPR-dCas protein on said carrier; and eluting the target RNA and a target RNA-binding factor selected from the group consisting of genomic DNA, protein, and non-target RNA from said complex. [Effects of the Invention]

[0008]

[0009]

[0010]

[0011] Furthermore, according to the present invention, it is possible to purify target RNA within cells and analyze the intracellular environment.

[0012]

[0013]

[0014] [Brief explanation of the drawing]

[0015] [Figure 1] A method for purifying RNA complexes: Trans RNA Immunoprecipitation (TRIP). [Figure 2] Application to the analysis of RNA function modifications: Precision RNA Sequence Modification (PRSM). [Figure 3] Construction of a photoactivated Cas13 (PAdCas13) system. [Figure 4] Construction of a photoactivated Cas13 (PAdCas13) system. [Figure 5] Construction of a photoactivated Cas13 (PAdCas13) system. [Figure 6] Target lncRNAs with high selectivity. [Figure 7] An example of RNA visualization targeting human XIST RNA. Using GFP-labeled dCas13, the nuclear localization of XIST RNA was confirmed in living cells. [Figure 8] TRIP method implementation model diagram. [Figure 9] An overview of the TRIP method targeting the untranslated region of SARS-CoV-2. [Figure 10] Transcript from pGL3-SARS-CoV-2.ORF1-Promoter. [Figure 11] Protocol for ChIP (Chromatin immunoprecipitation). [Figure 12] Performance evaluation 1 of the TRIP analysis method targeting SARS-CoV-2. [Figure 13] Performance evaluation 2 of the TRIP analysis method targeting SARS-CoV-2. [Figure 14] Types of RNAs bound by SARS-CoV-2-derived RNA in human cells; ontology enrichment analysis. [Figure 15] Performance evaluation 3 of the TRIP analysis method targeting SARS-CoV-2. [Figure 16] Performance evaluation 4 of the TRIP analysis method targeting SARS-CoV-2. [Figure 17] Data showing that the hydrogen bond of S565 is involved in the binding of atorvastatin and rosuvastatin to HMG-CoA. [Figure 18] Performance evaluation 5 of the TRIP analysis method targeting SARS-CoV-2. [Figure 19] Overview of the DNA immunoprecipitation used in the TRIP method. [Figure 20A] Verification test of the photosensitive switching system. [Figure 20B] Performance evaluation test of PA-dCas13 protein. (A) dCas13 protein carrying a guide RNA targeting lncRNA XIST is expressed in the nuclear fraction; (B) western blotting was performed for each cell fraction to confirm that dCas13 translocates to the site (nucleus) where the guide RNA-targeted RNA localizes. [Figure 21] Overview of the example verifying whether the binding region of XIST RNA is associated with the histone code. [Figure 22A]Application to ChIP-seq analysis. Left figure: Visualization of whether the binding region of XIST RNA on DNA has regularity around the transcription start region of a gene. The second column from the left shows the results of a TRIP test targeting XIST. The first column from the left shows the negative test without using crRNA. The third, fourth, and fifth columns from the left show the accumulation diagram targeting histone modifications. The right figure is a graph of the accumulation region from the transcription start region in the left figure. The top line shows the results of a TRIP test targeting XIST. It shows an accumulation pattern similar to H3K4me3 in the third row, suggesting that XIST is a transcription regulator. [Figure 22B] Properties of target RNA (XIST) accumulating at the transcription start site (TSS). The target, defined by a guide RNA, was set to long noncoding RNA XIST, and chromatin immunoprecipitation was performed, comparing it with existing technologies. The present invention enabled detection with high resolution using one or two guide RNAs. The ability to instantly switch ON / OFF and compare within the RNA molecule are innovative features that are difficult to achieve not only with existing technologies but also with the original dCas13 protein. X-axis: Distance from the transcription start site (right end is 5,000 bp, left end is -5,000 bp), Y-axis: Average accumulation value at the transcription start region. [Figure 23] RNA structure analysis data, structural prediction diagrams, and predicted protein groups that bind to the XIST sequence were used as references when designing the XIRT crRNA. [Figure 24] This study validated the functional control of XIST RNA by dCas13. By introducing dCas13 to a specific sequence of XIST RNA in cells into which crRNAs corresponding to the crRNA numbers shown in Figure 23 (upper diagram) were introduced, it was confirmed whether the induction of H3 (histone H3) onto the genome via XIST RNA was inhibited. In cells into which crRNAs 1, 2, 3, and 5 were introduced, the binding of H3 to the genomic region was inhibited, indicating that targeting crRNAs 1, 2, 3, and 5 inhibits XIST function. [Figure 25] Applications using the photoactivation Cas13 (PAdCas13) system. RNA phenotypic analysis using SC-sequence. [Figure 26] Repair of dystrophin gene mutations, a causative factor of Duchenne muscular dystrophy, at the preRNA level. Development of exon-skip therapies for Duchenne muscular dystrophy. [Figure 27A] pLKO5.U6.crRNA(gene name).tRFP.v1. [Figure 27B] pLKO5.U6.crRNA(gene name).tRFP.v1. [Figure 28A] pENTR1A.dCas13a.GFP. [Figure 28B] pENTR1A.dCas13a.GFP. [Figure 28C] pENTR1A.dCas13a.GFP. [Figure 28D] pENTR1A.dCas13a.GFP. [Figure 29A] pGL3-SARS-CoV-2.ORF1-Promoter. [Figure 29B] pGL3-SARS-CoV-2.ORF1-Promoter. [Figure 29C] pGL3-SARS-CoV-2.ORF1-Promoter. [Figure 30] esiRNA cDNA target sequences in which translation repression via the virus-derived 5'UTR was confirmed. [Figure 31] PD-1 pre-mRNA from PD-1-positive T cells / NK cells. [Figure 32] A guide RNA that targets introns 1 and 2 of PD-1 pre-mRNA. [Figure 33] Creation of PD-1 deactivation lymphocytes using dCas13. Successful creation of PD-1 expression-regulating T cells using comprehensive RNA targeting analysis. This enables the development of ex-vivo pharmaceuticals using the guide RNA sequence and dCas13 protein contained within cells in the region shown in Figure (a): providing safer cell-based therapeutics than CAR-T cell therapy (Kymriah). [Figure 34]We observed enhanced translational activity in luciferase RNA fused with 5'UTR RNA gene information derived from SARS-CoV-2. [Modes for carrying out the invention]

[0016] RNAs targeted for isolation, purification, functional elucidation, and prevention or treatment of diseases in this invention include lncRNAs, RNAs derived from RNA viruses including SARS-CoV-2 RNA, and RNAs with splicing abnormalities.

[0017] In the present invention, examples of CRISPR-dCas proteins having a helix region, capable of forming a complex with guide RNA and target RNA, and lacking nuclease activity, or CRISPR-dCas proteins having nuclease activity, include Cas5 / dCas5, Cas6 / dCas6, Cas7 / dCas7, Cas9 / dCas9, Cas10 / dCas10, Cas11 / dCas11, and Cas13 / dCas13, with Cas13 / dCas13 being preferred.

[0018] The CRISPR-dCas protein and the CRISPR-Cas protein have a helix region. When the helix region is cleaved at any point, it separates into an N-domain on the N-terminal side from the cleavage site and a C-domain on the C-terminal side from the cleavage site, forming two polypeptides: (N-domain)-(factor 1) with the N-domain bound to factor 1, and (factor 2)-(C-domain) with the C-domain bound to factor 2. These polypeptides may be linked with a linker containing a protease recognition sequence or a linker containing a self-cleaving peptide to form a single protein (CRISPR-dCas / Cas protein) represented as (N domain)-(factor 1)-(linker containing a protease recognition sequence)-(factor 2)-(C domain) or (N domain)-(factor 1)-(linker containing a self-cleaving peptide)-(factor 2)-(C domain), or they may be a set of polypeptides containing (N domain)-(factor 1) and polypeptides containing (factor 2)-(C domain) (CRISPR-dCas / Cas protein derivative set). This set is the "CRISPR-dCas / Cas protein derivative set having two parts". A single protein represented as (N domain)-(factor 1)-(linker containing a protease recognition sequence)-(factor 2)-(C domain) or (N domain)-(factor 1)-(linker containing a self-cleaving peptide)-(factor 2)-(C domain) is cleaved by the protease recognition sequence or self-cleaving peptide to form a set of polypeptides: one containing (N domain)-(factor 1) and the other containing (factor 2)-(C domain). When factor I and factor II bind in response to a stimulus, they form a complex represented by (N domain)-(factor I)-(non-covalent bond)-(factor II)-(C domain). This complex, along with a protein represented by (N domain)-(factor I)-(linker containing a protease recognition sequence)-(factor II)-(C domain) or (N domain)-(factor I)-(linker containing a self-cleaving peptide)-(factor II)-(C domain), may together be referred to herein as a "CRISPR-dCas / Cas protein derivative."In the absence of a stimulus, the non-covalent bond in the complex is cleaved, resulting in a set of polypeptides: one containing the (N domain)-(factor 1) and the other containing the (factor 2)-(C domain). The formation and cleavage of the non-covalent bond between factor 1 and factor 2 are reversible depending on the presence or absence of a stimulus. Stimuli that promote the binding of factor 1 and factor 2 include physical stimuli such as light (white light, blue light, etc.), heat (high temperature, low temperature), pH (acidic, neutral, alkaline), and pressure, as well as ligand-receptor combinations and chemical substances. When the stimulus is light, one of factor 1 and factor 2 may be an nMAG and the other may be a pMAG. "Non-covalent bond" includes the binding of factor 1 and factor 2; for example, if factor 1 and factor 2 are an nMAG and a pMAG, the binding of nMAG and pMAG is included in "non-covalent bond".

[0019] The protease recognition sequence may be cleaved by an intracellular protease, or a protease that can be cleaved by the protease recognition sequence may be introduced into the cell together with the CRISPR-dCas / Cas protein. The length of the linker can be appropriately selected by those skilled in the art, but is preferably a peptide consisting of 3 to 100 amino acids, more preferably 5 to 60 amino acids. Various protease recognition sequences known in the art can be used as the protease recognition sequence (J. Biol. Chem., Vol. 283, No. 30, p20897, 2008). Preferably, the protease recognition sequence is an intracellular protease recognition sequence in a cell into which a CRISPR-dCas / Cas protein derivative or a set of protein derivatives is introduced. Examples of intracellular protease recognition sequences include amino acid sequences recognized by intracellular proteases Furin, PC7, and PACE4. The linker may be a peptide consisting of a protease recognition sequence, or it may include further amino acid sequences at the N-terminal and / or C-terminal end of the protease recognition sequence. Examples of self-cleaving peptides include those described in the literature (Szymczak-Workman, Andrea L et al. “Design and construction of 2A peptide-linked multicistronic vectors.” Cold Spring Harbor protocols vol. 2012,2 199-204. 1 Feb. 2012, doi:10.1101 / pdb.ip067876), which is incorporated herein by reference in its entirety. Examples of self-cleaving peptides include, but are not limited to, T2A, P2A, E2A, and F2A.

[0020] The N domain and C domain may or may not have some amino acids in the helix region, the amino acid sequence of the helix region may be modified, new amino acid sequences may be added or inserted, and they may have amino acid sequences derived from a protease recognition sequence, a self-cleaving peptide, or a linker.

[0021] The present invention encompasses polynucleotides or complementary chains thereof that encode CRISPR-dCas / Cas protein derivatives, or sets of CRISPR-dCas / Cas protein derivatives. Examples of such polynucleotides include: (i) Polynucleotides encoding (N domain)-(factor 1)-(linker containing protease recognition sequence)-(factor 2)-(C domain) or (N domain)-(factor 1)-(linker containing self-cleaving peptide)-(factor 2)-(C domain); (ii) A polynucleotide encoding a polypeptide containing (N domain)-(factor 1) and a polynucleotide encoding a polypeptide containing (factor 2)-(C domain) are linked via a suitable spacer nucleotide sequence; one polynucleotide (iii) A set of two polynucleotides, one polynucleotide encoding a polypeptide containing an (N domain)-(factor 1) and the other polynucleotide encoding a polypeptide containing a (factor 2)-(C domain).

[0022] The vector of the present invention includes any of (i) to (iii) above, where (i) and (ii) are a single vector, and (iii) is a set of two types of vectors.

[0023] Either plasmid vectors or viral vectors may be used as vectors. Examples of viral vectors include adeno-associated virus vectors, adenovirus vectors, retrovirus vectors, and lentivirus vectors. Examples of plasmid vectors include entry vectors for gateway systems (such as pENTR), donor vectors for genetic recombination, and destination vectors for parallel transfer.

[0024] The vector may encode a CRISPR-dCas / Cas protein derivative, or a polynucleotide encoding a set of CRISPR-dCas / Cas protein derivatives, or its complementary strand, as well as at least one guide RNA.

[0025] The transformants of the present invention are produced by transforming host cells with the vector of the present invention. Examples of hosts include mammals such as humans, mice, rats, rabbits, guinea pigs, hamsters, goats, dogs, and monkeys. If the host cells are ES cells or fertilized eggs of a mammal other than a human, non-human mammals can be produced from the transformed cells, and the transformants of the present invention include non-human mammals.

[0026] The target RNA purification carrier of the present invention comprises a solid-phase carrier on which multiple CRISPR-dCas (dead Cas) proteins capable of forming complexes with guide RNA and target RNA, and lacking nuclease activity, are supported. Examples of CRISPR-dCas (dead Cas) proteins lacking nuclease activity include dCas5, dCas6, dCas7, dCas9, dCas10, dCas11, and dCas13, with dCas13 being preferred.

[0027] Known solid supports for supporting CRISPR-dCas proteins include diatomaceous earth, activated carbon, alumina, titanium dioxide, cross-linked starch particles, cellulose polymers, chitin, and chitosan derivatives.

[0028] For immobilizing CRISPR-dCas proteins onto solid phase supports, known methods for protein immobilization such as physical adsorption, ionic bonding, inclusion, and covalent bonding can be used, but among these, covalent bonding is preferable due to its superior long-term stability. Various methods can be used to covalently bond dCas proteins, including using compounds containing aldehyde groups such as formaldehyde, glyoxal, and glutaraldehyde, using polyfunctional acylation agents, and crosslinking sulfhydryl groups. For target RNA purification, it is preferable to pack the support into a column reactor.

[0029] Figure 1 illustrates a method for functionally analyzing target RNA in cells or cell lysates using the CRISPR-dCas / Cas protein derivative of the present invention. In Figure 1, photoactivated dCas13 is used as the CRISPR-dCas protein. Inactivated dCas13 is dCas13 before irradiation with blue or white light (in the dark), and activated dCas13 is dCas13 after irradiation with blue or white light (in the light). Before light irradiation, crRNA, inactivated dCas13, and target RNA exist separately, but when activated dCas13 is generated by light irradiation, a crRNA-activated dCas13-target RNA complex is formed. The target RNA may be bound to DNA, other RNA, proteins, etc.

[0030] Figure 1 shows examples of target RNA being complexed with DNA, RNA, or protein. When DNA or RNA is bound to the target RNA, the DNA or RNA to which the target RNA binds can be identified by separating it from the target RNA and performing real-time PCR (qPCR) next-generation sequencing (NGS) analysis. Similarly, when a protein is bound to the target RNA, the protein to which the target RNA binds can be identified by separating it from the target RNA and performing proteomic analysis. In this way, by analyzing the DNA, RNA, and protein that bind to the target RNA, the function of the target RNA can be elucidated. Furthermore, the sequence of the target RNA can be determined by combining qPCR and NGS analysis. These analyses allow for the analysis of the intracellular environment. For example, Figure 2 shows the results of the analysis of the intracellular environment after differentiation induction of iPS cells.

[0031] crRNA sequences can be designed based on crRNA algorithms that predict higher-order RNA structure. A target RNA purification carrier can have numerous crRNAs bound to a CRISPR-dCas protein, and by sequencing numerous target RNAs in a sample bound to this carrier, information about the target RNA can be obtained. For example, a group of lncRNAs (long noncoding RNAs) expressed during a disease can be determined from a sample derived from diseased cells. Alternatively, if the sequences of a group of lncRNAs expressed during a disease have already been determined, effective drug candidates can be screened by monitoring how their expression patterns change in response to candidate drug compounds. Since many relationships between diseases and lncRNAs are known, crRNAs can be designed using such information as a reference.

[0032] In one preferred embodiment of the present invention, a photoactivated dCas protein system (PAdCas system) can be used to observe the expression, distribution, and movement of lncRNAs within cells.

[0033] Figure 2 shows an example of applying the cells of the present invention to PRSM (Precision RNA Sequence Modification).

[0034] Figures 3-5 show an example of the PAdCas system of the present invention. In the dark, the PAdCas system is divided into an N-terminal portion containing the guide RNA recognition domain (corresponding to ILwaCas13a.NN MAG in Figures 3-5) and a second portion containing the HEPN domain (corresponding to ILwaCas13a.CP MAG in Figures 3-5). When cells containing these are irradiated with blue or white light, the nMAG and pMAG formation changes, and they bind to form an active dCas protein. The complex formed by the binding of this active dCas to intracellular crRNA and target RNA can be detected by labeling the active dCas (for example, with a fluorescent protein such as green fluorescent protein). The nucleotide and amino acid sequences of nMAG and pMAG are known from the literature (Nature Communications volume 6, Article number: 6256 (2015)).

[0035] The CRISPR-Cas / dCas proteins used in this invention, which can form complexes with guide RNA and target RNA and have or do not have RNA nuclease activity, can control the RNA splicing mechanism. For example, in patients with Duchenne muscular dystrophy in whom a stop codon has been introduced into exon 44, Duchenne muscular dystrophy can be treated by introducing Cas13 and guide RNA for skipping exon 44 into the cell.

[0036] Tables 1-4 below show diseases primarily caused by RNA splicing abnormalities, such as Duchenne muscular dystrophy.

[0037] [Table 1]

[0038] [Table 2]

[0039] [Table 3]

[0040] [Table 4]

[0041] The present invention further provides a therapeutic agent for COVID-19 that can suppress the proliferation of the novel coronavirus in the bodies of mammals, including humans.

[0042] Research by the inventors has revealed that the protein of the novel coronavirus is biosynthesized using the lipid metabolic pathway in mammals, particularly humans. The inventors tested lipid metabolism regulators as substances that can suppress the biosynthesis of the novel coronavirus protein in the lipid metabolic pathway, thereby preventing viral infection, and further suppressing viral replication even if infection occurs, thereby alleviating the symptoms of COVID-19 and preventing severe illness. They found that administering lipid metabolism regulators to mammals such as humans, especially humans who have not been administered lipid metabolism regulators, can prevent COVID-19 infection, and even if infected, it can suppress or at least delay the progression from mild to moderate, or from moderate to severe. By taking lipid metabolism regulators before infection with the novel coronavirus, the replication of the novel coronavirus in the body can be suppressed, thus preventing the onset of infection. Lipid metabolism regulators have low toxicity, making them particularly useful for preventing COVID-19 infection. The dosage of lipid metabolism regulators is approximately 1 to 150 mg per day for adult humans, which can be administered in 1 to 4 divided doses per day.

[0043] Examples of lipid metabolism regulators include, but are not limited to, mevastatin, atorvastatin, pravastatin, rosuvastatin, fluvastatin, and lovastatin. Lipid metabolism regulators that have HMG-CoA reductase inhibitory activity are preferred. Preferred lipid metabolism regulators include atorvastatin and rosuvastatin.

[0044] By applying the present invention to NK cells, PD-1 can be suppressed. For example, pleural or peritoneal dissemination can be treated by taking pleural or peritoneal fluid from a patient, suppressing PD-1 in the NK cells contained in the pleural or peritoneal fluid, and returning it to the pleural or peritoneal fluid.

[0045] For example, an anti-PD-1 antibody can be combined with the dCas13 complex of the present invention.

[0046] By suppressing the maturation of normal PD-1 mRNA using an anti-PD1 antibody and the dCas13 complex of the present invention, PD-1-expressing T cells can be activated not only by the antibody but also by eliminating PD-1 expression from within the T cells themselves, thereby enabling more effective treatment of cancer. Specifically, dCas13 targeting PD-1 pre-mRNA can be expressed in human lymphocytes to control splicing in a manner dependent on external stimuli. [Examples]

[0047] The present invention will be described in more detail below based on examples. Example 1: RNA complex purification method using trans RNA immunoprecipitation (TRIP) (Figure 1) The RNA target sequence recognition and binding system known as the CRISPR-dCas protein (e.g., CRISPR-dCas 13, sometimes abbreviated as "dCas13") recognizes the target gene and forms a complex with a guide RNA (crRNA) containing 28 nucleotides of the target sequence and a dCas protein without nuclease activity designed to split into two, such as a combination of pMAG and nMag that can bind upon light irradiation. For example, in Figure 1, in the case of dCas13 split into pMAG and nMag, inactivated dCas13 corresponds to the state before light irradiation where pMAG and nMAG are separated, and activated dCas13 corresponds to the state after light irradiation where pMAG and nMAG are bound. The inactivated dCas13 and activated dCas13 may be in a system where the bound and unbound states are reversibly changed by temperature, for example, using a heat shock protein. The bifid dCas protein is in an inactive state, but can be reactivated by external stimuli such as light or heat. Here, reactivation means that the CRISPR-dCas protein forms a complex with the target RNA and crRNA. Using TRIP technology, it is possible to extract the complex of the target RNA, crRNA, and dCas13. The complex may contain various factors such as other RNAs, DNA, and proteins that bind to the target RNA. In some cases, it is also possible to remove specific RNAs (such as ribosomal RNA) from the cell extract. In Figure 1, "Turn ON & immunoprecipitation & wash" means that inactivated dCas13 is converted to activated dCas13 by light irradiation (Turn ON), the resulting complex is immunoprecipitated, and then washed to remove components other than the complex.Subsequent extraction separates the target RNA from the DNA, protein, or other RNA bound to it. The DNA / RNA can then be sequenced using next-generation sequencing, and the protein can undergo proteomic analysis.

[0048] In Figure 1, TRIP technology is applied to a cell extract. TRIP technology allows for RNA labeling using Cas13 or dCas13 to be applied either inside cells or in a cell extract.

[0049] Example 2 (1) Application to the analysis of RNA function modification (Precision RNA Sequence Modification (PRSM)) By utilizing the fact that target RNA labeling with Cas13 or dCas13 itself modifies the function of the targeted RNA, comprehensive analysis of RNA function at the cell level into which the crRNA library has been introduced becomes possible. The functional modification of the target RNA can be regulated by switching from inactive-Cas13 / dCas13 to active-Cas13 / dCas13 in response to external stimuli, allowing the phenomenon to be observed in cells into which crRNA and Cas13 or dCas13 have been introduced at the timing desired by the researcher.

[0050] Figure 2 illustrates the application of Droplet-Sequencing (Drop-Seq). When Droplet-Sequencing is performed at the examiner's desired timing on a cell population into which an arbitrary crRNA (crRNA library in Figure 2) and Cas13 / dCas13 have been introduced and switched to Active-Cas13 / dCas13, the function of the RNA targeted by the crRNA is modified by Active-Cas13 / dCas13. As a result, each cell exhibits a different phenotype depending on the introduced crRNA sequence, allowing for the analysis of transcript expression levels as a cell-specific phenotype using next-generation sequencing. Using single-cell RNA-seq (scRNA-seq) technology allows tracking the trajectories of various cell lines, enabling the identification of the phenotype of the target RNA and the sequences responsible for its function by using a pre-designed crRNA library at the molecular and cell biology levels. The beads in Figure 2 have numerous complementary sequences (e.g., antisense polynucleotides of the target RNA or guide RNA) bound to them, and the target RNA binds to these beads.

[0051] Droplet sequencing is a well-known technique (References: Shapiro E, et.al. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet. 2013 Sep;14(9):618-30. doi: 10.1038 / nrg3542. Epub 2013 Jul 30. PMID: 23897237. Alizadeh AA, et.al. Toward understanding and exploiting tumor heterogeneity. Nat Med. 2015 Aug;21(8):846-53. doi: 10.1038 / nm.3915. PMID: 26248267; PMCID: PMC4785013.). Figure 2 shows an embodiment of the present invention in which Droplet-Sequencing is combined with the introduction of crRNA and photoactivated dCas13 (PA-dCas13) into iPS cells (iPSCs). PA-dCas13 and crRNA may be introduced into iPS cells by known methods that can introduce proteins and RNA into cells, such as liposomes and lipofectamines. Alternatively, a plasmid vector or viral vector encoding PA-dCas13 and crRNA may be introduced into iPS cells to express crRNA and photoactivated dCas13 (PA-dCas13) within the cells.

[0052] (2) Construction of a photoactivated Cas13 (PAdCas13) system The structure of the Cas13 protein was analyzed, and it was split into two peptides at the Helical1 region, which is expected to have little effect on the recognition of target RNAs such as HEPN-1 and HEPN-2, and on binding to crRNA (Figure 3). Each of the split peptides was further divided into two peptides: the N-terminal peptide (Cas13a.N:REC LOBE) and the C-terminal peptide (Cas13a.C:NUC LOBE) of dCas13.

[0053] In this example, by adding photosensitively binding peptides (pMAG and nMAG) to the C-terminus of dCas13a.N and the N-terminus of dCas13a.C, respectively, we created a mechanism in which the peptides, which were split into two by light (blue light) stimulation, recombine and regain their original function of forming a complex with target RNA under the induction of the crRNA present in Cas13 (Figures 4 and 5). The methods that can be used to regain the function of the split Cas13 / dCas13 peptides are not limited to physical stimuli such as light stimulation, temperature, and pressure, but can also be substituted with chemical stimuli such as ion gradients and drug stimulation.

[0054] Example 3: RNA purification test using crRNA targeting human XIST RNA Using the RNA ChIP method, XIST RNA and RNAs that bind to XIST were purified from cell extracts by immunoprecipitation via dCas13 (TRIP method). Next-generation sequencing confirmed that XIST RNA could be extracted with high purity (Figure 6). pENTR1A.dCas13a.GFP (Figure 28A) was prepared as an entry vector compatible with Gateway®-specific recombination, and recombination into a mammalian cell expression vector was performed. A lentiviral vector constitutively expressing crRNA targeting XIST was created by inserting the XIST gene sequence into pLKO5.U6.crRNA(gene name).tRFP.v1 (Figure 27A).

[0055] Figure 7 shows an example of RNA visualization targeting human XIST RNA, where the nuclear localization of XIST RNA in living cells was confirmed using GFP-labeled photoactivated dCas13 (PAdCas13).

[0056] Figure 8 is a model diagram of the TRIP method implementation. Figure 9 shows an overview of the interaction verification test between SARS-CoV-2 derived RNA and human intracellular factors.

[0057] The secondary structure of the virus-derived RNA sequence is predicted, and a targetable crRNA is designed. Alternatively, a synthetic RNA expression vector, pGL3-SARS-CoV-2.ORF1-Promoter (Figure 29A), is prepared by fusing the virus-derived RNA with a tag sequence (luciferase sequence) that can be targeted by the crRNA. In this study, a lentiviral vector constitutively expressing a crRNA targeting the luciferase gene was created by inserting the XIST gene sequence into pLKO5.U6.crRNA(gene name).tRFP.v1. In virus-infected cells (or infection model cells), crRNA targeting the viral RNA or crRNA targeting the tag sequence and dCas13 are expressed, and after conversion to Activate-Cas13 / dCas13 at an arbitrary time, the virus-derived RNA and its complex are purified using the TRIP method. Next-generation sequencing is performed for DNA / RNA. Proteomic analysis is performed for proteins. Figure 10 shows an overview of the TRIP method actually used, targeting the untranslated region of SARS-CoV-2. We attempted to elucidate the mechanism by which untranslated sequences derived from SARS-CoV-2 synthesize their own proteins within human cells.

[0058] Figure 11 shows the structural analysis of a fusion RNA of a tag sequence (RNA of the luciferase protein in this figure) and the untranslated region of SARS-CoV-2, as well as the design method for the crRNA sequence for the TRIP method. It can be seen that the untranslated region of SARS-CoV-2 exhibits a unique structure.

[0059] Figure 12 shows an overview of the RNA immunoprecipitation used in the TRIP method.

[0060] Figure 12 shows qPCR analysis to confirm the purity of the RNA recovered by the TRIP method. HEK293T cells and A549 cells were introduced with either the untranslated region sequence or only the tag sequence of Tag-fused SARS-CoV-2 shown in Figures 9 and 10. The TRIP method was then performed targeting the tag sequence, and the recovery efficiency of the untranslated region sequence of SARS-CoV-2 was compared with the recovered RNA. The RNA sequence could only be recovered in cells that had been introduced with Tag-fused SARS-CoV-2 and subjected to Cas13-mediated immunoprecipitation.

[0061] Figure 13 shows an example of performance evaluation of a TRIP analysis method targeting SARS-CoV-2. In Figure 13, the arrows indicate gene groups involved in lipid metabolism obtained by immunoprecipitation. As shown in Figure 13, it was found that the RNA of the untranslated region of SARS-CoV-2 has specificity for binding to RNA sequences within human cells and significantly binds to intracellular metabolic mechanisms.

[0062] Figure 14 shows another example of performance evaluation of a TRIP analysis method targeting SARS-CoV-2. Ontology enrichment analysis revealed that the function of the RNA group recovered in Figure 13 is related to lipid metabolism.

[0063] Figure 15 shows another example of performance evaluation of a TRIP analysis method targeting SARS-CoV-2. This study investigated whether introducing only the untranslated region sequence or the tag sequence of Tag-fused SARS-CoV-2 into cells caused changes in gene expression in lipid metabolism mechanisms.

[0064] We investigated whether the transcriptional activity of lipid metabolism-related genes extracted by GO analysis is altered by the updating of SARS-CoV-2 5'UTR gene expression.

[0065] ACAA2, HMGcs, FADS1 / 2, and SCD are all factors involved in lipid metabolism and cholesterol metabolism. When pGL3-5'UTR was expressed in HEK293T and A549 cells, a significant change in gene expression was observed compared to when pGL3 was expressed.

[0066] Figure 16(b) is an interpretation diagram of the results in Figure 15(a). In the system with increased expression of SARS-CoV-2-derived 5'UTR, metabolism to the PUFA system was enhanced in A549 cells, and cholesterol metabolism was enhanced in HEK293T cells.

[0067] Figure 16(c) shows an attempt to suppress HMGcs using commercially available statins. Statins suppressed the expression of Luciferase protein, which was elevated by the SARS-CoV-2-derived 5'UTR. However, no inhibition of cell proliferation or repression of Luciferase RNA transcription was observed.

[0068] Figure 16(d) shows that HMGcs and ACAA2 expression were suppressed using esiRNA, and the suppression of Luciferase protein expression, which was elevated by the 5'UTR derived from SARS-CoV-2, was confirmed. The range of sequences targeted by esiRNA (MISSION® esiRNA: composed of a heterogeneous pool of several hundred 21bp siRNAs) is shown in Figure 17.

[0069] The above findings indicate that lipid metabolism regulation suppresses the expression of Luciferase protein, which is elevated by the 5'UTR derived from SARS-CoV-2. No toxicity to cell lines was observed in any of the suppression systems, nor was suppression of Luciferase mRNA expression observed. Therefore, it is shown that the suppression of Luciferase protein expression, which is elevated by the 5'UTR derived from SARS-CoV-2, is due to translational repression of the Luciferase protein.

[0070] As shown in Figure 17, the fact that atorvastatin and rosuvastatin have many binding sites (S565) when they bind to HMG-CoA is thought to be one of the reasons why they have an inhibitory effect on the proliferation of SARS-CoV-2.

[0071] Figure 18 shows another example of performance evaluation of the TRIP analysis method targeting SARS-CoV-2, illustrating the rules governing the formation of complexes within host cells, including human cells, by the untranslated region sequence of SARS-CoV-2. For the untranslated region sequence of SARS-CoV-2 (CoV19 in the figure) to bind with human-derived RNA, certain sequences are required on the human side, suggesting the existence of a protein that mediates the binding of the SARS-CoV-2 untranslated region sequence to human RNA.

[0072] Figure 19 shows an overview of the DNA immunoprecipitation method used in the TRIP method, illustrating the procedure for extracting and analyzing DNA that binds to XIST RNA.

[0073] Figure 20 shows the demonstration test of the photosensitive switching system. Purification of the XIST-DNA complex via dCas13-crRNA was possible only in cells that were photostimulated. The generated genomic DNA was detected by qPCR against known XIST RNA binding regions.

[0074] Figure 21 shows an overview of an example in which, in addition to Figure 19, the binding region of XIST RNA was investigated to determine whether it is related to the histone code.

[0075] Left panel of Figure 22A: A visualization of whether the binding region of XIST RNA on DNA exhibits regularity around the transcription start region of a gene. The second column from the left shows the results of a TRIP test targeting XIST. The first column from the left shows the negative test without using crRNA. The third, fourth, and fifth columns from the left show the accumulation diagram targeting histone modifications. The right panel is a graph of the accumulation region from the transcription start region in the left panel. The top row shows the results of a TRIP test targeting XIST. It shows an accumulation pattern similar to that of H3K4me3 in the third row, suggesting that XIST is a transcription regulator.

[0076] Figure 22B shows the results of DNA sequencing of genomic DNA sequences targeted by XIST, extracted from gRNA-dCas13-mediated pull down (TRIP) targeting the lncRNA XIST, confirming its accumulation at the transcription start region. The isolated DNA is known to interact with XIST RNA and to be involved in gene transcription regulation in conjunction with histone modifications. The results of this study demonstrate that we were able to extract XIST target sequences with higher accumulation levels (peak height) and higher degradation levels (peak narrowness) than existing technologies (e.g., ChIRP, reference: Mol Cell. 2011 Nov 18;44(4):667-78).

[0077] Figure 23 shows the RNA structure analysis data, structural prediction diagram, and predicted protein group binding to the XIST sequence that were used as references in designing the XIST crRNA.

[0078] Figure 24 shows the results of a functional regulation test of XIST RNA by dCas13. In Figure 23, we investigated whether inducing dCas13 at a specific sequence of XIST RNA in cells into which crRNAs corresponding to the crRNA numbers in the upper diagram were introduced would inhibit the induction of H3 (histone H3) onto the genome via XIST RNA. In cells into which crRNAs 1, 2, 3, and 5 were introduced, the binding of H3 to the genomic region was inhibited, indicating that targeting crRNAs 1, 2, 3, and 5 inhibits the function of XIST.

[0079] Figure 25 shows applications using a photoactivated Cas13 (PAdCas13) system. The applications shown in Figure 24 enable comprehensive RNA function analysis (Precision RNA Sequence Modification (PRSM) method in Figure 2). One example of its application is the ability to inhibit the sequence-specific function of a target RNA at a timing desired by the researcher during differentiation induction studies using iPS cells.

[0080] Figure 26 shows the preRNA-level repair of dystrophin gene mutations, a causative factor of (Duchenne) muscular dystrophy. As part of our investigation into the potential for regulating RNA splicing mechanisms and therapeutic applications by Cas13 / dCas13, we attempted to regulate dystrophin gene splicing. Diseases primarily caused by abnormal RNA splicing are shown in the attached figure.

[0081] Example 4: PD-1 pre-mRNA of PD-1-positive T cells / NK cells (Figure 31) T cells, which have the ability to recognize and eliminate cancer cells, express PD-1, causing them to mistakenly identify cancer cells as self-cells. However, by using dCas13, it is possible to intentionally suppress PD-1 expression from T cells surrounding cancer cells, thereby creating genetically unmodified chimeric antigen receptor T cells that specifically attack cancer cells, leading to the development of novel immunotherapies (Figure 31).

[0082] The target was the expression of exon 2 of PD-1 pre-mRNA, and the aim was to control the process from PD-1 pre-mRNA to mature mRNA by altering the structure of the functional sequences of splicing regulatory proteins that bind to introns 1 and 2 of PD-1. The sequences of guide RNAs targeting each sequence are shown (Figure 32).

[0083] Creation of genetically unmodified chimeric antigen receptor T cells with enhanced tumor cell attack capabilities. Figure 33 (left): Human CD8+ T cell line EBT-8, which expresses PD-1, was introduced with a PA-dCas13 constitutive expression system, followed by the introduction of guide RNAs targeting PD-1 pre-mRNA. Guide RNA introduction was performed using a lentiviral vector, and all of the aforementioned PD-1 target guide RNAs 1-18 were introduced simultaneously. In the region labeled a in Figure 33 (right panel), selective suppression of PD-1 expression was confirmed in EBT-8.

[0084] We will create chimeric antigen receptor T cells with successfully suppressed expression using lymphocytes derived from patient ascites fluid, and evaluate their efficacy and safety in an animal model of peritoneal dissemination.

[0085] Example 5 We observed enhanced translational activity in luciferase RNA fused with 5'UTR RNA gene information derived from SARS-CoV-2 (Figure 34). Figure 34a: In the SARS-CoV-2 RNA genome, the 5'UTR gene region corresponds to a 261nt region located on the 3' side of the ORF1A sequence. The sequence of this 5'UTR region was inserted by fusing it to the 3' side of the Kozak sequence in the Luciferase RNA sequence of the pGL3-promoter vector.

[0086] Figure 34b: We compared and measured whether the expression of Luciferase protein increased in a dose-dependent manner with the introduction of pGL3-promoter or pGL3-5'UTR vector in HEK293T and A549 cells. We observed increased expression of Luciferase protein fused with 5'UTR RNA gene information derived from SARS-CoV-2 in both HEK293T and A549 cells. This suggests that the 5'UTR derived from SARS-CoV-2 contains a sequence that enhances the translational activity of mRNA fused to it in human cells.

[0087] Figure 34c: The expression level of Luciferase mRNA was checked using qPCR in the experiment performed in Figure 34b to see if it had changed. No upregulation of Luciferase mRNA was observed. Based on these results, we obtained evidence that the 5'UTR derived from SARS-CoV-2 contains a sequence that enhances the translational activity of mRNA fused within human cells. The 5'UTR derived from SARS-CoV-2 is expected to be useful in molecular genetics research as a novel IRES-like sequence that enhances the translation efficiency of any mRNA.

Claims

1. A step of forming a complex on a target RNA purification carrier, which comprises a CRISPR-dCas (dead Cas) protein capable of forming a complex with guide RNA and target RNA and lacking nuclease activity, supported on a solid phase carrier, by reacting the guide RNA and target RNA with the carrier, thereby forming a complex containing the target RNA, the guide RNA, and the CRISPR-dCas protein, and A method for purifying target RNA, comprising the step of eluting the target RNA and a target RNA binding factor selected from the group consisting of genomic DNA, protein, and non-target RNA from the complex.

2. The purification method according to claim 1, wherein the CRISPR-dCas protein is dCas13.

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