RNA expression modulating or editing method via regulation of Cas13 protein

Abstract

The present invention relates to a method for regulating or editing RNA expression through the chemical or optogenetic regulation of Cas13 protein activity. Specifically, to regulate the activity of Cas13 in a CRISPR-Cas13 system, fragments of the Cas13 protein are generated to enable recombination, and a protein capable of chemical or optogenetic binding is attached to each fragment. It was confirmed that the Cas13 protein can be activated by treatment with a small molecule compound or light irradiation. Accordingly, the method of the present invention can be usefully employed for the treatment of diseases by regulating disease-related RNA expression or editing RNA mutations.

Description

Technology Field

[0001] The present invention relates to a method for regulating or editing RNA expression through chemical or optogenetic regulation of the activity of Cas13 protein. Background Technology

[0003] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) distributed at periodic intervals constitute a CRISPR-Cas system that confers adaptive immunity in many bacteria in combination with associated sequences (cas). The CRISPR-Cas system consists of arrays of short DNA repeats interposed by hypervariable sequences that provide adaptive immunity against invasive genetic elements, such as phages and plasmids, through sequence-specific targeting and interference. Typically, invasive DNA sequences are acquired as new "spacers" and, paired with CRISPR repeats, are inserted into the CRISPR locus as new repeat-spacer units. Subsequently, the repeat-spacer array is transcribed into long pre-CRISPR RNA (pre-crRNA), which is processed into small interfering CRISPR RNAs (crRNA) that induce sequence-specific recognition. In particular, crRNA induces nucleases as complementary targets for sequence-specific nucleic acid cleavage mediated by Cas endonucleases. The CRISPR-Cas system is identified in half (~46%) of bacteria and the majority (~90%) of archaea.

[0004] The Cas system is currently being applied in many therapeutic fields by targeting cellular DNA and replacing it with an appropriate guide molecule to perform DNA sequence editing. In addition to the Cas9 system capable of targeting DNA, several Cas proteins have been discovered, among which the Cas13 protein targets single-stranded RNA.

[0005] The CRISPR-Cas13 system was discovered as a system that targets RNA, unlike Cas9, and was first reported in 2016 under the name C2C2. Existing CRISPR-Cas13 systems are mainly composed of four subtypes, all of which are characterized by having two HEPN domains (higher eukaryotes and prokaryotes nucleotide-binding domains) with RNase activity. Similar to Cas9, Cas13 forms a complex with crRNA to cleave target ssRNA, thereby rendering foreign genes non-functional.

[0006] The CRISPR-Cas13 system is currently being applied in various fields. There are studies disclosing that Cas13α expression can be induced by inserting a promoter capable of binding to NF-κB, which is overexpressed in cancer (Molecular Therapy: Oncolytics Vol. 19 December 2020); studies disclosing that cleavage efficiency varies depending on the crRNA spacer in glioma cells (Adv. Sci. 2019, 6, 1901299); studies applying the CRISPR-Cas13 system as an RNA virus therapeutic (Molecular Therapy: Nucleic Acids Vol. 19 March 2020); and studies disclosing that only mutant K-Ras in cancer cells is cleaved specifically by the K-RAS gene and spacer (Cancer Letters, Volume 431, 1 September 2018, Pages 171-181). Furthermore, given the reduction of neurons in neurodegenerative diseases such as Parkinson's disease, there is potential for treating neurological disorders through a system that converts glia to neuron via Cas13d delivery. The disclosed literature (Cell, Volume 181, Issue 3, 30 April 2020, Pages 590-603.) and the CRISPR-Cas system have been reported to be effectively applied in zebrafish (Developmental Cell, Volume 54, Issue 6, 28 September 2020, Pages 805-817.). However, the Cas13 system has the limitation that it cannot regulate expression.

[0007] Optogenetics is a biological technology that allows for the control of cells in biological tissues using light; a representative example is the genetic engineering of nerve cells to express light-responsive ion channels. As a technology combining optics and genetics, optogenetics enables the regulation and observation of the activity of individual nerve cells in biological tissues, and even in freely moving animals, allowing for real-time verification of the effects induced by the regulation of neural activity. The primary component required for optogenetics is light-responsive protein. Optogenetic actuators such as channelrhodopsin, halorodopsin, and archrhodopsin are used to regulate neural activity, while optogenetic sensors such as GCaMP, which detects changes in calcium concentration; synaptopHluorin, which detects the secretion of neuronal vesicles; GluSnFRs, which detect neurotransmitters; and Arclightning (ASAP1), which detects cell membrane potential, are used to optically record neural activity. Optogenetics allows for the selective modulation or recording of neural activity in specific genetically classified neurons, and because it utilizes light, target location and time can be precisely controlled. Therefore, using optogenetics offers the potential to effectively regulate the CRISPR-Cas13 system within cells through the control of intensity, time, and space.

[0008] Furthermore, chemical genetics refers to the regulation of cellular or physiological functions of proteins by disrupting them using small molecule compounds, as a powerful method utilizing small molecule regulators to provide molecular standards for various biological processes. An example of regulating protein function with small molecule compounds via chemical genetics is disclosed in which a fusion protein containing the heterodimer-forming proteins FRB and FKBP can form various oligomers upon the addition of the compound rapamycin (J Biosci Bioeng. 2016 Jul;122(1):40-6.).

[0009] Accordingly, the inventors completed the present invention by generating fragments of the Cas13 protein capable of recombination to regulate the activity of Cas13 in a CRISPR-Cas13 system and connecting a protein capable of chemogenetic or optogenetic binding to each fragment, thereby confirming that the Cas13 protein can be activated by treatment with a small molecule compound or light irradiation. The problem to be solved

[0011] The object of the present invention is to provide a method for regulating or editing RNA expression through chemical or optogenetic regulation of the activity of Cas13 protein. means of solving the problem

[0013] To achieve the above objective,

[0014] The present invention provides a method for regulating the activity of a Cas13 protein, comprising the step of treating a compound or light irradiating a first fragment comprising an N-terminal fragment of a Cas13 protein and a first regulatory protein; and a second fragment comprising a second regulatory protein and a C-terminal fragment of a Cas13 protein.

[0015] In addition, the present invention comprises the step of 1) preparing a vector comprising a construct comprising a sequence encoding an N-terminal fragment of a Cas13 protein and a sequence encoding a first regulatory protein; and a vector comprising a construct comprising a second fragment comprising a sequence encoding a second regulatory protein and a sequence encoding a C-terminal fragment of a Cas13 protein;

[0016] 2) a step of injecting a crRNA (CRISPR RNA) capable of hybridizing to the vector and target RNA sequence into a mammalian cell; and

[0017] 3) a step of treating with a compound or light irradiating; provides a method for regulating the expression of target RNA.

[0018] In addition, the present invention comprises the step of 1) preparing a vector comprising a construct comprising a sequence encoding an N-terminal fragment of a Cas13 protein and a sequence encoding a first regulatory protein; and a construct comprising a second fragment comprising a sequence encoding a second regulatory protein, a sequence encoding a C-terminal fragment of a Cas13 protein, and a sequence encoding an RNA editing protein.

[0019] 2) a step of injecting a crRNA (CRISPR RNA) capable of hybridizing to the vector and target RNA sequence into a mammalian cell; and

[0020] 3) a step of treating with a compound or light irradiating; provides a method for editing a target RNA sequence. Effects of the invention

[0022] The present invention relates to a method for regulating or editing RNA expression through the chemical or optogenetic regulation of Cas13 protein activity. Specifically, to regulate the activity of Cas13 in a CRISPR-Cas13 system, fragments of the Cas13 protein are generated to enable recombination, and a protein capable of chemical or optogenetic binding is attached to each fragment. It was confirmed that the Cas13 protein can be activated by treatment with a small molecule compound or light irradiation. Accordingly, the method of the present invention can be usefully employed for the treatment of diseases by regulating disease-related RNA expression or editing RNA mutations. Brief explanation of the drawing

[0024] Figure 1 shows the structure of the predicted PspCas13b protein, with eight candidate fragments of Cas13b indicated. Figure 2 is a schematic diagram showing the cleavage sites of candidate Cas13b fragments, which are recombinable Cas13b proteins and are expected to exhibit Cas13b activity by conserving two HEPN domains with RNase activity. Figure 3 is a schematic diagram showing a construct in which each regulatory protein is connected by a linker (GS16) using the FKBP (FK506-binding protein)-FRB (FKBP-rapamycin-binding) system, which is known to be dimerized by rapamycin, so as not to affect Cas13b protein function. Figure 4 is a schematic diagram showing a construct in which each regulatory protein is connected by a linker (GS16) using an nMag (negative Mag)-pMag (positive Mag) system known to be dimerized by blue light. Figure 5 shows Cas13 activity measured by double luciferase assay after treatment with 500 nM rapamycin (error bars, mean ± sem, n=5, Student's two-sided t-test was performed). Figure 6 is a schematic diagram showing the crRNA-mediated RNA degradation activity of Cas13, in which rapamycin treatment induces heteromerization between FRB and FKBP, causing the split Cas13 fragment to recombine. Figure 7 shows the results of the second screening of the Cas13 splitting site near residues 351 and 352 (error bars, mean ± sem, n=3, Student's two-sided t-test was performed). Figure 8 is a schematic diagram showing the crRNA-mediated RNA degradation activity of Cas13 (paCas13), in which blue light treatment induces heteromerization between pMag and nMag, causing the split Cas13 fragments to recombine. Figure 9 shows the decrease in luciferase activity upon light stimulation using a paCas13 fragment fused with a photo-induced dimerization domain (pMag: nMag and pMag: nMagHigh1). Figure 10 is a schematic diagram of a paCas13 candidate based on the Cas13 splitting site near residues 351 and 352. Figure 11 shows photo-induced RNA knockdown of endogenous KRAS, PPIB, and STAT3 in HEK293T cells transfected with paCas13-1, paCas13-2, or paCas13-3 and multiple crRNA (error bars, mean ± sem, n = 3). Figure 12 is a schematic diagram of RNA regulation engineered by coupling padCas13 (including H133A and H1058A mutations that inactivate the HEPN RNase domain of Cas13b) with ADAR2DD. Figure 13 is a schematic diagram of an A-to-I RNA edit reporter analysis, in which a single G-to-A mutation is introduced into the firefly luciferase coding sequence and a W417X (X = STOP) codon switch occurs, resulting in the loss of the measurable firefly luciferase signal. Figure 14 shows the analysis results of the padCas13 editor over time under light conditions quantified by the reconstruction of firefly luciferase signals (D: dark, L: light). Figure 15 shows a standard curve for modifying Sanger sequencing chromatograms with fluorescence and PCR bias. Figure 16 shows sequencing chromatograms under Dark (Top) or Light (Bottom) conditions. Figure 17 is a figure showing the percentage of RNA edited and normalized by RT-PCR-Sanger sequencing (D: dark, L: light). Figure 18 shows the percentage of RNA editing in transfected cells containing only crRNA by RT-PCR-Sanger sequencing. Figure 19 shows the deep-sequencing quantification (n = 1) of A-to-I RNA editing by the padCas13 editor under light stimulation for 24 hours. Figure 20 is a figure showing the RNA-seq readout results surrounding the target site (1250A → I) of Firefly luciferase editing. Figure 21 is a schematic diagram of the EGFP indicator for light-induced C-to-U RNA editing activity. Figure 22 shows representative fluorescence microscopy images of HEK293T cells co-transfected with padCas13-2 editor, EGFP indicator, and target crRNA. Figure 23 shows a representative flow cytometry plot of transfected with the padCas13-2 editor [X-axis: FITC-A (log scale), Y-axis: SSC-A (linear scale)]. Figure 24 is a figure showing the quantification of EGFP H66Y restored by the padCas13 editor under conditions. Figure 25 shows the A-to-I RNA editing rate by the padCas13-1 or padCas13-2 editor under light stimulation for 24 hours (error bars, mean ± sem, n = 3). Figure 26 shows the C-to-U RNA editing rate by the padCas13-1 or padCas13-2 editor under light stimulation for 24 hours (error bars, mean ± sem, n = 3). Specific details for implementing the invention

[0025] The present invention will be described in detail below.

[0027] The present invention provides a method for regulating the activity of a Cas13 protein, comprising the step of treating a compound or light irradiating a first fragment comprising an N-terminal fragment of a Cas13 protein and a first regulatory protein; and a second fragment comprising a second regulatory protein and a C-terminal fragment of a Cas13 protein.

[0028] The above Cas13 protein is a type of Cas protein. Cas proteins are CRISPR-associated proteins that are enzymes capable of recognizing and cleaving nucleic acids, such as DNA or RNA, when they have a double strand or a single strand (dsDNA / RNA and ssDNA / RNA). Specifically, they can recognize and cleave double-stranded or single-stranded nucleic acids bound to crRNA or guide RNA. That is, endonuclease function is activated by recognizing that crRNA is bound to a target site. Additionally, as endonuclease function is activated, they may possess exonuclease activity capable of non-specifically cleaving double-stranded and / or single-stranded DNA and / or RNA.

[0029] The Cas13 protein can be any one protein selected from the group consisting of Cas13a, Cas13b, Cas13c, and Cas13d, and can naturally recognize and cleave RNA, and is known as "C2c2" in bacteria. Cas13 is a class 2, type VI CRISPR protein that is activated by recognizing ssRNA targets. Leptotrichia wadei Cas13a found in and Prevotella sp. Cas13b found in P5-125 is representative, and these two do not require specific motifs like PAM.

[0030] The Cas13b protein is associated with one or more functional domains, and the effector protein contains one or more mutations within the HEPN domain, so that the complex can transmit epigenetic modifiers or transcriptional or translational activation or inhibition signals. The complex may be formed in vitro or in vitro, introduced into a cell or in contact with RNA; or formed in vivo.

[0031] The above Cas13b protein is not limited, but in a specific embodiment of the present invention, the Cas13b protein is PspCas13b.

[0032] PspCas13b is the most highly expressed protein in animal cells and has been verified to have an effective KD effect. Its structure consists of two HEPN domains with RNase activity, crRNA, and a domain that interacts with target RNA.

[0034] A linker may be inserted between the N-terminal fragment of the Cas13 protein and the first regulatory protein; and between the second regulatory protein and the C-terminal fragment of the Cas13 protein.

[0035] The above linker includes glycine (G) and serine (S), and may have an amino acid sequence in which glycine and serine are repeated 16 times (GSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGS, SEQ ID NO. 21).

[0037] The N-terminal fragment of the Cas13 protein may be a fragment up to the 272nd (Sequence No. 1), 286th (Sequence No. 3), 351st (Sequence No. 5), 480th (Sequence No. 7), 550th (Sequence No. 9), 624th (Sequence No. 11), 725th (Sequence No. 13), or 928th (Sequence No. 15) amino acid from the N-terminus of the Cas13 protein, and the fragment includes one HEPN domain having RNase activity.

[0038] The C-terminal fragment of the above Cas13 protein may be a fragment up to the 861st (Sequence No. 2), 847th (Sequence No. 4), 782nd (Sequence No. 6), 653rd (Sequence No. 8), 583rd (Sequence No. 10), 509th (Sequence No. 12), 408th (Sequence No. 14), or 205th (Sequence No. 16) amino acid from the C-terminal of the Cas13 protein, and the fragment includes one HEPN domain having RNase activity.

[0040] The treatment with the above compound or light irradiation induces dimerization of the first regulatory protein and the second regulatory protein.

[0041] The above compound treatment refers to chemogenetically regulating the cellular or physiological functions of proteins by using low-molecular-weight compounds to disrupt them.

[0042] The above compound is not limited to, but in a specific embodiment of the present invention, the compound is rapamycin.

[0043] The above light irradiation refers to a biological technology capable of controlling the cells of biological tissues using light.

[0044] The light is not limited to, but in a specific embodiment of the present invention, the light is blue light.

[0046] The combination of the first regulatory protein and the second regulatory protein is not limited, but in a specific embodiment of the present invention, the combination of the first regulatory protein and the second regulatory protein is FKBP (SEQ No. 18)-FRB (SEQ No. 17), nMag (SEQ No. 19)-pMag (SEQ No. 20), or nMagH (SEQ No. 74)-pMag (SEQ No. 20).

[0047] Rapamycin, an antifungal antibiotic, simultaneously binds to the 12-kDa FK506 binding protein (FKBP) and the FKBP-rapamycin binding (FRB) domain, mediating the formation of a tight heterodimer (K d = 2.5 nM). Various cellular functions, such as gene expression and protein translocation, can be artificially regulated by the conditional dimerization of the protein of interest fused to FKBP or FRB through the addition of rapamycin.

[0048] nMag-pMag are designed to recognize each other based on electrostatic interactions, preventing homomerization and inducing light-induced heterodimerization. They are known to exert spatially and temporally precise control over various signaling proteins in mammalian cells. The nMag-pMag system has a small size similar to FKBP-FRB and has the advantage of being able to regulate the rate of dissociation reactions. nMagH (nMagHigh1) is a variant in which the 135th methionine (M) and 165th methionine of nMag are mutated to isoleucine (I).

[0050] In addition, the present invention comprises the step of 1) preparing a vector comprising a construct comprising a sequence encoding an N-terminal fragment of a Cas13 protein and a sequence encoding a first regulatory protein; and a vector comprising a construct comprising a second fragment comprising a sequence encoding a second regulatory protein and a sequence encoding a C-terminal fragment of a Cas13 protein;

[0051] 2) a step of injecting a crRNA (CRISPR RNA) capable of hybridizing to the vector and target RNA sequence into a mammalian cell; and

[0052] 3) a step of treating with a compound or light irradiating; provides a method for regulating the expression of target RNA.

[0054] The above mammalian cell may be an isolated human cell or a mammalian cell other than a human.

[0056] The above crRNA includes a guide sequence and a direct repeat sequence capable of hybridizing to a target RNA sequence.

[0057] The above crRNA is CRISPR RNA and may be a single-strand RNA. Additionally, crRNA can bind to tracrRNA to activate a CRISPR-associated protein, and crRNA can be used in a bound form to tracrRNA. In this case, crRNA may have a sequence complementary to a gene sequence specifically present in the target.

[0058] The above crRNA may be an RNA composed of 15 to 40 nucleic acids. In this case, the polynucleotide may be composed of 18 to 30 or 20 to 25 nucleic acids. In one embodiment, the crRNA may be composed of 20 nucleic acids. Additionally, the crRNA may include an additional sequence at the 3' end to enable the CRISPR-associated protein to be active.

[0059] crRNA contains a spacer sequence and a direct repeat (DR) sequence or a derivative thereof.

[0060] Includes. In a preferred embodiment, the spacer sequence or a derivative thereof comprises a seed sequence, said seed sequence is important for recognition and / or hybridization of the sequence at the target locus. In a preferred embodiment of the present invention, the crRNA is a short crRNA that can be associated with a monomorphic DR sequence.

[0061] crRNA comprises bulge, hairpin, or stem loop structures. The loop is preferably GAAA, but is not limited to this sequence, nor is it actually limited to a length of only 4 bp. In fact, the preferred loop-forming sequence used in hairpin structures is 4 nucleotides long and most preferably has the sequence GAAA. However, longer or shorter loop sequences may also be used, and surrogate sequences may also be used. The sequence preferably comprises a nucleotide triple (e.g., AAA) and an additional nucleotide (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG.

[0062] The above crRNA may be chemically modified. Examples of chemical modification include, but are not limited to, the incorporation of 2'-O-methyl (M), 2'-O-methyl 3'-phosphorothioate (MS), or 2'-O-methyl 3'thio-pace (MSP) at one or more terminal nucleotides. Such chemically modified crRNA may have increased stability and increased activity compared to the unmodified crRNA, although the on-target specificity versus off-target specificity is unpredictable.

[0063] Chemically modified crRNA includes, without limitation, RNA having lock nucleic acid (LNA) nucleotides containing a phosphothioate linkage and a methylene bridge between the 2' and 4' carbons of a ribose ring.

[0065] A vector may be used to synthesize a polynucleotide encoding the Cas13b protein and crRNA used in the method of the present invention, or to regulate the expression of a target RNA or to edit a target RNA. As used herein, a "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted to induce replication of the inserted segment. Generally, a vector is capable of replication when associated with appropriate control elements.

[0066] Generally, the term "vector" refers to a nucleic acid molecule capable of delivering another nucleic acid to which it is connected. Vectors include, without limitation, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that do not have free ends (e.g., circular) or contain one or more free ends; nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which a virus-derived DNA or RNA sequence is encapsulated in a vector within a virus

[0067] Viral vectors exist (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also contain polynucleotides possessed by the virus for transfection into host cells. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors of bacterial replication origin and episomal mammalian vectors). Other vectors (e.g., non-episosomal mammalian vectors) are incorporated into the host cell's genome upon introduction into the host cell and thereby replicate along with the host genome. Furthermore, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Conventional expression vectors useful for recombinant DNA technology often exist in the form of plasmids. A recombinant expression vector may contain the nucleic acid of the present invention in a form suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vector contains one or more regulatory elements, which may be selected based on the host cell to be used for expression and are operably linked to the nucleic acid sequence to be expressed. Within the recombinant expression vector, "operably linked" is intended to mean that the target nucleotide sequence is linked to the regulatory element in a manner that enables the expression of the nucleotide sequence (e.g., within an in vitro transcription / translation system, or within the host cell if the vector is introduced into the host cell).

[0069] The above vector may be any one selected from the group consisting of plasmids and viruses.

[0070] Specific examples of plasmid DNA include commercial plasmids such as pCMV3, pET28a, pUC57, and pET. Other examples of plasmids that can be used in the present invention include Escherichia coli-derived plasmids (pUC57, pCMV3, pET28a, pET, pGEX, pQE, pDEST, and pCOLD), and Bacillus subtilis ( Bacillus subtilis There are )-derived plasmids (pUB110 and pTP5) and yeast-derived plasmids (YEp13, YEp24, and YCp50). Since the protein expression levels and modifications of these plasmids vary depending on the host cell, the host cell most suitable for the purpose can be selected and used.

[0071] Specific examples of viruses include adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, modified vaccinia virus ankara (MVA), herpes simplex virus, or baculovirus.

[0073] In carrying out any method disclosed herein, a suitable vector may be introduced into a cell or embryo through one or more methods known in the art, including but not limited to microinjection, electroporation, sonoporation, bio-physics, calcium phosphate-mediated transfection, cation transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, dedicated formulation-enhanced absorption of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes or artificial virions.

[0074] In some methods, the vector is introduced into the cell by microinjection. The vector or vectors can be microinjected into the nucleus or cytoplasm. In some methods, the vector or vectors can be introduced into the cell by nucleofection.

[0075] Vectors can be designed for the expression of CRISPR transcripts (e.g., nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells, e.g., Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells.

[0076] Recombinant expression vectors can be transcribed and translated in vitro using, for example, a T7 promoter regulatory sequence and T7 polymerase.

[0077] The above Cas13 protein forms a complex with the above crRNA. The complex may be in the form of a ribonucleoprotein (RNP) and may pass through the cell membrane in the form of a ribonucleoprotein to be delivered into the cell or into the body. Additionally, the complex may be delivered into the cell or into the body by being contained in a conventional RNA carrier.

[0079] In addition, the present invention comprises the step of 1) preparing a vector comprising a construct comprising a sequence encoding an N-terminal fragment of a Cas13 protein and a sequence encoding a first regulatory protein; and a construct comprising a second fragment comprising a sequence encoding a second regulatory protein, a sequence encoding a C-terminal fragment of a Cas13 protein, and a sequence encoding an RNA editing protein.

[0080] 2) a step of injecting a crRNA (CRISPR RNA) capable of hybridizing to the vector and target RNA sequence into a mammalian cell; and

[0081] 3) a step of treating with a compound or light irradiating; provides a method for editing a target RNA sequence.

[0082] The above Cas13 protein may be a deadCas13 protein with RNase activity removed.

[0083] The above deadCas13 protein is inactivated by mutating two histidines of HEPN to alanine (H133A / H1058A) so that the HEPN domain having RNase activity cannot function.

[0084] The RNA editing protein above may be ADAR1 (Adenosine deaminase Acting on RNA) or ADAR2 (Adenosine deaminase Acting on RNA), but is not limited thereto. In a specific embodiment of the present invention, the ADAR protein is ADAR2, and the deaminase domain of ADAR2 that is involved in A-to-I RNA editing (ADAR2 276-702) and the deaminase domain of ADAR2 that has been mutated V16S / E488Q / V351G / S486A / T375A / S370C / P462A / N597I / L332I / I398V / K350I / M383L / D619G / S582T / V440I / S495N / K418E / S661T to enable C-to-U RNA editing were used.

[0086] In a specific embodiment of the present invention, a fragment of Cas13b was designed in which two HEPN domains having RNase activity are conserved and recombinable, and which is expected to exhibit Cas13b activity (Figs. 1 and 2). A flexible regulated construct (see Fig. 3) was designed by connecting each regulatory protein with a linker (GS16) using the FKBP (FK506-binding protein)-FRB (FKBP-rapamycin-binding) system, which is known to be dimerized by Rapamycin, so as not to affect Cas13b protein function, and a flexible regulated construct (Fig. 4) was designed by connecting each regulatory protein with a linker (GS16) using the nMag-pMag system, which is known to be dimerized by blue light, so as not to affect Cas13b protein function. We confirmed that the activity of the Cas13b protein can be regulated by rapamycin by observing a decrease in luciferase activity upon treatment with rapamycin (see Fig. 5), and developed a chemo-inducible RNA regulatory module utilizing this (see Fig. 6). Furthermore, we confirmed that Cas13b can be activated by irradiation with blue light, and that the expression of the target RNA can be reduced by crRNA capable of hybridizing to the target RNA with Cas13b activated by said blue light (Figs. 8 to 11). Additionally, as a result of removing RNase activity and attaching ADAR2, an RNA editing regulatory protein, to the C-terminus of Cas13b, A-to-I RNA mutations induced by blue light in Firefly reporters, in which normal proteins are not produced due to nonsense mutations, It was confirmed that editing is possible (Figs. 12 to 20). In addition, a fluorescent protein-based indicator was designed and it was confirmed that C-to-U base editing can be performed by blue light (Figs. 21 to 24). Furthermore, it was confirmed that the photo-induced padCas13 editor can edit endogenous transcripts (Figs. 25 to 26).

[0088] Therefore, the method of the present invention can be usefully employed in the treatment of diseases by regulating disease-related RNA expression or editing RNA mutations.

[0090] The present invention will be explained in detail below through the following examples and experimental examples.

[0091] However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

[0093] < Examples 1> Prediction of candidate fragments of Cas13b protein capable of recombination through PspCas13b protein restructuring

[0094] PspCas13b, which is known to undergo potent expression in animal cells to regulate Cas13b activity while targeting endogenous RNA in animal cells ( Prevotella sp. The P5-125 Cas13b system was used. However, since the structure of PspCas13b was not known, the structure of PspCas13b was reconstructed using the UCSF Chimera tool with the structure of PbuCas13b, which had already been identified (see Fig. 1). Two HEPN domains with RNase activity were preserved and recombined to design a fragment of Cas13b that is expected to exhibit Cas13b activity.

[0095] As a result, eight candidate fragments of the PspCas13b protein were predicted, as shown in Figures 1 and 2.

[0097] <Example 2> Design of a construct capable of chemically controlling the activity of PspCas13b protein

[0098] A construct was designed to chemically control the activity of the PspCas13b protein based on the fragment of the recombinable Cas13b protein predicted in Example 1 above.

[0099] Specifically, as shown in Fig. 3, a construct was designed using the FKBP (FK506-binding protein)-FRB (FKBP-rapamycin-binding) system, which is known to dimerize by rapamycin. The cDNA encoding the Cas13b fragment of Example 1 fused with the HIV nuclear export signal was amplified in pC0046-EF1a-PspCas13b-NES-HIV (Addgene plasmid 103862). Each regulatory protein was linked with a linker (GS16) to enable flexible regulation so as not to affect Cas13b protein function. The encoding DNA sequence of the FKBP-FRB domain was synthesized in Twist Bioscience. All Cas13 fragments were synthesized using Gibson Assembly. PCR fragments for Gibson Assembly were amplified using Phusion Hot-Start II DNA polymerase (Thermofisher). Chemo-inducible Cas13 constructs based on the FKBP-FRB domain and Cas13 fragments (N-terminus and C-terminus) were cloned into the XhoI / XmaI and BsrGI / NotI sites of the EGFP-N1 plasmid (Clonetech), respectively.

[0101] <Example 3> Designing a construct to enable optogenetic regulation of PspCas13b protein activity

[0102] A construct was designed to enable optogenetic control of the activity of the PspCas13b protein based on the fragment of the recombinable Cas13b protein predicted in Example 1 above.

[0103] Specifically, as shown in Figure 4, a construct was designed using the nMag-pMag system, which is known to dimerize by blue light. Since FKBP and FRB are 107 amino acids and 93 amino acids long, respectively, a Magnet system of similar size (150 amino acids) was used to avoid steric hindrance. Two combinations were used: pMag:nMag and pMag:nMagH (nMag with M135I and M165I mutations). To construct padCas13, an inactivated HEPN RNase domain (including H133A and H1058A mutations) was amplified from pC0049-EF1a-dPspCas13b-NES-HIV (Addgene 103865). Each regulatory protein was connected via a linker (GS16) to enable fluid regulation without affecting Cas13b protein function. The encoding DNA sequences of the pMag:nMag and pMag:nMagH domains were synthesized in Twist Bioscience. All Cas13 fragments were synthesized using Gibson Assembly. PCR fragments for Gibson Assembly were amplified using Phusion Hot-Start II DNA polymerase (Thermofisher). Photo-inducible Cas13 constructs based on the pMag:nMag or pMag:nMagH domains and Cas13 fragments (N-terminus and C-terminus) were cloned into the XhoI / XmaI and BsrGI / NotI sites of the EGFP-N1 plasmid (Clonetech), respectively.

[0105] <Example 4> Production of crRNA

[0106] Fluc Reporter, GFP indicator, KRAS, PPIB, STAT3crRNAs targeting A-to-I or C-to-U RNA editing sites were generated by annealing oligocloning using the pC0043-PspCas13b crRNA backbone (Bbsl site of Addgene plasmid 103854). All crRNA plasmids were cloned using T4 DNA ligase (Elpis Biotech). Multiplexed crRNAs were constructed using Gibson assembly with three fragment ligations.

[0108] <Experimental Example 1> Confirmation of whether Cas13b protein is activated by rapamycin

[0109] A Luciferase reporter assay was performed to determine whether the Cas13b protein is activated by rapamycin using the construct designed in Example 2 above.

[0110] Specifically, to evaluate changes in protein levels, HEK293T cells (ATCC) were co-transfected with 10 ng of the dual luciferase reporter plasmid (Promega), 75 ng of each inducible Cas13 fragment expressing the plasmid, and 100 ng of the crRNA expression plasmid. 16 hours prior to transfection, cells were plated in 96-well plates (Corning) and allowed to grow until 80% confluency was reached. The following day, 10 μL of Opti-MEM containing 0.6 μL lipofectamine LTX and 0.26 μL PLUS reagent was mixed with all transfected DNA plasmids, followed by the addition of a total of 20 μL of Opti-MEM I reduced serum medium (ThermoFisher) per well. All solutions were incubated for 5 minutes before being slowly added to the cells. Treatment with 500 nM rapamycin was performed for 24 hours prior to analysis, and after 48 hours, luciferase readouts for Firefly luciferase (experimental group) and Renilla luciferase (control group) were sequentially measured using the Dual-Glo Luciferase Assay System (Promega) with a Tecan plate reader in accordance with the manufacturer's instructions. The experiment was repeated three times, and the target RNA was Firefly The decrease in transcripts was confirmed by comparing with the expression values ​​at the protein level of the control group, Renilla.

[0111] As a result, as shown in Figure 5, it was confirmed that among the Cas13b protein fragments, N550 / C551, N624 / C625, and N725 / C726 could reconstitute in cells even without rapamycin stimulation. Additionally, it was confirmed that luciferase expression decreased to an intermediate level in the absence of rapamycin, and the background activity was not due to residual nuclease activity of the individual Cas13 fragments. Based on these results, a chemo-inducible RNA regulatory module capable of reconstituting Cas13 fragments through rapamycin treatment was developed (Figure 6). For subsequent experiments, the N351 / C352 fragment, which showed a stark difference before and after induction by rapamycin, was used.

[0112] To select a more efficient fragmentation site, a second screening of Cas13 fragmentation sites was performed near residues 351 and 352, and N351 / C352, N351 / C350, and N353 / C352 paCas13 were named paCas13-1, paCas13-2, and paCas13-3, respectively (Fig. 7).

[0114] <Experimental Example 2> Confirmation of whether the Cas13b protein is activated by blue light

[0115] <2-1> Characterization of Photoactivable Cas13 (paCas13)

[0116] A Luciferase reporter assay was performed to determine whether the Cas13b protein is activated by light stimulation using the construct designed in Example 3 above.

[0117] To test the photo-induced RNA targeting of paCas13, the target RNA knockdown activity of the paCas13 fragment was evaluated using a dual luciferase system under blue light stimulation for 24 hours with an LED plate emitting 488 nm blue light (Fig. 8). The specific luciferase reporter assay is the same as in Example 4.

[0118] As a result, as shown in Figure 9, each paCas13 construct exhibited significant luciferase knockdown upon blue light induction, similar to the rapamycin-inducible construct. Efficient targeted mRNA cleavage was observed in pMag combined with nMagH (nMagHigh1) rather than nMag. Therefore, all subsequent experiments utilized a system with pMag:nMagH.

[0119] In addition, photo-induced candidates were selected based on the screening results of Example 4 (Fig. 10), and endogenous transcripts previously targeted by the Cas13 system, KRAS, PPIB and STAT3 Experiments were conducted to confirm the function of paCas13 for RNA interference by targeting it.

[0120] Specifically, a single crRNA expression vector containing multiple crRNAs was used to create a system capable of achieving efficient and easy multiple RNA modulation.

[0121] As a result, as shown in Figure 11, significant knockdown of the target transcript by paCas13 under light stimulation was observed.

[0122] The above results indicate that the paCas13 system can disrupt endogenous transcripts through light stimulation.

[0124] <2-2> Optogenetic RNA Regulation by paCas13

[0125] To further demonstrate the utility of paCas13, a photoactivated and reversible RNA editing system (hereinafter referred to as padCas13) was constructed by fusing a regulatory domain to the padCas13 fragment (Fig. 12).

[0126] Programmable RNA editing systems utilizing catalytically inactive Cas13 (dCas13) to deliver the catalytic domain of ADAR2 (adenosine deaminase acting on RNA type 2) to specific transcripts in mammalian cells have been known from numerous studies. ADAR2 hydrolytically deaminates adenosine in double RNA to convert it into inosine, and inosine pairs with cytosine and exhibits properties similar to guanosine. In the present invention, the padCas13 system was constructed using the ADAR2 system.

[0128] <2-2-1> Photo-induced A-to-I RNA editing by paCas13

[0129] To confirm the photo-induced RNA editing of the present invention, A-to-I (adenine to inosine) editing was performed, followed by a luciferase reporter assay.

[0130] Specifically, the catalytic deaminase domain of ADAR2 (ADAR2DD) for A-to-I editing was amplified in pC0050-CMV-dPspCas13b-longlinker-ADAR2DD(wt) (Addgene plasmid 103866). The padCas13 editor was designed by fusing ADAR2DD to the C-terminus of the Cas13 fragment. To verify the activity of the padCas13 editor, a nonsense mutant [W417X (UGG→UAG)] was introduced Fluc An RNA editing reporter for the gene was designed. This mutation can be modified to a wild-type codon through A-to-I editing, which can be detected as the restoration of the firefly luciferase signal (Fig. 13). The specific luciferase reporter assay is the same as in Example 4.

[0131] As a result of observing the A-to-I editing efficiency induced by light stimulation over time, as shown in Figure 14, when blue light was turned on, luciferase activity by restoration increased as the duration of light stimulation increased. When the light was turned off after 6 hours of stimulation, it was confirmed that the increased activity of luciferase by restoration returned to the baseline within 24 hours.

[0132] To further verify the restoration of luciferase activity, Sanger sequencing was performed after reverse transcription (RT)-polymerase chain reaction (PCR).

[0133] Specifically, to evaluate changes in RNA levels after transfection with the paCas13 system, total RNA was isolated from HEK293T cells, and changes in RNA levels were quantified using RT-qPCR. Total RNA isolation was performed using the PureLink RNA Mini Kit (Invitrogen). After RNA isolation, it was reverse transcribed into cDNA using the gDNA eraser (Takara) and the PrimeScript RT Reagent Kit. All qPCR reactions were performed in a 20 μL volume using SYBR Green Master Mix (Toyobo) and amplified on a CFXmaestro 96 instrument (Bio-rad). qPCR primers were designed to span the crRNA target site. Expression levels were calculated using the cycle threshold (Ct) value of GAPDH and the Ct value of the gene of interest. The relative expression level of a gene is 2 △Ct It was determined as follows, where △Ct was calculated as Ct(gene of interest) - Ct(GAPDH). The relative expression level of the target gene was obtained by normalizing the expression level of the target gene in cells treated with on-target crRNA to the expression level in cells treated with non-targeting (NT) crRNA.

[0134] Sanger sequencing was performed at Cosmogenetech after PCR amplifying cDNA products using target-specific primers with Phusion DNA polymerase (Thermofisher). Editing efficiency was calculated by analyzing the height of each Sanger peak at the target site using Indigo software (https: / / www.gear-genomics.com / indigo / ). Sanger sequencing standard curves were generated from the sequencing results of multiple intentionally mixed DNA samples. Standard curves were used to correct for editing efficiency that may be altered by PCR bias and sequencing bias.

[0135] As a result of measuring RNA editing levels over a period of up to 48 hours with and without light stimulation, it was confirmed that A-to-I RNA editing occurred over time in the presence of light stimulation, as shown in Fig. 15. In addition, it was confirmed that the RNA editing level of the target site by the padCas13 editor steadily increased over time (Figs. 16 and 17). Since cells transfected with crRNA alone also exhibited a moderate A-to-I editing rate, the background activity observed in the dark was considered to be an RNA editing background unrelated to padCas13 (Fig. 18).

[0136] The above results suggest that the padCas13 editor of the present invention enables reversible RNA editing in an optically dependent manner.

[0138] To evaluate the efficiency and specificity of the padCas13 system of the present invention, Fluc Transcriptome-wide RNA sequencing (RNA-seq) analysis was performed on cells transfected with the padCas13 editor targeting the W417X mutation.

[0139] As a result, as shown in Figure 19, it was confirmed that the target editing rate under 24-hour light stimulation was 16%.

[0140] While the padCas13 editor was able to induce targeted A-to-I editing, many "A" sites surrounding the target site are accessible via the padCas13 editor's fusion ADAR2. These sites also bind to crRNA to form RNA duplex structures, which are the natural editing target constructs of the endogenous ADAR2 enzyme. Therefore, the target mutation site ( Fluc RNA sequencing was performed to analyze the adenosine surrounding W417X.

[0141] Specifically, HEK293T cells were plated in 24-well plates (SPL) and co-transfected with 250 ng of each paCas13 fragment and 300 ng of crRNA expressing the plasmid. After photostimulation, total RNA was extracted using the PureLink RNA mini kit (Invitrogen), followed by DNase treatment in the column for 15 minutes (Invitrogen). The stranded mRNA library was prepared using the TruSeq stranded mRNA library kit (Illumina) and sequenced on an Illumina NextSeq550 with 76 nt paired-end reads. Approximately 130 million total reads per condition were demultiplexed. The raw sequencing data were processed using PIGx RNA-seq (https: / / github.com / BIMSBbioinfo / pigx_rnaseq).

[0142] As a result, as shown in Figure 20, no additional A-to-I editing was observed in adenosine sites other than the target.

[0143] These results indicate that the padCas13 editor can perform photo-induced and highly specific A-to-I RNA editing.

[0145] <2-2-2> Photo-induced C-to-U RNA editing by paCas13

[0146] A photo-induced C-to-U RNA editing system was constructed by fusing the padCas13 fragment into ADAR2DD, which is capable of cytidine deamination. To verify the activity of the padCas13 editor for C-to-U base editing, a fluorescent protein-based indicator was designed (Fig. 21), and fluorescence imaging and flow cytometry were performed.

[0147] Specifically, for C-to-U RNA editing, the evolved ADAR2DD (including E488Q / V351G / S486A / T375A / S370C / P462A / N597I / L332I / I398V / K350I / M383L / D619G / S452T / S48) mutant pC0079 RESCUE-S (Addgene plasmid 130662) was amplified. To develop this system, the Y66H green-to-blue mutation [Y66H(UAC→CAC)] was introduced into the EGFP-N1 plasmid of GFP by Gibson Assembly. Additionally, a crRNA targeting the target CAC codon that causes C-to-U conversion was designed. Under light stimulation, the padCas13 editor can perform the C-to-U conversion of the Y66H mutant and restore GFP fluorescence. To enhance the effect of C-to-U RNA editing, two vectors were cloned by Gibson assembly following extended PCR cloning. The first vector expresses the N-terminus of a pMag-conjugated dCas13 fragment and a crRNA linked to a GFP indicator via a T2A self-cleaving peptide. Each sequence is controlled by the hU6 promoter and the EFS (EF1a short) promoter, respectively. The second vector expresses the C-terminus of a dCas13 fragment fused with nMagH and ADAR2DD under the control of the EFS promoter.

[0148] Live-cell imaging was performed using a Nikon A1R confocal microscope mounted on a Nikon Eclipse Ti body equipped with a Nikon CFI PlanApochromat VC 60X / 1.4 numerical aperture (NA) and digital-zooming Nikon imaging software (NIS-elements AR 64-bit version 3.21). The Chamlide TC system placed on the microscope stage was operated at 37°C and 10% CO₂. 2 It was used to maintain the environmental conditions of the (Live Cell Instruments). A custom 96-well 488nm LED array (Live Cell Instruments) was used to irradiate uniform blue light in all wells.

[0149] To perform flow cytometry analysis, 3 x 10⁶ GFP indicators were used 5 HEK293T cells were seeded into 6-well plates (SPL). After 24 hours, the cells were co-transfected with the GFP indicator and padCas13 editor plasmid using Lipofectamine LTX (Invitrogen) according to the manufacturer's manual. The GFP+ ratio of the cells indicates editing efficiency. Transfected cells were analyzed by BD FACSLSRFortessa 24 hours after photostimulation. Only single viable cells were used for fluorescence analysis. Flow cytometry results were analyzed using FlowJo X (v. 10.0.7).

[0150] When the padCas13 editor and target crRNA or non-target crRNA (NT, negative control) crRNA were transfected with a GFP indicator and photoinducibility was tested upon blue light stimulation for 24 hours, it was found that the padCas13 editor induced C-to-U RNA editing to generate BFP / GFP double-positive cells under photostimulation, as shown in Fig. 22. It was confirmed that GFP restoration by C-to-U editing increased from 4.95% to 42% under blue light illumination compared to dark conditions with target crRNA (Figs. 23 and 24).

[0151] Through the above results, it was confirmed that the padCas13 system of the present invention has light-dependent RNA editing activity suitable for both A-to-I and C-to-U base editing.

[0153] <2-2-3> Editing Photo-induced Endogenous Transcriptomes by padCas13

[0154] We evaluated whether the light-induced padCas13 editor of the present invention can be applied to editing endogenous transcripts.

[0155] As a result, as shown in FIGS. 25 and 26, it was confirmed that the padCas13 editor can mediate A-to-I and CU editing of all subjects tested in an optically induced manner.

[0156] These results indicate a high likelihood of using padCas13 to improve diseases. In particular, it is highly likely to improve disease states requiring the temporal regulation of disease-related RNA molecules.

Claims

Claim 1 i) a first fragment comprising an N-terminal fragment of PspCas13b protein and a first regulatory protein; A method for regulating the activity of a PspCas13b protein, comprising the step of: ii) inducing dimerization of a first fragment and a second fragment by treating a compound or light irradiating a second fragment comprising a C-terminal fragment of the PspCas13b protein and a second regulatory protein, wherein the combination of the first regulatory protein and the second regulatory protein is FKBP-FRB or nMagH-pMag, and the N-terminal fragment of the PspCas13b protein and the C-terminal fragment of the PspCas13b protein are dimerized into any one combination selected from the group consisting of: a) a combination of a fragment up to the 286th amino acid from the N-terminus of the PspCas13b protein and a fragment up to the 847th amino acid from the C-terminus of the PspCas13b protein; b) a fragment up to the 351st amino acid from the N-terminus of the PspCas13b protein and a) a combination of fragments from the C-terminus to the 782nd amino acid of the PspCas13b protein; c) a combination of fragments from the N-terminus to the 351st amino acid of the PspCas13b protein and fragments from the C-terminus to the 784th amino acid of the PspCas13b protein; and d) a combination of fragments from the N-terminus to the 353rd amino acid of the PspCas13b protein and fragments from the C-terminus to the 782nd amino acid of the PspCas13b protein. Claim 2 delete Claim 3 A method for regulating the activity of a PspCas13b protein according to claim 1, wherein a linker is inserted between the N-terminal fragment of the PspCas13b protein and a first regulatory protein; and between the second regulatory protein and a C-terminal fragment of the PspCas13b protein. Claim 4 A method for regulating the activity of PspCas13b protein according to claim 3, wherein the linker comprises glycine (G) and serine (S). Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 A method for regulating the activity of PspCas13b protein, wherein, in claim 1, the compound is Rapamycin. Claim 10 A method for regulating the activity of PspCas13b protein, wherein, in claim 1, the light is blue light. Claim 11 delete Claim 12 1) a step of preparing a first vector comprising a first nucleic acid construct comprising a sequence encoding an N-terminal fragment of a PspCas13b protein and a sequence encoding a first regulatory protein; and a second vector comprising a second nucleic acid construct comprising a sequence encoding a C-terminal fragment of a PspCas13b protein and a sequence encoding a second regulatory protein; 2) a step of injecting a crRNA (CRISPR RNA) capable of hybridizing to said vector and target RNA sequence into a mammalian cell; and 3) a step of inducing dimerization of an expression product generated in a first vector and an expression product generated in a second vector by compound treatment or light irradiation; a method for regulating the expression of a target RNA comprising: a) a combination of a fragment from the N-terminus to the 286th amino acid of the PspCas13b protein and a fragment from the C-terminus to the 847th amino acid of the PspCas13b protein; b) a combination of a fragment from the N-terminus to the 351st amino acid of the PspCas13b protein and a fragment from the C-terminus to the 782nd amino acid of the PspCas13b protein; c) A combination of the fragment from the N-terminus of the PspCas13b protein up to the 351st amino acid and the fragment from the C-terminus of the PspCas13b protein up to the 784th amino acid; and d) a combination of the fragment from the N-terminus of the PspCas13b protein up to the 353rd amino acid and the fragment from the C-terminus of the PspCas13b protein up to the 782nd amino acid. Claim 13 A method for regulating the expression of a target RNA according to claim 12, wherein the crRNA comprises a guide sequence capable of hybridizing to a target RNA sequence and a direct repeat sequence. Claim 14 In paragraph 12, a method for controlling the expression of target RNA, wherein the vector is a virus or a plasmid. Claim 15 A method for controlling the expression of target RNA according to claim 14, wherein the virus is an adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, modified vaccinia virus ankara (MVA), herpes simplex virus, or baculovirus. Claim 16 1) a first vector comprising a first nucleic acid construct comprising a sequence encoding an N-terminal fragment of a PspCas13b protein and a sequence encoding a first regulatory protein; and a second vector comprising a second nucleic acid construct comprising a sequence encoding a C-terminal fragment of a PspCas13b protein, a sequence encoding a second regulatory protein, and a sequence encoding an RNA editing protein; 2) a step of injecting a crRNA (CRISPR RNA) capable of hybridizing to said vector and target RNA sequence into a mammalian cell; A method for editing a target RNA sequence comprising: 3) a step of inducing dimerization of an expression product generated in a first vector and an expression product generated in a second vector by compound treatment or light irradiation; wherein the combination of the first regulatory protein and the second regulatory protein is FKBP-FRB or nMagH-pMag, and the N-terminal fragment of the PspCas13b protein and the C-terminal fragment of the PspCas13b protein are dimerized into any one combination selected from the group consisting of: a) a combination of a fragment from the N-terminus to the 286th amino acid of the PspCas13b protein and a fragment from the C-terminus to the 847th amino acid of the PspCas13b protein; b) a combination of a fragment from the N-terminus to the 351st amino acid of the PspCas13b protein and a fragment from the C-terminus to the 782nd amino acid of the PspCas13b protein; c) PspCas13b protein A combination of the fragment from the N-terminus to the 351st amino acid and the fragment from the C-terminus to the 784th amino acid of the PspCas13b protein; and d) a combination of the fragment from the N-terminus to the 353rd amino acid of the PspCas13b protein and the fragment from the C-terminus to the 782nd amino acid of the PspCas13b protein. Claim 17 delete Claim 18 A method for editing a target RNA sequence according to claim 16, wherein the RNA editing protein is ADAR (Adenosine deaminase Acting on RNA).

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