Gene editing systems and their applications

Abstract

This disclosure discloses inhibitors of CTGF RNA, MITF RNA, and SRD5A2 RNA, and their uses, wherein the inhibitors are gene editing systems. The gene editing systems of this disclosure can be used to knock down CTGF RNA, MITF RNA, or SRD5A2 RNA for scar repair, skin whitening, reduction or removal of melasma, prevention or treatment of melanoma, and / or prevention or treatment of male pattern baldness. Furthermore, this disclosure discloses a gene editing system that targets AR RNA encoded by the androgen receptor gene, which can be used for the treatment of diseases such as male pattern baldness.

Description

[Technical Field]

[0001] This application claims priority to Chinese Patent Application No. 2023109694981, filed on 2 August 2023, and PCT Patent Application No. PCT / CN2024 / 071775, filed on 11 January 2024, and incorporates the full texts of these applications by reference.

[0002] (Technical field) This invention belongs to the field of gene editing technology, and more specifically, relates to gene editing systems and their applications. [Background technology]

[0003] Wound healing is a complex cellular activity that generally progresses in an orderly manner through four stages: hemostasis, inflammation, proliferation, and remodeling. Of these, proliferation and remodeling are particularly important in determining scar formation because they involve the production and rearrangement of the extracellular matrix (ECM). However, due to the strong contractility of myofibroblasts, the healing process involving scar formation can easily result in the formation of a disordered, dense, and collagen-rich matrix, potentially disrupting the skin's natural structure. Fibrotic tissue leads to an uncontrolled excessive accumulation of subcutaneous proteins, thickening the skin surface and creating an irregular, uneven, and uneven state, thus forming scars. Connective tissue growth factor (CTGF, also known as CCN2) is a major mediator of scar formation.

[0004] Melanin production is a biosynthetic pathway in melanocytes that produces melanin, involving a series of complex enzyme-catalyzed and chemocatalyzed reactions. Five signaling pathways primarily regulate it, with microphthalmia-associated transcription factors (MITFs) being the ultimate target of multiple signaling pathways and a major regulator of melanin production.

[0005] Androgenetic alopecia (AGA, also known as male pattern baldness or juvenile alopecia) is the most common type of hair loss, characterized by gradual progression from adolescence to middle age. Under the influence of androgens, scalp hair follicles gradually atrophy and shrink, eventually degenerating into thin, vellus hairs, which are visually perceived as hair loss. While androgens are a major cause of AGA, blood androgen levels are within the normal range in many AGA patients, suggesting that AGA may be associated with increased androgen receptor gene expression and / or increased 5α-reductase gene expression within the hair follicles.

[0006] The androgen receptor (AR, also known as NR3C4 [nuclear receptor subfamily 3, group C, member 4]) is a type of nuclear receptor that is activated when it binds to testosterone or dihydrotestosterone in the cytoplasm, and then translocates to the nucleus.

[0007] Multiple genome-wide association studies (GWAS) have shown that the androgen receptor (AR) gene and the ectodysplasin-A2 receptor (EDA2R) gene on the X chromosome are major susceptibility genes for androgenetic alopecia (AGA).

[0008] Furthermore, the SRD5A2 gene encodes 3-oxo-5α-steroid 4-dehydrogenase 2 (type 2 5α-reductase, 5αR2), one of the three isozymes of 5α-reductase. SRD5A2 is a key enzyme in androgen metabolism, catalyzing the conversion of testosterone to dihydrotestosterone (DHT), a potent AR agonist. Elevated SRD5A2 levels have been detected in the scalp area affected by AGA, and finasteride, an SRD5A2 inhibitor, is used as a treatment for AGA patients. [Overview of the Initiative]

[0009] A first aspect of this disclosure provides a CTGF RNA, MITF RNA, or SRD5A2 RNA inhibitor, wherein the inhibitor is a gene editing system.

[0010] In some embodiments of the present disclosure, the gene editing system knocks down the level of CTGF RNA, MITF RNA, or SRD5A2 RNA, or inhibits the translation of CTGF RNA, MITF RNA, or SRD5A2 RNA.

[0011] In some embodiments of this disclosure, the gene editing system knocks down the level of CTGF RNA, MITF RNA, or SRD5A2 RNA.

[0012] In some embodiments of this disclosure, the gene editing system includes: (a) a gRNA containing a guide sequence that hybridizes with the target RNA, or a polynucleotide sequence encoding the gRNA, and (b) An RNA guide nuclease, or a polynucleotide sequence encoding the RNA guide nuclease.

[0013] The gRNA can form a complex with the nuclease and guide the complex to bind to the target RNA in a sequence-specific manner, and the target RNA is CTGF RNA, MITF RNA, or SRD5A2 RNA.

[0014] In some embodiments of the present disclosure, the gRNA can form a complex with the nuclease, which can then guide the complex to bind to and cleave the target RNA.

[0015] In some embodiments of this disclosure, the target RNA is CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of this disclosure, the target RNA is CTGF pre-mRNA, MITF pre-mRNA, or SRD5A2 pre-mRNA, or CTGF mRNA, MITF mRNA, or SRD5A2 mRNA. In some embodiments of this disclosure, the target RNA is CTGF mRNA, MITF mRNA, or SRD5A2 mRNA. In some embodiments of this disclosure, the target RNA is mammalian CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of this disclosure, the target RNA is human CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of this disclosure, the target RNA is human CTGF mRNA, MITF mRNA, or SRD5A2 mRNA.

[0016] In some embodiments of this disclosure, the target RNA sequence is the sequence shown in SEQ ID NO: 14, 22, or 35.

[0017] In some embodiments of this disclosure, the target RNA sequence is, for example, the nucleotides from position 494 to 785 of the sequence shown in SEQ ID NO: 14, the nucleotides from position 404 to 606 of the sequence shown in SEQ ID NO: 22, or the nucleotides from position 484 to 820 of the sequence shown in SEQ ID NO: 35.

[0018] In some embodiments of this disclosure, the target RNA sequence is a nucleotide sequence from nucleotides at positions 494, 604, 678, or 761 to 518, 628, 702, or 785 of the sequence shown in, for example, SEQ ID NO: 14 (human CCN2 mRNA, NCBI NM_001901.4).

[0019] In some embodiments of this disclosure, the target RNA sequence is a nucleotide sequence from the 404th, 429th, 453rd, 479th, 509th, 546th, or 582nd nucleotide of the sequence shown in, for example, SEQ ID NO: 22 (Human MITF mRNA, NCBI NM_001354607.2) to the 428th, 453rd, 477th, 503rd, 533rd, 570th, or 606th nucleotide.

[0020] In some embodiments of this disclosure, the target RNA sequence is a nucleotide sequence from the 484th, 524th, 547th, 585th, 635th, 721st, 755th, or 796th nucleotides of the sequence shown in, for example, SEQ ID NO: 35 (human SRD5A2 mRNA, NCBI XM_011533072.3), to the 508th, 548th, 571st, 609th, 659th, 745th, 779th, or 820th nucleotides.

[0021] In some embodiments of this disclosure, the guide sequence and the target RNA have at least 80% sequence identity. In some embodiments of this disclosure, the guide sequence and the target RNA have at least 85%, at least 90%, at least 95%, or 100% sequence identity. Furthermore, in some embodiments of this disclosure, the guide sequence and the target RNA have 100% sequence identity.

[0022] In some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 14, 22, or 35 have at least 80% sequence identity. In some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 14, 22, or 35 have at least 85%, at least 90%, at least 95%, or 100% sequence identity. Furthermore, in some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 14, 22, or 35 have 100% sequence identity.

[0023] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with the sequence shown in any one of SEQ ID NOs: 5-13, 23-32, 36-57. Further, in some embodiments of the present disclosure, the guide sequence has 100% sequence identity with the sequence shown in any one of SEQ ID NOs: 5-13, 23-32, 36-57. In some embodiments of the present disclosure, the guide sequence is the sequence shown in any one of SEQ ID NOs: 5-13, 23-32, 36-57.

[0024] In some embodiments of the present disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with the sequence shown in any one of SEQ ID NOs: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, 54. In some embodiments of the present disclosure, the guide sequence is the sequence shown in any one of SEQ ID NOs: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, 54.

[0025] In some embodiments of the present disclosure, the gRNA includes a guide sequence and a backbone sequence.

[0026] The backbone sequence interacts with the RNA-guided nuclease.

[0027] In some embodiments of the present disclosure, the backbone sequence is a direct repeat sequence.

[0028] In some embodiments of the present disclosure, the co-directed repeat sequence includes a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown, for example, SEQ ID NO: 2 or 3.

[0029] In some embodiments of the present disclosure, the RNA guide nuclease may be selected from Cas9, Cas12, Cas13, TnpB, IscB, IsrB, Fancor nuclease, or fragments thereof (including, but not limited to, fragments of nucleic acid-binding domains).

[0030] In some embodiments of this disclosure, the RNA guide nuclease is a Cas protein.

[0031] In some embodiments of this disclosure, the gRNA can form a CRISPR complex with a Cas protein, which can then be guided to bind to the target RNA in a sequence-specific manner.

[0032] In some embodiments of this disclosure, the gRNA can form a CRISPR complex with a Cas protein, which can then guide the CRISPR complex to sequence-specifically bind to and cleave the target RNA.

[0033] In some embodiments of this disclosure, the RNA guide nuclease is a Cas9 protein, a Cas12 protein, or a Cas13 protein.

[0034] In some embodiments of this disclosure, the RNA guide nuclease is a Cas13 protein. In some embodiments, the Cas13 protein is preferably a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein.

[0035] In some embodiments of the present disclosure, the amino acid sequence of the Cas13 protein has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1.

[0036] In some embodiments of this disclosure, the Cas13 protein includes the sequence shown in SEQ ID NO: 1.

[0037] In some embodiments of this disclosure, the RNA guide nuclease includes any one or more of the following: an intracellular localization signal, a deaminase domain, a translation activation domain, a translation inhibition domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0038] In some embodiments of this disclosure, the Cas protein comprises any one or more of the following: an intracellular localization signal, a deaminase domain, a translation activation domain, a translation inhibition domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0039] In some embodiments of this disclosure, the intracellular localization signal may be selected from nuclear localization signals and nuclear export signal sequences.

[0040] In some embodiments of this disclosure, the gene editing system includes: (a) gRNA, or a polynucleotide sequence encoding the gRNA, and (b) Cas13 protein, or a polynucleotide sequence encoding the Cas13 protein.

[0041] The gRNA can form a complex with the Cas13 protein, and the complex can bind to and cleave the target RNA.

[0042] In some embodiments of the present disclosure, the polynucleotide sequence encoding the gRNA is ligated to a first control sequence, which is used to control the expression of the gRNA.

[0043] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA guide nuclease is ligated to a second regulatory sequence, which is used to control the expression of the RNA guide nuclease.

[0044] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA guide nuclease is ligated to a control sequence, which is used to control the expression of the RNA guide nuclease.

[0045] In some embodiments of the present disclosure, the polynucleotide sequence encoding the gRNA is ligated to a control sequence, which is used to control the expression of the gRNA.

[0046] In some embodiments of this disclosure, the regulatory sequence that controls RNA guide nuclease expression and the regulatory sequence that controls gRNA expression are the same or different.

[0047] In some embodiments of this disclosure, the control sequence is a promoter sequence. In some embodiments of this disclosure, the control sequence is an enhancer sequence. In some embodiments of this disclosure, the control sequence is both a promoter and an enhancer sequence.

[0048] The gene editing systems described herein can be introduced into cells (or cell-free systems) in a variety of non-limiting manner. For example, they can be introduced (i) as mRNA and gRNA encoding an RNA guide nuclease, (ii) as part of a single vector or plasmid, or as multiple vectors or plasmids, (iii) as isolated RNA guide nuclease and gRNA, or (iv) as an RNP complex consisting of RNA guide nuclease and gRNA.

[0049] In some embodiments of this disclosure, the complex reduces the level of the target RNA in a mammal (e.g., in a human body).

[0050] In some embodiments of this disclosure, the complex reduces the level of the target RNA within a cell, for example, the level of the target RNA within a cell expressing the target RNA.

[0051] In some embodiments of this disclosure, the complex reduces the level of the target RNA in cells by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the target RNA level can be measured using methods well known to those skilled in the art. For example, without limiting these, untreated cells including the qPCR method of the Examples or cells treated with a gene editing system targeting a non-mammalian genome can be used as a negative control, and the knockdown level of the target RNA in the test group can be calculated by comparing it to the negative control.

[0052] In some embodiments of the present invention, the number of off-target genes when the complex binds to and cleaves the target RNA is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by a method commonly used by those skilled in the art. In some embodiments, the number of off-target genes is determined by genes with differential expression determined by RNA sequencing.

[0053] In some embodiments of this disclosure, the complex reduces the protein levels encoded by the target RNA in an animal body (e.g., in a human body).

[0054] In some embodiments of the present disclosure, the complex reduces intracellular levels of the protein encoding the target RNA after contact with the cell. In some embodiments of the present disclosure, the protein encoding the target RNA is a CTGF protein, a MITF protein, or an SRD5A2 protein. In some embodiments of the present disclosure, the complex reduces intracellular levels of the CTGF protein, MITF protein, or SRD5A2 protein.

[0055] In some embodiments of this disclosure, the complex reduces intracellular levels of CTGF protein, MITF protein, or SRD5A2 protein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the level of the target RNA-coding protein can be measured using methods well known to those skilled in the art. Without limiting these, the knockdown levels of CTGF protein, MITF protein, or SRD5A2 protein in the test group can be calculated by comparing them to the negative control group, using untreated cells or cells treated with gene editing systems targeting non-mammalian genomes as negative controls, including but not limited to ELISA, Western Blotting, etc.

[0056] A second aspect of this disclosure provides gRNA (guide RNA, gRNA) for gene editing systems.

[0057] In some embodiments of this disclosure, the gRNA includes a guide sequence that hybridizes with a target RNA, wherein the target RNA is CTGF RNA, MITF RNA, or SRD5A2 RNA.

[0058] In some embodiments of this disclosure, the gRNA includes a guide sequence and a backbone sequence. The backbone sequence interacts with an RNA guide nuclease. The backbone sequence is a sequence that is normally invariant within the gRNA molecule when designing the gRNA molecule. For example, the backbone sequence may refer to a portion of the gRNA molecule other than the guide sequence. In some embodiments of this disclosure, the backbone sequence is a direct repeat (DR).

[0059] In some embodiments of this disclosure, the guide sequence and the target RNA have at least 80% sequence identity. In some embodiments of this disclosure, the guide sequence and the target RNA have at least 85%, at least 90%, at least 95%, or 100% sequence identity. Furthermore, in some embodiments of this disclosure, the guide sequence and the target RNA have 100% sequence identity.

[0060] In some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 14, 22, or 35 have at least 80% sequence identity. In some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 14, 22, or 35 have at least 85%, at least 90%, at least 95%, or 100% sequence identity. Furthermore, in some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 14, 22, or 35 have 100% sequence identity.

[0061] In some embodiments of this disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence of the sequence shown in SEQ ID NO: 14, the sequence of the sequence shown in SEQ ID NO: 22, the sequence of

[0062] In some embodiments of this disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequence from position 494, 604, 678, or 761 of the sequence shown in SEQ ID NO: 14 to position 518, 628, 702, or 785.

[0063] In some embodiments of this disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequences from position 404, 429, 453, 479, 509, 546, or 582 of the sequence shown in SEQ ID NO: 22, to position 428, 453, 477, 503, 533, 570, or 606.

[0064] In some embodiments of this disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity with the nucleotide sequences from position 484, 524, 547, 585, 635, 721, 755, or 796 of the sequence shown in SEQ ID NO: 35 to position 508, 548, 571, 609, 659, 745, 779, or 820.

[0065] In some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 5-13, 23-32, 36-57 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity. Furthermore, in some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 5-13, 23-32, 36-57 have 100% sequence identity. In some embodiments of this disclosure, the guide sequence includes the sequence represented by any one of SEQ ID NO: 5-13, 23-32, 36-57. In some embodiments of this disclosure, the guide sequence is the sequence represented by any one of SEQ ID NO: 5-13, 23-32, 36-57.

[0066] In some embodiments of this disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of the sequences represented by SEQ ID NO: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, or 54. In some embodiments of this disclosure, the guide sequence includes any one of the sequences represented by SEQ ID NO: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, or 54. In some embodiments of this disclosure, the guide sequence is any one of the sequences represented by SEQ ID NO: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, or 54.

[0067] In some embodiments of the present disclosure, the backbone sequence is a co-directed repeat sequence, the co-directed repeat sequence includes a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 2 or 3.

[0068] In some embodiments of this disclosure, the co-directed repeat sequence includes, for example, the sequence shown in SEQ ID NO: 2 or 3.

[0069] In some embodiments of the present disclosure, the guide array is located at the 3' end or 5' end of the co-directional repeating array. In some embodiments of the present disclosure, the guide array is located at the 3' end of the co-directional repeating array. In some embodiments of the present disclosure, the guide array is located at the 5' end of the co-directional repeating array.

[0070] In some embodiments of this disclosure, the gRNA includes an aptamer sequence.

[0071] In some embodiments of this disclosure, the aptamer sequence is inserted into the loop of a stem-loop structure in the secondary structure of the co-directed repeat sequence of the gRNA.

[0072] In some embodiments of the Disclosure, the gRNA comprises modified nucleotides. These modifications include, but are not limited to, a 2'-O-methyl group, a 2'-O-methyl-3'-thiophosphate group, or a 2'-O-methyl-3'-thioPACE modification. In some embodiments of the Disclosure, the gRNA comprises modified nucleotides, which are selected from deoxyribonucleotides and roq nucleic acids (LNAs). In some embodiments of the Disclosure, the gRNA comprises at least one chemically modified nucleotide. In some embodiments of the Disclosure, the gRNA is a hybrid RNA-DNA guide, i.e., some RNA nucleotides in the gRNA are substituted with DNA nucleotides. In some embodiments of the Disclosure, the gRNA is a hybrid RNA-LNA (roq nucleic acid) guide, i.e., some RNA nucleotides in the gRNA are substituted with LNA nucleotides.

[0073] In some embodiments of this disclosure, the target RNA is located in the cell nucleus and / or cytoplasm of a eukaryotic cell.

[0074] In some embodiments of this disclosure, the gRNA can form a complex (also referred to as a gene editing complex) with an RNA guide nuclease, which can then guide the complex to bind to the target RNA in a sequence-specific manner.

[0075] In some embodiments of this disclosure, the gRNA can form a complex (also referred to as a gene editing complex) with an RNA guide nuclease, which can then guide the complex to sequence-specifically bind to and cleave the target RNA.

[0076] In some embodiments of this disclosure, the complex reduces the level of the target RNA in a mammal (e.g., in a human body).

[0077] A third aspect of this disclosure provides an isolated nucleic acid which encodes the gRNA disclosed herein.

[0078] A fourth aspect of this disclosure provides a vector comprising a polynucleotide sequence encoding the gRNA disclosed herein, and a regulatory sequence used to control the expression of the guide RNA.

[0079] In some embodiments of this disclosure, the vector is an adeno-associated virus (AAV) vector.

[0080] In some embodiments of this disclosure, the control sequence is a promoter sequence. In some embodiments of this disclosure, the control sequence is an enhancer sequence. In some embodiments of this disclosure, the control sequence is both a promoter and an enhancer sequence.

[0081] In some embodiments of this disclosure, the control sequence is a U6 promoter or an eye-specific promoter.

[0082] In some embodiments of this disclosure, the eye-specific promoter may be selected from: a retinal cleavage protein promoter, a K12 promoter, a rhodopsin promoter, a rod cell-specific promoter, a cone cell-specific promoter, a rhodopsin kinase promoter, a GRK1 promoter, an interphotoreceptor retinoid-binding protein proximal (IRBP) promoter, and an opsin promoter (e.g., a red opsin promoter, a blue opsin promoter, etc.).

[0083] In some embodiments of this disclosure, the promoter is a chicken β-actin protein (CB) promoter. The chicken β-actin protein promoter may be a short chicken β-actin protein promoter or a long chicken β-actin protein promoter. In some embodiments of this disclosure, the promoter (e.g., chicken β-actin protein promoter) includes an enhancer sequence, such as a cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence may be a short CMV enhancer sequence or a long CMV enhancer sequence. In some embodiments of this disclosure, the promoter includes a long CMV enhancer sequence and a long chicken β-actin protein promoter. In some embodiments of this disclosure, the promoter includes a short CMV enhancer sequence and a short chicken β-actin protein promoter. However, those skilled in the art will understand that a short CMV enhancer is used with a long CB promoter, and a long CMV enhancer is used with a short CB promoter. In some embodiments of this disclosure, the promoter is a CBh promoter.

[0084] In some embodiments of this disclosure, the promoter is a CBh promoter.

[0085] In some embodiments of the present disclosure, the control array includes an HRE enhancer element (hypoxia response element).

[0086] In some embodiments, the control array includes NRS elements and HRE enhancer elements arranged in tandem.

[0087] A fifth aspect of this disclosure provides a vector system, wherein the vector system comprises a polynucleotide sequence encoding the gRNA disclosed herein and a first regulatory sequence that controls the expression of the gRNA, and a polynucleotide sequence encoding the RNA guide nuclease and a second regulatory sequence that controls the expression of the RNA guide nuclease.

[0088] In some embodiments of the present disclosure, the vector system includes one or more vectors.

[0089] In some embodiments of the present disclosure, the vector system comprises a plurality of vectors, wherein a polynucleotide sequence encoding the gRNA and a first regulatory sequence controlling the expression of the gRNA are located on the first vector, and a polynucleotide sequence encoding the RNA guide nuclease and a second regulatory sequence controlling the expression of the RNA guide nuclease are located on the second vector.

[0090] In some embodiments of this disclosure, the control sequence is a promoter sequence. In some embodiments of this disclosure, the control sequence is an enhancer sequence. In some embodiments of this disclosure, the control sequence is both a promoter and an enhancer sequence.

[0091] A sixth aspect of the present disclosure provides an adeno-associated virus (AAV) vector, wherein the adeno-associated virus (AAV) vector comprises a nucleic acid encoding an RNA guide nuclease and the DNA of the gRNA of the present disclosure.

[0092] A seventh aspect of the present disclosure provides lipid nanoparticles (LNPs), wherein the lipid nanoparticles (LNPs) include the gRNA and the mRNA encoding the RNA guide nuclease of the present disclosure.

[0093] An eighth aspect of the present disclosure provides a lentiviral vector comprising the gRNA and mRNA encoding an RNA guide nuclease of the present disclosure. Optionally, the lentiviral vector may be pseudotyped with an envelope protein. Optionally, the mRNA encoding the RNA guide nuclease is ligated to an aptamer sequence.

[0094] A ninth aspect of the present disclosure provides a ribonucleoprotein complex (RNP) formed by the gRNA and RNA guide nuclease of the present disclosure.

[0095] A tenth aspect of the present disclosure provides a virus-like particle (VLP), wherein the virus-like particle (VLP) comprises a ribonucleoprotein complex (RNP) formed by the gRNA and RNA guide nuclease of the present disclosure. Optionally, the RNA guide nuclease comprises fusion with a gag protein.

[0096] An eleventh aspect of this disclosure provides cells, which include the aforementioned inhibitors, gRNAs, nucleic acids, vectors, and / or vector systems of this disclosure. Optionally, the cells are eukaryotic cells.

[0097] A twelfth aspect of this disclosure provides a pharmaceutical composition comprising an inhibitor, gRNA, nucleic acid, vector and / or vector system of this disclosure.

[0098] In some embodiments of this disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0099] A thirteenth aspect of this disclosure provides applications in the manufacture of reagents that achieve any one of the following in the inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells and / or pharmaceutical compositions of this disclosure: To cleave or nicking one or more target RNA molecules, to activate or increase one or more target RNA molecules, to activate or inhibit the translation of one or more target RNA molecules, to inactivate one or more target RNA molecules, to visualize, label or detect one or more target RNA molecules, to bind to one or more target RNA molecules, to transport one or more target RNA molecules, and to mask one or more target RNA molecules.

[0100] In some embodiments of this disclosure, applications are provided in the manufacture of reagents that achieve any one of the following in the inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells and / or pharmaceutical compositions of this disclosure: Cleavage of one or more target RNA molecules, inhibit translation of one or more target RNA molecules, or bind to one or more target RNA molecules.

[0101] In some embodiments of this disclosure, the inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells and / or pharmaceutical compositions of this disclosure are provided for use in the manufacture of reagents that achieve any one of the following: Binding to one or more target RNA molecules.

[0102] In some embodiments of this disclosure, applications are provided in the manufacture of reagents that achieve any one of the following in the inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells and / or pharmaceutical compositions of this disclosure: The act of cleaving one or more types of RNA molecules.

[0103] In some embodiments of this disclosure, the target RNA is CTGF pre-mRNA, MITF pre-mRNA, or SRD5A2 pre-mRNA, or CTGF mRNA, MITF mRNA, or SRD5A2 mRNA (i.e., mature mRNA). In some embodiments of this disclosure, the target RNA is CTGF mRNA, MITF mRNA, or SRD5A2 mRNA. In some embodiments of this disclosure, the target RNA is mammalian CTGF RNA, MITF RNA, or SRD5A2 RNA, which may be selected from, for example, human, rat, mouse, or non-human primate CTGF RNA, MITF RNA, or SRD5A2 RNA, such as monkey, dog, pig, or rabbit. In some embodiments of this disclosure, the target RNA is human CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of this disclosure, the target RNA is human CTGF mRNA, MITF mRNA, or SRD5A2 mRNA.

[0104] A fourteenth aspect of the present disclosure provides a method for diagnosing, treating or preventing a target RNA-related disease or condition, and administering an effective amount of the said inhibitor, gRNA, nucleic acid, vector, vector system, adeno-associated virus (AAV) vector, lipid nanoparticle (LNP), lentiviral vector, ribonucleoprotein complex (RNP), virus-like particles (VLP), eukaryotic cells and / or pharmaceutical composition of the present disclosure to a sample of a subject or to a subject as needed.

[0105] In some embodiments of this disclosure, the target RNA-related disease or condition refers to a disease or condition resulting from the abnormal high expression of the target RNA.

[0106] In some embodiments of this disclosure, the target RNA is CTGF pre-mRNA, MITF pre-mRNA, or SRD5A2 pre-mRNA, or CTGF mRNA, MITF mRNA, or SRD5A2 mRNA (immediately mature mRNA). In some embodiments of this disclosure, the target RNA is CTGF mRNA, MITF mRNA, or SRD5A2 mRNA. In some embodiments of this disclosure, the target RNA is human CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of this disclosure, the target RNA is human CTGF mRNA, MITF mRNA, or SRD5A2 mRNA.

[0107] In some embodiments of this disclosure, diseases associated with the target RNA include melanoma, male pattern baldness, and scarring.

[0108] In some embodiments of this disclosure, pathological conditions associated with the target RNA include melanoma, male pattern baldness, and scar formation.

[0109] A fifteenth aspect of the present disclosure provides applications in the manufacture of agents for the diagnosis, treatment, or prevention of diseases or conditions related to target RNA, using the aforementioned inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells, and / or pharmaceutical compositions of the present disclosure.

[0110] In some embodiments of this disclosure, the disease or condition associated with the target RNA refers to a disease or condition resulting from the abnormal high expression of the target RNA.

[0111] In some embodiments of this disclosure, the target RNA is CTGF pre-mRNA, MITF pre-mRNA, or SRD5A2 pre-mRNA, or CTGF mRNA, MITF mRNA, or SRD5A2 mRNA. In some embodiments of this disclosure, the target RNA is CTGF mRNA, MITF mRNA, or SRD5A2 mRNA. In some embodiments of this disclosure, the target RNA is human CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of this disclosure, the target RNA is human CTGF mRNA, MITF mRNA, or SRD5A2 mRNA.

[0112] In some embodiments of this disclosure, diseases associated with the target RNA include melanoma, male pattern baldness, and scarring.

[0113] In some embodiments of this disclosure, pathological conditions associated with the target RNA include melanoma, male pattern baldness, and scar formation.

[0114] A sixteenth aspect of this disclosure provides the aforementioned inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), and / or applications in the manufacture of cosmetics in eukaryotic cells.

[0115] In some embodiments of the present disclosure, the cosmetic can be used for scar repair, skin whitening, reduction or removal of melasma, prevention or treatment of melanoma, and / or prevention or treatment of male pattern baldness.

[0116] A 17th aspect of the present disclosure provides a cosmetic, which comprises the aforementioned inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), and / or eukaryotic cells.

[0117] In some embodiments of the present disclosure, the cosmetic can be used for scar repair, skin whitening, reduction or removal of melasma, prevention or treatment of melanoma, and / or prevention or treatment of male pattern baldness.

[0118] In some embodiments of this disclosure, the inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), cells, pharmaceutical compositions, or cosmetics described herein can be administered to a subject (e.g., a human or an animal) in an effective amount. An effective amount means an amount that can produce the desired function or activity in a human and / or animal and is tolerable by the human and / or animal.

[0119] In some embodiments of the present disclosure, the inhibitors, guide RNAs, nucleic acids, vectors, vector systems, adeno-associated virus vectors, lipid nanoparticles, lentiviral vectors, ribonucleoprotein complexes, virus-like particles, cells, pharmaceutical compositions, or cosmetics of the present disclosure may contain "pharmaceutically, cosmetically, chemically, or biologically acceptable" components. Examples include pharmaceutically acceptable excipients, various cosmetically acceptable excipients, thickeners, or diluents.

[0120] An eighteenth aspect of this disclosure provides the use of a gene editing system in the manufacture of a pharmaceutical composition for the diagnosis, treatment, or prevention of male pattern baldness, wherein the gene editing system knocks down AR RNA levels or inhibits the translation of AR RNA.

[0121] In some embodiments of this disclosure, the gene editing system knocks down AR RNA levels or inhibits AR RNA translation.

[0122] In some embodiments of this disclosure, the gene editing system knocks down AR RNA levels.

[0123] In some embodiments of this disclosure, the gene editing system inhibits the translation of AR RNA.

[0124] In some embodiments of this disclosure, the gene editing system includes: gRNA containing a guide sequence that hybridizes with AR RNA or a polynucleotide sequence of the gRNA, and RNA guide nuclease or a polynucleotide sequence encoding the nuclease, The gRNA can form a complex with the nuclease and guide the complex to sequence-specific binding to the AR RNA.

[0125] In some embodiments of this disclosure, the gene editing system includes: gRNA containing a guide sequence that hybridizes with AR RNA, and RNA guide nuclease, The gRNA can form a complex with the nuclease and guide the complex to sequence-specific binding to the AR RNA.

[0126] In some embodiments of this disclosure, the gene editing system includes: A polynucleotide sequence encoding a gRNA containing a guide sequence that hybridizes with AR RNA, and a polynucleotide sequence encoding an RNA guide nuclease. The gRNA can form a complex with the nuclease and guide the complex to sequence-specific binding to the AR RNA.

[0127] In some embodiments of this disclosure, the AR RNA is pre-mRNA and / or mature mRNA.

[0128] In some embodiments of this disclosure, the AR RNA is human pre-mRNA and / or mature mRNA.

[0129] In some embodiments of this disclosure, the AR RNA sequence is selected from the sequences shown in SEQ ID NO: 394-396.

[0130] In some embodiments of this disclosure, the AR RNA is the sequence registered in the NCBI database as NM_000044.6 (SEQ ID NO: 394), i.e., “transcript variant 1”.

[0131] In some embodiments of this disclosure, the AR RNA is the sequence registered in the NCBI database as NM_001011645.3 (SEQ ID NO: 395), i.e., “transcript variant 2”.

[0132] In some embodiments of this disclosure, the AR RNA is a sequence (SEQ ID NO: 396) registered in the Ensembl database as ENST00000374690.9.

[0133] In some embodiments of this disclosure, the gRNA guides the complex to bind to and cleave the AR RNA.

[0134] In some embodiments of the present disclosure, the complex, after contact with a cell containing AR RNA, reduces the intracellular level of the AR RNA or AR protein (androgen receptor encoded by AR RNA) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0135] In some embodiments of the present disclosure, the complex, after contact with cells containing AR RNA, reduces the intracellular level of the AR RNA by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0136] In some embodiments of the present disclosure, the complex, after contact with cells containing AR RNA, reduces the intracellular level of the AR protein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0137] In some embodiments of this disclosure, the cells are eukaryotic cells. In some embodiments of this disclosure, the cells are mammalian cells. In some embodiments of this disclosure, the cells are human cells.

[0138] In some embodiments of the present disclosure, the number of off-target genes when the complex binds to and cleaves the target RNA (the AR RNA) is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1.

[0139] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 394-396 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0140] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 394-396 have 100% sequence identity.

[0141] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in SEQ ID NO: 394 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0142] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in SEQ ID NO: 394 have 100% sequence identity.

[0143] In some embodiments of this disclosure, the guide sequence of the gRNA and the nucleotide sequence consisting of nucleotides from position 2826 to 2990 of the sequence shown in SEQ ID NO: 394 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0144] For human targets, gRNAs were designed targeting NCBI's NM_001011645.3 (transcript variant 2, mRNA; SEQ ID NO: 395) and Ensembl's transcript ENST00000374690.9 (SEQ ID NO: 396), and their guide sequences are shown in Table 9.

[0145] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0146] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 71-378 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0147] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 71-80 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0148] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 71-80 have 100% sequence identity.

[0149] In some embodiments of this disclosure, the gRNA comprises a guide sequence and a co-directing repeat sequence, the co-directing repeat sequence interacting with an RNA guide nuclease.

[0150] In some embodiments of this disclosure, the RNA guide nuclease is a Cas protein. Optionally, the RNA guide nuclease is a Cas9 protein, a Cas12 protein, or a Cas13 protein.

[0151] In some embodiments of this disclosure, the RNA guide nuclease is a Cas13 protein. Furthermore, optionally, the RNA guide nuclease is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein.

[0152] In some embodiments of this disclosure, the Cas13 protein comprises the sequence shown in SEQ ID NO: 1.

[0153] In some embodiments of this disclosure, the RNA guide nuclease includes any one or more of the following: an intracellular localization signal, a deaminase domain, a translation activation domain, a translation inhibition domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0154] In some embodiments of this disclosure, the intracellular localization signal is a nuclear localization signal and / or nuclear export signal sequence.

[0155] In some embodiments of this disclosure, the intracellular localization signal is a mitochondrial localization signal or a chloroplast localization signal sequence.

[0156] In some embodiments of the present disclosure, the RNA guide nuclease comprises a nuclear localization signal and / or nuclear export signal sequence, as well as an optional deaminase domain, a translation activation domain, or a translation inhibition domain.

[0157] In some embodiments of this disclosure, the RNA guide nuclease-coding polynucleotide sequence is linked to a control sequence 1 that controls its expression, and the gRNA-coding polynucleotide sequence is linked to a control sequence 2 that controls its expression.

[0158] In some embodiments of this disclosure, the control sequence promoter sequence. In some embodiments of this disclosure, the control sequence enhancer sequence. In some embodiments of this disclosure, the control sequence promoter and enhancer sequences.

[0159] In some embodiments of this disclosure, the control sequence CMV promoter, CMV enhancer, CBh promoter, U6 promoter, and specificity promoter are selected.

[0160] In some embodiments of this disclosure, the gene editing system includes: A gRNA containing a guide sequence that hybridizes with AR RNA, or a polynucleotide sequence encoding the gRNA, and a Cas13 protein, or a polynucleotide sequence encoding the Cas13 protein. The gRNA forms a CRISPR complex with the Cas13 protein, and the complex can guide the AR RNA to sequence-specifically bind and cleave.

[0161] In some embodiments of this disclosure, the coding sequence is linked to a regulatory sequence that controls its expression.

[0162] In some embodiments of this disclosure, the polynucleotide sequence encoding the Cas13 protein is ligated to a regulatory sequence 1 that controls its expression, and the polynucleotide sequence encoding the gRNA is ligated to a regulatory sequence 2 that controls its expression.

[0163] In some embodiments of this disclosure, at least two gRNAs (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more) are expressed in tandem under the control of the same regulatory sequence. Furthermore, the crRNA precursor (pre-crRNA) obtained by tandem expression is processed by the Cas13 protein to become a mature gRNA molecule.

[0164] In some embodiments of this disclosure, the polynucleotide sequence encoding the gRNA and the polynucleotide sequence encoding the RNA guide nuclease are located on the same vector.

[0165] The gene editing systems described herein can be introduced into cells (or cell-free systems) in a variety of non-limiting manner. For example, they can be introduced (i) as mRNA and gRNA encoding an RNA guide nuclease, (ii) as part of a single vector or plasmid, or as multiple vectors or plasmids, (iii) as isolated RNA guide nuclease and gRNA, or (iv) as an RNP complex consisting of RNA guide nuclease and gRNA.

[0166] A 19th aspect of this disclosure provides a gene editing system that knocks down AR RNA levels or inhibits AR RNA translation.

[0167] In some embodiments of this disclosure, the gene editing system knocks down AR RNA levels or inhibits AR RNA translation.

[0168] In some embodiments of this disclosure, the gene editing system knocks down AR RNA levels.

[0169] In some embodiments of this disclosure, the gene editing system inhibits the translation of AR RNA.

[0170] In some embodiments of this disclosure, the gene editing system includes: A gRNA containing a guide sequence that hybridizes with AR RNA, or a polynucleotide sequence encoding the gRNA, and an RNA guide nuclease, or a polynucleotide sequence encoding the nuclease. The gRNA can form a complex with the nuclease and guide the complex to sequence-specifically bind to the AR RNA.

[0171] In some embodiments of this disclosure, the gene editing system includes: gRNA containing a guide sequence that hybridizes with AR RNA, and RNA guide nuclease, The gRNA forms a complex with the nuclease and can guide the complex to sequence-specifically bind to the AR RNA.

[0172] In some embodiments of this disclosure, the gene editing system includes: A polynucleotide sequence encoding a gRNA containing a guide sequence that hybridizes with AR RNA, and a polynucleotide sequence encoding an RNA guide nuclease. The gRNA can form a complex with the nuclease and guide the complex to sequence-specifically bind to the AR RNA.

[0173] In some embodiments of this disclosure, the AR RNA is pre-mRNA and / or mature mRNA.

[0174] In some embodiments of this disclosure, the AR RNA is human pre-mRNA and / or mature mRNA.

[0175] In some embodiments of this disclosure, the AR RNA sequence is selected from the sequences shown in any of SEQ ID NO: 394-396.

[0176] In some embodiments of this disclosure, the AR RNA is the sequence registered in the NCBI database as NM_000044.6 (SEQ ID NO: 394), i.e., “transcript variant 1”.

[0177] In some embodiments of this disclosure, the AR RNA is the sequence registered in the NCBI database as NM_001011645.3 (SEQ ID NO: 395), i.e., “transcript variant 2”.

[0178] In some embodiments of this disclosure, the AR RNA is a sequence (SEQ ID NO: 396) registered in the Ensembl database as ENST00000374690.9.

[0179] In some embodiments of this disclosure, the gRNA guides the complex to bind to and cleave the AR RNA.

[0180] In some embodiments of the present disclosure, the complex, after contact with a cell containing AR RNA, reduces the intracellular level of the AR RNA or AR protein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0181] In some embodiments of the present disclosure, the complex, after contact with cells containing AR RNA, reduces the intracellular level of the AR RNA by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0182] In some embodiments of the present disclosure, the complex, after contact with cells containing AR RNA, reduces the intracellular level of the AR protein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0183] In some embodiments of this disclosure, the cells are eukaryotic cells. In some embodiments of this disclosure, the cells are mammalian cells. In some embodiments of this disclosure, the cells are human cells.

[0184] The reduction in RNA levels can be measured using methods well known to those skilled in the art. These methods include, but are not limited to, qPCR methods, such as RT-qPCR. The knockdown level of target RNA in the test group can be calculated by comparing it to the negative control, using the target RNA (AR RNA) level in untreated cells or cells treated with a gene editing system targeting a non-mammalian genome as a negative control. After editing with the same Cas protein (e.g., by expressing the same Cas protein) and gRNAs containing different guide sequences, the difference in target RNA levels can be compared between the test group and the negative control group. For example, the test group can be edited using C13-2 and the gRNAs of this disclosure, and the negative control group can be edited using C13-2 and gRNAs targeting, for example, bacterial genomes. The test group can also be compared with well-known editing tools such as CasRx+gRNA editing tools.

[0185] In some embodiments of this disclosure, the number of off-target genes when the complex binds to and cleaves the target RNA (the AR RNA) is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by methods well known to those skilled in the art. In some embodiments, the number of off-target genes is determined as the intersection (common portion) of the differentially expressed gene set identified by RNA sequencing and the off-target gene set predicted by the program. In some embodiments, the number of off-target genes is determined as the intersection (common portion) of the differentially expressed gene set with reduced expression identified by RNA sequencing and the off-target gene set predicted by the program. Off-target gene prediction programs well known to those skilled in the art may be used with standard parameter settings to predict off-target genes. As a non-limiting example, the EMBOSS-water program can be used to make predictions for the entire genome and cDNA sequence of a target species (e.g., Homo sapiens or Mus musculus), set parameters (e.g., gap_open=10, gap_extend=0.5), perform alignment on the forward and reverse strands of the gRNA guide sequence, and filter the prediction results to obtain potential target genes (including target genes and / or off-target genes).

[0186] The decrease in the protein level encoded by the target RNA (the AR RNA) can be measured using methods well known to those skilled in the art, including, but not limited to, ELISA and Western blotting. The level of AR protein in the test group can be calculated by comparing it to the negative control, using untreated cells or cells treated with a gene editing system targeting a non-mammalian genome.

[0187] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 394-396 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0188] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 394-396 have 100% sequence identity.

[0189] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in SEQ ID NO: 394 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0190] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in SEQ ID NO: 394 have 100% sequence identity.

[0191] In some embodiments of this disclosure, the guide sequence of the gRNA and the nucleotide sequence consisting of nucleotides from position 2826 to 2990 of the sequence shown in SEQ ID NO: 394 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0192] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 71-378 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0193] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 71-80 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0194] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 71-80 have 100% sequence identity.

[0195] In some embodiments of this disclosure, the gRNA comprises a guide sequence and a co-directing repeat sequence, the co-directing repeat sequence and an RNA guide nuclease interact.

[0196] In some embodiments of this disclosure, the RNA guide nuclease is a Cas protein. Optionally, the RNA guide nuclease is a Cas9 protein, a Cas12 protein, or a Cas13 protein.

[0197] In some embodiments of this disclosure, the RNA guide nuclease Cas13 protein is used. Furthermore, optionally, the RNA guide nuclease Cas13a protein, Cas13b protein, Cas13c protein, or Cas13d protein is used.

[0198] In some embodiments of this disclosure, the Cas13 protein comprises the sequence shown in SEQ ID NO: 1.

[0199] In some embodiments of this disclosure, the RNA guide nuclease includes any one or more of the following: an intracellular localization signal, a deaminase domain, a translation activation domain, a translation inhibition domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0200] In some embodiments of this disclosure, the intracellular localization signal is a nuclear localization signal and / or nuclear export signal sequence.

[0201] In some embodiments of this disclosure, the intracellular localization signal is a mitochondrial localization signal or a chloroplast localization signal sequence.

[0202] In some embodiments of the present disclosure, the RNA guide nuclease comprises a nuclear localization signal and / or nuclear export signal sequence, as well as an optional deaminase domain, a translation activation domain, or a translation inhibition domain.

[0203] In some embodiments of this disclosure, the polynucleotide sequence encoding the RNA guide nuclease is ligated to a control sequence 1 that controls its expression, and the polynucleotide sequence encoding the gRNA is ligated to a control sequence 2 that controls its expression.

[0204] In some embodiments of this disclosure, the control sequence is a promoter sequence. In some embodiments of this disclosure, the control sequence is an enhancer sequence. In some embodiments of this disclosure, the control sequence is both a promoter and an enhancer sequence.

[0205] In some embodiments of this disclosure, the control sequence is selected from a CMV promoter, a CMV enhancer, a CBh promoter, a U6 promoter, and a specificity promoter.

[0206] In some embodiments of this disclosure, the gene editing system includes: A gRNA containing a guide sequence that hybridizes with an AR RNA, or a polynucleotide sequence encoding the gRNA, and a Cas13 protein, or a polynucleotide sequence encoding the Cas13 protein.

[0207] The gRNA forms a CRISPR complex with the Cas13 protein, and the complex can guide the sequence-specific binding and cleavage of the AR RNA.

[0208] In some embodiments of this disclosure, the encoded sequence is linked to a control sequence that controls its expression.

[0209] In some embodiments of this disclosure, the polynucleotide sequence encoding the Cas13 protein is ligated to a regulatory sequence 1 that controls its expression, and the polynucleotide sequence encoding the gRNA is ligated to a regulatory sequence 2 that controls its expression.

[0210] In some embodiments of this disclosure, at least two gRNAs (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more) are expressed in tandem under the control of the same regulatory sequence. Furthermore, the crRNA precursor (pre-crRNA) obtained by tandem expression is processed by the Cas13 protein to become a mature gRNA molecule.

[0211] In some embodiments of this disclosure, the polynucleotide sequence encoding the gRNA and the polynucleotide sequence encoding the RNA guide nuclease are located on the same vector.

[0212] The gene editing systems described herein can be introduced into cells (or cell-free systems) in a variety of non-limiting manner. For example, they can be introduced (i) as mRNA and gRNA encoding an RNA guide nuclease, (ii) as part of a single vector or plasmid, or as multiple vectors or plasmids, (iii) as isolated RNA guide nuclease and gRNA, or (iv) as an RNP complex consisting of RNA guide nuclease and gRNA.

[0213] A 20th aspect of this disclosure provides a gRNA (guide RNA, gRNA) for a gene editing system, which includes a guide sequence that hybridizes with an AR RNA.

[0214] In some embodiments of this disclosure, the AR RNA is pre-mRNA or mature mRNA.

[0215] In some embodiments of this disclosure, the AR RNA is human AR RNA. In some embodiments of this disclosure, the AR RNA is human mRNA.

[0216] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 394-396 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0217] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 394-396 have 100% sequence identity.

[0218] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in SEQ ID NO: 394 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0219] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in SEQ ID NO: 394 have 100% sequence identity.

[0220] In some embodiments of this disclosure, the guide sequence of the gRNA and the nucleotide sequence consisting of nucleotides from position 2826 to 2990 of the sequence shown in SEQ ID NO: 394 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0221] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 71-378 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0222] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 71-80 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0223] In some embodiments of this disclosure, the guide sequence of the gRNA and the sequence shown in any one of SEQ ID NO: 71-80 have 100% sequence identity.

[0224] In some embodiments of the present disclosure, the gRNA can form a complex (also referred to as a gene editing complex) with an RNA guide nuclease, which can then guide the complex to bind to the AR RNA in a sequence-specific manner.

[0225] In some embodiments of the present disclosure, the gRNA can form a complex (also referred to as a gene editing complex) with an RNA guide nuclease, which can then guide the complex to bind to the AR RNA in a sequence-specific manner.

[0226] In some embodiments of this disclosure, the complex reduces the level of the AR RNA in a mammal (e.g., in a human body).

[0227] In some embodiments of this disclosure, the complex reduces the level of AR RNA within a cell after contact with the cell containing the AR RNA, for example, the level of target RNA within a cell expressing the target RNA.

[0228] In some embodiments of this disclosure, the complex reduces the intracellular level of the AR RNA by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the AR RNA level can be measured using methods well known to those skilled in the art. Without limiting these, the AR RNA knockdown level in the test group can be calculated by comparing it to the negative control, using untreated cells or cells treated with a gene editing system targeting a non-mammalian genome as a negative control, including, for example, the qPCR method described in the examples.

[0229] In some embodiments of the present invention, the number of off-target genes when the complex binds to and cleaves the AR RNA is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by methods well known to those skilled in the art. In some embodiments, the number of off-target genes is determined as the intersection (common area) between the differentially expressed gene set identified by RNA sequencing and the off-target gene set predicted by the program. As a non-limiting example, the EMBOSS-water program can be used to perform predictions on the entire genome and cDNA sequence of a target species (Homo sapiens / Mus musculus), set parameters (e.g., gap_open=10, gap_extend=0.5), perform alignment on the forward and reverse strands of the gRNA guide sequence, and filter the prediction results to obtain potential target genes (including target genes and off-target genes).

[0230] In some embodiments of this disclosure, the complex reduces the level of the protein encoded by the AR RNA in an animal body (e.g., a human body).

[0231] In some embodiments of the present disclosure, the complex reduces the level of the protein encoded by the AR RNA in cells after contact with cells containing the target RNA. In some embodiments of the present disclosure, the protein encoded by the AR RNA is an AR protein. In some embodiments of the present disclosure, the complex reduces the level of the AR protein in cells.

[0232] In some embodiments of this disclosure, the complex reduces intracellular AR protein levels by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the AR RNA-coding protein level can be measured using methods well known to those skilled in the art, including, but not limited to, ELISA and Western blotting. Using untreated cells or cells treated with gene editing systems targeting non-mammalian genomes as negative controls, the knockdown level of AR protein in the test group can be calculated by comparing it to the negative control group.

[0233] In some embodiments of this disclosure, the gRNA comprises a guide sequence and a backbone sequence, the backbone sequence and the RNA guide nuclease interacting with each other. In some embodiments of this disclosure, the backbone sequence is a direct repeat (DR) sequence.

[0234] A 21st aspect of the present disclosure provides isolated nucleic acids, any one of which encodes the gRNA.

[0235] A 22nd aspect of the present disclosure provides a vector comprising any one of the present disclosures, a polynucleotide sequence encoding the gRNA, and a regulatory sequence that controls the expression of the gRNA.

[0236] In some embodiments of this disclosure, the vector is an adeno-associated virus (AAV) vector.

[0237] In some embodiments of this disclosure, the control sequence is a promoter sequence. In some embodiments of this disclosure, the control sequence is an enhancer sequence. In some embodiments of this disclosure, the control sequence is both a promoter and an enhancer sequence.

[0238] In some embodiments of this disclosure, the control sequence is selected from a CMV promoter, a CMV enhancer, a CBh promoter, a U6 promoter, and a specificity promoter.

[0239] In some embodiments of this disclosure, the promoter is a chicken β-actin protein (CB) promoter. The chicken β-actin protein promoter may be a short chicken β-actin protein promoter or a long chicken β-actin protein promoter. In some embodiments of this disclosure, the promoter (e.g., chicken β-actin protein promoter) includes an enhancer sequence, e.g., a cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence may be a short CMV enhancer sequence or a long CMV enhancer sequence. In some embodiments of this disclosure, the promoter includes a long CMV enhancer sequence and a long chicken β-actin protein promoter. In some embodiments of this disclosure, the promoter includes a short CMV enhancer sequence and a short chicken β-actin protein promoter. However, those skilled in the art will understand that a short CMV enhancer is used with a long CB promoter, and a long CMV enhancer is used with a short CB promoter. In some embodiments of this disclosure, the promoter is a CBh promoter.

[0240] In some embodiments of this disclosure, the promoter is a CBh promoter.

[0241] In some embodiments of the present disclosure, the control array includes an HRE enhancer element (hypoxia response element).

[0242] In some embodiments of the present disclosure, the control array includes tandem-arranged NRS elements and HRE enhancer elements (hypoxia response elements).

[0243] A 23rd aspect of the present disclosure provides a vector system comprising a polynucleotide sequence encoding the gRNA of the present disclosure, a second regulatory sequence controlling the expression of the gRNA, and a polynucleotide sequence encoding the RNA guide nuclease and a first regulatory sequence controlling the expression of the RNA guide nuclease.

[0244] In some embodiments of the present disclosure, the vector system includes one or more vectors.

[0245] In some embodiments of the present disclosure, the vector system comprises a plurality of vectors, wherein a polynucleotide sequence encoding the gRNA and a second regulatory sequence controlling the expression of the gRNA are located on the second vector, and a polynucleotide sequence encoding the RNA guide nuclease and a first regulatory sequence controlling the expression of the RNA guide nuclease are located on the first vector.

[0246] In some embodiments of this disclosure, the control sequence is a promoter sequence. In some embodiments of this disclosure, the control sequence is an enhancer sequence. In some embodiments of this disclosure, the control sequence is both a promoter and an enhancer sequence.

[0247] A 24th aspect of the present disclosure provides an adeno-associated virus (AAV) vector, wherein the adeno-associated virus (AAV) vector comprises a nucleic acid encoding an RNA guide nuclease and the DNA of the gRNA of the present disclosure.

[0248] A 25th aspect of the present disclosure provides lipid nanoparticles (LNPs), wherein the lipid nanoparticles (LNPs) include the gRNA and the mRNA encoding the RNA guide nuclease of the present disclosure.

[0249] A 26th aspect of this disclosure provides a lentiviral vector comprising the gRNA and mRNA encoding an RNA guide nuclease as described in this disclosure. Optionally, the lentiviral vector may be pseudotyped with an envelope protein. Optionally, the mRNA encoding the RNA guide nuclease is ligated to an aptamer sequence.

[0250] A 27th aspect of the present disclosure provides a ribonucleoprotein complex (RNP) formed by the gRNA and RNA guide nuclease of the present disclosure.

[0251] A 28th aspect of this disclosure provides a virus-like particle (VLP), wherein the VLP comprises a ribonucleoprotein complex (RNP) formed by the gRNA and RNA guide nuclease of this disclosure. Optionally, the RNA guide nuclease is fused with a gag protein.

[0252] A 29th aspect of this disclosure provides a eukaryotic cell comprising the gene editing systems, gRNAs, nucleic acids, vectors, and / or vector systems of this disclosure. Optionally, the eukaryotic cell is a mammalian cell. Furthermore, optionally, the eukaryotic cell is a human cell.

[0253] In some embodiments of the present disclosure, the eukaryotic cell includes the gene editing system of the present disclosure.

[0254] A 30th aspect of the present disclosure provides a pharmaceutical composition comprising any one of the gene editing systems, gRNA, nucleic acids, vectors, and / or vector systems of the present disclosure.

[0255] In some embodiments of the present disclosure, the pharmaceutical composition comprises any one of the gene editing systems described herein.

[0256] In some embodiments of the present disclosure, the pharmaceutical composition comprises any one of the gRNAs, nucleic acids, vectors, and / or vector systems described herein.

[0257] In some embodiments of this disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0258] A 31st aspect of this disclosure provides applications in the manufacture of reagents that achieve any one of the following in any of the gene editing systems, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells, or pharmaceutical compositions of this disclosure: Cleaving one or more types of AR RNA molecules, nicking one or more types of AR RNA molecules, activating or increasing one or more types of AR RNA molecules, activating or inhibiting the translation of one or more types of AR RNA molecules, inactivating one or more types of AR RNA molecules, visualizing, labeling or detecting one or more types of AR RNA molecules, binding to one or more types of AR RNA molecules, transporting one or more types of AR RNA molecules, and masking one or more types of AR RNA molecules.

[0259] In some embodiments of this disclosure, applications are provided in the manufacture of reagents that achieve any one of the following in any one of the gene editing systems, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells, or pharmaceutical compositions of this disclosure: Cleavage of AR RNA molecules, inhibition of AR RNA translation, and binding to AR RNA molecules.

[0260] In some embodiments of this disclosure, applications are provided for any one of the gene editing systems, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells, or pharmaceutical compositions used in the production of reagents used for cleaving AR RNA molecules.

[0261] In some embodiments of this disclosure, applications are provided in the manufacture of reagents used for binding AR RNA molecules or to AR RNA molecules in any one of the gene editing systems, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells, or pharmaceutical compositions of this disclosure.

[0262] In some embodiments of this disclosure, the AR RNA is pre-mRNA or mature mRNA. In some embodiments of this disclosure, the AR RNA is mature mRNA. In some embodiments of this disclosure, the AR RNA is human AR RNA. In some embodiments of this disclosure, the AR RNA is human AR mRNA.

[0263] A 32nd aspect of the present disclosure provides a method for diagnosing, treating or preventing a disease or condition, and administering an effective amount of the gene editing system, gRNA, nucleic acid, vector, vector system, adeno-associated virus (AAV) vector, lipid nanoparticle (LNP), lentiviral vector, ribonucleoprotein complex (RNP), virus-like particles (VLP), eukaryotic cells and / or pharmaceutical composition of the present disclosure to a sample of a subject or to a subject as needed.

[0264] In some embodiments of the present disclosure, a method is provided for diagnosing, treating or preventing a disease or condition, and for administering an effective amount of the gene editing system, eukaryotic cells and / or pharmaceutical composition of the present disclosure to a sample of a subject or to a subject as needed.

[0265] In some embodiments of this disclosure, methods are provided for diagnosing, treating or preventing a disease or condition, and for using an effective amount of the gene editing system of this disclosure on a sample of a subject or on a subject as needed.

[0266] In some embodiments of this disclosure, the disease or condition is an AR RNA-related disease or condition. Optionally, the disease or condition refers to a disease or condition resulting from abnormally high expression of AR RNA.

[0267] In some embodiments of this disclosure, the AR RNA is pre-mRNA or mature mRNA. In some embodiments of this disclosure, the AR RNA is mature mRNA. In some embodiments of this disclosure, the AR RNA is human AR RNA. In some embodiments of this disclosure, the AR RNA is human AR mRNA.

[0268] In some embodiments of this disclosure, the disease or condition is male pattern baldness. [Brief explanation of the drawing]

[0269] [Figure 1] Figure 1 shows gRNAs designed to target CTGF(CCN2)RNA. [Figure 2] Figure 2 shows the CTGF RNA levels after editing using C13-2 and gRNA combinations (detected by qPCR using CTGF-my primers). [Figure 3] Figure 3 shows the CTGF RNA levels after editing using C13-2 and gRNA combinations (using CTGF-11 primers). [Figure 4]Figure 4 shows the designed gRNA targeting MITF RNA. [Figure 5] Figure 5 shows the MITF RNA levels after editing by the combination of C13-2 and gRNA. [Figure 6] Figure 6 shows the designed gRNA targeting SRD5A2 RNA. [Figure 7] Figure 7 shows the SRD5A2 RNA levels after editing by the combination of C13-2 and gRNA (detected by qPCR using Primer 2). [Figure 8] [[ID=1】Figure 8 shows the SRD5A2 RNA levels after editing by the combination of C13-2 and gRNA (detected by qPCR using Primer 3). [Figure 9] Figure 9 shows the CTGF RNA levels after editing by the combination of CasRx and gRNA (CTGF-my primer). [Figure 10] Figure 10 shows the CTGF RNA levels after editing by the combination of CasRx and gRNA (CTGF-ll primer). [Figure 11] Figure 11 shows the MITF RNA levels after editing by the combination of CasRx and gRNA. [Figure 12] Figure 12 shows the SRD5A2 RNA levels after editing by the combination of CasRx and gRNA (Primer 2). [Figure 13] Figure 13 shows the screening results of gRNA. [Figure 14] Figure 14 shows the comparative measurement results between the Cas13 system and shRNA. [Figure 15] Figure 15 shows the measurement results of C13-2 protein and CasRx in the same batch of tests. [Figure 16] Figure 16 shows the measurement results of the AR protein expression levels in the edited cells.

Modes for Carrying Out the Invention

[0270] The present invention will be described below using examples, but will not be limited to the scope of the examples described. In the following examples, test methods for which specific conditions are not described will follow conventional methods and conditions, or will be selected according to the product description.

[0271] Definition:

[0272] For example, as used herein, the term “gene editing system” means a protein, nucleic acid, or combination thereof that, when introduced into a cell, can modify an endogenous target nucleic acid sequence (e.g., target RNA). Gene editing systems may include, but are not limited to, CRISPR-Cas systems, TALEN systems, ZFN systems, etc. Specifically, gene editing systems may include RNA guide nucleases and gRNAs. Gene editing systems well known to those skilled in the art include, but are not limited to, the following: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12f / CasZ, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, C sn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Cs b3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1 Systems comprising Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, Cas13f, TnpB, IscB, IsrB, Fancor, or fragments thereof (e.g., nucleic acid-binding domain fragments). Also includes systems comprising Cas9, Cas12, Cas13, TnpB, IscB, IsrB, Fancor nuclease, or fragments thereof.

[0273] An example of a gene editing system used in the methods described herein is the clustered regularly spaced short palindromic repeat (CRISPR) / CRISPR-related (Cas) nuclease system. This system is a nuclease system adapted for mammalian genome engineering by modifying a bacterial-derived system, and typically includes a CRISPR-related nucleic acid cleavage enzyme (e.g., Cas nuclease) and gRNA (gRNA).

[0274] For example, in this specification, the term “knockdown” refers to a measurable reduction in the level of target RNA in a gene-modified cell compared to the level of target RNA in a control cell. For example, the reduction in the level of target RNA in the gene-modified cell compared to the level of target RNA in a control cell may be 0%, ≥5%, ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or ≥95%. Those skilled in the art will readily understand, based on the description herein, a method for knocking out target RNA or a portion thereof using gene-editing inhibitory techniques.

[0275] In this specification, "inhibitor" means an agent that selectively inhibits target RNA, for example, by reducing the expression level of target RNA or inhibiting the translation of target RNA. Inhibitors can reduce the target RNA level by contacting the target RNA with a gene editing system and cleaving the target RNA, among other things.

[0276] In this specification, "gene editing system gRNA," "gRNA," "guide RNA," and "gRNA" can be used interchangeably. A gRNA is a molecule that forms a complex with an RNA guide nuclease in a gene editing system and guides the complex to a target sequence in a sequence-specific manner. A gRNA contains a guide sequence that can hybridize with the target sequence. When the RNA guide nuclease is a Cas protein, particularly Cas13, the guide RNA usually contains a codirectional repeat sequence ligated to the guide sequence.

[0277] In this specification, the terms “guide sequence” and “target domain” are used interchangeably and refer to a sequence of nucleotides in gRNA that is partially or completely complementary to a target sequence in target RNA and can hybridize to the target sequence via base pairing facilitated by an RNA guide nuclease. In the present invention, complete complementarity between the guide sequence and the target sequence is not required; it is sufficient if there is sufficient complementarity to induce hybridization and the formation of a gene editing complex.

[0278] Suitable direct repeat (DR) sequences can be found in the CRISPR locus structure of prokaryotes (e.g., bacteria or archaea) and obtained by experimental screening. They can also be obtained by sequence modification or optimization based on these findings. Non-limiting examples include deletions, substitutions, or additions of 1, 2, 3, 4, or more complementary base pairs in the complementary double-stranded region of the secondary structure of a DR sequence, as well as deletions, substitutions, or additions of nucleotides on the loop of a stem-loop structure (e.g., insertion of an aptamer sequence into the loop). Direct repeat sequences are typically several tens of nucleotides long, and some of them are anti-complementary to each other. This means that secondary structures such as stem-loop structures (often called hairpin structures) are formed within the RNA molecule, while other parts are non-structural. Direct repeat sequences are the constant portion of guide RNA molecules, possessing a strong secondary structure that facilitates the interaction between RNA guide nucleases and guide RNA molecules.

[0279] In this specification, “hybridization” or “hybridizing” means the process by which fully or partially complementary polynucleotide chains associate under appropriate hybridization conditions to form a double-stranded structure or region thereof, including association by hydrogen bonding between nucleic acids. In this specification, “hybridization” also includes cases where the double-stranded structure or region thereof contains one or more bulges or mismatches. The intensity of hybridization and the strength of the bonding between nucleic acids are influenced by factors such as the degree of complementarity between nucleic acids, the stringency of the relevant conditions, and the Tm of the resulting hybrid. Hydrogen bonds are commonly formed between adenine and thymine, adenine and uracil, or cytosine and guanine, but other non-classical base pairs can also form hydrogen bonds. Modified nucleotides are expected to be able to form hydrogen bonds that enable or facilitate hybridization via non-classical pathways.

[0280] For example, as used herein, the term “target RNA” refers to a polynucleotide containing a target sequence, and means a specific sequence or its counter-complementary sequence that is required to be bound, targeted, or modified using a gene editing system. For example, this can be a complete mature mRNA molecule or a pre-mRNA molecule, or a fragment thereof.

[0281] In this specification, “target sequence” means a short sequence within a target RNA molecule that may be fully or partially complementary to the guide sequence of the gRNA molecule. The gene editing complex can be sequence-specifically positioned at the target sequence by the guide sequence and can exert the desired function at or near that location. The target sequence is often several tens of nucleotides (nt) long, for example, about 10nt, 20nt, 30nt, 40nt, 50nt, or 60nt.

[0282] In this specification, "cleavage" refers to the cleavage of a covalent bond (e.g., a covalent phosphodiester bond) in the ribosylphosphodiester backbone of a polynucleotide. .

[0283] The sequence-specific binding ability of a gRNA-guided complex to target RNA can be evaluated by any suitable assay. For example, sufficient components of a gene editing system (including the gRNA to be evaluated) to form a gene editing complex may be provided to a host cell having the corresponding target RNA molecule, and then, for example, a vector encoding the components of the complex may be transfected, after which preferential cleavage at the target sequence may be evaluated. Similarly, cleavage of the target RNA sequence can also be evaluated in vitro. For example, the target RNA, components of a gene editing complex (including a control gRNA different from the gRNA to be evaluated and the gRNA under test) may be prepared, and the binding ability to target RNA or the target RNA cleavage rate may be compared between the gRNA to be evaluated and the control gRNA. The ability of the gRNA to guide the complex to cleave the target RNA can also be evaluated by the assays described above.

[0284] In this specification, “RNA guide nuclease” means a polypeptide that is sequence-specifically guided by gRNA to a target RNA sequence and can form a complex with gRNA hybridized to the target sequence. RNA guide nucleases can cleave the target sequence, causing a strand break. While RNA guide nucleases can cleave the target sequence upon binding, the term “RNA guide nuclease” also includes inactive RNA guide nucleases that bind to the target sequence but do not cleave it. RNA guide nucleases described herein include, but are not limited to, wild-type RNA guide nucleases (e.g., C13-2, CasRx, etc.), their variants (e.g., variants with complete loss of cleavage activity, variants with partial loss of cleavage activity, variants with improved cleavage activity, variants with reduced off-target effects, variants with reduced side-cut effects), or functional fragments or fusion proteins thereof.

[0285] As used herein, the term "Cas protein" refers to a CRISPR-associated (Cas) polypeptide or protein. A Cas protein can be guided to a target sequence in a target RNA and bind to and optionally cleave the target RNA by forming a complex or a functional combination with one or more types of gRNAs.

[0286] As used herein, the term "sequence identity (identity or percent identity)" refers to the degree of match between two polypeptide sequences or two nucleic acid sequences. When comparing two sequences, a position is considered identical if the same base or amino acid residue is present at that position (e.g., if one position in each of two DNA molecules is occupied by adenine, or if one position in each of two polypeptides is occupied by lysine). The "percent identity" between two sequences is the value obtained by multiplying by 100% the number of matching positions shared by the two sequences divided by the number of positions compared. For example, if 6 out of 10 positions of two sequences match, the sequence identity of the two sequences is 60%. Usually, the two sequences are compared to obtain the maximum sequence identity. Such alignment can be performed using generally publicly available or commercially available alignment algorithms and programs such as ClustalΩ, MAFFT, Probcons, T-Coffee, Probalign, BLAST, etc., which can be reasonably selected by those skilled in the art. Those skilled in the art can determine parameters suitable for sequence alignment. Examples include algorithms necessary to achieve an optimal or best alignment for the entire length of the sequences to be compared, and algorithms necessary to achieve an optimal or best alignment for local portions of the sequences to be compared.

[0287] In this specification, “regulatory sequence” includes expression regulatory elements such as promoters, enhancers, and internal ribosome entry sites (IRESs). These may include transcription termination signals (e.g., poly-A addition signals) and poly-U sequences. Regulatory sequences include elements that guide the continuous expression of nucleotide sequences in many types of host cells, and elements that guide the expression of nucleotide sequences only in specific host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily guide expression in target tissues such as muscle, nerve cells, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., nerve cells, lymphocytes). Regulatory sequences can also guide expression in time-dependent ways, such as in a cell cycle-dependent or developmental stage-dependent manner, and this guidance may or may not be tissue-specific or cell-type specific. The term “regulatory sequence” also includes enhancer elements such as WPRE, CMV enhancers, SV40 enhancers, and the intron sequence between exons 2 and 3 of rabbit β-globulin. Those skilled in the art will understand that the design of an expression vector depends on factors such as the selection of the host cells to be transformed and the desired expression level. The vector can be introduced into host cells to generate the RNA guide nuclease and / or gRNA described herein.

[0288] As used herein, the term “promoter” has the meaning commonly understood by those skilled in the art.

[0289] As used herein, the term “enhancer” has the meaning commonly understood by those skilled in the art.

[0290] In this specification, when referring to nucleotide sequences / DNA / RNA encoding proteins, RNA, or gene editing complexes, such coding sequences may be codon-optimized. For example, such coding sequences may be codon-optimized for expression in eukaryotic, mammalian, or human cell environments.

[0291] In this specification, the term "codon optimization" refers to the following process: modifying the codons of a desired gene in such a way that the modified codons improve the expression of the polypeptide sequence while maintaining the same polypeptide sequence encoded by that gene. For example, when expressing a human polypeptide in E. coli, expression can usually be improved by replacing codons that are frequently used in humans with codons that are expressed more efficiently in E. coli.

[0292] In this specification, the term “pharmaceutically acceptable excipient” refers to a diluent, adjuvant, drug carrier, or other excipient administered together with the active ingredient. Its selection depends on the intended use and the intended method of administration. The excipient must not be incompatible with the active ingredient, for example, by causing undesirable biological effects or harmful interactions with other components of the pharmaceutical composition. Pharmaceutical compositions may be manufactured by methods well known in the pharmaceutical manufacturing field.

[0293] When referring to RNA sequences, the letter "T" in the sequence can be used interchangeably with "U". When referring to "guide sequences", the letter "T" in the sequence can be used interchangeably with "U". When referring to "co-direction repeat sequences", the letter "T" in the sequence can be used interchangeably with "U".

[0294] For example, as used herein, the term “cosmetics” refers to products applied to any part of the surface of the human body (skin, hair, nails, lips, etc.) by application, spraying, misting, or other similar methods, for the purpose of cleansing, fragrance, altering appearance, correcting, caring for, or maintaining good condition. The aforementioned cosmetics include the various products listed in the “National Standard of the People’s Republic of China - Glossary of Cosmetic Terms” (GB / T 27578-2011).

[0295] In this specification, a “pharmaceutically, cosmetically, chemically, or biologically acceptable” ingredient means an ingredient that does not cause excessive adverse side effects (e.g., toxicity, irritation, or allergic reactions) in humans and / or animals, i.e., has a reasonable benefit-risk ratio. Examples include pharmaceutically acceptable excipients, various cosmetically acceptable excipients, thickeners, or diluents. For example, the cosmetics of this disclosure may contain liquids such as water, saline solution, glycerin, or ethanol. Furthermore, auxiliary substances such as fillers, lubricants, flow aids, wetting agents, or emulsifiers, fragrances, and pH buffers may be present.

[0296] SHRNA

[0297] In some embodiments of this disclosure, the gRNA forms a complex (also referred to as a gene editing complex) with an RNA guide nuclease, which can sequence-specifically bind to and cleave a specific sequence of the target RNA (CTGF RNA, MITF RNA, SRD5A2 RNA, or AR RNA). The gRNA comprises a guide sequence and a backbone sequence. The backbone sequence interacts with the RNA guide nuclease. The backbone sequence is a sequence within the gRNA molecule that is not typically modified during the design of the gRNA molecule. For example, the backbone sequence may refer to a part of the gRNA molecule other than the guide sequence. In some embodiments of this disclosure, the backbone sequence is a codirectional repeat (direct repeat, DR) sequence.

[0298] In some embodiments of this disclosure, the guide sequence and the target RNA have at least 80% sequence identity. In some embodiments of this disclosure, the guide sequence and the target RNA have at least 85%, at least 90%, at least 95%, or 100% sequence identity. Furthermore, in some embodiments of this disclosure, the guide sequence and the target RNA have 100% sequence identity.

[0299] In some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 14, 22, or 35 have at least 80% sequence identity. In some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 14, 22, or 35 have at least 85%, at least 90%, at least 95%, or 100% sequence identity. Furthermore, in some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 14, 22, or 35 have 100% sequence identity.

[0300] In some embodiments of this disclosure, the guide sequence and the nucleotide sequences of, for example, the nucleotides from positions 494 to 785 of the sequence shown in SEQ ID NO: 14, the nucleotides from positions 404 to 606 of the sequence shown in SEQ ID NO: 22, or the nucleotides from positions 484 to 820 of the sequence shown in SEQ ID NO: 35 have at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0301] In some embodiments of this disclosure, the guide sequence and the nucleotide sequences from nucleotide position 494, 604, 678, or 761 of the sequence shown in, for example, SEQ ID NO: 14, to nucleotide position 518, 628, 702, or 785 have at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0302] In some embodiments of this disclosure, the guide sequence and the nucleotide sequences from nucleotides at positions 404, 429, 453, 479, 509, 546, or 582 of the sequence shown in, for example, SEQ ID NO: 22, to nucleotides at positions 428, 453, 477, 503, 533, 570, or 606 have at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0303] In some embodiments of this disclosure, the guide sequence and the nucleotide sequences from nucleotides at positions 484, 524, 547, 585, 635, 721, 755, or 796 of the sequence shown in, for example, SEQ ID NO: 35, up to nucleotides at positions 508, 548, 571, 609, 659, 745, 779, or 820 have at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0304] In some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 5-13, 23-32, 36-57 have at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity. Furthermore, in some embodiments of this disclosure, the guide sequence and the sequence represented by any one of SEQ ID NO: 5-13, 23-32, 36-57 have 100% sequence identity. In some embodiments of this disclosure, the guide sequence includes the sequence represented by any one of SEQ ID NO: 5-13, 23-32, 36-57. In some embodiments of this disclosure, the guide sequence is the sequence represented by any one of SEQ ID NO: 5-13, 23-32, 36-57.

[0305] In some embodiments of this disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of the sequences represented by SEQ ID NO: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, or 54. In some embodiments of this disclosure, the guide sequence includes any one of the sequences represented by SEQ ID NO: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, or 54. In some embodiments of this disclosure, the guide sequence is any one of the sequences represented by SEQ ID NO: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, or 54.

[0306] In some embodiments of the present disclosure, the guide sequence comprises 20 to 40, 20 to 35, 20 to 30, or 25 to 30 nucleotides.

[0307] In some embodiments of the present disclosure, the guide sequence hybridizes with the target RNA, and the mismatch is 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer nucleotides.

[0308] In some embodiments of this disclosure, the identity between the guide sequence and the target RNA sequence is 100%, i.e., fully complementary.

[0309] In some embodiments of the present disclosure, the backbone sequence is a co-directed repeat sequence, which includes a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown, for example, SEQ ID NO: 2 or 3.

[0310] In some embodiments of this disclosure, the co-directed repeat sequence includes, for example, the sequence shown in SEQ ID NO: 2 or 3.

[0311] In some embodiments of the present disclosure, the guide array is located at the 3' end or 5' end of the co-directional repeating array. In some embodiments of the present disclosure, the guide array is located at the 3' end of the co-directional repeating array. In some embodiments of the present disclosure, the guide array is located at the 5' end of the co-directional repeating array.

[0312] In some embodiments of this disclosure, the gRNA includes an aptamer sequence.

[0313] In some embodiments of this disclosure, the aptamer sequence is inserted into the loop of a stem-loop structure in the secondary structure of the co-directed repeat sequence of the gRNA.

[0314] In some embodiments of this disclosure, the gRNA comprises a modified nucleotide. Such modifications include, but are not limited to, a 2'-O-methyl group, a 2'-O-methyl-3'-thiophosphate group, or a 2'-O-methyl-3'-thioPACE modification. In some embodiments of this disclosure, the gRNA comprises a modified nucleotide, which is selected from deoxyribonucleotides and roch nucleic acids (LNAs). In some embodiments of this disclosure, the gRNA comprises at least one chemically modified nucleotide. Chemically modified gRNAs described in Hendel et al., Nat. Biotechnol. 33(9):985-989 (2015) are incorporated herein by reference in their entirety.

[0315] In some embodiments of this disclosure, the gRNA is a hybrid RNA-DNA guide, i.e., some RNA nucleotides in the gRNA are substituted with DNA nucleotides. In some embodiments of this disclosure, the gRNA is a hybrid RNA-LNA (Lock Nucleic Acid) guide, i.e., some RNA nucleotides in the gRNA are substituted with LNA nucleotides. Hybrid RNA-DNA guide polynucleotides are described in WO2016 / 123230, which is incorporated herein by reference in its entirety.

[0316] In some embodiments of this disclosure, the target RNA is located in the cell nucleus and / or cytoplasm of a eukaryotic cell.

[0317] In some embodiments of this disclosure, the gRNA can form a complex (also referred to as a gene editing complex) with an RNA guide nuclease, which can then guide the complex to bind to the target RNA in a sequence-specific manner.

[0318] In some embodiments of the present disclosure, the gRNA can form a complex (also referred to as a gene editing complex) with an RNA guide nuclease, which can then guide the complex to sequence-specifically bind to and cleave the target RNA.

[0319] In some embodiments of this disclosure, the complex reduces the level of the target RNA within the cell.

[0320] In some embodiments of this disclosure, the complex reduces the level of target RNA within a cell after contact with the cell containing the target RNA. For example, it reduces the level of target RNA in a cell expressing the target RNA.

[0321] In some embodiments of this disclosure, the complex reduces the level of the target RNA in cells by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the target RNA level can be measured using methods well known to those skilled in the art, including, but not limited to, the qPCR method described in the examples, such as the RT-qPCR method. The target RNA knockdown level in the test group can be calculated by comparing it to the negative control using untreated cells or cells treated with a gene editing system targeting a non-mammalian genome as a negative control. After editing with the same Cas protein (e.g., by expressing the same Cas protein) and gRNAs containing different guide sequences, the difference in target RNA levels can be compared between the test group and the negative control group. For example, the test group can be edited using C13-2 and the gRNAs disclosed herein, while the negative control group can be edited using C13-2 and, for example, gRNAs that target bacterial genomes. The test group can also be compared with well-known editing tools such as CasRx+gRNA editing tools.

[0322] In some embodiments of the present disclosure, the complex reduces the level of the target RNA in a cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%. In some embodiments of the present disclosure, the complex reduces the level of the target RNA in a cell by at least 40%. In some embodiments of the present disclosure, the complex reduces the level of the target RNA in a cell by at least 80%. In some embodiments of the present disclosure, the complex reduces the level of the target RNA in a cell by at least 85%. In some embodiments of the present disclosure, the complex reduces the level of the target RNA in a cell by at least 90%.

[0323] In some embodiments of the present invention, the number of off-target genes when the complex binds to and cleaves the target RNA is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by methods well known to those skilled in the art. In some embodiments, the number of off-target genes is determined as the intersection (common portion) of the differentially expressed gene set identified by RNA sequencing and the off-target gene set predicted by the program. As a non-limiting example, the EMBOSS-water program can be used to perform predictions on the entire genome and cDNA sequence of a target species (Homo sapiens / Mus musculus), set parameters (e.g., gap_open=10, gap_extend=0.5), perform alignment on the forward and reverse strands of the gRNA guide sequence, and filter the prediction results to obtain potential target genes (including target genes and off-target genes).

[0324] In some embodiments of the present disclosure, the complex reduces the protein levels of the target RNA-encoded protein in cells after contact with cells containing the target RNA. In some embodiments of the present disclosure, the protein encoded by the target RNA is a CTGF protein, a MITF protein, an SRD5A2 protein, or an AR protein. In some embodiments of the present disclosure, the complex reduces the levels of the CTGF protein, MITF protein, an SRD5A2 protein, or an AR protein in cells.

[0325] In some embodiments of this disclosure, the complex reduces intracellular levels of CTGF protein, MITF protein, SRD5A2 protein, or AR protein by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the level of the target RNA-coding protein can be measured using methods well known to those skilled in the art. The knockdown levels of CTGF protein, MITF protein, SRD5A2 protein, or AR protein in a test group can be calculated by comparing them to a negative control, using untreated cells or cells treated with gene editing systems targeting non-mammalian genomes, including but not limited to ELISA, Western blotting, etc., as a negative control.

[0326] In some embodiments of this disclosure, the RNA guide nuclease is a Cas protein. In some embodiments, the cleavage activity of the RNA guide nuclease is completely or partially lost.

[0327] In some embodiments of this disclosure, the RNA guide nuclease is a Cas protein.

[0328] In some embodiments of this disclosure, the RNA guide nuclease is a Cas protein, and the gRNA comprises a guide sequence and a codirectional repeat sequence.

[0329] In some embodiments of this disclosure, the Cas protein is a Cas9 protein, a Cas12 protein, or a Cas13 protein.

[0330] In some embodiments of this disclosure, the Cas9 protein is selected from SpCas9, SaCas9, Nme2Cas9, Nme3Cas9, CjCas9, NmCas9, and FnCas9.

[0331] In some embodiments of this disclosure, the Cas12 protein is selected from Cas12a protein, Cas12b protein, Cas12c protein, Cas12d protein, Cas12e protein, Cas12f protein, Cas12g protein, Cas12h protein, Cas12i protein, Cas12j protein, Cas12k protein, Cas12l protein, and Cas12m protein.

[0332] In some embodiments of this disclosure, the Cas protein is the Cas13 protein.

[0333] In some embodiments of this disclosure, the Cas protein is Cas13a, Cas13b, Cas13c, or Cas13d. Also in some embodiments of this disclosure, the Cas protein is Cas13a, Cas13b, or Cas13c, in which the RxxxxH motif present in the catalytic center of the two HEPN domains is mutated, and these Cas proteins have completely or partially lost cleavage activity. In some embodiments, the Cas protein is a CasRx protein. In some embodiments, the Cas protein is a dCasRx having mutations in both of the two HEPN domains (R239A, H244A in HEPN-1 and R858A, H863A in HEPN-2).

[0334] In some embodiments of this disclosure, the Cas protein includes, for example, the sequence shown in SEQ ID NO: 1.

[0335] In some embodiments of the present disclosure, the guide array is located at the 3' end or 5' end of the co-directional repeating array. In some embodiments of the present disclosure, the guide array is located at the 3' end of the co-directional repeating array. In some embodiments of the present disclosure, the guide array is located at the 5' end of the co-directional repeating array.

[0336] In some embodiments of this disclosure, the co-directed repeating sequence includes, for example, the sequence shown in SEQ ID NO: 2.

[0337] target RNA

[0338] The gene editing systems and combinations described herein can be used to target one or more target RNA molecules, for example, target RNA molecules present in a biological sample. In some embodiments of this disclosure, the target RNA is pre-mRNA or mRNA (mature mRNA).

[0339] In some embodiments of this disclosure, the target RNA is CTGF RNA, MITF RNA, or SRD5A2 RNA or a fragment thereof. In some embodiments of this disclosure, the target RNA is CTGF pre-mRNA, MITF pre-mRNA, or SRD5A2 pre-mRNA, CTGF mRNA, MITF mRNA, or SRD5A2 mRNA, or a fragment thereof. In some embodiments of this disclosure, the target RNA is CTGF mRNA, MITF mRNA, or SRD5A2 mRNA, or a fragment thereof. In some embodiments of this disclosure, the target RNA is CTGF pre-mRNA, MITF pre-mRNA, or SRD5A2 pre-mRNA, or a fragment thereof. In some embodiments of this disclosure, the target RNA is CTGF mRNA, MITF mRNA, or SRD5A2 mRNA. In some embodiments of this disclosure, the target RNA is mammalian CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of this disclosure, the target RNA is human CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of this disclosure, the target RNA is human CTGF mRNA, MITF mRNA, or SRD5A2 mRNA.

[0340] In some embodiments of this disclosure, the target RNA sequence is the sequence shown in SEQ ID NO: 14, 22, or 35.

[0341] In some embodiments of this disclosure, the target RNA sequence is, for example, the nucleotides from position 494 to 785 of the sequence shown in SEQ ID NO: 14, the nucleotides from position 404 to 606 of the sequence shown in SEQ ID NO: 22, or the nucleotides from position 484 to 820 of the sequence shown in SEQ ID NO: 35.

[0342] In some embodiments of this disclosure, the target RNA sequence is a nucleotide sequence from nucleotides at positions 494, 604, 678, or 761 to nucleotides at positions 518, 628, 702, or 785 of the sequence shown in, for example, SEQ ID NO: 14 (human CCN2 mRNA, NCBI NM_001901.4).

[0343] In some embodiments of this disclosure, the target RNA sequence is a nucleotide sequence from nucleotides at positions 404, 429, 453, 479, 509, 546, or 582 of the sequence shown in, for example, SEQ ID NO: 22 (human MITF mRNA, NCBI NM_001354607.2) to nucleotides at positions 428, 453, 477, 503, 533, 570, or 606.

[0344] In some embodiments of this disclosure, the target RNA sequence is a nucleotide sequence from nucleotides at positions 484, 524, 547, 585, 635, 721, 755, or 796 of the sequence shown in SEQ ID NO: 35 (human SRD5A2 mRNA, NCBI XM_011533072.3) to nucleotides at positions 508, 548, 571, 609, 659, 745, 779, or 820.

[0345] Where this specification refers to transcripts of CTGF mRNA, MITF mRNA, or SRD5A2 mRNA as target nucleic acids (e.g., NCBI accessions NM_001901.4, NM_001354607.2, XM_011533072.3), the gRNAs of this disclosure are not intended to be limited to those nucleic acid molecules only. Those skilled in the art will understand that the gRNAs of this disclosure may target other nucleic acid molecules, such as human CTGF pre-mRNA, MITF pre-mRNA, or SRD5A2 pre-mRNA, or other transcripts of CTGF mRNA, MITF mRNA, or SRD5A2 mRNA.

[0346] In some embodiments of this disclosure, the expression level of CTGF RNA, MITF RNA, or SRD5A2 RNA is reduced by using the gene editing system of this disclosure. For example, an RNA-guided nuclease is used to bind to and cleave CTGF RNA, MITF RNA, or SRD5A2 RNA under the guidance of gRNA. In some embodiments of this disclosure, the expression level of intracellular CTGF RNA, MITF RNA, or SRD5A2 RNA can be reduced using the gene editing system of this disclosure.

[0347] In some embodiments of this disclosure, the target RNA is AR RNA. In some embodiments of this disclosure, the target RNA is human AR RNA. In some embodiments of this disclosure, the target RNA is human mRNA.

[0348] In some embodiments of this disclosure, the target RNA is an AR RNA or a fragment thereof.

[0349] In some embodiments of this disclosure, the target RNA is AR mRNA or a fragment thereof.

[0350] In some embodiments of this disclosure, the target RNA is an AR pre-mRNA or a fragment thereof.

[0351] In some embodiments of this disclosure, the target RNA is selected from the sequences represented by SEQ ID NO: 394-396.

[0352] In some embodiments of this disclosure, the expression level of AR RNA can be reduced by using the gene editing system of this disclosure. For example, by using an RNA guided nuclease to contact and cleave the AR RNA under the guidance of gRNA. In some embodiments of this disclosure, the expression level of AR RNA in cells can be reduced by using the gene editing system of this disclosure.

[0353] Aptamer / Aptamer Sequence

[0354] In some embodiments of the present disclosure, the guide polynucleotide further comprises an aptamer sequence. In some embodiments of the present disclosure, the aptamer sequence is inserted into a loop of the guide polynucleotide. In some embodiments of the present disclosure, the aptamer sequence is bonded to the end of the guide polynucleotide. In some embodiments of the present disclosure, the aptamer sequence comprises an MS2 aptamer sequence, a PP7 aptamer sequence, or a Qβ aptamer sequence.

[0355] Linker protein (Adaptor protein)

[0356] In some embodiments of the present disclosure, the gene editing system further comprises a linker protein and a fusion protein including a fusion domain, or a nucleic acid encoding the fusion protein, wherein the linker protein can bind to an aptamer sequence.

[0357] In some embodiments of the present disclosure, the linker protein comprises an MS2 phage coat protein (MCP), a PP7 phage coat protein (PCP), or a Qβ phage coat protein (QCP). In some embodiments of the present disclosure, the fusion domain comprises a cytosine deaminase domain, an adenosine deaminase domain, a translation activation domain, a translation inhibition domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, or an affinity tag or reporter tag domain.

[0358] RNA guide nuclease

[0359] In some embodiments of this disclosure, when the term “RNA guide nuclease” is used, it may refer in a narrow sense to the RNA guide nuclease itself, or to a fusion protein obtained by covalently bonding or fusing an RNA guide nuclease with another domain.

[0360] In some embodiments of this disclosure, the RNA guide nuclease is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12f / CasZ, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, C sx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, Cas13f, TnpB, IscB, IsrB, Fancor, or fragments thereof (such as nucleic acid-binding domain fragments as non-limiting examples) may be selected.

[0361] In some embodiments of this disclosure, the RNA guide nuclease may be Cas9, Cas12, Cas13, TnpB, IscB, IsrB, Fancor nuclease, or fragments thereof; however, it may be arbitrarily selected from nucleic acid binding domain fragments including these.

[0362] In some embodiments of this disclosure, the RNA guide nuclease is a Cas protein.

[0363] In some embodiments of this disclosure, the RNA guide nuclease is the Cas13 protein.

[0364] In some embodiments of the present disclosure, the RNA guide nuclease may be selected from SpCas9, SaCas9, Nme2Cas9, Nme3Cas9, CjCas9, NmCas9, FnCas9, PpnCas9, FrCas9, SauCas9, SauriCas9, ScaCas9, St1Cas9, BlatCas9, CdiCas9, GeoCas9, their fragments, and their variants or fragments of variants.

[0365] In some embodiments of this disclosure, the RNA guide nuclease is AsCpf1, enAsCas12a (addgene plasmid #196724), dFnCas12a (addgene plasmid #136379), ErCas12a, LbCas12a D832A, LbCas12a H759A, LbCas12a E795L, FnCas12a3, FnCas12a D917A, AsCas12a R1226A, AsCas12a D908A, AsCas12a E174R / S542R, AsCas12a (S542R / K548V / N552R), PrCas12a, PxCas12a, PcCas12a, PdCas12a, Mb2Cas12a, Mb3Cas12a, MlCas12a, CMaCas12 a, CMtCas12a, HkCas12a, Lb5Cas12a, ErCas12a, TsCas12a, FnCpf1, LbCas12a, ttHsCas12a, AaCas12b, AaCas12b D570A, AaCas12b Q119F / E475R / E758R, BhCas12b, BvCas12b, BrCas12b, AkCas12b, AmCas12b, BsCas12b, OspCas12c, Cas12c2(addgene plasmid #183072), Cas12c_4(addgene plasmid #183071), Cas12c1(addgene plasmid #120872), CasY.1 (from Katanobacteria), CasY.2 (from Vogelbacteria), CasY.3 (from Vogelbacteria), CasY.4 (from Parcubacteria), CasY.5 (from Komeilibacteria), CasY.6 (from Kerfeldbacteria), PlmCasX, DpbCasX, Un1Cas12f, CnCas12f1, enRhCas12f1, AsCas12f1, SpaCas12f1, Cas12g1 (addgene plasmid #120879), Cas12h from WO2021113522A1 (SEQ ID NO: 1), Cas12i1 (addgene plasmid #171670), Cas12i2 (addgene plasmid#188275)、Cas12i1(adjacent plasmid #120882)、Cas12i2(adjacent plasmid #120883) while CN111757889B is Cas12f.4 / Cas12f.5 / Cas12f.6 ofC as12i dSiCas12i(D1049A), SiCas12i, Si2Cas12i, WiCas1 2i , Wi2Cas12i , Wi3Cas12i , SaCas12i , Sa2Cas12i , Sa3Cas12i , WaCas12i , Wa2Cas12i , xCas12i , hfCas12Max , Cas12i - Max ( addgene plasmid #188276)、Cas12i1 D647A(addgene plasmid #171671)、Cas12i-HiFi(addgene plasmid #188269)、Cas12i1 D647A、Cas12j3(addgene plasmid #188497)、Cas12j2(addgene plasmid #188498); #181787); C552A)、AcCas12n、dAcCas12n(D240)、TnpB Actinomadura_cellulosilytica_strain_DSM_45823、TnpB Actinomadura_namibiensis_strain_DSM_44197、TnpB Actinomadura_umbrina_strain_DSM_43927_$、TnpB Actinoplanes_lobatus_strain_DSM_43150 (TnpB-1 and TnpB-2)、TnpB Alicyclobacillus_macrosporagiidus_strain_DSM_17980、TnpBHaloactinospora_alba_Strain_DSM_45015, TnpB Lipingzhangella_halophila_strain_DSM_102030, TnpB Meiothermus_Silvanus_DSM_9946, TnpB QNFX01000004, ISDra2 TnpB (PDB: 8H1J), KraIscB-1, AwaIscB, OgeuIscB, GtFz1 (derived from Guillardia theta), SpuFz1 (derived from Spizellomyces punctatus), NlovFz2 (derived from Percolozoa Naegleria lovaniensis), MmeFz2 (derived from Mercenaria mercenaria), their fragments, and their variants or fragments of variants may be selected.

[0366] In some embodiments of this disclosure, the RNA guide nuclease is selected from wild-type RNA guide nucleases (including, but not limited to, CasRx, C13-2, etc.), their variants (including, but not limited to, variants with complete loss of cleavage activity, variants with partial loss of cleavage activity, variants with improved cleavage activity, variants with reduced off-target / off-target effects), or functional fragments or fusion proteins thereof.

[0367] In some embodiments of this disclosure, the RNA guide nuclease comprises one or more fusion domains of any of the following: an intracellular localization signal, a deaminase domain, a translation activation domain, a translation inhibition domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0368] In some embodiments of this disclosure, the RNA guide nuclease includes an intracellular localization signal.

[0369] In some embodiments of this disclosure, the RNA guide nuclease comprises an intracellular localization signal and a deaminase domain.

[0370] In some embodiments of this disclosure, the intracellular localization signal may be selected from nuclear localization signals and nuclear export signals.

[0371] In some embodiments of this disclosure, the RNA guide nuclease is fused with at least one intracellular localization signal. Exemplary intracellular localization signals include, for example, organelle localization signals such as nuclear localization signals (NLS), nuclear export signals (NES), or mitochondrial localization signals.

[0372] In some embodiments of this disclosure, the RNA guide nuclease is fused with at least one heterologous NLS. In some embodiments of this disclosure, the RNA guide nuclease is fused with at least two NLSs. In some embodiments of this disclosure, the RNA guide nuclease is fused with at least three NLSs. In some embodiments of this disclosure, the RNA guide nuclease is fused with at least one N-terminal NLS and at least one C-terminal NLS. In some embodiments of this disclosure, the guide nuclease is fused with at least two C-terminal NLSs. In some embodiments of this disclosure, the RNA guide nuclease is fused with at least two N-terminal NLSs.

[0373] In some embodiments, the NLS is SPKKKRKVEAS (SEQ ID NO: 397), GPKKKRKVAAA (SEQ ID NO: 398), PKKKRKV (SEQ ID NO: 399), KRPAATKKAGQAKKKK (SEQ ID NO: 400), PAAKRVKLD (SEQ ID NO: 401), RQRRNELKRSP (SEQ ID NO: 402), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 403), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 404), VSRKRPRP (SEQ ID NO: 405), PPKKARED (SEQ ID NO: 406), POPKKKPL (SEQ ID NO: 407), SALIKKKKKMAP (SEQ ID NO: It is selected independently from 408), DRLRR (SEQ ID NO: 409), PKQKKRK (SEQ ID NO: 410), RKLKKKIKKL (SEQ ID NO: 389), REKKKFLKRR (SEQ ID NO: 392), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 379), and RKCLQAGMNLEARKTKK (SEQ ID NO: 386).

[0374] In some embodiments of this disclosure, the RNA guide nuclease is fused with a heterogeneous NES. In some embodiments of this disclosure, the RNA guide nuclease is fused with at least two NESs. In some embodiments of this disclosure, the RNA guide nuclease is fused with at least three NESs. In some embodiments of this disclosure, the RNA guide nuclease is fused with at least one N-terminal NES and at least one C-terminal NES. In some embodiments of this disclosure, the RNA guide nuclease is fused with at least two C-terminal NESs. In some embodiments of this disclosure, the RNA guide nuclease is fused with at least two N-terminal NESs.

[0375] In some embodiments of this disclosure, the NES is independently selected from adenovirus type 5 E1B NES, HIV Rev NES, MAPK NES, or PTK2 NES.

[0376] In some embodiments of this disclosure, the RNA guide nuclease is fused with an NLS and a NES, and a cleavable linker may be present between the NLS and the NES. In some embodiments of this disclosure, in the production cell system, the NES may facilitate the encapsulation of the RNA guide nuclease into a delivery particle (e.g., a virus-like particle (VLP)). In some embodiments of this disclosure, the NLS may be exposed by cleavage of the linker in the target cell, thereby promoting the nuclear localization of the RNA guide nuclease. Exemplary fusion domains (e.g., fused heterologous protein domains) include RNA cleavage domains (e.g., PIN endonuclease domain, NYN domain, SOT1-derived SMR domain, or Staphylococcus Domains that affect RNA stability (e.g., tristetraprolin (TTP) or UPF1, EXOSC5 and STAU1-derived domains), domains that edit nucleotides or ribonucleotides (e.g., cytosine deaminase, PPR protein, adenosine deaminase, ADAR family proteins or APOBEC family proteins), translation activation domains (e.g., eIF4E and other translation initiation factors, yeast poly(A) binding protein or GLD2-derived domains), translation inhibition domains (e.g., Pumilio or FBF PUF protein, deadenylationase, CAF1, Argonaute protein), RNA methylation domains (e.g., m6A methyltransferase factor (METTL14, METTL3 or WTAP, etc.)-derived domains), RNA demethylation domains (e.g., human alkylation repair homolog 5), domains that affect splicing (e.g., SRSF1 RS-rich domain, hnRNP Examples include the glycy-rich domain of A1, the alanine-rich motif of RBM4, or the proline-rich motif of DAZAP1; tags that enable affinity purification or immunoprecipitation (e.g., FLAG, HA, biotin, or HALO tags); and domains that enable proximity-dependent protein labeling and identification (e.g., biotin ligase (BirA, etc.) or peroxidase (APEX2, etc.)), thereby enabling biotinylation of target DNA-interacting proteins.

[0377] In some embodiments of this disclosure, the deaminase domain comprises an adenosine deaminase domain. In some embodiments of this disclosure, an RNA guide nuclease (including, but not limited to, a Cas13 protein having a mutant HEPN domain or a Cas13 protein with deactivated catalytic activity) that is maintained, partially inactivated, or completely inactivated in nuclease activity is covalently bonded or fused with an adenosine deaminase domain to guide A-to-I deaminase activity in RNA transcripts within mammalian cells. Cox et al., Science 358(6366):1019-1027(2017) describes an ADAR2-based adenosine deaminase domain targeting A-to-I RNA editing, which is incorporated herein by reference. In some other embodiments, the adenosine deaminase domain is covalently bonded or fused to an adapter protein capable of binding to an aptamer sequence inserted or added within the guide polynucleotide, thereby enabling the adenosine deaminase domain to non-covalently bind to an RNA guide nuclease that forms a complex with the guide polynucleotide.

[0378] In some embodiments of this disclosure, the deaminase domain comprises a cytosine deaminase domain. In some embodiments, an RNA guide nuclease (including, but not limited to, a Cas13 protein having a mutated HEPN domain or a Cas13 protein with deactivated catalytic activity) that is maintained, partially inactivated, or completely inactivated in nuclease activity is covalently bonded or fused to the cytosine deaminase domain to guide C-to-U deaminase activity in RNA transcripts within mammalian cells. Abudayyeh et al., Science 365(6451):382-386 (2019) describes an ADAR2-based cytosine deaminase domain targeting C-to-U RNA editing, which is incorporated herein by reference. In some other embodiments, the cytosine deaminase domain is covalently bonded or fused to an adapter protein capable of binding to an aptamer sequence inserted or added within the guide polynucleotide, thereby enabling the cytosine deaminase domain to non-covalently bind to an RNA guide nuclease that forms a complex with the guide polynucleotide.

[0379] In some embodiments of this disclosure, the fusion domain comprises a splicing factor domain. In some embodiments, a Cas13 protein having a mutated HEPN domain, or a Cas13 protein with inactivated catalytic activity, is covalently bound or fused to the splicing factor domain to guide alternative splicing of RNA transcripts in mammalian cells. The splicing factor domain includes, but is not limited to, the RS-rich domain of SRSF1, the glycine-rich domain of hnRNPA1, the alanine-rich motif of RBM4, or the proline-rich motif of DAZAP1. In some other embodiments, the splicing factor domain is covalently bound or fused to an adapter protein capable of binding to an aptamer sequence inserted or added within the gRNA, thereby enabling the splicing factor domain to non-covalently bind to a Cas13 protein that forms a complex with the gRNA.

[0380] In some embodiments of this disclosure, an RNA guide nuclease is covalently bound to or fused to a translation activation domain. In some embodiments, an RNA guide nuclease that retains nuclease activity, is partially inactivated, or is completely inactivated (including, but not limited to, a Cas13 protein with mutant HEPN or a Cas13 protein with deactivated catalytic activity) is covalently bound to or fused to a translation activation domain to activate or increase the expression of a target RNA. The translation activation domain includes, but is not limited to, eIF4E and other translation initiation factors, a yeast poly(A) binding protein, or a GLD2 domain. In some other embodiments, the translation activation domain is covalently bound to or fused to an adapter protein capable of binding to an aptamer sequence inserted or added to the guide polynucleotide, thereby enabling the translation activation domain to non-covalently bind to an RNA guide nuclease that forms a complex with the guide polynucleotide.

[0381] In some embodiments of this disclosure, an RNA guide nuclease is covalently bound to or fused to a translational repression domain. In some embodiments, an RNA guide nuclease that is active, partially inactivated, or completely inactivated (including, but not limited to, a Cas13 protein having a mutant HEPN domain, or a Cas13 protein with deactivated catalytic activity) is covalently bound to or fused to a translational repression domain to inhibit or reduce the expression of a target RNA. Translational repression domains include, but are not limited to, Pumilio or FBF PUF proteins, deadenosylase, CAF1, and Argonaute proteins. In other embodiments, the translational repression domain is covalently bound to or fused to an adapter protein capable of binding to an aptamer sequence inserted or added to the guide polynucleotide, thereby enabling the translational repression domain to non-covalently bind to an RNA guide nuclease that forms a complex with the guide polynucleotide.

[0382] In some embodiments of this disclosure, an RNA guide nuclease is covalently bound to or fused to an RNA methylation domain. In some embodiments, an RNA guide nuclease that is active, partially inactivated, or completely inactivated (including, but not limited to, a Cas13 protein having a mutant HEPN domain, or a Cas13 protein with deactivated catalytic activity) is covalently bound to or fused to an RNA methylation domain to methylate a target RNA. The RNA methylation domain includes, but is not limited to, an m6A domain such as METTL14, METTL3, or WTAP. In other embodiments, the RNA methylation domain is covalently bound to or fused to an adapter protein capable of binding to an aptamer sequence inserted or added to the guide polynucleotide, thereby enabling the RNA methylation domain to non-covalently bind to an RNA guide nuclease that forms a complex with the guide polynucleotide.

[0383] In some embodiments of this disclosure, an RNA guide nuclease is covalently bound to or fused to an RNA demethylation domain. In some embodiments, an RNA guide nuclease that is active, partially inactivated, or completely inactivated (including, but not limited to, a Cas13 protein having a mutant HEPN domain, or a Cas13 protein with deactivated catalytic activity) is covalently bound to or fused to an RNA demethylation domain to demethylate the target RNA. The RNA methylation domain includes, but is not limited to, human alkylation repair homolog 5 (ALKBH5). In other embodiments, the RNA demethylation domain is covalently bound to or fused to an adapter protein capable of binding to an aptamer sequence inserted or added to the guide polynucleotide, thereby enabling the RNA demethylation domain to non-covalently bind to an RNA guide nuclease that forms a complex with the guide polynucleotide.

[0384] In some embodiments of this disclosure, an RNA guide nuclease is covalently bound to or fused to a ribonuclease domain. In some embodiments, the nuclease is covalently bound to or fused to a ribonuclease domain, but retains nuclease activity, is partially inactivated, or is completely inactivated (including, but not limited to, a Cas13 protein having a mutant HEPN domain, or a Cas13 protein with inactivated catalytic activity), and cleaves target RNA. The ribonuclease domain includes, but is not limited to, a PIN endonuclease domain, a NYN domain, an SMR domain derived from SOT1, or an RNase domain derived from a Staphylococcus nuclease.

[0385] In some embodiments of this disclosure, the RNA guide nuclease is covalently bound to or fused to an affinity tag, affinity domain, reporter tag, or reporter domain. In some embodiments, the RNA guide nuclease is covalently bound to or fused to a reporter domain, such as a fluorescent protein. Reporter domains include, but are not limited to, GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, and BFP. In some embodiments, the RNA guide nuclease is covalently bound to or fused to an affinity tag, such as a purification tag. Affinity tags include, but are not limited to, HA tags, His tags (e.g., 6-His), Myc tags, E tags, S tags, calmodulin tags, FLAG tags, GST tags, MBP tags, Halo tags, or biotin.

[0386] In some embodiments of this disclosure, the RNA guide nuclease may be covalently linked to the fusion domain with or without a linker sequence. That is, they may be directly covalently linked or covalently linked via a linker sequence. Typically, the linker sequence consists of 1 to 100, 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids.

[0387] In some embodiments of this disclosure, affinity tags and affinity domains can be used interchangeably, and reporter tags and reporter domains can also be used interchangeably.

[0388] In some embodiments of this disclosure, the gRNA can form a complex with the RNA guide nuclease and guide the complex to sequence-specifically bind to a target RNA.

[0389] In some embodiments of this disclosure, the gRNA can form a complex with the RNA guide nuclease and guide the complex to sequence-specifically bind to a target RNA.

[0390] Cas protein

[0391] In some embodiments of this disclosure, the Cas protein is Cas9 protein, Cas12 protein, or Cas13 protein. In some embodiments, the Cas protein is Cas12a protein, Cas12b protein, Cas12c protein, Cas12d protein, Cas12e protein, Cas12f protein, Cas12g protein, Cas12h protein, Cas12i protein, Cas12j protein, or Cas12k protein.

[0392] In some embodiments of this disclosure, the Cas protein may be selected from SpCas9, SaCas9, Nme2Cas9, Nme3Cas9, CjCas9, NmCas9, FnCas9, PpnCas9, FrCas9, SauCas9, SauriCas9, ScaCas9, St1Cas9, BlatCas9, CdiCas9, GeoCas9, their fragments, and their variants or fragments of variants. The Cas12 protein may be selected from AsCpf1, LbCas12a, AkCas12b, AacCas12b, PlmCasX, DpbCasX, Cas12i1, Cas12i2, Cas12i3, and Casφ-2.

[0393] In some embodiments of this disclosure, the Cas protein is AsCpf1, enAsCas12a (addgene plasmid #196724), dFnCas12a (addgene plasmid #136379), ErCas12a, LbCas12a D832A, LbCas12a H759A, LbCas12a E795L, FnCas12a3, FnCas12a D917A, AsCas12a R1226A, AsCas12a D908A, AsCas12a E174R / S542R, AsCas12a (S542R / K548V / N552R), PrCas12a, PxCas12a, PcCas12a, PdCas12a, Mb2Cas12a, Mb3Cas12a, MlCas12a, CMaCas12 a, CMtCas12a, HkCas12a, Lb5Cas12a, ErCas12a, TsCas12a, FnCpf1, LbCas12a, ttHsCas12a, AaCas12b, AaCas12b D570A, AaCas12b Q119F / E475R / E758R, BhCas12b, BvCas12b, BrCas12b, AkCas12b, AmCas12b, BsCas12b, OspCas12c, Cas12c2(addgene plasmid #183072), Cas12c_4(addgene plasmid #183071), Cas12c1(addgene plasmid #120872), CasY.1 (from Katanobacteria), CasY.2 (from Vogelbacteria), CasY.3 (from Vogelbacteria), CasY.4 (from Parcubacteria), CasY.5 (from Komeilibacteria), CasY.6 (from Kerfeldbacteria), PlmCasX, DpbCasX, Un1Cas12f, CnCas12f1, enRhCas12f1, AsCas12f1, SpaCas12f1, Cas12g1 (addgene plasmid #120879), Cas12h of WO2021113522A1, Cas12i1 (addgene plasmid #171670), Cas12i2 (addgene plasmid #188275), Cas12i1 (addgene plasmid#120882), Cas12i2 (addgene plasmid #120883); the following Cas12i proteins are named Cas12f.4 / Cas12f.5 / Cas12f.6 in CN111757889B; dSiCas12i (D1049A), SiCas12i, Si2Cas12i, WiCas12i, Wi2Cas12i, Wi3Cas12i, SaCas12i, Sa2Cas12i, Sa3Cas12i, WaCas12i, Wa2Cas12i, xCas12i, hfCas12Max, Cas12i-Max (addgene plasmid #188276), Cas12i1 D647A (addgene plasmid #171671), Cas12i-HiFi (addgene plasmid #188269), Cas12i1 D647A, Cas12j3(addgene plasmid #188497), Cas12j2(addgene plasmid #188498), AsCas12j-2(addgene plasmid #191655), Cas12j-8(addgene plasmid #194966), ShCas12k, N7Cas12k, AcCas12k, Cas12k-TniQ(addgene plasmid #181787), Cas12k-TnsC(addgene plasmid #181789), Cas12l, MmCas12m, MmCas12m ΔZF (H549A, C552A), dCas12m-ΔZF (D485A, H549A, C552A), AcCas12n, dAcCas12n(D240), TnpB Actinomadura_cellulosilytica_strain_DSM_45823, TnpB Actinomadura_namibiensis_strain_DSM_44197, TnpB Actinomadura_umbrina_strain_DSM_43927_$, TnpB Actinoplanes_lobatus_strain_DSM_43150 (TnpB-1 and TnpB-2), TnpB Alicyclobacillus_macrosporagidus_strain_DSM_17980, TnpBHaloactinospora_alba_Strain_DSM_45015, TnpB Lipingzhangella_halophila_strain_DSM_102030, TnpB Meiothermus_Silvanus_DSM_9946, TnpB QNFX01000004, ISDra2 TnpB (PDB: 8H1J), KraIscB-1, AwaIscB, OgeuIscB, GtFz1 (derived from Guillardia theta), SpuFz1 (derived from Spizellomyces punctatus), NlovFz2 (derived from Percolozoa Naegleria lovaniensis), MmeFz2 (derived from Mercenaria mercenaria), their fragments, and their variants or fragments of variants may be selected.

[0394] In some embodiments of this disclosure, the Cas protein is Cas13a, Cas13b, Cas13c, or Cas13d. In some embodiments of this disclosure, the Cas protein is Cas13a, Cas13b, Cas13c, or Cas13d having mutations in both HEPN domains, and these Cas proteins have completely or partially lost cleavage activity. For example: LwaCas13a, LsCas13a, LbuCas13a, dLbuCas13a(R472A / H477A / R1048A / H1053A), TccCas13a, LneCas13a(LneC2c2), LbmCas13a, LbnCas13a, PpCas13a, LbfCas13a, CgCas13a, Cg2Cas13a, PspCas13b, PspCas13b H133A / H1058A, PbuCas13b, PgiCas13b, BzCas13b, RanCas13b, PguCas13b, dPguCas13b(H151A / H1121A), Cas13bt1, Examples include Cas13bt3, CcaCas13b, MisCas13b, Hgm4Cas13b, Pba4Cas13b, Bba2Cas13b, CasRx, dCasRx(R239A / H244A / R858A / H863A), CasRx_N2V8(A134V,A140V, A141V,A143V), RspCas13d, and C13-2.

[0395] In some embodiments of this disclosure, the Cas13 protein is the Cas13d protein.

[0396] In some embodiments of the present disclosure, the Cas13 protein and the CasRx or C13-2 protein have sequence identity of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0397] In some embodiments of this disclosure, the Cas13 protein is a CasRx protein. In some embodiments, the Cas13 protein is a dCasRx having mutations in both of its two HEPN domains (R239A, H244A in HEPN-1 and R858A, H863A in HEPN-2).

[0398] In some embodiments of this disclosure, the Cas13 protein comprises the sequence shown in SEQ ID NO: 1.

[0399] In some embodiments of this disclosure, the amino acid sequence of the Cas13 protein and the sequence shown in SEQ ID NO: 1 have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0400] In some embodiments of this disclosure, the Cas13 protein is the C13-2 protein. In some embodiments, the Cas13 protein is the dead C13-2 protein.

[0401] In some embodiments of this disclosure, the Cas13 protein contains one, two, three, four, five, or six mutations at the corresponding positions of amino acid residues R210, H215, R750, H755, R785, and / or H790 of the reference protein shown in SEQ ID NO: 1(C13-2). In some embodiments, the Cas13 protein has a mutation to A (alanine) at the corresponding positions of amino acid residues R210, H215, R750, H755, R785, and / or H790 of the reference protein shown in SEQ ID NO: 1.

[0402] In some embodiments of this disclosure, the Cas13 protein contains mutations at the corresponding positions of amino acid residues R210 and H215 of the reference protein shown in SEQ ID NO: 1. In some embodiments, the Cas13 protein contains mutations at the corresponding positions of amino acid residues R750 and H755 of the reference protein shown in SEQ ID NO: 1. In some embodiments, the Cas13 protein contains mutations at the corresponding positions of amino acid residues R785 and H790 of the reference protein shown in SEQ ID NO: 1.

[0403] In some embodiments of this disclosure, the Cas13 protein contains mutations at the corresponding positions of amino acid residues R210, H215, R750, and H755 of the reference protein shown in SEQ ID NO: 1.

[0404] In some embodiments of this disclosure, the Cas13 protein contains mutations at the corresponding positions of amino acid residues R750, H755, R785, and H790 of the reference protein shown in SEQ ID NO: 1.

[0405] In some embodiments of this disclosure, the Cas13 protein contains mutations at the corresponding positions of amino acid residues R210, H215, R785 and / or H790 of the reference protein shown in SEQ ID NO: 1.

[0406] In some embodiments of this disclosure, the Cas13 protein contains mutations at the corresponding positions of amino acid residues R210, H215, R750, H755, R785, and H790 of the reference protein shown in SEQ ID NO: 1.

[0407] In some embodiments of this disclosure, the corresponding positions of R210, R750, or R785 are mutated to A. In some embodiments, the corresponding positions of H215, H755, or H790 are mutated to A. In some embodiments of this disclosure, the corresponding positions of R210, H215, R750, H755, R785, and H790 are all mutated to A.

[0408] In some embodiments of this disclosure, the Cas13 protein is obtained by introducing mutations into the RxxxxH motif at positions 210-215, the RxxxxH motif at positions 750-755, and / or the RxxxxH motif at positions 785-790 of the sequence shown in SEQ ID NO: 1.

[0409] In some embodiments of this disclosure, the Cas13 protein is obtained by introducing one, two, three, four, five, or six mutations at the R210, H215, R750, H755, R785, and / or H790 positions of the sequence shown in SEQ ID NO: 1. In some embodiments, the Cas13 protein is obtained by mutating the R210, H215, R750, H755, R785, and / or H790 positions of the sequence shown in SEQ ID NO: 1 to A (alanine).

[0410] In some embodiments of this disclosure, the Cas13 protein is obtained by mutating the positions of R210, H215, R785 and H790 in the sequence shown in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by mutating the positions of R210, H215, R750 and H755 in the sequence shown in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by mutating the positions of R750, H755, R785 and H790 in the sequence shown in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by shifting the positions of R210, H215, R750, H755, R785 and H790 in the sequence shown in SEQ ID NO: 1 to A.

[0411] In some embodiments of this disclosure, the Cas13 protein contains any one or more mutations at the corresponding positions of the following amino acid residues of the reference protein shown in SEQ ID NO: 1, compared to the reference protein shown in SEQ ID NO: 1: R11, N34, R35, R47, R58, R63, R64, N68, N87, N265, N274, R276, R290, R294, N299, N303, R308, R314, R320, R328, N332, R341, N346, R358, N372, N383, N390, N394, R47+R290, R47+R314, R290+R314, R47+R290+R314 , R308+N68, N394+N68, N87+N68, R308+N265, N394+N265, N87+N265, R308+N68+N265, N87+N68+N265, T7, A16, S260, A263, M266, N274, F2 88, M302, N303, L304, V305, I311, D313, H324, P326, H327, N332, N346, T353, T360, E365, A373, M380, S382, K395, Y396, D402, D411, S418.

[0412] In some embodiments of this disclosure, the Cas13 protein is obtained by introducing one or more mutations of any kind at the following positions in the sequence shown in SEQ ID NO: 1: R11, N34, R35, R47, R58, R63, R64, N68, N87, N265, N274, R276, R290, R294, N299, N303, R308, R314, R320, R328, N332, R341, N346, R358, N372, N383, N390, N394, R47+R290, R47+R314, R290+R314, R47+R290+R314, R30 8+N68, N394+N68, N87+N68, R308+N265, N394+N265, N87+N265, R308+N68+N265, N87+N68+N265, T7, A16, S260, A263, M266, N274, F288 , M302, N303, L304, V305, I311, D313, H324, P326, H327, N332, N346, T353, T360, E365, A373, M380, S382, K395, Y396, D402, D411, S418.

[0413] In some embodiments of the present disclosure, the C13-2 protein, C13-2 protein analogs, C13-2 protein variants, and their functional fragments or fusion proteins form complexes with co-directional repeat sequences, including, for example, the sequence shown in SEQ ID NO: 2 or 3 of the present disclosure, and as a result, can specifically bind to the target RNA of the present disclosure under the guidance of said sequence.

[0414] In some embodiments of this disclosure, the Cas protein comprises any one or more of the following fusion domains: an intracellular localization signal, a deaminase domain, a translation activation domain, a translation inhibition domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0415] In some embodiments of this disclosure, the Cas protein includes an intracellular localization signal.

[0416] In some embodiments of this disclosure, the Cas protein comprises an intracellular localization signal and a deaminase domain.

[0417] In some embodiments of this disclosure, the intracellular localization signal may be selected from nuclear localization signals and nuclear export signals.

[0418] In some embodiments of this disclosure, the Cas protein may be covalently linked with or without a linker sequence. That is, they may be directly covalently linked or covalently linked via a linker sequence. Typically, the linker sequence consists of 1 to 100, 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids.

[0419] In some embodiments of this disclosure, the gRNA can form a CRISPR complex with the Cas protein and guide the CRISPR complex to sequence-specifically bind to a target RNA.

[0420] In some embodiments of this disclosure, a gRNA can form a CRISPR complex with a Cas protein, and the CRISPR complex can guide sequence-specific binding and cleavage of a target RNA.

[0421] Nucleotide sequences encoding gRNA or RNA guide nucleases

[0422] In some embodiments of this disclosure, the nucleotide sequence encoding the RNA guide nuclease is a plasmid. In some embodiments of this disclosure, the nucleotide sequence encoding the RNA guide nuclease is part of a viral vector genome, such as the DNA genome of an AAV vector flanked by ITRs. In some embodiments of this disclosure, the nucleotide sequence encoding the RNA guide nuclease is mRNA.

[0423] In some embodiments of this disclosure, the nucleotide sequence encoding the gRNA or RNA guide nuclease is codon-optimized.

[0424] In some embodiments of this disclosure, the nucleotide sequence encoding the gRNA is DNA. In some embodiments, the DNA sequence is codon-optimized.

[0425] In some embodiments of this disclosure, the nucleotide sequence encoding the RNA guide nuclease is DNA or mRNA. In some embodiments, the DNA sequence is codon-optimized. In some embodiments, the mRNA sequence is codon-optimized.

[0426] In some embodiments of this disclosure, codon optimization is performed for the purpose of expression in a desired cell type. In some embodiments, codon optimization is performed for the purpose of expression in a eukaryotic cell environment. In some embodiments, codon optimization is performed for the purpose of expression in a mammalian cell environment. In some embodiments, codon optimization is performed for the purpose of expression in a human cell environment.

[0427] Typically, codon optimization may involve modifying a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon in the original sequence (e.g., around 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more) with a codon that is more frequently used in the host cell and maintains the original amino acid sequence. Codon optimization computational procedures or algorithms are publicly known, and codon optimization can be performed using tools or algorithms such as ExpOptim, Codon OptimWiz, NGTM Codon, Codon optimization, Synthetic Gene Designer, and DNAWorks.

[0428] vector

[0429] Vectors may include any type of nucleotide, such as DNA or RNA, but are not limited to these. Nucleotides may be single-stranded or double-stranded, and may include naturally occurring, unnatural, or modified nucleotides. Suitable vectors include expression vectors (e.g., plasmids or viruses).

[0430] In some embodiments of this disclosure, the recombinant vector may include a control sequence. The control sequence may include, for example, transcription and translation start codons, stop codons, etc. These control sequences may have specificity depending on the type of host cell into which the vector is introduced (e.g., bacteria, fungi, plants, or animals).

[0431] In some embodiments of this disclosure, the recombinant vector may optionally include gene vector elements (nucleic acids), such as a selective labeling region, a lac operator, a CMV promoter, a CAG promoter, a tac promoter, a T7 RNA polymerase promoter, an SP6 RNA polymerase promoter, an SV40 promoter, an IRES sequence, a WPRE element, an ITR sequence, a FLAG tag coding region, a c-myc tag coding region, a polyHis tag coding region, an HA tag coding region, an MBP tag coding region, a GST tag coding region, a ployA coding region, an SV40 polyadenylation signal, an SV40 origin of replication, a Col E1 origin of replication, a loxP site, or a Cre recombinase coding region.

[0432] In some embodiments of this disclosure, the coding sequence in the vector is codon-optimized to be expressed in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., mammalian cells, human cells).

[0433] control array

[0434] In some embodiments of the present disclosure, the control sequence includes one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof.

[0435] In some embodiments of this disclosure, the control sequence is a U6 promoter or an eye-specific promoter.

[0436] In some embodiments of this disclosure, the control sequence is a CBh promoter. In some embodiments, the CBh promoter includes a CMV enhancer sequence. In some embodiments, the CBh promoter includes a chicken β-actin promoter. In some embodiments, the CBh promoter includes a hybrid intron.

[0437] In some embodiments of the present disclosure, the control array includes HRE enhancer elements (hypoxia response elements). In some embodiments, the control array includes at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine HRE enhancer elements arranged in tandem. In some embodiments, the control array includes two, three, four, five, six, seven, eight, or nine HRE enhancer elements arranged in tandem.

[0438] In some embodiments of this disclosure, the regulatory sequence includes a neuron restrictive silencer (NRS) derived from the human synapsin gene. In some embodiments, the regulatory sequence includes an NRS and an HRE enhancer. In some embodiments, the regulatory sequence includes a tandem arrangement of the NRS and HRE enhancer. To enhance the regulatory effect, the tandem arrangement of the NRS and HRE enhancer may be present multiple times. In some embodiments, the regulatory sequence includes a tandem arrangement of the NRS and HRE enhancer that is repeated at least two, three, four, five, or six times consecutively. In some embodiments, the regulatory sequence includes a tandem arrangement of the NRS and HRE enhancer that is repeated two, three, four, five, or six times consecutively.

[0439] promoter

[0440] In some embodiments of the present disclosure, the vector comprises a pol III promoter (e.g., U6 and H1 promoters), a pol II promoter (e.g., an LTR promoter for Rous sarcoma virus (RSV), which is a reverse transcription virus (optionally including an RSV enhancer), a cytomegalovirus (CMV) promoter (optionally including a CMV enhancer), an SV40 promoter, a dihydrofolate reductase promoter, a β-actin promoter, a phosphoglycerate kinase (PGK) promoter, an EF1α promoter), or a pol III promoter and a pol II promoter.

[0441] In some embodiments of this disclosure, the promoter is a constitutive promoter, constitutively active and not regulated by external signals or molecules. Not limited examples of suitable constitutive promoters include the CMV, RSV, SV40, EF1α, CAG, and β-actin promoters. In some embodiments of this disclosure, the promoter is an inducible promoter, regulated by external signals or molecules (e.g., transcription factors).

[0442] In some embodiments of this disclosure, the promoter is a tissue-specific promoter used to drive tissue-specific expression of the Cas13 protein. Suitable muscle-specific promoters include, but are not limited to, CK8, MHCK7, myosin promoter (Mb), desmin promoter, muscle creatine kinase promoter (MCK) and its variants, and SPc5-12 synthesis promoter. Suitable immune cell-specific promoters include, but are not limited to, the B29 promoter (B cells), CD14 promoter (mononuclear cells), CD43 promoter (white cells and platelets), CD68 (macrophages), and SV40 / CD43 promoter (white cells and platelets). Suitable hematopoietic cell-specific promoters include, but are not limited to, the CD43 promoter (white cells and platelets), CD45 promoter (hematopoietic cells), INF-β (hematopoietic cells), WASP promoter (hematopoietic cells), SV40 / CD43 promoter (white cells and platelets), and SV40 / CD45 promoter (hematopoietic cells). Appropriate pancreas-specific promoters include, but are not limited to, the elastase-1 promoter. Appropriate endothelial cell-specific promoters include, but are not limited to, the Fit-1 promoter and the ICAM-2 promoter. Appropriate nerve tissue / cell-specific promoters include, but are not limited to, the GFAP promoter (astrocytes), the SYN1 promoter (neurons), and the NSE / RU5' promoter (mature neurons). Appropriate nerve tissue / cell-specific promoters include the GFAP promoter and the SYN1 promoter. Appropriate kidney-specific promoters include, but are not limited to, the NphsI promoter (podocytes). Appropriate bone-specific promoters include, but are not limited to, the OG-2 promoter (osteoblasts, odontoblasts). Appropriate lung-specific promoters include, but are not limited to, the SP-B promoter (lung). Appropriate liver-specific promoters include, but are not limited to, the SV40 / Alb promoter. Appropriate cardiac-specific promoters include, but are not limited to, α-MHC.

[0443] In some embodiments of this disclosure, the promoter is an eye-specific promoter.

[0444] In some embodiments of this disclosure, the eye-specific promoter may be selected from a retinal split protein promoter, a K12 promoter, a rhodopsin promoter, a rod cell-specific promoter, a cone cell-specific promoter, a rhodopsin kinase promoter, a GRK1 promoter, an interphotoreceptor retinoid-binding protein proximal (IRBP) promoter, and an opsin promoter (e.g., a red opsin promoter, a blue opsin promoter, etc.).

[0445] In some embodiments of this disclosure, the promoter is a chicken β-actin protein (CB) promoter. The chicken β-actin protein promoter is either a short chicken β-actin protein promoter or a long chicken β-actin protein promoter. In some embodiments of this disclosure, the promoter (e.g., chicken β-actin protein promoter) includes an enhancer sequence, such as a cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence can be either a short CMV enhancer sequence or a long CMV enhancer sequence. In some embodiments of this disclosure, the promoter includes a long CMV enhancer sequence and a long chicken β-actin protein promoter. In some embodiments of this disclosure, the promoter includes a short CMV enhancer sequence and a short chicken β-actin protein promoter. However, those skilled in the art will understand that a short CMV enhancer can be used with a long CB promoter, and that a long CMV enhancer can be used with a short CB promoter. In some embodiments of this disclosure, the promoter is a CBh promoter.

[0446] Enhancer

[0447] In some embodiments of the present disclosure, the enhancer is selected from WPRE, CMV enhancer, SV40 enhancer, and intron sequences between exons 2 and 3 of rabbit β-globulin.

[0448] In some embodiments of this disclosure, the enhancer may be located upstream of the promoter element. Alternatively, the enhancer may be located downstream of or within the coding sequence controlled by the promoter and may retain its function. Therefore, the enhancer or a portion thereof may be included in the transcript (RNA sequence) of the coding sequence.

[0449] In some embodiments of this disclosure, the enhancer may be located upstream or downstream of the coding sequence controlled by the promoter within a range of 100 bp, 200 bp, 300 bp, 400 bp, 500 bp or more.

[0450] In some embodiments of this disclosure, the enhancer enhances the expression of the coding sequence to a level higher than the enhanced expression provided by the promoter.

[0451] Vector System

[0452] In some embodiments of this disclosure, the RNA guide nuclease or the polynucleotide sequence encoding the gRNA is optimized for expression in eukaryotic cells.

[0453] In some embodiments of this disclosure, the RNA guide nuclease or the polynucleotide sequence encoding the gRNA is optimized for expression in mammalian cells.

[0454] In some embodiments of this disclosure, the RNA guide nuclease or the polynucleotide sequence encoding the gRNA is optimized for expression in human cells.

[0455] In some embodiments of this disclosure, the RNA guide nuclease or the polynucleotide sequence encoding the gRNA is optimized for expression in prokaryotic cells.

[0456] In some embodiments of this disclosure, the RNA guide nuclease or the polynucleotide sequence encoding the gRNA is optimized for expression in bacterial cells.

[0457] In some embodiments of this disclosure, the RNA guide nuclease or the nucleic acid molecule encoding the gRNA is a plasmid. In some embodiments of this disclosure, the RNA guide nuclease or the nucleic acid molecule encoding the gRNA is part of a viral vector genome, a non-limiting example being the DNA genome of an AAV vector flanked by ITRs. In some embodiments of this disclosure, the RNA guide nuclease or the nucleic acid molecule encoding the gRNA is mRNA.

[0458] Adeno-associated virus (AAV) vector (AAV vector)

[0459] The delivery of the CRISPR-Cas system via AAV vectors is described in Maeder et al., Nature Medicine 25:229-233 (2019), which is incorporated herein by reference in its entirety. In some embodiments of this disclosure, the AAV vector comprises an ssDNA genome containing an RNA guide nuclease and a sequence encoding a gRNA flanked by an ITR.

[0460] In some embodiments of this disclosure, the gRNA or gene editing system described herein is packaged within the capsid of an AAV vector, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, AAV PHP.B2, AAV PHP.B3, AAV PHP.A, AAV PHP.eB, AAV PHP.eS, AAV2.7m8, AAV8.7m8, AAV ShH10, AAVrh10, or AAVrh74.

[0461] In some embodiments of this disclosure, the gRNA or gene editing system described herein is packaged within the capsid of AAV2, AAV5, AAV6, AAV8, AAV9, or AAV PHP.eB.

[0462] In some embodiments of this disclosure, the AAV vectors described herein may be selected from AAV2 / 2, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2 / 10, AAV2 / 11, AAV2 / 12, AAV2 / 13, AAV2 / PHP.B, AAV2 / PHP.B2, AAV2 / PHP.B3, AAV2 / PHP.A, AAV2 / PHP.eB, AAV2 / PHP.eS, AAV2 / 2.7m8, AAV2 / 8.7m8, AAV2 / ShH10, AAV2 / rh10, and AAV2 / rh74.

[0463] In some embodiments of this disclosure, the AAV vectors described herein may be selected from AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 8, AAV2 / 9, and AAV2 / PHP.eB.

[0464] In some embodiments of this disclosure, the gene editing system described herein is packaged in an AAV vector, the AAV vector comprising a tissue-specific modified capsid, for example, a modified ocular tissue-specific capsid.

[0465] Lipid nanoparticles (LNPs)

[0466] The full text of Gillmore et al., N. Engl. J. Med., 385:493-502 (2021), describing LNP delivery in the CRISPR-Cas system, is incorporated herein by reference. In some embodiments of this disclosure, the lipid nanoparticles (LNPs) comprise four components in addition to the RNA payload (Cas13 mRNA and gRNA): cationic or ionizable lipids, cholesterol, cofactor lipids, and PEG lipids. In some embodiments of this disclosure, the cationic or ionizable lipids include cKK-E12, C12-200, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA, FTT5, Moderna SM-102, and Intellia LP01. In some embodiments of this disclosure, the PEG lipids include PEG-2000-C-DMG, PEG-2000-DMG, or ALC-0159. In some embodiments of this disclosure, the auxiliary lipid includes DSPC. The components of LNP are described in Paunovska et al., Nature Reviews Genetics 23:265-280 (2022), which is incorporated herein by reference in its entirety.

[0467] lentiviral vectors

[0468] In some embodiments of the present disclosure, the lentiviral vector may be pseudotyped with an homogeneous or heterogeneous envelope protein, such as VSV-G. In some embodiments of the present disclosure, mRNA encoding an RNA guide nuclease is ligated to the aptamer sequence.

[0469] RNP complex (ribonucleoprotein complex (RNP))

[0470] In some embodiments of this disclosure, RNP complexes (ribonucleoprotein complexes (RNPs)) can be delivered to eukaryotic cells, mammalian cells, or human cells by microinjection or electroporation. In some embodiments of this disclosure, the ribonucleoprotein complexes (RNPs) can be packaged in virus-like particles (VLPs) and delivered in vivo to mammalian or human subjects.

[0471] Virus-like particles (VLPs)

[0472] In some embodiments of this disclosure, modified virus-like particles (VLPs) can be pseudotyped with homogeneous or heterogeneous envelope proteins, such as VSV-G. In some embodiments of this disclosure, the RNA guide nuclease is fused to a gag protein (e.g., MLVgag) via a cleavable linker, and when the linker is cleaved in a target cell, an NLS located between the linker and the RNA guide nuclease is exposed. In some embodiments of this disclosure, the fusion protein comprises a gag protein (e.g., MLVgag) (e.g., in the 5' to 3' direction), one or more NESs, a cleavable linker, one or more NLSs, and an RNA guide nuclease. In some embodiments of this disclosure, the RNA guide nuclease is fused to a second dimerization domain fused to a membrane protein and to a first dimerization domain capable of dimerization or heterodimerization, and dimerization is promoted in the presence of a ligand, enriching the RNA guide nuclease or fusion protein in the VLP.

[0473] cell

[0474] The cells of this disclosure may be isolated cells. The cells of this disclosure (e.g., cells used to generate cell-free systems) may be eukaryotic or prokaryotic cells. Non-limiting examples of such cells include bacteria, archaea, plants, fungi, yeasts, insects, and mammalian cells. Specific examples include Lactobacillus, Lactococcus, Bacillus (e.g., Bacillus subtilis), Escherichia (e.g., Escherichia coli), Clostridium, Saccharomyces, Pichia (e.g., Saccharomyces cerevisiae or Pichia pastoris), Kluyveromyces lactis, Salmonella typhimurium, Drosophila cells, Caenorhabditis elegans cells, Xenopus laevis cells, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian cell lines (e.g., HeLa cells, bone marrow-derived cell lines, and lymphoid cell lines).

[0475] In some embodiments of this disclosure, the cells are prokaryotic cells, such as bacterial cells (e.g., Escherichia coli). In some embodiments of this disclosure, the cells are eukaryotic cells, such as mammalian cells or human cells. In some embodiments of this disclosure, the cells may be primary eukaryotic cells, stem cells, tumor / cancer cells, circulating tumor cells (CTCs), blood cells (e.g., T cells, B cells, NK cells, Tregs, etc.), hematopoietic stem cells, specialized immune cells (e.g., tumor-infiltrating lymphocytes or tumor-suppressor lymphocytes, etc.), or stromal cells in the tumor microenvironment (e.g., cancer-associated fibroblasts, etc.). In some embodiments of this disclosure, the cells may be brain cells or nerve cells of the central or peripheral nervous system (e.g., neurons, astrocytes, microglia, retinal ganglion cells, rod / cone cells, etc.).

[0476] Diseases or conditions related to target RNA

[0477] In some embodiments of this disclosure, the target RNA-related disease or condition refers to a disease or condition caused by the abnormal high expression of a target RNA such as CTGF RNA, MITF RNA, SRD5A2 RNA, or AR RNA.

[0478] The gene editing systems that target CTGF RNA, MITF RNA, SRD5A2 RNA, or AR RNA according to this disclosure can effectively knock down CTGF RNA, MITF RNA, SRD5A2 RNA, or AR RNA. Therefore, these gene editing systems can be used for the prevention, treatment, or diagnosis of these diseases.

[0479] In some embodiments of this disclosure, the pharmaceutical composition is administered in vivo to a human subject. The pharmaceutical composition can be administered via any effective route of administration to deliver a therapeutically effective amount of the pharmaceutical composition to a subject. Examples of routes of administration, but not limited to, include, intravenous infusion, intravenous injection, intraperitoneal injection, intramuscular injection, intratumor injection, subcutaneous injection, intradermal injection, intracardiac injection, intravascular injection, intracerebellar injection, intraocular injection, subretinal injection, intravitreous injection, anterior chamber injection, intratympanic injection, intranasal injection, and inhalation.

[0480] In some embodiments of the present disclosure, the gene editing system or pharmaceutical composition of the present disclosure can be delivered to a subject in need using an appropriate delivery method to achieve scar repair, skin whitening, reduction or elimination of melasma, prevention or treatment of melanoma, and / or reduction of androgen receptor expression, ultimately leading to the prevention or treatment of male pattern baldness.

[0481] In some embodiments of this disclosure, MITF expression can be reduced by using the inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells, and / or pharmaceutical compositions of this disclosure, thereby reducing melanin expression, reducing or eliminating melasma, achieving a skin whitening effect, and / or treating melanoma. The reference Yi X, et al. MITF-siRNA formulation is a safe and effective therapy for human melasma[J]. Molecular Therapy, 2011, 19(2): 362-371. describes MITF-siRNA formulations as a safe and effective therapy for human melasma, and the full text is incorporated herein by reference.

[0482] In some embodiments of this disclosure, the expression of SRD5A2 can be reduced by using the inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells, and / or pharmaceutical compositions of this disclosure, thereby preventing or treating hair loss due to male pattern baldness. The reference Khantham C, et al. Antioxidation, Anti-Inflammation, and Regulation of SRD5A Gene Expression of Oryza sativa cv. Bue Bang 3 CMU Husk and Bran Extracts as Androgenetic Alopecia Molecular Treatment Substances. Plants. 2022; 11(3):330. https: / / doi.org / 10.3390 / plants11030330 describes how plant extracts can reduce SRD5A2 expression in AGA, and the full text is incorporated herein by reference.

[0483] cosmetics

[0484] In some embodiments of the present disclosure, the cosmetic can be used for scar repair, skin whitening, reduction or removal of melasma, prevention or treatment of melanoma, and / or prevention or treatment of male pattern baldness.

[0485] In some embodiments of this disclosure, the expression of CTGF can be reduced by using the inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells, and / or pharmaceutical compositions of this disclosure, thereby suppressing excessive collagen expression during wound healing, inhibiting excessive fibrosis of wounds leading to scar formation, and ultimately achieving the objectives of scar repair and / or skin rejuvenation. The reference Cho KH, et al. Local delivery of CTGF siRNA with poly (sorbitol-co-PEI) reduces scar contraction in cutaneous wound healing[J]. Tissue Engineering and Regenerative Medicine, 2017, 14(3): 211-220 describes CTGF-targeted siRNA that reduces scar contraction during cutaneous wound healing, and the full text is incorporated herein by reference.

[0486] In some embodiments of this disclosure, the use of the inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells, and / or pharmaceutical compositions of this disclosure reduces MITF expression, reduces melanin expression, reduces or eliminates melasma, provides a whitening effect, and / or treats melanoma. The reference Yi X, et al. MITF-siRNA formulation is a safe and effective therapy for human melasma[J]. Molecular Therapy, 2011, 19(2): 362-371. describes MITF-targeted siRNA for the treatment of melasma, and its full text is incorporated herein by reference.

[0487] In some embodiments of this disclosure, the expression of SRD5A2 can be reduced by using the inhibitors, gRNAs, nucleic acids, vectors, vector systems, adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs), lentiviral vectors, ribonucleoprotein complexes (RNPs), virus-like particles (VLPs), eukaryotic cells, and / or pharmaceutical compositions of this disclosure, thereby preventing or treating hair loss due to male pattern baldness. The reference Khantham C, et al. Antioxidation, Anti-Inflammation, and Regulation of SRD5A Gene Expression of Oryza sativa cv. Bue Bang 3 CMU Husk and Bran Extracts as Androgenetic Alopecia Molecular Treatment Substances. Plants. 2022; 11(3):330. https: / / doi.org / 10.3390 / plants11030330 describes a method by which plant extracts suppress SRD5A2 expression in AGA, and the full text is incorporated herein by reference.

[0488] Based on well-known knowledge in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain various preferred embodiments of the present invention.

[0489] The present invention is further illustrated by the following examples, but is not limited to the scope of the examples described below. Test methods not specifically described in the following examples were carried out according to conventional methods and conditions or in accordance with the product instructions. [Examples]

[0490] In the following examples, a newly discovered highly active Cas13 protein, namely C13-2 (also known as CasRfg.4), was used to verify the effectiveness of targeting each target RNA.

[0491] Amino acid sequence of C13-2 protein (SEQ ID NO: 1): MSKDKKTKAKRMGVKALLAHGEDKLTMTTFGKGNRSKIEFTEGYHGRALETPKHFGIRGFEVRRIDENVDLCGDLEEGKTIEALLVNPSEKVGEDYLKLKGTLEKRFFGREFPHDNIRIQLIYNILDIYKILGMNVADILYALGNMQDTELDIDMFGQSLNNEDNLKECLKRMRPYMGYFGDIFKISPKGENIADREHNKKVLRCISVLRNATAHDKQDEYPWFKSSDIYETKIFKADMWKIIKDQYREKIKKVNKDFLSKNAVNMAILFDLLNARDVEQKKQITDEFYRFTIRKDGKNLGMNLVKIREIIIDRYASGLRDKKHDPHRQKINVIADFLIFRALSQNQGIIDKTVSSLRLTKDEEEKDHVYQNAAELVWGMVSNCLTPYFNDPKNKYILKYKDAKTPGDFEDWITSKISEDDGEPFVKVLSFLCNFLEGKEINELLT AYIHKFECIQDFLNVISSLGENVQFQPRFALFNNASFAQNVAVQLRILASIGKMKPDLTEAKRPLYKAAIRMLCPPEKWEKYTSDEWLEKNMLLNSEDRKNDKKKKQVNPFRNFIAGNVIESRRRFMYLVRYSKPKAVRAIMQNRSIVNYVLHRLPSEQVHRYASVFPENFADLEQEIDFLTKKLFEFFSFEELLHEKDVILNNSRSHKPSLEIERLKAIGLYL SVAYIAIKNIVKANARYYIAFAVFERDKELVKAKDARIQTKIPETDFPDYFCLTQYYLDRDEEKKFPGDPRDKEAFFEHLRKTKRHFSKQWREWLNEKIADAKSSQATGLLLEARNDVEHLNVLRAIPDYIQDFRHGEKGETAMNSYFELYHYLMQRLMLKNTELDLSHWSGWIMRSGRPDRLIQIAFVSLAYNLPYRNLTKEHHFDDTVLQKIREKESLD

[0492] C13-2 corresponding DR(SEQ ID NO: 2) sequence: GGAAGATAACTCTACAAACCTGTAGGGTTCTGAGAC.

[0493] The applicant further screened and obtained another DR sequence that showed good efficacy, also known as DR-hf2 (SEQ ID NO: 3). Its sequence is as follows: GGAAGATAACTCTACAAACCTGTAGAGTTCTGAGAC.

[0494] Example 1. Verification of the editing efficiency of the endogenous CTGF gene.

[0495] 1. Construction of an editing vector targeting the endogenous CTGF gene. The C13-2-BsaI plasmid (SEQ ID NO: 4) containing a general-purpose crRNA backbone expression cassette was synthesized by an external contractor.

[0496] We designed gRNAs targeting human CCN2 mRNA (NCBI NM_001901.4, SEQ ID NO: 14). For example, these are shown in Table 1 and Figure 1. As shown in Table 1 and Figure 1, we designed gRNAs targeting human CCN2 mRNA (NCBI NM_001901.4, SEQ ID NO: 14). [Table 2]

[0497] Sense and antisense strands of the DNA sequence were synthesized. The sense strand was a sequence obtained by adding "agac" to the 5' end of the guide sequence shown in Table 1, and the antisense strand was a sequence obtained by adding "aaaa" to the 5' end of the reverse complementary sequence of the same guide sequence. Both strands were annealed to obtain a double-stranded DNA fragment with sticky ends corresponding to the target site of CTGF RNA.

[0498] A primer annealing reaction system is shown in Table 2 below, for example. The primers were incubated in a PCR instrument at 95°C for 5 minutes, then immediately removed and incubated on ice for 5 minutes to allow the primers to anneal to each other and form double-stranded DNA with sticky ends.

[0499] [Table 3]

[0500] After digestion with restriction enzyme Bsa I using the synthesized C13-2-BsaI plasmid, the annealed product and the purified and recovered backbone were ligated with T4. After transformation into E. coli, positive clones were selected, and the target plasmid, i.e., the validation vector (expressing gRNA targeting C13-2 protein and CTGF RNA), was extracted and used for subsequent tests.

[0501] 2. Transfection of validation vector into 293T cells We used a 293T cell line (293T-CTGF cells) that highly expresses CTGF.

[0502] Cell line construction: A vector, Lv-CTGF-T2a-GFP (SEQ ID NO: 15), was constructed to overexpress the CTGF and EGFP genes. Here, CTGF and EGFP were linked by a 2A peptide. The Lv-CTGF-T2a-GFP plasmid was packaged as a lentivirus and introduced into 293T cells to create a cell line that stably overexpresses the CTGF gene.

[0503] 293T-CTGF cells were transfected with validation vectors having different target sites. The negative control group was transfected with the C13-2-BsaI vector. Transfection was performed in 24-well plates according to the manufacturer's instructions for Lipofectamine 2000 (Thermo).

[0504] 3, qPCR analysis Intracellular RNA was extracted from 293T-CTGF cells 72 hours after transfection using the SteadyPure Universal RNA Extraction Kit (AG21017), and the RNA concentration was measured using a micro-spectrophotometer. The RNA products were reverse transcribed using the Evo M-MLV Mix Kit with gDNA Clean for qPCR, and the resulting cDNA was subjected to qPCR using the SYBR Green Premix Pro Taq HS qPCR Kit, etc.

[0505] The primers used for qPCR were as follows: CTGF detection (primer my): GCGTGTGCACCGCCAAAGAT (SEQ ID NO: 16) AACGTCCATGCTGCACAGGG(SEQ ID NO: 17). CTGF detection (primer 11): CAGCATGGACGTTCGTCTG (SEQ ID NO: 18) AACCACGGTTTGGTCCTTGG(SEQ ID NO: 19). Internal control GAPDH detection: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 20) GAAGGGGTCATTGATGGCAAC(SEQ ID NO: 21).

[0506] The reaction system was prepared according to the instructions for the SYBR® Green Premix Pro Taq HS qPCR Kit, and QuantStudio TM 5. Detection was performed using a Real-Time PCR System.

[0507] In this study, the relative quantitative method is 2^ -ΔΔCt The target RNA level was calculated using a specific method. An example of the calculation method is shown below: △Ct = Ct (CTGF) - Ct (GAPDH) △△Ct = △Ct(Sample to be tested, e.g., C13-2-CTGF-g2) - △Ct(C13-2-BsaI) 2^ -ΔΔCt =2^(-△△Ct)

[0508] According to the calculation method above, CTGF 2^ -ΔΔCt The values ​​were calculated. The test was repeated multiple times, and the average value of the results was calculated.

[0509] As shown in Figures 2 and 3, Figure 2 shows the detection results using the CTGF-my primer, and Figure 3 shows the detection results using the CTGF-11 primer.

[0510] qPCR results showed that the combination of Cas13 protein with g6, g7, g8, and g10 gRNAs significantly improved CTGF RNA editing efficiency and effectively suppressed CTGF expression by a statistically significant difference (P<0.0001). The editing efficiency was in the order of g8 > g10 > g6 > g7. The combination of Cas13 protein with g2, g3, and g4 gRNAs resulted in very low CTGF RNA editing efficiency or failure to knock down CTGF RNA.

[0511] Example 2. Verification of the editing efficiency of the endogenous MITF gene. The editing efficiency was verified using the same method as in Example 1.

[0512] 1. Construction of an editing vector targeting the endogenous MITF gene. We designed gRNAs targeting human MITF mRNA (NCBI NM_001354607.2, SEQ ID NO: 22). These are shown, for example, in Table 3 and Figure 4. [Table 4]

[0513] Fragments targeting the MITF RNA target site were obtained by primer annealing in the same manner as in Example 1.

[0514] After digestion with restriction enzyme Bsa I using the C13-2-BsaI plasmid, the annealed product and the purified and recovered backbone were ligated with T4. After transformation into E. coli, positive clones were selected, and the target plasmid, i.e., the validation vector (expressing gRNA targeting C13-2 protein and MITF RNA), was extracted and used for subsequent tests.

[0515] 2. Transfection of 293T cells using a validation vector. 293T cells were transfected using validation vectors with different target sites. The negative control group was transfected with the C13-2-BsaI vector.

[0516] Transfection was performed in a 24-well plate according to the manufacturer's instructions for Lipofectamine 2000 (Thermo).

[0517] 3, qPCR analysis Detection by qPCR was performed 72 hours after transfection using the same method as in Example 1. A separate 293T blank control group was also established: MITF RNA levels in untreated 293T cells (not transfected with plasmids) were detected by qPCR.

[0518] The primers used for qPCR are as follows: MITF detection: GCCTCCAAGCCTCCGATAAG (SEQ ID NO: 33) GCACTCTCTGTTGCATGAACT(SEQ ID NO: 34). Internal standard GAPDH detection: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 20) GAAGGGGTCATTGATGGCAAC(SEQ ID NO: 21).

[0519] According to the calculation method above, 2^ -ΔΔCtThe edited target RNA level was calculated using a specific method. The experiment was repeated multiple times, and the average value was calculated. See Figure 5, for example.

[0520] qPCR results showed that combinations of Cas13 protein and g1, g2, g3, g4, g5, g6, and g7 gRNAs efficiently edited MITF RNA and effectively suppressed MITF expression by a statistically significant difference (P<0.0001). The editing efficiency of the Cas13 protein combination with g2, g3, g4, g6, and g7 gRNAs exceeded 90%, the editing efficiency of the g1 and g5 gRNA combinations was 80% and 71%, respectively, and the editing efficiency of the g8, g9, and g10 gRNA combination was less than 35%.

[0521] Example 3. Verification of the editing efficiency of the endogenous SRD5A2 gene.

[0522] The editing efficiency was verified using the same method as in Example 1.

[0523] 1. Construction of editing vectors and control vectors targeting the endogenous CTGF gene. We designed gRNAs targeting human SRD5A2 mRNA (NCBI XM_011533072.3, SEQ ID NO: 35). These are shown, for example, in Table 4 and Figure 6.

[0524] [Table 5]

[0525] Fragments targeting the SRD5A2 RNA target site were obtained by primer annealing using the same method as in Example 1.

[0526] After digesting the C13-2-BsaI plasmid with the restriction enzyme Bsa I, the annealed product and the replicated and recovered backbone were ligated with T4. After transformation into E. coli, positive clones were selected, and the target plasmid, i.e., the validation vector (expressing gRNA targeting C13-2 protein and SRD5A2 RNA), was extracted and used for subsequent testing.

[0527] Control vectors expressing shRNA-1 and shRNA-2, which target SRD5A2 RNA, were also constructed. For example, these are shown in Table 5.

[0528] [Table 6]

[0529] shRNA fragments targeting the SRD5A2 target site are prepared using primer annealing, and the primers used are shown in Table 6, for example.

[0530] [Table 7]

[0531] The pAAV-CMV-EGFP (SEQ ID NO: 58), the backbone of the shRNA expression vector, was synthesized by an external contractor. The backbone was double-digested with restriction enzymes Bsa I and Not I, and the annealed product, along with the purified and recovered backbone, was ligated with T4. After transformation into E. coli, positive clones were selected, and the target plasmid was extracted. This plasmid was able to express shRNA-1 and shRNA-2. It was then used in subsequent tests.

[0532] 2. Transfection of 293T cells using a validation vector. Construction of a 293T cell line (293T-SRD5A2) overexpressing SRD5A2: A vector, Lv-SRD5A2-T2a-GFP (described in SEQ ID NO 65), was constructed to overexpress the SRD5A2 gene and the EGFP gene. Here, SRD5A2 and EGFP were linked by a 2A peptide. The Lv-SRD5A2-T2a-GFP plasmid was packaged as a lentivirus and introduced into 293T cells to create a 293T-SRD5A2 cell line that stably overexpresses SRD5A2.

[0533] 293T-SRD5A2 cells were transfected with validation vectors and shRNA control vectors having different target sites. The negative control group was transfected with the C13-2-BsaI vector.

[0534] Transfection was performed in a 24-well plate according to the instructions from the Lipofectamine 2000 manufacturer.

[0535] 3, qPCR analysis Detection by qPCR was performed 72 hours after transfection using the same method as in Example 1. A separate 293T-NC control group was also established: SRD5A2 RNA levels were detected by qPCR in untreated 293T cells (which had high SRD5A2 expression levels and had not been transfected with gene editing plasmids or shRNA plasmids).

[0536] The primers used for qPCR are as follows: SRD5A2 detection (primer 2, P2): ACTGCTCAATCGAGGGAGG (SEQ ID NO: 66) CACCCAAGCTAAACCGTATGTC(SEQ ID NO: 67). SRD5A2 detection (primer 3, P3): CGGTTTAGCTTGGGTGTCTTC (SEQ ID NO: 68) CCGAGGAAATTGGCTCCAGAA(SEQ ID NO: 69). Internal standard GAPDH detection: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 20) GAAGGGGTCATTGATGGCAAC(SEQ ID NO: 21).

[0537] The edited target RNA level is 2^ -ΔΔCt The results were calculated using the specified method. C13-2-BsaI was used as a negative control group, with its level set to 1.00. The test was repeated multiple times, and the average value of the results was calculated. See, for example, Figures 7 and 8. Figure 7 shows the detection results using primer 2 (P2). Figure 8 shows the detection results using primer 3 (P3).

[0538] In Figure 7, all gRNA test groups effectively suppressed target RNA expression by a statistically significant difference (P<0.01) compared to the negative control group. The editing efficiency (>90%) of the h1, h7, h9, and h11 gRNA test groups was higher than that of shRNA-1 and shRNA-2. The h9 and h11 gRNA test groups showed a statistically significant difference (P<0.05) compared to shRNA-1.

[0539] In Figure 8, the h3, h5, h8, h9, and h19 gRNA test groups all effectively suppressed target RNA expression compared to the negative control group, showing a statistically significant difference (P<0.01). The editing efficiency of the h3, h5, h8, and h9 gRNA test groups was higher than that of shRNA-1. The editing efficiency of the h19 gRNA test group was 95%, which was higher than that of shRNA-1 and shRNA-2.

[0540] Example 4. The editing efficiency of the gRNA of the present invention was verified using CTGF targeting other Cas13 molecules. The editing efficiency was verified using the same method as described in the previous example.

[0541] The CasRx-BsaI plasmid was synthesized by an external contractor. Its sequence is shown, for example, in SEQ ID NO: 70. Various CasRx-CTGF gRNA plasmids were constructed using guide sequence-related primer annealing, BsaI digestion, and T4 ligation, enabling the expression of gRNAs targeting CasRx and CTGF. Transfection was performed on 293T cell lines that highly express CTGF, and knockdown efficiency was detected using qPCR.

[0542] The results are shown in Figures 9 and 10. The combination of CasRx with CTGF-g6, g7, g8, and g10 gRNAs also showed high editing efficiency.

[0543] Example 5. Verification of editing efficiency by using the gRNA of the present invention in combination with other Cas13-targeting MITFs. The editing efficiency was verified using the same method as described in the previous example.

[0544] Using guide sequence-related primer annealing, BsaI digestion of CasRx-BsaI plasmids, and T4 ligation, various CasRx-MITF gRNA plasmids were constructed, enabling the expression of gRNAs targeting CasRx and MITF. Transfection was performed on 293T cells, and knockdown efficiency was detected by qPCR after 72 hours.

[0545] The results are shown in Figure 11. High editing efficiency was also demonstrated with the combination of CasRx and MITF-g3, g4, g6, and g7 gRNAs.

[0546] Example 6. Off-target detection analysis of C13-2 in combination with gRNA editing MITF Cells from Example 5 were collected 48 hours after transfection, and total RNA was extracted. The cells were submitted to an external service provider for RNA-seq analysis (n=3 per group). An lncRNA strand-specific library was used, with a sequencing data volume of 16G and the PE150 sequencing method.

[0547] RNAseq analysis principle:

[0548] 1. Data quality control (QC) was performed using fastqc and multiqc, and low-quality reads were removed using fastp.

[0549] 2. rRNA-derived reads were removed by mapping to human rRNA sequences, and then the rRNA-removed reads were aligned to the hg38 reference genome using Hisat2.

[0550] 3. After alignment, gene expression levels were quantified using Kallisto software, and then differential expression analysis (e.g., vs. 293T-NC) was performed using sleuth software. Genes with |b|>0.5, qval<0.05, and mean_obs>1 (or >2) were identified as differentially expressed genes (DEGs).

[0551] 4. Using EMBOSS Water software, sgRNA sequences were aligned to reference cDNA. Transcripts with 18 or more alignments, 6 or fewer mismatches, and a minimum of 8 consecutive pairs were considered predicted off-target transcripts, and the corresponding genes were considered off-target genes.

[0552] 5. The common regions between the gene set with significantly reduced expression and the predicted off-target gene set were extracted to obtain the off-target gene set.

[0553] RNASeq result analysis: The edited MITF gene expression levels in each test group were analyzed using the unedited 293T-NC as a baseline. The results are shown in Table 7.

[0554] [Table 8]

[0555] The results of the off-target gene analysis are shown in Table 8.

[0556] [Table 9]

[0557] Experimental results showed that the gRNAs of MITF-g3, MITF-g4, and MITF-g7 underwent editing only at MITF target sites, with no off-target effects observed. One off-target gene was identified in the MITF-g6 gRNA.

[0558] Example 7. Verification of editing efficiency by using the gRNA of the present invention in combination with other Cas13 target SRD5A2. Editing efficiency was detected using the same method as in the previously described embodiment.

[0559] Various CasRx-SRD5A2 gRNA plasmids were constructed by annealing oligonucleotides related to the guide sequence, digesting the CasRx-BsaI plasmid with BsaI, and ligating it with T4 DNA ligase. These plasmids can express gRNAs targeting CasRx and SRD5A2. Transfection was performed on 293T cell lines overexpressing SRD5A2, and knockdown efficiency was measured by RT-qPCR after 72 hours.

[0560] As a result, high editing efficiency was observed even in combinations of CasRx with SRD5A2-h1, h7, h9, and h11 gRNAs, as shown in Figure 12.

[0561] Example 8. Verification of editing efficiency of endogenous AR genes. By using the Cas13 tool, specifically the C13-2 protein, in combination with gRNA (crRNA), it was possible to target human AR mRNA (NCBI Reference Sequence: NM_000044.6, SEQ ID NO: 394) for gene editing. Reducing AR expression leads to a decrease in androgen receptor expression, ultimately contributing to the treatment of male pattern baldness.

[0562] (1) Construction of a validation vector targeting the endogenous AR gene A C13-2-BsaI plasmid (SEQ ID NO: 4) with a general-purpose crRNA backbone expression cassette was synthesized by an external contractor.

[0563] Sense and antisense strands of the DNA sequence were synthesized. The sense strand was a sequence obtained by adding "agac" to the 5' end of the guide sequence shown in Table 9, and the antisense strand was a sequence obtained by adding "aaaa" to the 5' end of the reverse complementary sequence of the same guide sequence. Both strands were annealed to obtain a double-stranded DNA fragment with sticky ends corresponding to the target site of the AR RNA.

[0564] A primer annealing reaction system is shown in the table below, for example. The primers were incubated in a PCR instrument at 95°C for 5 minutes, then immediately removed and incubated on ice for 5 minutes to allow the primers to anneal to each other and form double-stranded DNA with sticky ends.

[0565] [Table 10]

[0566] The C13-2-BsaI plasmid was digested using Bsa I restriction enzyme, and the backbone was purified and recovered. The annealing product was ligated with T4 ligase to obtain a validation vector plasmid (CMV-C13-2-U6-gRNA). After transformation into E. coli, positive clones were selected, the plasmid was extracted, and used for subsequent testing.

[0567] (2) Transfection of validation vector into 293T cells 293T cells were transfected with validation vectors and a control vector (C13-2-BsaI) having different target site sequences.

[0568] Transfection was performed in a 24-well plate according to the manufacturer's instructions for Lipofectamine 2000 (Thermo).

[0569] (3)qPCR analysis RNA was extracted from cells 72 hours after transfection using the SteadyPure Universal RNA Extraction Kit (AG21017), and the RNA concentration was measured using an ultra-micro spectrophotometer. The RNA products were reverse transcribed using the Evo M-MLV Mix Kit with gDNA Clean for qPCR (AG11728), and the resulting cDNA was subjected to qPCR using the SYBR Green Premix Pro Taq HS qPCR Kit, etc.

[0570] The primers used for qPCR were as follows: AR detection (primer 4): GACGACCAGATGGCTGTCATT (SEQ ID NO: 380) GGGCGAAGTAGAGCATCCT(SEQ ID NO: 381) AR detection (primer 5): CCAGGGACCATGTTTTGCC (SEQ ID NO: 382) CGAAGACGACAAGATGGACAA(SEQ ID NO: 383) Internal standard GAPDH detection: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 384) GAAGGGGTCATTGATGGCAAC(SEQ ID NO: 385).

[0571] The reaction system was prepared according to the instructions for the SYBR® Green Premix Pro Taq HS qPCR Kit (Rox Plus) and QuantStudio TM 5. Detection was performed using a Real-Time PCR System.

[0572] In this study, the relative quantitative method is 2^ -ΔΔCt The target RNA level was calculated using a specific method. An example of the calculation method is shown below: △Ct = Ct (AR) - Ct (GAPDH) ΔΔCt = ΔCt (test vector example C13-2-AR-h1) - ΔCt (control C13-2-BsaI) 2^ -ΔΔCt = 2^(-ΔΔCt)

[0573] The 2^ value of the edited AR RNA calculated by the above calculation method is shown in Table 11, Table 12, and Figure 13 below. -ΔΔCt The values are shown in Table 11, Table 12, and Figure 13 below.

[0574] [[ID=1-4]]

Table 11

[0576] [[ID=2-B]]

Table 12

[0577] *NC indicates the blank control of untransfected 293T cells of the plasmid.

[0578] From the qPCR results, the gRNA combined with C13-2 can reduce the AR RNA expression level.

[0579] Example 9. Verification of the editing effect of different Cas13s [[ID=4,B]]

[0580] (1) Construction of a verification vector targeting the endogenous AR gene[[ID=4,D]] A plurality of control groups were set up to compare the AR RNA editing effects of C13-2 + gRNA, CasRx + gRNA, and shRNA.

[0581] The CasRx-BsaI plasmid (SEQ ID NO: 70), which contains a general-purpose crRNA backbone expression cassette, the control vector shRNA-asiAR72 plasmid (SEQ ID NO: 387), and the shRNA-ARstart plasmid (SEQ ID NO: 388) were synthesized by an external contractor.

[0582] The shRNA-asiAR72 plasmid expresses shRNA, and its target sequence is GTTCACTTTTGACCTGCTAAT (SEQ ID NO: 390), located in exon 8 of the AR gene.

[0583] The shRNA-ARstart plasmid expresses shRNA, and its target sequence is GACCTACCGAGGAGCTTTC (SEQ ID NO: 391), located in exon 1 of the AR gene.

[0584] A CasRx+gRNA vector (CMV-CasRx-U6-gRNA) was constructed according to the method of Example 9. Sense and antisense strands of the DNA sequence were synthesized. The sense strand had "aaac" added to the 5' end of the guide sequence shown in Table 9, and the antisense strand had "aaaa" added to the 5' end of the reverse complementary sequence of the guide sequence. Both strands were annealed to obtain a double-stranded DNA fragment with sticky ends corresponding to the target site of the AR RNA. Subsequently, the CasRx-BsaI plasmid was digested with BsaI, and the resulting fragment was ligated using T4 DNA ligase. E. coli was transformed, positive colonies were selected, and the plasmid was extracted for subsequent testing.

[0585] (2) Transfection of 293T cells using validation vectors 293T cells were transfected with validation vectors and control vectors having different target sites. 293T cells that had not undergone plasmid transfection were used as a blank control (NC group).

[0586] Transfection was performed in a 24-well plate according to the manufacturer's instructions for Lipofectamine 2000 (Thermo).

[0587] (3)qPCR analysis Cells 72 hours after transfection were detected by qPCR using the same method as in Example 8. The relative quantitative method was 2^ -ΔΔCt The target RNA level was calculated using the specified method.

[0588] Tables 13 and 14, and Figure 14, show the comparative measurement results with shRNA (all are averages of three replicated experiments). The AR RNA knockdown efficiency by AR-h2, AR-13, and AR-h8 gRNAs was superior to that of shRNA.

[0589] [Table 13]

[0590] [Table 14]

[0591] Tables 15, 16, and Figure 15 show the measurement results using CasRx in the same test batch (all were repeated tests three times, and the average value of the results was calculated). Both C13-2 and CasRx were able to knock down AR RNA by combining different gRNAs, and C13-2 showed superior editing efficiency compared to CasRx.

[0592] [Table 15]

[0593] [Table 16]

[0594] (4) Detection of protein levels (WB) Cells were harvested 72 hours after transfection, digested with trypsin, centrifuged, and washed with PBS. 100 μL of chilled RIPA lysis solution was added, and the cells were dissolved on ice for 30 minutes. 5× sample buffer was added, the mixture was heated in a boiling water bath for 5 minutes, then centrifuged at maximum rotation speed for 1 minute. The supernatant was collected and subjected to protein electrophoresis. After electrophoresis, the cells were transferred to a PVDF membrane and subjected to Western blotting (WB). Internal controls were detected using GAPDH antibody (Cell Signaling, #5174), and target protein expression was detected using AR antibody (Abcam, ab133273). A goat anti-rabbit secondary antibody (Sigma-Aldrich, A0545) was used as the secondary antibody.

[0595] The results of the Western Blow (WB) test are shown in Figure 16. As shown in the figure, the C13-2 and CasRx test groups showed a significant reduction in AR protein expression compared to the C13-2-BsaI and CasRx-BsaI control groups.

[0596] Example 10. Off-target detection

[0597] Control vector construction Since 293T cells do not contain the EGFP sequence, a C13-2-GFP vector targeting EGFP was constructed according to the method of Example 8 and used as a negative control. The GFP-targeting spacer sequence is tgccgttcttctgcttgtcggccatgatat (SEQ ID NO: 393).

[0598] RNA-seq analysis Validation vectors expressing C13-2 and each of the gRNAs AR-h2, AR-h4, AR-h8, or AR-h9 were constructed according to the method of Example 8 and used as test groups. These were transfected into 293T cells along with the negative control group, and total RNA samples were extracted after 48 hours for RNA-seq analysis (n=3 in each group). An lncRNA strand-specific library was used, with a sequencing data volume of 16G and the sequencing method PE150.

[0599] RNAseq analysis principle Data quality control (QC) was performed using fastqc and multiqc, and low-quality reads were removed using fastp.

[0600] rRNA-derived reads were removed by mapping to human rRNA sequences, and then the reads were aligned to the hg38 reference genome using Hisat2.

[0601] After alignment, gene expression levels were quantified using Kallisto software, followed by differential expression analysis (e.g., vs. 293T-NC) using sleuth software. Genes with |b|>0.5, qval<0.05, and mean_obs>1 (or >2) were identified as differentially expressed genes (DEGs).

[0602] In some embodiments of this disclosure, the guide sequence hybridizes to the target RNA, and the mismatch is 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer nucleotides.

[0603] We extracted the common regions between the gene set with significantly reduced expression and the predicted off-target gene set to obtain the off-target gene set.

[0604] RNASeq result analysis

[0605] The analysis results are shown in Table 17 below. [Table 17]

[0606] RNA-seq analysis revealed that when C13-2 was edited in combination with the gRNAs AR-h2, AR-h4, AR-h8, and AR-h9, no off-target sequences related to the guide sequence were detected.

[0607] Although specific embodiments of the present invention have been described above, those skilled in the art will understand that these are merely illustrative and that various changes or modifications can be made without departing from the principles and spirit of the present invention. Accordingly, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An inhibitor of CTGF RNA, MITF RNA, or SRD5A2 RNA, characterized in that the inhibitor is a gene editing system; Optionally, the gene editing system may knock down the level of CTGF RNA, MITF RNA, or SRD5A2 RNA, or inhibit the translation of CTGF RNA, MITF RNA, or SRD5A2 RNA.

2. A guide RNA for a gene editing system, comprising a guide sequence for hybridization with a target RNA, wherein the target RNA is CTGF RNA, MITF RNA, or SRD5A2 RNA; The target RNA is optionally CTGF pre-mRNA, MITF pre-mRNA, or SRD5A2 pre-mRNA, or CTGF mRNA, MITF mRNA, or SRD5A2 mRNA; The target RNA is optionally mammalian CTGF RNA, MITF RNA, or SRD5A2 RNA.

3. A pharmaceutical composition characterized by comprising the inhibitor described in claim 1 or the guide RNA described in claim 2; Optionally, the pharmaceutical composition may contain pharmaceutically acceptable excipients.

4. Use of the inhibitor according to claim 1 or the guide RNA according to claim 2 in the manufacture of a pharmaceutical product for the diagnosis, treatment, or prevention of a disease or condition related to a target RNA; Optionally, a disease or condition related to target RNA refers to a disease or condition caused by abnormally high expression of target RNA; Optionally, the target RNA is CTGF premRNA, MITF premRNA, or SRD5A2 premRNA, or CTGF mRNA, MITF mRNA, or SRD5A2 mRNA; Optionally, the target RNA is human CTGF RNA, MITF RNA, or SRD5A2 RNA; Optionally, the target RNA sequence is the sequence shown in Sequence ID No. 14, 22, or 35; The target RNA sequence is optionally nucleotides 494–785 of the sequence shown in SEQ ID NO: 14, nucleotides 404–606 of the sequence shown in SEQ ID NO: 22, or nucleotides 484–820 of the sequence shown in SEQ ID NO:

35.

5. Use of the inhibitor according to claim 1 or the guide RNA according to claim 2 in the manufacture of cosmetics.

6. A cosmetic comprising the inhibitor described in claim 1 or the guide RNA described in claim 2.

7. Use of a gene editing system in the manufacture of a pharmaceutical product for the diagnosis, treatment, or prevention of male pattern baldness, wherein the gene editing system knocks down androgen receptor (AR) RNA levels or inhibits the translation of AR RNA.

8. A gene editing system characterized by knocking down AR RNA levels or inhibiting AR RNA translation.

9. A gene editing system guide RNA characterized by containing a guide sequence that hybridizes with AR RNA.

10. A pharmaceutical composition characterized by comprising the gene editing system described in claim 2.

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