Gene editing system and application thereof

Abstract

The invention provides an inhibitor of CTGF RNA, MITF RNA and SRD5A2RNA and application of the inhibitor. The inhibitor is a gene editing system. The gene editing system can knock down CTGF RNA, MITF RNA or SRD5A2RNA, and can be used for repairing scars, whitening skin, reducing or eliminating chloasma, preventing or treating melanoma, and / or preventing or treating androgenetic alopecia. The invention further provides a gene editing system, the gene editing system targets the AR RNA coded by the androgen receptor gene, and the gene editing system can be used for treating androgenetic alopecia and other diseases.

Description

Gene editing systems and their applications

[0001] This application claims priority to Chinese Patent Application No. 2023109694981 filed on August 2, 2023, and PCT Patent Application No. PCT / CN2024 / 071775 filed on January 11, 2024. The entire contents of the above patent applications are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of gene editing technology, and specifically relates to a gene editing system and its application. Background Art

[0003] Wound healing is a complex cellular activity. Generally speaking, wound healing proceeds in an organized manner, including four stages: hemostasis, inflammation, proliferation and remodeling. Among them, proliferation and remodeling are particularly important in determining scar formation because they are related to the production and reorganization of the extracellular matrix (ECM). However, due to the high contractility of myofibroblasts, the healing process of scar formation will largely produce a disorganized, dense, collagen-rich matrix, thereby destroying the natural structure of the skin. Fibrotic tissue causes uncontrolled excessive accumulation of proteins under the skin, making the skin surface thick, irregular and uneven, forming scars. Connective tissue growth factor (CTGF, connective tissue growth factor, also known as CCN2) is a key mediator of scar formation.

[0004] Melanogenesis is a biosynthetic pathway for the production of melanin in melanocytes, involving a complex series of enzymatic and chemical reactions. Five major signaling pathways are involved in its regulation, among which microphthalmia-associated transcription factor (MITF) is the ultimate target of multiple signaling pathways and a major regulator of melanogenesis.

[0005] Androgenetic alopecia (AGA), also known as male pattern baldness or premature balding, is a progressive hair loss that occurs gradually in young adults and is the most common type of hair loss. Under the influence of androgens, hair follicles on the scalp gradually shrink and shrink, eventually degenerating into small vellus hairs, which appear as baldness to the naked eye. Androgens are a key factor in the development of AGA, but nearly all AGA patients have normal levels of circulating androgens. AGA may be associated with elevated expression of the androgen receptor gene and / or 5α-reductase gene in hair follicles.

[0006] 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 the male hormones testosterone or dihydrotestosterone bind to it in the cytoplasm and then transported into the nucleus.

[0007] Several genome-wide association studies (GWAS) have shown that the androgen receptor (AR) gene and the ectodysplasim-A2 receptor (EDA2R) gene on the X chromosome are the most important susceptibility genes for AGA.

[0008] Furthermore, the SRD5A2 gene encodes 3-oxo-5α-steroid 4-dehydrogenase 2, also known as 5α-reductase type 2 (5αR2), one of three isoenzymes of 5α-reductase. SRD5A2 is a key enzyme in androgen metabolism, catalyzing the synthesis of the potent AR agonist dihydrotestosterone (DHT) from testosterone. Elevated SRD5A2 levels have been detected in areas of the scalp affected by androgenic alopecia (AGA), and the SRD5A2 inhibitor finasteride is used as a treatment for AGA patients.

[0009] Summary of the Invention

[0010] A first aspect of the present disclosure provides a CTGF RNA, MITF RNA or SRD5A2 RNA inhibitor, which is a gene editing system.

[0011] 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.

[0012] In some embodiments of the present disclosure, the gene editing system knocks down the level of CTGF RNA, MITF RNA, or SRD5A2 RNA.

[0013] In some embodiments of the present disclosure, the gene editing system comprises:

[0014] (a) a guide RNA comprising a guide sequence that hybridizes to a target RNA, or a polynucleotide sequence encoding the guide RNA; and

[0015] (b) an RNA-guided nuclease, or a polynucleotide sequence encoding the RNA-guided nuclease;

[0016] The guide RNA is capable of forming a complex with the nuclease and guiding the complex to bind to the target RNA in a sequence-specific manner. The target RNA is CTGF RNA, MITF RNA or SRD5A2 RNA.

[0017] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with the nuclease and guiding the complex to bind to and cleave the target RNA.

[0018] In some embodiments of the present disclosure, the target RNA is CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of the present 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 the present disclosure, the target RNA is CTGF mRNA, MITF mRNA, or SRD5A2 mRNA. In some embodiments of the present disclosure, the target RNA is mammalian CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of the present disclosure, the target RNA is human CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of the present disclosure, the target RNA is human CTGF mRNA, MITF mRNA, or SRD5A2 mRNA.

[0019] In some embodiments of the present disclosure, the target RNA sequence is a sequence as shown in SEQ ID NO: 14, 22 or 35.

[0020] In some embodiments of the present disclosure, the target RNA sequence is nucleotides 494 to 785 of the sequence shown in SEQ ID NO: 14, nucleotides 404 to 606 of the sequence shown in SEQ ID NO: 22, or nucleotides 484 to 820 of the sequence shown in SEQ ID NO: 35.

[0021] In some embodiments of the present disclosure, the target RNA sequence is a nucleotide sequence of nucleotide 494, 604, 678 or 761 to nucleotide 518, 628, 702 or 785 of the sequence shown in SEQ ID NO: 14 (human CCN2 mRNA, NCBI NM_001901.4).

[0022] In some embodiments of the present disclosure, the target RNA sequence is a nucleotide sequence from nucleotide 404, 429, 453, 479, 509, 546 or 582 to nucleotide 428, 453, 477, 503, 533, 570 or 606 of the sequence shown in SEQ ID NO: 22 (human MITF mRNA, NCBI NM_001354607.2).

[0023] In some embodiments of the present disclosure, the target RNA sequence is a nucleotide sequence from nucleotide 484, 524, 547, 585, 635, 721, 755 or 796 to nucleotide 508, 548, 571, 609, 659, 745, 779 or 820 of the sequence shown in SEQ ID NO: 35 (human SRD5A2 mRNA, NCBI XM_011533072.3).

[0024] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity with the target RNA. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity with the target RNA. Further, in some embodiments of the present disclosure, the guide sequence has 100% sequence identity with the target RNA.

[0025] In some embodiments of the disclosure, the guide sequence has at least 80% sequence identity to the sequence shown in any one of SEQ ID NOs: 14, 22, or 35. In some embodiments of the disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 14, 22, or 35. Further, in some embodiments of the disclosure, the guide sequence has 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 14, 22, or 35.

[0026] 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 to 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 to 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.

[0027] In some embodiments of the disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, and 54. In some embodiments of the 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, and 54.

[0028] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a backbone sequence. The backbone sequence interacts with the RNA-guided nuclease.

[0029] In some embodiments of the present disclosure, the backbone sequence is a direct repeat sequence.

[0030] In some embodiments of the present disclosure, the direct repeat sequence comprises 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 to the sequence shown in SEQ ID NO: 2 or 3.

[0031] In some embodiments of the present disclosure, the RNA-guided nuclease is selected from Cas9, Cas12, Cas13, TnpB, IscB, IsrB, Fancor nuclease, or fragments thereof (including but not limited to nucleic acid binding domain fragments).

[0032] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas protein.

[0033] In some embodiments of the present disclosure, the guide RNA is capable of forming a CRISPR complex with the Cas protein and guiding the sequence-specific binding of the CRISPR complex to the target RNA.

[0034] In some embodiments of the present disclosure, the guide RNA is capable of forming a CRISPR complex with the Cas protein and guiding the CRISPR complex to bind to and cleave the target RNA.

[0035] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas9 protein, a Cas12 protein, or a Cas13 protein.

[0036] In some embodiments of the present disclosure, the RNA-guided 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.

[0037] 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 to the sequence shown in SEQ ID NO: 1.

[0038] In some embodiments of the present disclosure, the Cas13 protein comprises the sequence shown in SEQ ID NO: 1.

[0039] In some embodiments of the present disclosure, the RNA-guided nuclease comprises any one or more of the following: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0040] In some embodiments of the present disclosure, the Cas protein comprises any one or more of the following: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0041] In some embodiments of the present disclosure, the subcellular localization signal is selected from a nuclear localization signal and a nuclear export signal sequence.

[0042] In some embodiments of the present disclosure, the gene editing system comprises:

[0043] (a) a guide RNA, or a polynucleotide sequence encoding the guide RNA; and

[0044] (b) Cas13 protein, or a polynucleotide sequence encoding the Cas13 protein;

[0045] The guide RNA is capable of forming a complex with the Cas13 protein and guiding the complex to bind to and cut the target RNA.

[0046] In some embodiments of the present disclosure, the polynucleotide sequence encoding the guide RNA is linked to a first regulatory sequence, and the first regulatory sequence is used to regulate the expression of the guide RNA.

[0047] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA-guided nuclease is linked to a second regulatory sequence, which is used to regulate the expression of the RNA-guided nuclease.

[0048] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA-guided nuclease is linked to a regulatory sequence, and the regulatory sequence is used to regulate the expression of the RNA-guided nuclease.

[0049] In some embodiments of the present disclosure, the polynucleotide sequence encoding the guide RNA is linked to a regulatory sequence, and the regulatory sequence is used to regulate the expression of the guide RNA.

[0050] In some embodiments of the present disclosure, the regulatory sequence that regulates expression of the RNA-guided nuclease is the same as or different from the regulatory sequence that regulates expression of the guide RNA.

[0051] In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter and enhancer sequence.

[0052] The gene editing systems described herein can be introduced into cells (or cell-free systems) in a variety of non-limiting ways: (i) as mRNA encoding an RNA-guided nuclease and a guide RNA, (ii) as part of a single vector or plasmid, or divided into multiple vectors or plasmids, (iii) as separate RNA-guided nucleases and guide RNAs, or (iv) as an RNP complex of an RNA-guided nuclease and a guide RNA.

[0053] In some embodiments of the present disclosure, the complex reduces the level of the target RNA in a mammal (eg, a human).

[0054] In some embodiments of the disclosure, the complex reduces the level of the target RNA in a cell, such as a cell expressing the target RNA.

[0055] In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the 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 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 can be tested using conventional methods in the art; including but not limited to the qPCR method described in the Examples, untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as negative controls to calculate the target RNA knockdown level of the experimental group compared to the negative control.

[0056] 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 conventional methods in the art. In some embodiments, the number of off-target genes is determined by differentially expressed genes determined by RNA sequencing.

[0057] In some embodiments of the present disclosure, the complex reduces the level of the protein encoded by the target RNA in an animal (eg, a human).

[0058] In some embodiments of the present disclosure, upon contact of the complex with a cell containing a target RNA, the complex reduces the level of a protein encoded by the target RNA in the cell. In some embodiments of the present disclosure, the protein encoded by the target RNA is CTGF protein, MITF protein, or SRD5A2 protein. In some embodiments of the present disclosure, the complex reduces the level of CTGF protein, MITF protein, or SRD5A2 protein in the cell.

[0059] In some embodiments of the present disclosure, the complex reduces the level of CTGF protein, MITF protein, or SRD5A2 protein 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 level of protein encoded by the target RNA can be tested using conventional methods in the art, including but not limited to ELISA and Western Blotting. Untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as negative controls to calculate the knockdown level of CTGF protein, MITF protein, or SRD5A2 protein in the experimental group compared to the negative control group.

[0060] A second aspect of the present disclosure provides a gene editing system guide RNA (gRNA).

[0061] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence that hybridizes to a target RNA, and the target RNA is CTGF RNA, MITF RNA, or SRD5A2 RNA.

[0062] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a backbone sequence. The backbone sequence interacts with the RNA-guided nuclease. The backbone sequence is the sequence that generally remains unchanged in the guide RNA molecule when designing the guide RNA molecule. For example, the backbone sequence can refer to the portion of the guide RNA molecule other than the guide sequence. In some embodiments of the present disclosure, the backbone sequence is a direct repeat (DR).

[0063] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity with the target RNA. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity with the target RNA. Further, in some embodiments of the present disclosure, the guide sequence has 100% sequence identity with the target RNA.

[0064] In some embodiments of the disclosure, the guide sequence has at least 80% sequence identity to the sequence shown in any one of SEQ ID NOs: 14, 22, or 35. In some embodiments of the disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 14, 22, or 35. Further, in some embodiments of the disclosure, the guide sequence has 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 14, 22, or 35.

[0065] In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the nucleotide sequence of nucleotides 494 to 785 of the sequence set forth in SEQ ID NO: 14, nucleotides 404 to 606 of the sequence set forth in SEQ ID NO: 22, or nucleotides 484 to 820 of the sequence set forth in SEQ ID NO: 35.

[0066] In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95% or 100% sequence identity with the nucleotide sequence of nucleotide 494, 604, 678 or 761 to nucleotide 518, 628, 702 or 785 of the sequence set forth in SEQ ID NO: 14.

[0067] In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95% or 100% sequence identity to the nucleotide sequence of nucleotide 404, 429, 453, 479, 509, 546 or 582 to nucleotide 428, 453, 477, 503, 533, 570 or 606 of the sequence set forth in SEQ ID NO: 22.

[0068] In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95% or 100% sequence identity to the nucleotide sequence of nucleotide 484, 524, 547, 585, 635, 721, 755 or 796 to nucleotide 508, 548, 571, 609, 659, 745, 779 or 820 of the sequence set forth in SEQ ID NO: 35.

[0069] 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 comprises 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.

[0070] In some embodiments of the disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, and 54. In some embodiments of the disclosure, the guide sequence comprises any one of SEQ ID NOs: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, and 54. In some embodiments of the disclosure, the guide sequence is any one of SEQ ID NOs: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, and 54.

[0071] In some embodiments of the present disclosure, the backbone sequence is a direct repeat sequence, which comprises 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 to the sequence shown in SEQ ID NO: 2 or 3.

[0072] In some embodiments of the present disclosure, the direct repeat sequence comprises a sequence as shown in SEQ ID NO: 2 or 3. In some embodiments of the present disclosure, the direct repeat sequence consists of a sequence as shown in SEQ ID NO: 2 or 3.

[0073] In some embodiments of the present disclosure, the guide sequence is located at the 3' end or 5' end of the direct repeat sequence. In some embodiments of the present disclosure, the guide sequence is located at the 3' end of the direct repeat sequence. In some embodiments of the present disclosure, the guide sequence is located at the 5' end of the direct repeat sequence.

[0074] In some embodiments of the present disclosure, the guide RNA comprises an aptamer sequence.

[0075] In some embodiments of the present disclosure, the aptamer sequence is inserted into the loop of the stem-loop structure of the direct repeat sequence secondary structure of the guide RNA.

[0076] In some embodiments of the present disclosure, the guide RNA comprises modified nucleotides. The modifications include, but are not limited to, 2'-O-methyl, 2'-O-methyl-3'-phosphorothioate or 2'-O-methyl-3'-thio PACE modifications. In some embodiments of the present disclosure, the guide RNA comprises modified nucleotides selected from deoxyribonucleotides and locked nucleic acids (LNA). In some embodiments, the guide RNA comprises at least one chemically modified nucleotide. In some embodiments, the guide RNA is a hybrid RNA-DNA guide, i.e., some RNA nucleotides in the guide RNA are replaced by DNA nucleotides. In some embodiments, the guide RNA is a hybrid RNA-LNA (locked nucleic acid) guide, i.e., some RNA nucleotides in the guide RNA are replaced by LNA nucleotides.

[0077] In some embodiments of the present disclosure, the target RNA is located in the nucleus and / or cytoplasm of a eukaryotic cell.

[0078] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease (also referred to as a gene editing complex) and guiding the sequence-specific binding of the complex to the target RNA.

[0079] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease and directing the complex to bind to and cleave the target RNA.

[0080] In some embodiments of the present disclosure, the complex reduces the level of the target RNA in a mammal (eg, a human).

[0081] A third aspect of the present disclosure provides an isolated nucleic acid encoding a guide RNA according to the present disclosure.

[0082] The fourth aspect of the present disclosure provides a vector comprising a polynucleotide sequence encoding the guide RNA according to the present disclosure, and a regulatory sequence for regulating the expression of the guide RNA.

[0083] In some embodiments of the present disclosure, the vector is an adeno-associated virus vector.

[0084] In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter and enhancer sequence.

[0085] In some embodiments of the present disclosure, the regulatory sequence is a U6 promoter or an eye-specific promoter.

[0086] In some embodiments of the present disclosure, the eye-specific promoter is selected from the group consisting of: a retinoschisis protein promoter, a K12 promoter, a rhodopsin promoter, a rod-specific promoter, a cone-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.).

[0087] In some embodiments of the present disclosure, the promoter is a chicken β-actin (CB) promoter. The chicken β-actin promoter can be a short chicken β-actin promoter or a long chicken β-actin promoter. In some embodiments, the promoter (e.g., chicken β-actin promoter) comprises an enhancer sequence, such as a cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence can be a short CMV enhancer sequence or a long CMV enhancer sequence. In some embodiments, the promoter comprises a long CMV enhancer sequence and a long chicken β-actin promoter. In some embodiments, the promoter comprises a short CMV enhancer sequence and a short chicken β-actin promoter. However, those skilled in the art will appreciate that a short CMV enhancer can be used together with a long CB promoter, and a long CMV enhancer can be used together with a short CB promoter. In some embodiments of the present disclosure, the promoter is a CBh promoter.

[0088] In some embodiments of the present disclosure, the promoter is a CBh promoter.

[0089] In some embodiments of the disclosure, the regulatory sequence comprises an HRE enhancer element.

[0090] In some embodiments of the present disclosure, the regulatory sequence comprises an NRS element and an HRE enhancer element in tandem.

[0091] The fifth aspect of the present disclosure provides a vector system, wherein the vector system comprises a polynucleotide sequence encoding the guide RNA disclosed herein and a first regulatory sequence that regulates the expression of the guide RNA; and a polynucleotide sequence encoding the RNA-guided nuclease and a second regulatory sequence that regulates the expression of the RNA-guided nuclease.

[0092] In some embodiments of the present disclosure, the vector system comprises one or more vectors.

[0093] In some embodiments of the present disclosure, the vector system comprises multiple vectors, the polynucleotide sequence encoding the guide RNA and the first regulatory sequence regulating the expression of the guide RNA are located on the first vector, and the polynucleotide sequence encoding the RNA-guided nuclease and the second regulatory sequence regulating the expression of the RNA-guided nuclease are located on the second vector.

[0094] In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter and enhancer sequence.

[0095] A sixth aspect of the present disclosure provides an adeno-associated viral vector, wherein the adeno-associated viral vector comprises DNA encoding an RNA-guided nuclease and the guide RNA of the present disclosure.

[0096] A seventh aspect of the present disclosure provides a lipid nanoparticle, wherein the lipid nanoparticle comprises the guide RNA described in the present disclosure and an mRNA encoding the RNA-guided nuclease.

[0097] An eighth aspect of the present disclosure provides a lentiviral vector, wherein the lentiviral vector comprises the guide RNA disclosed herein and an mRNA encoding an RNA-guided nuclease; optionally, the lentiviral vector is pseudotyped with an envelope protein; optionally, the mRNA encoding the RNA-guided nuclease is linked to an aptamer sequence.

[0098] A ninth aspect of the present disclosure provides a ribonucleoprotein complex, wherein the ribonucleoprotein complex is formed by the guide RNA and the RNA-guided nuclease described in the present disclosure.

[0099] The tenth aspect of the present disclosure provides a virus-like particle, wherein the virus-like particle comprises a ribonucleoprotein complex formed by the guide RNA and the RNA-guided nuclease described in the present disclosure; optionally, the RNA-guided nuclease is fused to the gag protein.

[0100] The eleventh aspect of the present disclosure provides a cell, comprising the inhibitor, guide RNA, nucleic acid, vector and / or vector system described in the present disclosure; optionally, the cell is a eukaryotic cell.

[0101] A twelfth aspect of the present disclosure provides a pharmaceutical composition comprising the inhibitor, guide RNA, nucleic acid, vector and / or vector system described in the present disclosure.

[0102] In some embodiments of the present disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0103] A thirteenth aspect of the present disclosure provides use of the inhibitor, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell and / or pharmaceutical composition according to the present disclosure in any of the following or in the preparation of an agent for achieving any of the following schemes:

[0104] Cleave or nick one or more target RNA molecules, activate or upregulate one or more target RNA molecules, activate or inhibit translation of one or more target RNA molecules, inactivate one or more target RNA molecules, visualize, label or detect one or more target RNA molecules, bind one or more target RNA molecules, transport one or more target RNA molecules, and mask one or more target RNA molecules.

[0105] In some embodiments of the present disclosure, there is provided use of the inhibitor, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell and / or pharmaceutical composition according to the present disclosure in any of the following or in the preparation of an agent for achieving any of the following schemes:

[0106] Cleave one or more target RNA molecules, inhibit translation of one or more target RNA molecules, bind to one or more target RNA molecules.

[0107] In some embodiments of the present disclosure, there is provided use of the inhibitor, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell and / or pharmaceutical composition according to the present disclosure in any of the following or in the preparation of an agent for achieving any of the following schemes:

[0108] Binds to one or more target RNA molecules.

[0109] In some embodiments of the present disclosure, there is provided use of the inhibitor, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell and / or pharmaceutical composition according to the present disclosure in any of the following or in the preparation of an agent for achieving any of the following schemes:

[0110] Cleave one or more target RNA molecules.

[0111] In some embodiments of the present 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 the present disclosure, the target RNA is CTGF mRNA, MITF mRNA or SRD5A2 mRNA. In some embodiments of the present disclosure, the target RNA is mammalian CTGF RNA, MITF RNA or SRD5A2 RNA, for example, CTGF RNA, MITF RNA or SRD5A2 RNA selected from humans, rats, mice, non-human primates such as monkeys, dogs, pigs, rabbits, etc. In some embodiments of the present disclosure, the target RNA is human CTGF RNA, MITF RNA or SRD5A2 RNA. In some embodiments of the present disclosure, the target RNA is human CTGF mRNA, MITF mRNA or SRD5A2 mRNA.

[0112] A fourteenth aspect of the present disclosure provides a method for diagnosing, treating or preventing a disease or condition associated with a target RNA, by administering an effective amount of an inhibitor, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell and / or pharmaceutical composition according to the present disclosure to a sample of a subject in need or to a subject in need.

[0113] In some embodiments of the present disclosure, the disease or disorder associated with the target RNA refers to a disease or disorder caused by abnormally high expression of the target RNA.

[0114] In some embodiments of the present 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 the present disclosure, the target RNA is CTGF mRNA, MITF mRNA or SRD5A2 mRNA. In some embodiments of the present disclosure, the target RNA is human CTGF RNA, MITF RNA or SRD5A2 RNA. In some embodiments of the present disclosure, the target RNA is human CTGF mRNA, MITF mRNA or SRD5A2 mRNA.

[0115] In some embodiments of the present disclosure, the diseases associated with the target RNA include: melanoma, androgenic alopecia, and scar formation.

[0116] In some embodiments of the present disclosure, the target RNA-related diseases include melanoma, androgenic alopecia, and scar formation.

[0117] A fifteenth aspect of the present disclosure provides the use of the inhibitors, guide RNAs, nucleic acids, vectors, vector systems, adeno-associated viral vectors, lipid nanoparticles, lentiviral vectors, ribonucleoprotein complexes, virus-like particles, eukaryotic cells and / or pharmaceutical compositions according to the present disclosure in the preparation of a medicament for diagnosing, treating or preventing a disease or condition associated with a target RNA.

[0118] In some embodiments of the present disclosure, the disease or disorder associated with the target RNA refers to a disease or disorder caused by abnormally high expression of the target RNA.

[0119] In some embodiments of the present 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 the present disclosure, the target RNA is CTGF mRNA, MITF mRNA, or SRD5A2 mRNA. In some embodiments of the present disclosure, the target RNA is human CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of the present disclosure, the target RNA is human CTGF mRNA, MITF mRNA, or SRD5A2 mRNA.

[0120] In some embodiments of the present disclosure, the diseases associated with the target RNA include: melanoma, androgenic alopecia, and scar formation.

[0121] In some embodiments of the present disclosure, the target RNA-related diseases include melanoma, androgenic alopecia, and scar formation.

[0122] A sixteenth aspect of the present disclosure provides the use of the inhibitor, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle and / or eukaryotic cell according to the present disclosure in the preparation of cosmetics.

[0123] In some embodiments of the present disclosure, the cosmetic can be used to repair scars, whiten skin, reduce or eliminate melasma, prevent or treat melanoma, and / or prevent or treat androgenic alopecia.

[0124] A seventeenth aspect of the present disclosure provides a cosmetic comprising the inhibitor, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle and / or eukaryotic cell according to the present disclosure.

[0125] In some embodiments of the present disclosure, the cosmetic can be used to repair scars, whiten skin, reduce or eliminate melasma, prevent or treat melanoma, and / or prevent or treat androgenic alopecia.

[0126] In some embodiments of the present disclosure, the inhibitors, guide RNAs, nucleic acids, vectors, vector systems, adeno-associated viral vectors, lipid nanoparticles, lentiviral vectors, ribonucleoprotein complexes, virus-like particles, cells, pharmaceutical compositions, or cosmetics described herein can be administered to a subject, such as a human or animal, in an effective amount. The effective amount refers to an amount that can produce a function or activity in a human and / or animal and is acceptable to the human and / or animal.

[0127] In some embodiments of the present disclosure, the inhibitors, guide RNAs, nucleic acids, vectors, vector systems, adeno-associated viral vectors, lipid nanoparticles, lentiviral vectors, ribonucleoprotein complexes, virus-like particles, cells, pharmaceutical compositions, or cosmetics described herein may further comprise pharmaceutically, cosmetically, chemically, or biologically acceptable ingredients, such as pharmaceutically acceptable excipients, cosmetically acceptable excipients, thickeners, or diluents.

[0128] An eighteenth aspect of the present disclosure provides use of a gene editing system in preparing a medicament for diagnosing, treating or preventing androgenic alopecia, wherein the gene editing system:

[0129] Knock down AR RNA levels or inhibit AR RNA translation.

[0130] In some embodiments of the present disclosure, the gene editing system knocks down AR RNA levels or inhibits the translation of AR RNA.

[0131] In some embodiments of the present disclosure, the gene editing system knocks down AR RNA levels.

[0132] In some embodiments of the present disclosure, the gene editing system inhibits translation of AR RNA.

[0133] In some embodiments of the present disclosure, the gene editing system comprises:

[0134] a guide RNA comprising a guide sequence that hybridizes to an AR RNA, or a polynucleotide sequence of said guide RNA, and an RNA-guided nuclease, or a polynucleotide sequence encoding said nuclease,

[0135] The guide RNA is capable of forming a complex with the nuclease and directing the complex to sequence-specific binding to the AR RNA.

[0136] In some embodiments of the present disclosure, the gene editing system comprises:

[0137] a guide RNA comprising a guide sequence that hybridizes to the AR RNA, and an RNA-guided nuclease;

[0138] The guide RNA is capable of forming a complex with the nuclease and directing the complex to sequence-specific binding to the AR RNA.

[0139] In some embodiments of the present disclosure, the gene editing system comprises:

[0140] a polynucleotide sequence encoding a guide RNA comprising a guide sequence that hybridizes to the AR RNA, and a polynucleotide sequence encoding an RNA-guided nuclease;

[0141] The guide RNA is capable of forming a complex with the nuclease and directing the complex to sequence-specific binding to the AR RNA.

[0142] In some embodiments of the present disclosure, the AR RNA is pre-mRNA and / or mature mRNA.

[0143] In some embodiments of the present disclosure, the AR RNA is human pre-mRNA and / or mature mRNA.

[0144] In some embodiments of the present disclosure, the AR RNA sequence is selected from any one of SEQ ID NOs: 394-396.

[0145] In some embodiments of the present disclosure, the AR RNA is the sequence numbered NM_000044.6 in the NCBI database (SEQ ID NO: 394), namely "transcript variant 1".

[0146] In some embodiments of the present disclosure, the AR RNA is the sequence numbered NM_001011645.3 in the NCBI database (SEQ ID NO: 395), namely "transcript variant 2".

[0147] In some embodiments of the present disclosure, the AR RNA is the sequence numbered ENST00000374690.9 in the Ensembl database (SEQ ID NO: 396).

[0148] In some embodiments of the present disclosure, the guide RNA guides the complex to bind to and cleave the AR RNA.

[0149] In some embodiments of the present disclosure, after contacting the complex with a cell containing AR RNA, the level of the AR RNA or AR protein (androgen receptor encoded by AR RNA) in the cell is reduced 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%.

[0150] In some embodiments of the present disclosure, upon contact of the complex with a cell comprising AR RNA, the level of the AR RNA in the cell is reduced 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%.

[0151] In some embodiments of the present disclosure, upon contact of the complex with a cell containing AR RNA, the level of the AR protein in the cell is reduced 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%.

[0152] In some embodiments of the present disclosure, the cell is a eukaryotic cell. In some embodiments of the present disclosure, the cell is a mammalian cell. In some embodiments of the present disclosure, the cell is a human cell.

[0153] 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.

[0154] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 394-396.

[0155] In some embodiments of the present disclosure, the guide sequence of the guide RNA has 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 394-396.

[0156] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in SEQ ID NO: 394.

[0157] In some embodiments of the present disclosure, the guide sequence of the guide RNA has 100% sequence identity to the sequence shown in SEQ ID NO: 394.

[0158] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the nucleotide sequence consisting of nucleotides 2826 to 2990 of the sequence shown in SEQ ID NO: 394.

[0159] The inventors designed gRNA for NCBI's NM_001011645.3 transcript variant 2, mRNA (SEQ ID NO: 395) and Ensembl database transcript ENST00000374690.9 (SEQ ID NO: 396), and their guide sequences are shown in Table 9.

[0160] Table 9. Designed gRNA

[0161] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 71-378.

[0162] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 71-80.

[0163] In some embodiments of the present disclosure, the guide sequence of the guide RNA has 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 71-80.

[0164] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a direct repeat sequence, and the direct repeat sequence interacts with the RNA-guided nuclease.

[0165] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas protein; optionally, the RNA-guided nuclease is a Cas9 protein, a Cas12 protein, or a Cas13 protein.

[0166] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas13 protein. Further optionally, the RNA-guided nuclease is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein.

[0167] In some embodiments of the present disclosure, the Cas13 protein comprises the sequence shown in SEQ ID NO: 1.

[0168] In some embodiments of the present disclosure, the RNA-guided nuclease comprises any one or more of the following: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0169] In some embodiments of the present disclosure, the subcellular localization signal is a nuclear localization signal and / or a nuclear export signal sequence.

[0170] In some embodiments of the present disclosure, the subcellular localization signal is a mitochondrial localization signal or a chloroplast localization signal sequence.

[0171] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a nuclear localization signal and / or a nuclear export signal sequence, and optionally a deaminase domain, a translation activation domain, or a translation repression domain.

[0172] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA-guided nuclease is linked to a regulatory sequence 1 that regulates its expression, and the polynucleotide sequence encoding the guide RNA is linked to a regulatory sequence 2 that regulates its expression.

[0173] In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter and enhancer sequence.

[0174] In some embodiments of the present disclosure, the regulatory sequence is selected from the group consisting of a CMV promoter, a CMV enhancer, a CBh promoter, a U6 promoter, and a specific promoter.

[0175] In some embodiments of the present disclosure, the gene editing system comprises:

[0176] A guide RNA comprising a guide sequence that hybridizes to the AR RNA or a polynucleotide sequence encoding the guide RNA, and a Cas13 protein or a polynucleotide sequence encoding the Cas13 protein;

[0177] The guide RNA is capable of forming a CRISPR complex with the Cas13 protein and guiding the complex sequence to specifically bind to and cut AR RNA.

[0178] In some embodiments, the coding sequence is linked to regulatory sequences that control its expression.

[0179] In some embodiments, the polynucleotide sequence encoding the Cas13 protein is connected to a regulatory sequence 1 that regulates its expression, and the polynucleotide sequence encoding the guide RNA is connected to a regulatory sequence 2 that regulates its expression.

[0180] In some embodiments, at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirty, fourteen, fifteen, or more) guide RNAs are expressed in tandem under the control of the same regulatory sequence. Further, tandem expression yields a pre-crRNA, which is then processed by the Cas13 protein to yield a mature guide RNA molecule.

[0181] In some embodiments, the polynucleotide sequence encoding the guide RNA and the polynucleotide sequence encoding the RNA-guided nuclease are located on the same vector.

[0182] In some embodiments of the present disclosure, the gene editing system is introduced into a cell or cell-free system in any of the following ways: (i) as mRNA encoding an RNA-guided nuclease and a guide RNA, (ii) as part of a single vector or plasmid, or divided into multiple vectors or plasmids, (iii) as separate RNA-guided nucleases and guide RNAs, or (iv) as an RNP complex of an RNA-guided nuclease and a guide RNA.

[0183] A nineteenth aspect of the present disclosure provides a gene editing system, wherein:

[0184] Knock down AR RNA levels or inhibit AR RNA translation.

[0185] In some embodiments of the present disclosure, the gene editing system knocks down AR RNA levels or inhibits the translation of AR RNA.

[0186] In some embodiments of the present disclosure, the gene editing system knocks down AR RNA levels.

[0187] In some embodiments of the present disclosure, the gene editing system inhibits translation of AR RNA.

[0188] In some embodiments of the present disclosure, the gene editing system comprises:

[0189] a guide RNA comprising a guide sequence that hybridizes to the AR RNA, or a polynucleotide sequence encoding the guide RNA, and an RNA-guided nuclease, or a polynucleotide sequence encoding the nuclease;

[0190] The guide RNA is capable of forming a complex with the nuclease and directing the complex to sequence-specific binding to the AR RNA.

[0191] In some embodiments of the present disclosure, the gene editing system comprises:

[0192] a guide RNA comprising a guide sequence that hybridizes to the AR RNA, and an RNA-guided nuclease;

[0193] The guide RNA is capable of forming a complex with the nuclease and directing the complex to sequence-specific binding to the AR RNA.

[0194] In some embodiments of the present disclosure, the gene editing system comprises:

[0195] a polynucleotide sequence encoding a guide RNA comprising a guide sequence that hybridizes to the AR RNA, and a polynucleotide sequence encoding an RNA-guided nuclease;

[0196] The guide RNA is capable of forming a complex with the nuclease and directing the complex to sequence-specific binding to the AR RNA.

[0197] In some embodiments of the present disclosure, the AR RNA is pre-mRNA and / or mature mRNA.

[0198] In some embodiments of the present disclosure, the AR RNA is human pre-mRNA and / or mature mRNA.

[0199] In some embodiments of the present disclosure, the AR RNA sequence is selected from any one of SEQ ID NOs: 394-396.

[0200] In some embodiments of the present disclosure, the AR RNA is the sequence numbered NM_000044.6 in the NCBI database (SEQ ID NO: 394), namely "transcript variant 1".

[0201] In some embodiments of the present disclosure, the AR RNA is the sequence numbered NM_001011645.3 in the NCBI database (SEQ ID NO: 395), namely "transcript variant 2".

[0202] In some embodiments of the present disclosure, the AR RNA is the sequence numbered ENST00000374690.9 in the Ensembl database (SEQ ID NO: 396).

[0203] In some embodiments of the present disclosure, the guide RNA guides the complex to bind to and cleave the AR RNA.

[0204] In some embodiments of the present disclosure, upon contact of the complex with a cell containing AR RNA, the level of the AR RNA or AR protein in the cell is reduced 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%.

[0205] In some embodiments of the present disclosure, upon contact of the complex with a cell comprising AR RNA, the level of the AR RNA in the cell is reduced 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%.

[0206] In some embodiments of the present disclosure, upon contact of the complex with a cell containing AR RNA, the level of the AR protein in the cell is reduced 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%.

[0207] In some embodiments of the present disclosure, the cell is a eukaryotic cell. In some embodiments of the present disclosure, the cell is a mammalian cell. In some embodiments of the present disclosure, the cell is a human cell.

[0208] The reduction in the RNA level can be tested using conventional methods in the art; including but not limited to qPCR methods, such as RT-qPCR methods. The target RNA (the AR RNA) level of untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be tested as a negative control, and the target RNA knockdown level of the experimental group compared to the negative control is calculated. The experimental group and the negative control group can be edited using the same Cas protein (e.g., by expressing the same Cas protein) and gRNA containing different guide sequences to compare the difference in target RNA levels; for example, the experimental group is edited using C13-2 and a gRNA as claimed in the present disclosure, and the negative control group is edited using C13-2 and a gRNA targeting, for example, a bacterial genome. The experimental group can also be compared with known editing tools, for example, compared with the CasRx+gRNA editing tool.

[0209] In some embodiments of the present disclosure, the number of off-target genes when the complex binds and cuts 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 conventional methods in the art. In some embodiments, the number of off-target genes is determined by taking the intersection of the differentially expressed gene set determined by RNA sequencing and the off-target gene set predicted by the program. In some embodiments, the number of off-target genes is determined by taking the intersection of the differentially expressed gene set determined by RNA sequencing and the off-target gene set predicted by the program. Off-target genes can be predicted using off-target gene prediction programs known in the art under conventional parameter settings. As a non-limiting example, the program predicts a target gene using the EMBOSS-water program in the whole genome and whole cDNA sequence of the target species (e.g., Homo sapiens or Mus musculus, etc.), setting the parameters to gap_extend = 0.5 & gap_extend = 10, using the forward and reverse strands of the gRNA guide sequence for alignment, filtering the prediction results, and obtaining predicted potential target genes (including on-target genes and / or off-target genes).

[0210] The reduction in the level of protein encoded by the target RNA (the AR RNA) can be tested using conventional methods in the art; including but not limited to ELISA and Western Blotting. Untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as negative controls, and the level of AR protein in the experimental group is calculated compared to the negative control group.

[0211] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 394-396.

[0212] In some embodiments of the present disclosure, the guide sequence of the guide RNA has 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 394-396.

[0213] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in SEQ ID NO: 394.

[0214] In some embodiments of the present disclosure, the guide sequence of the guide RNA has 100% sequence identity with the sequence shown in SEQ ID NO: 394.

[0215] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the nucleotide sequence consisting of nucleotides 2826 to 2990 of the sequence shown in SEQ ID NO: 394.

[0216] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 71-378.

[0217] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 71-80.

[0218] In some embodiments of the present disclosure, the guide sequence of the guide RNA has 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 71-80.

[0219] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a direct repeat sequence, and the direct repeat sequence interacts with the RNA-guided nuclease.

[0220] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas protein; optionally, the RNA-guided nuclease is a Cas9 protein, a Cas12 protein, or a Cas13 protein.

[0221] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas13 protein. Further optionally, the RNA-guided nuclease is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein.

[0222] In some embodiments of the present disclosure, the Cas13 protein comprises the sequence shown in SEQ ID NO: 1.

[0223] In some embodiments of the present disclosure, the RNA-guided nuclease comprises any one or more of the following: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0224] In some embodiments of the present disclosure, the subcellular localization signal is a nuclear localization signal and / or a nuclear export signal sequence.

[0225] In some embodiments of the present disclosure, the subcellular localization signal is a mitochondrial localization signal or a chloroplast localization signal sequence.

[0226] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a nuclear localization signal and / or a nuclear export signal sequence, and optionally a deaminase domain, a translation activation domain, or a translation repression domain.

[0227] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA-guided nuclease is linked to a regulatory sequence 1 that regulates its expression, and the polynucleotide sequence encoding the guide RNA is linked to a regulatory sequence 2 that regulates its expression.

[0228] In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter and enhancer sequence.

[0229] In some embodiments of the present disclosure, the regulatory sequence is selected from the group consisting of a CMV promoter, a CMV enhancer, a CBh promoter, a U6 promoter, and a specific promoter.

[0230] In some embodiments of the present disclosure, the gene editing system comprises:

[0231] A guide RNA comprising a guide sequence that hybridizes to the AR RNA or a polynucleotide sequence encoding the guide RNA, and a Cas13 protein or a polynucleotide sequence encoding the Cas13 protein;

[0232] The guide RNA is capable of forming a CRISPR complex with the Cas13 protein and guiding the complex sequence to specifically bind to and cut AR RNA.

[0233] In some embodiments, the coding sequence is linked to regulatory sequences that control its expression.

[0234] In some embodiments, the polynucleotide sequence encoding the Cas13 protein is connected to a regulatory sequence 1 that regulates its expression, and the polynucleotide sequence encoding the guide RNA is connected to a regulatory sequence 2 that regulates its expression.

[0235] In some embodiments, at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirty, fourteen, fifteen, or more) guide RNAs are expressed in tandem under the control of the same regulatory sequence. Further, tandem expression yields a pre-crRNA, which is then processed by the Cas13 protein to yield a mature guide RNA molecule.

[0236] In some embodiments, the polynucleotide sequence encoding the guide RNA and the polynucleotide sequence encoding the RNA-guided nuclease are located on the same vector.

[0237] In some embodiments of the present disclosure, the gene editing system is introduced into a cell or cell-free system in any of the following ways: (i) as mRNA encoding an RNA-guided nuclease and a guide RNA, (ii) as part of a single vector or plasmid, or divided into multiple vectors or plasmids, (iii) as separate RNA-guided nucleases and guide RNAs, or (iv) as an RNP complex of an RNA-guided nuclease and a guide RNA.

[0238] The twentieth aspect of the present disclosure provides a gene editing system guide RNA (gRNA), which comprises a guide sequence that hybridizes with AR RNA.

[0239] In some embodiments of the present disclosure, the AR RNA is pre-mRNA or mature mRNA. In some embodiments of the present disclosure, the AR RNA is mature mRNA.

[0240] In some embodiments of the present disclosure, the AR RNA is human AR RNA. In some embodiments of the present disclosure, the AR RNA is human mRNA.

[0241] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 394-396.

[0242] In some embodiments of the present disclosure, the guide sequence of the guide RNA has 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 394-396.

[0243] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in SEQ ID NO: 394.

[0244] In some embodiments of the present disclosure, the guide sequence of the guide RNA has 100% sequence identity to the sequence shown in SEQ ID NO: 394.

[0245] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the nucleotide sequence consisting of nucleotides 2826 to 2990 of the sequence shown in SEQ ID NO: 394.

[0246] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 71-378.

[0247] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 71-80.

[0248] In some embodiments of the present disclosure, the guide sequence of the guide RNA has 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 71-80.

[0249] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease and directing sequence-specific binding of the complex to the AR RNA.

[0250] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with the nuclease and guiding the complex to bind to and cleave the AR RNA.

[0251] In some embodiments of the present disclosure, the complex reduces the level of the AR RNA in a mammal (eg, a human).

[0252] In some embodiments of the present disclosure, after contacting the complex with a cell containing AR RNA, the level of the AR RNA in the cell is reduced, for example, the level of the target RNA in a cell expressing the target RNA.

[0253] In some embodiments of the present disclosure, the complex reduces the level of the AR RNA in the 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 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 AR RNA can be tested using conventional methods in the art; including but not limited to the qPCR method described in the Examples, untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as negative controls to calculate the AR RNA knockdown level of the experimental group compared to the negative control.

[0254] In some embodiments of the present invention, the number of off-target genes when the complex binds to and cuts 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 conventional methods in the art. In some embodiments, the number of off-target genes is determined by taking the intersection of the differentially expressed gene set determined by RNA sequencing and the off-target gene set predicted by the program. As a non-limiting example, the program prediction method is as follows: using the EMBOSS-water program to predict in the whole genome and full cDNA sequence of the target species (Homo sapiens / Mus mu sculus), setting the parameters to gap_extend=0.5&gap_extend=10, using the forward and reverse strands of the gRNA guide sequence for comparison, filtering the prediction results, and obtaining predicted potential target genes (including on-target genes and off-target genes).

[0255] In some embodiments of the present disclosure, the complex reduces the level of the protein encoded by the AR RNA in an animal (eg, a human).

[0256] In some embodiments of the present disclosure, upon contact of the complex with a cell containing the target RNA, the level of the protein encoded by the AR RNA in the cell is reduced. 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 the cell.

[0257] In some embodiments of the present disclosure, the complex reduces the level of AR protein in the 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 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 protein encoded by the AR RNA can be tested using conventional methods in the art; including but not limited to ELISA and Western Blotting. Untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as negative controls to calculate the knockdown level of AR protein in the experimental group compared to the negative control group.

[0258] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a backbone sequence, wherein the backbone sequence interacts with the RNA-guided nuclease. In some embodiments of the present disclosure, the backbone sequence is a direct repeat sequence (DR).

[0259] The twenty-first aspect of the present disclosure provides an isolated nucleic acid encoding the guide RNA according to any one of the present disclosure.

[0260] The twenty-second aspect of the present disclosure provides a vector, comprising a polynucleotide sequence encoding the guide RNA according to any one of the present disclosure, and a regulatory sequence for regulating the expression of the guide RNA.

[0261] In some embodiments of the present disclosure, the vector is an adeno-associated virus vector.

[0262] In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter and enhancer sequence.

[0263] In some embodiments of the present disclosure, the regulatory sequence is selected from the group consisting of a CMV promoter, a CMV enhancer, a CBh promoter, a U6 promoter, and a specific promoter.

[0264] In some embodiments of the present disclosure, the promoter is a chicken β-actin (CB) promoter. The chicken β-actin promoter can be a short chicken β-actin promoter or a long chicken β-actin promoter. In some embodiments, the promoter (e.g., chicken β-actin promoter) comprises an enhancer sequence, such as a cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence can be a short CMV enhancer sequence or a long CMV enhancer sequence. In some embodiments, the promoter comprises a long CMV enhancer sequence and a long chicken β-actin promoter. In some embodiments, the promoter comprises a short CMV enhancer sequence and a short chicken β-actin promoter. However, those skilled in the art will appreciate that a short CMV enhancer can be used together with a long CB promoter, and a long CMV enhancer can be used together with a short CB promoter. In some embodiments of the present disclosure, the promoter is a CBh promoter.

[0265] In some embodiments of the present disclosure, the promoter is a CBh promoter.

[0266] In some embodiments of the disclosure, the regulatory sequence comprises an HRE enhancer element.

[0267] In some embodiments of the present disclosure, the regulatory sequence comprises an NRS element and an HRE enhancer element in tandem.

[0268] The twenty-third aspect of the present disclosure provides a vector system, wherein the vector system comprises a polynucleotide sequence encoding the guide RNA disclosed herein and a second regulatory sequence that regulates the expression of the guide RNA; and a polynucleotide sequence encoding the RNA-guided nuclease and a first regulatory sequence that regulates the expression of the RNA-guided nuclease.

[0269] In some embodiments of the present disclosure, the vector system comprises one or more vectors.

[0270] In some embodiments of the present disclosure, the vector system comprises multiple vectors, the polynucleotide sequence encoding the guide RNA and the second regulatory sequence regulating the expression of the guide RNA are located on the second vector, and the polynucleotide sequence encoding the RNA-guided nuclease and the first regulatory sequence regulating the expression of the RNA-guided nuclease are located on the first vector.

[0271] In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter and enhancer sequence.

[0272] The twenty-fourth aspect of the present disclosure provides an adeno-associated viral vector, wherein the adeno-associated viral vector comprises DNA encoding an RNA-guided nuclease and the guide RNA of the present disclosure.

[0273] The twenty-fifth aspect of the present disclosure provides a lipid nanoparticle, wherein the lipid nanoparticle comprises the guide RNA described in the present disclosure and an mRNA encoding the RNA-guided nuclease.

[0274] The twenty-sixth aspect of the present disclosure provides a lentiviral vector, wherein the lentiviral vector comprises the guide RNA disclosed herein and an mRNA encoding an RNA-guided nuclease; optionally, the lentiviral vector is pseudotyped with an envelope protein; optionally, the mRNA encoding the RNA-guided nuclease is linked to an aptamer sequence.

[0275] The twenty-seventh aspect of the present disclosure provides a ribonucleoprotein complex, wherein the ribonucleoprotein complex is formed by the guide RNA and the RNA-guided nuclease described in the present disclosure.

[0276] The twenty-eighth aspect of the present disclosure provides a virus-like particle, wherein the virus-like particle comprises a ribonucleoprotein complex formed by the guide RNA and the RNA-guided nuclease described in the present disclosure; optionally, the RNA-guided nuclease is fused to the gag protein.

[0277] The twenty-ninth aspect of the present disclosure provides a eukaryotic cell, which comprises the gene editing system, guide RNA, nucleic acid, vector and / or vector system described in the present disclosure; optionally, the eukaryotic cell is a mammalian cell; further optionally, the eukaryotic cell is a human cell.

[0278] In some embodiments of the present disclosure, the eukaryotic cell comprises the gene editing system described in the present disclosure.

[0279] The thirtieth aspect of the present disclosure provides a pharmaceutical composition, which comprises the gene editing system, guide RNA, nucleic acid, vector and / or vector system described in any one of the present disclosures.

[0280] In some embodiments of the present disclosure, the pharmaceutical composition comprises the gene editing system described in any one of the present disclosures.

[0281] In some embodiments of the present disclosure, the pharmaceutical composition comprises the guide RNA, nucleic acid, vector and / or vector system described in any one of the present disclosures.

[0282] In some embodiments of the present disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0283] The thirty-first aspect of the present disclosure provides a use of the gene editing system, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell or pharmaceutical composition according to any one of the present disclosure in any of the following or in the preparation of a reagent for implementing any of the following schemes:

[0284] Cleave or nick one or more AR RNA molecules, activate or upregulate one or more AR RNAs, activate or inhibit translation of one or more AR RNA molecules, inactivate one or more AR RNA molecules, visualize, label, or detect one or more AR RNA molecules, bind one or more AR RNA molecules, transport one or more AR RNA molecules, and mask one or more AR RNA molecules.

[0285] In some embodiments of the present disclosure, there is provided a use of the gene editing system, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell or pharmaceutical composition according to any one of the present disclosure in any of the following or in the preparation of a reagent for implementing any of the following schemes:

[0286] Cleaves AR RNA molecules, inhibits translation of AR RNA molecules, and binds to AR RNA molecules.

[0287] In some embodiments of the present disclosure, there is provided a use of a gene editing system, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell or pharmaceutical composition according to any one of the present disclosure in cleaving AR RNA molecules or preparing an agent for cleaving AR RNA molecules.

[0288] In some embodiments of the present disclosure, there is provided a use of a gene editing system, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell or pharmaceutical composition according to any one of the present disclosure in binding to AR RNA molecules or preparing an agent for binding to AR RNA molecules.

[0289] In some embodiments of the present disclosure, the AR RNA is pre-mRNA or mature mRNA. In some embodiments of the present disclosure, the AR RNA is mature mRNA. In some embodiments of the present disclosure, the AR RNA is human AR RNA. In some embodiments of the present disclosure, the AR RNA is human AR mRNA.

[0290] The thirty-second aspect of the present disclosure provides a method for diagnosing, treating or preventing a disease or condition, by administering an effective amount of the gene editing system, guide RNA, nucleic acid, vector, vector system, adeno-associated viral vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell and / or pharmaceutical composition according to the present disclosure to a sample of a subject in need or to a subject in need.

[0291] In some embodiments of the present disclosure, a method for diagnosing, treating, or preventing a disease or condition is provided, comprising administering an effective amount of the gene editing system, eukaryotic cell, and / or pharmaceutical composition according to the present disclosure to a sample of a subject in need thereof or to a subject in need thereof.

[0292] In some embodiments of the present disclosure, a method for diagnosing, treating, or preventing a disease or condition is provided, comprising administering an effective amount of the gene editing system according to the present disclosure to a sample of a subject in need thereof or to a subject in need thereof.

[0293] In some embodiments of the present disclosure, the disease or disorder is a disease or disorder associated with AR RNA; alternatively, the disease or disorder is a disease or disorder caused by abnormally high expression of AR RNA.

[0294] In some embodiments of the present disclosure, the AR RNA is pre-mRNA or mature mRNA. In some embodiments of the present disclosure, the AR RNA is mature mRNA. In some embodiments of the present disclosure, the AR RNA is human AR RNA. In some embodiments of the present disclosure, the AR RNA is human AR mRNA.

[0295] In some embodiments of the disclosure, the disease or condition is androgenic alopecia. BRIEF DESCRIPTION OF THE DRAWINGS

[0296] FIG1 shows the designed gRNA targeting CTGF (CCN2) RNA.

[0297] Figure 2 shows the CTGF RNA level after editing with C13-2 combined with gRNA (detected by qPCR using CTGF-my primers).

[0298] Figure 3 shows the CTGF RNA levels after editing with C13-2 in combination with gRNA (using CTGF-11 primers).

[0299] FIG4 shows the designed gRNA targeting MITF RNA.

[0300] FIG5 shows the MITF RNA level after editing by C13-2 in combination with gRNA.

[0301] FIG6 shows the designed gRNA targeting SRD5A2 RNA.

[0302] FIG7 shows the SRD5A2 RNA level after editing with C13-2 in combination with gRNA (detected by qPCR using primer 2).

[0303] FIG8 shows the SRD5A2 RNA level after editing with C13-2 in combination with gRNA (detected by qPCR using primer 3).

[0304] Figure 9 shows the CTGF RNA level after editing with CasRx and gRNA combination (CTGF-my primer)

[0305] Figure 10 shows the CTGF RNA level after editing with CasRx and gRNA combination (CTGF-11 primer)

[0306] FIG11 shows MITF RNA levels after combined editing by CasRx and gRNA.

[0307] FIG12 shows SRD5A2 RNA levels after editing by CasRx combined with gRNA (primer 2).

[0308] FIG13 shows the gRNA screening results.

[0309] FIG14 shows the comparative test results of the Cas13 system and shRNA.

[0310] FIG15 shows the test results of C13-2 protein and CasRx in the same batch of experiments.

[0311] FIG16 shows the test results of AR protein expression levels in edited cells. DETAILED DESCRIPTION

[0312] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0313] Definition section:

[0314] As used herein, the term "gene editing system" refers to a protein, nucleic acid, or a combination thereof that can modify an endogenous target nucleic acid sequence (e.g., a target RNA) when introduced into a cell. The gene editing system includes, but is not limited to, a CRISPR-Cas system, a TALEN system, and a ZFN system. Specifically, the gene editing system may include RNA-guided nucleases and guide RNAs. Many gene editing systems suitable for use in the present disclosure are known in the art, including, but not limited to, 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, Cas 12d / CasY, Cas12e / CasX, Cas12f / CasZ, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cs e4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cs , Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, 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 (non-limiting examples include nucleic acid binding domain fragments); and systems comprising Cas9, Cas12, Cas13, TnpB, IscB, IsrB, Fancor nucleases, or fragments thereof.

[0315] In some embodiments, the gene editing system used in the methods described herein is a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) nuclease system, which is an engineered nuclease system based on a bacterial system that can be used for mammalian genome engineering. Typically, the system includes a CRISPR-associated nuclease (e.g., a Cas nuclease) and a guide RNA (gRNA).

[0316] As used herein, the term "knockdown" refers to a measurable decrease in a target RNA in a genetically modified cell compared to the level of the target RNA in a control cell. For example, the level of the target RNA in the genetically modified cell is reduced by >0%, ≥5%, ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90% or ≥95% compared to the level of the target RNA in the control cell. Those skilled in the art will readily understand how to use gene editing-mediated inhibition techniques to knock down a target RNA or portion thereof based on the details described herein.

[0317] As used herein, the term "inhibitor" refers to selective inhibition of a target RNA, for example, reducing the expression of the target RNA, or inhibiting the translation of the target RNA. The inhibition can be artificially induced, for example, by contacting a gene editing system with the target RNA to cut the target RNA, thereby reducing the level of the target RNA.

[0318] As used herein, the terms "gene editing system guide RNA", "guide RNA", "guideRNA" and "gRNA" are used interchangeably. The term "guide RNA" is used to refer to a molecule in a gene editing system that forms a complex with an RNA-guided nuclease and guides the sequence-specific binding of the complex to a target sequence. The guide RNA comprises a guide sequence that can hybridize with the target sequence. When the RNA-guided nuclease is a Cas protein, especially a Cas13 protein, the guide RNA typically comprises a direct repeat sequence connected to the guide sequence.

[0319] As used herein, the terms "guide sequence" and "targeting domain" are used interchangeably and refer to a contiguous nucleotide sequence in a gRNA that has partial or full complementarity with a target sequence in a target RNA and can hybridize to the target sequence in the target RNA through base pairing promoted by an RNA-guided nuclease. Full complementarity between the guide sequence and the target sequence described in the present invention is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a gene editing complex.

[0320] Suitable direct repeat (DR) sequences may be present in the CRISPR locus structure of prokaryotes (such as bacteria and archaea) and obtained through experimental screening; they may also be obtained through sequence modification or optimization on this basis, non-limiting examples of which include the deletion, replacement or addition of 1, 2, 3, 4 or more complementary base pairs in the complementary double-stranded region of the secondary structure of the DR sequence, and the deletion, replacement or addition of nucleotides on the loop of the stem-loop structure of the secondary structure of the DR sequence (for example, an aptamer sequence may be inserted into the loop). The size of the direct repeat sequence is usually tens of nucleotides, and some of its fragments are reverse complementary to each other, which means that a secondary structure is formed inside the RNA molecule, such as a stem-loop structure (often called a hairpin structure), while other fragments are unstructured. The direct repeat sequence is a constant part of the guide RNA molecule, which contains a strong secondary structure, which is conducive to the interaction between the RNA-guided nuclease and the guide RNA molecule.

[0321] The term "hybridization" or "hybridization" refers to the process in which fully or partially complementary polynucleotide chains come together to form a double-stranded structure or region under suitable hybridization conditions, including the association between the nucleic acids caused by hydrogen bonds. As used herein, the term "hybridization" includes situations in which double-stranded structures or regions contain one or more protrusions or mispairings. The intensity of hybridization and hybridization (that is, the intensity of association between nucleic acids) is affected by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, and the Tm of the formed hybrid. Although hydrogen bonds are generally formed between adenine and thymine, adenine and uracil, or between cytosine and guanine, other non-classical base pairs can also form hydrogen bonds. It is contemplated that the modified nucleotides can form hydrogen bonds that allow or promote hybridization using non-classical approaches.

[0322] As used herein, the term "target RNA" refers to a polynucleotide containing a target sequence, representing a specific sequence or its reverse complement that one wishes to bind, target, or modify using a gene editing system. For example, this can be a complete mature mRNA molecule or pre-mRNA molecule, or a fragment thereof.

[0323] As used herein, the term "target sequence" refers to a short sequence in a target RNA molecule that is complementary (completely complementary or partially complementary) to the guide sequence of the gRNA molecule. The gene editing complex is specifically located at the target sequence through the guide sequence and performs the corresponding function at or near this position. The length of the target sequence is often tens of nucleotides (nt), for example, about 10nt, about 20nt, about 30nt, about 40nt, about 50nt, about 60nt.

[0324] As used herein, the terms "cleavage" and "cleaving" refer to breaking covalent bonds (eg, covalent phosphodiester bonds) in the ribosyl phosphodiester backbone of a polynucleotide.

[0325] The ability of the guide RNA to guide the complex to bind specifically to the sequence of the target RNA can be evaluated by any suitable assay. For example, the components of the gene editing system sufficient to form the gene editing complex, including the guide RNA to be tested, can be provided to a host cell with the corresponding target RNA molecule, for example, by transfection of a vector encoding the components of the gene editing complex, and then evaluating the preferential cutting within the target sequence. Similarly, the cutting of the target RNA sequence can be evaluated in a test tube by providing the target RNA, the components of the gene editing complex, including the guide RNA to be tested and a control guide RNA different from the test guide RNA, and comparing the ability to bind to the target RNA or the rate of cutting the target RNA between the guide RNA to be tested and the control guide RNA. The ability of the guide RNA to guide the complex to cut the target RNA can also be evaluated by the assay described above.

[0326] The term "RNA-guided nuclease" refers to a polypeptide that binds to a specific target RNA sequence in a sequence-specific manner, and the polypeptide is guided to the target RNA by a guide RNA that complexes with the polypeptide and hybridizes to the target sequence on the target RNA. The cleavage of the target sequence by the RNA-guided nuclease can result in chain breaks. Although RNA-guided nucleases can cleave target sequences upon binding, the term "RNA-guided nuclease" also includes RNA-guided nucleases that are capable of binding but not cleaving target sequences that are inactivated. The RNA-guided nucleases disclosed herein include, but are not limited to, wild-type RNA-guided nucleases (e.g., C13-2, CasRx, etc.), variants thereof (e.g., mutants with complete loss of cleavage activity, mutants with partial loss of cleavage activity, mutants with increased cleavage activity, mutants with reduced off-target effects, mutants with reduced side effects), or functional fragments or fusion proteins thereof.

[0327] As used herein, the term "Cas protein" is a CRISPR-associated (Cas) polypeptide or protein that, when complexed or functionally combined with one or more guide RNAs, can be guided to a target sequence in a target RNA and can sometimes subsequently bind to or cleave the target RNA.

[0328] As used herein, the term "sequence identity" (identity or percent identity) is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a certain position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or a certain position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent sequence identity" (percent identity) between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% sequence identity. Typically, comparisons are made when two sequences are aligned to produce maximum sequence identity. Such comparisons can be performed using published and commercially available alignment algorithms and programs, such as, but not limited to, ClustalΩ, MAFFT, Probcons, T-Coffee, Probalign, BLAST, which can be reasonably selected by one of ordinary skill in the art. Those skilled in the art can determine appropriate parameters for aligning sequences, including, for example, any algorithms needed to achieve better alignment or optimal comparison over the entire length of the sequences being compared, as well as any algorithms needed to achieve better alignment or optimal comparison over a portion of the sequences being compared.

[0329] As used herein, the term "regulatory sequence" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory sequences include those elements that direct the continuous expression of a nucleotide sequence in many types of host cells and those elements that direct the expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in desired tissues of interest such as muscle, neurons, bones, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., neuronal cells, lymphocytes). Regulatory sequences can also direct expression in a time-dependent manner such as a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue-specific or cell-type-specific. The term regulatory sequence also encompasses enhancer elements such as WPRE, CMV enhancer, SV40 enhancer, and the intron sequence between exons 2 and 3 of rabbit β-globin. Those skilled in the art will appreciate that the design of the expression vector can depend on factors such as the choice of host cells to be transformed, the desired expression level, and the like. The vector can be introduced into a host cell to produce the RNA-guided nuclease and / or guide RNA of the present invention.

[0330] As used herein, the term "promoter" has the meaning commonly recognized in the art.

[0331] As used herein, the term "enhancer" has the meaning commonly recognized in the art.

[0332] As used herein, when referring to a nucleotide sequence / DNA / RNA encoding a protein, RNA, or gene editing complex, the coding sequence may be codon-optimized, for example, for expression in a eukaryotic cell environment, for expression in a mammalian cell environment, or for expression in a human cell environment.

[0333] As used herein, the term "codon optimization" refers to the process of changing the codons of a given gene in such a way that the polypeptide sequence encoded by the gene remains the same, but the altered codons improve expression of the polypeptide sequence. For example, if the polypeptide is a human protein sequence and is expressed in E. coli, codon optimization of the DNA sequence to change the human codons to codons that are more efficiently expressed in E. coli will generally improve expression.

[0334] As used herein, the term pharmaceutically acceptable excipient refers to a diluent, adjuvant, pharmaceutical carrier or other excipient administered together with the active ingredient. Its selection depends on the method of administration of purposes and expectation. Excipient should not be incompatible with the active ingredient, for example, producing any undesirable biological effect or interacting with any other component of the pharmaceutical composition in a harmful manner. Pharmaceutical composition can be prepared by known methods in the field of pharmaceutical preparation.

[0335] When referring to an RNA sequence, "T" in the sequence can be used interchangeably with "U." When referring to a "guide sequence," "T" in the sequence can be used interchangeably with "U." When referring to a "direct repeat sequence," "T" in the sequence can be used interchangeably with "U."

[0336] As used herein, the term "cosmetics" refers to products that are applied to any part of the human body (skin, hair, nails, lips, etc.) by smearing, sprinkling, spraying, or other similar methods to cleanse, perfume, alter appearance, correct body odor, maintain skin, or preserve a healthy appearance. Cosmetics may include any of the products described in the "National Standard of the People's Republic of China—Terminology of Cosmetics" (GB / T 27578-2011).

[0337] As used herein, the term "pharmaceutically, cosmetically, chemically, or biologically acceptable" refers to substances that are suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio. Examples include pharmaceutically acceptable excipients and cosmetically acceptable excipients, thickeners, or diluents. For example, the cosmetics disclosed herein may contain liquids such as water, saline, glycerol, and ethanol; in addition, auxiliary substances such as fillers, lubricants, glidants, wetting agents or emulsifiers, fragrances, pH buffering substances, etc. may also be present.

[0338] guide RNA

[0339] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease (also referred to as a gene editing complex) and guiding the complex to specifically bind to the sequence of the target RNA (CTGF RNA, MITF RNA, SRD5A2 RNA, or AR RNA) and cut the target RNA, which comprises a guide sequence and a backbone sequence. The backbone sequence interacts with the RNA-guided nuclease. The backbone sequence is the sequence that generally remains unchanged in the guide RNA molecule when designing the guide RNA molecule. For example, the backbone sequence may refer to the portion of the guide RNA molecule other than the guide sequence. In some embodiments of the present disclosure, the backbone sequence is a direct repeat (DR).

[0340] In some embodiments of the present disclosure, the guide sequence has at least 80% sequence identity with the target RNA. In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity with the target RNA. Further, in some embodiments of the present disclosure, the guide sequence has 100% sequence identity with the target RNA.

[0341] In some embodiments of the disclosure, the guide sequence has at least 80% sequence identity to the sequence shown in any one of SEQ ID NOs: 14, 22, or 35. In some embodiments of the disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 14, 22, or 35. Further, in some embodiments of the disclosure, the guide sequence has 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 14, 22, or 35.

[0342] In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the nucleotide sequence of nucleotides 494 to 785 of the sequence set forth in SEQ ID NO: 14, nucleotides 404 to 606 of the sequence set forth in SEQ ID NO: 22, or nucleotides 484 to 820 of the sequence set forth in SEQ ID NO: 35.

[0343] In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95% or 100% sequence identity with the nucleotide sequence of nucleotide 494, 604, 678 or 761 to nucleotide 518, 628, 702 or 785 of the sequence set forth in SEQ ID NO: 14.

[0344] In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95% or 100% sequence identity to the nucleotide sequence of nucleotide 404, 429, 453, 479, 509, 546 or 582 to nucleotide 428, 453, 477, 503, 533, 570 or 606 of the sequence set forth in SEQ ID NO: 22.

[0345] In some embodiments of the present disclosure, the guide sequence has at least 85%, at least 90%, at least 95% or 100% sequence identity to the nucleotide sequence of nucleotide 484, 524, 547, 585, 635, 721, 755 or 796 to nucleotide 508, 548, 571, 609, 659, 745, 779 or 820 of the sequence set forth in SEQ ID NO: 35.

[0346] 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 comprises 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.

[0347] In some embodiments of the disclosure, the guide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, and 54. In some embodiments of the disclosure, the guide sequence comprises any one of SEQ ID NOs: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, and 54. In some embodiments of the disclosure, the guide sequence is any one of SEQ ID NOs: 8, 9, 10, 12, 23-29, 36, 38, 40, 42, 43, 44, 46, and 54.

[0348] In some embodiments of the disclosure, the guide sequence comprises 20-40, 20-35, 20-30, or 25-30 nucleotides.

[0349] In some embodiments of the present disclosure, the guide sequence hybridizes to the target RNA with no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch.

[0350] In some embodiments of the present disclosure, the guide sequence has 100% sequence identity with the target RNA, i.e., is fully complementary.

[0351] In some embodiments of the present disclosure, the backbone sequence is a direct repeat sequence, which comprises 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 to the sequence shown in SEQ ID NO: 2 or 3.

[0352] In some embodiments of the present disclosure, the direct repeat sequence comprises a sequence as shown in SEQ ID NO: 2 or 3. In some embodiments of the present disclosure, the direct repeat sequence consists of a sequence as shown in SEQ ID NO: 2 or 3.

[0353] In some embodiments of the present disclosure, the guide sequence is located at the 3' end or 5' end of the direct repeat sequence. In some embodiments of the present disclosure, the guide sequence is located at the 3' end of the direct repeat sequence. In some embodiments of the present disclosure, the guide sequence is located at the 5' end of the direct repeat sequence.

[0354] In some embodiments of the present disclosure, the guide RNA comprises an aptamer sequence.

[0355] In some embodiments of the present disclosure, the aptamer sequence is inserted into the loop of the stem-loop structure of the direct repeat sequence secondary structure of the guide RNA.

[0356] In some embodiments of the present disclosure, the guide RNA comprises modified nucleotides. The modifications include but are not limited to 2'-O-methyl, 2'-O-methyl-3'-thiophosphate or 2'-O-methyl-3'-thio PACE modifications. In some embodiments of the present disclosure, the guide RNA comprises modified nucleotides, and the modified nucleotides are selected from deoxyribonucleotides and locked nucleic acids (LNA). In some embodiments, the guide RNA comprises at least one chemically modified nucleotide. Chemically modified guide RNAs are described in Hendel et al., Nat. Biotechnol. 33 (9): 985-989 (2015), which is incorporated herein by reference in its entirety.

[0357] In some embodiments, the guide RNA is a hybrid RNA-DNA guide, i.e., some RNA nucleotides in the guide RNA are replaced by DNA nucleotides. In some embodiments, the guide RNA is a hybrid RNA-LNA (locked nucleic acid) guide, i.e., some RNA nucleotides in the guide RNA are replaced by LNA nucleotides. Hybrid RNA-DNA guide polynucleotides are described in WO2016 / 123230, which is incorporated herein by reference in its entirety.

[0358] In some embodiments of the present disclosure, the target RNA is located in the nucleus and / or cytoplasm of a eukaryotic cell.

[0359] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease (also referred to as a gene editing complex) and guiding the sequence-specific binding of the complex to the target RNA.

[0360] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease and directing the complex to bind to and cleave the target RNA.

[0361] In some embodiments of the disclosure, the complex reduces the level of the target RNA in the cell.

[0362] In some embodiments of the present disclosure, upon contacting the complex with a cell comprising the target RNA, the level of the target RNA in the cell is reduced, for example, the level of the target RNA in a cell expressing the target RNA.

[0363] In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the 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 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 can be tested using conventional methods in the art; including but not limited to qPCR methods as described in the examples, such as RT-qPCR methods. Untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as negative controls to calculate the target RNA knockdown level of the experimental group compared to the negative control. The experimental group and the negative control group can be edited using the same Cas protein (e.g., by expressing the same Cas protein) and gRNAs containing different guide sequences to compare the difference in target RNA levels; for example, the experimental group is edited using C13-2 and a gRNA as claimed in the present disclosure, and the negative control group is edited using C13-2 and a gRNA targeting, for example, a bacterial genome. The experimental groups can also be compared with known editing tools, such as CasRx+gRNA editing tools.

[0364] In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the 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 the cell by at least 40%. In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the cell by at least 80%. In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the cell by at least 85%. In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the cell by at least 90%.

[0365] In some embodiments of the present invention, the number of off-target genes when the complex binds to and cuts 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 conventional methods in the art. In some embodiments, the number of off-target genes is determined by differentially expressed genes determined by RNA sequencing. In some embodiments, the number of off-target genes is determined by taking the intersection of the differentially expressed gene set determined by RNA sequencing and the off-target gene set predicted by the program. As a non-limiting example, the program predicts the target gene using the EMBOSS-water program in the whole genome and full cDNA sequence of the target species (Homo sapiens / Mus musculus), setting the parameters to gap_extend=0.5&gap_extend=10, using the forward and reverse strands of the gRNA guide sequence for alignment, filtering the prediction results, and obtaining predicted potential target genes (including on-target genes and off-target genes).

[0366] In some embodiments of the present disclosure, upon contact of the complex with a cell containing a target RNA, the level of a protein encoded by the target RNA in the cell is reduced. In some embodiments of the present disclosure, the protein encoded by the target RNA is CTGF protein, MITF protein, SRD5A2 protein, or AR protein. In some embodiments of the present disclosure, the complex reduces the level of CTGF protein, MITF protein, SRD5A2 protein, or AR protein in the cell.

[0367] In some embodiments of the present disclosure, the complex reduces the level of CTGF protein, MITF protein, SRD5A2 protein, or AR protein 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 level of the target RNA-encoded protein can be tested using conventional methods in the art; including but not limited to ELISA and Western Blotting. Untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as negative controls to calculate the knockdown level of CTGF protein, MITF protein, SRD5A2 protein, or AR protein in the experimental group compared to the negative control group.

[0368] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas protein. In some embodiments, the cleavage activity of the RNA-guided nuclease is completely or partially lost.

[0369] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas protein.

[0370] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas protein, and the guide RNA comprises a guide sequence and a direct repeat sequence.

[0371] In some embodiments of the present disclosure, the Cas protein is Cas9 protein, Cas12 protein or Cas13 protein.

[0372] In some embodiments, the Cas9 protein is selected from SpCas9, SaCas9, Nme2Cas9, Nme3Cas9, CjCas9, NmCas9, and FnCas9.

[0373] In some embodiments, 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.

[0374] In some embodiments of the present disclosure, the Cas protein is a Cas13 protein.

[0375] In some embodiments of the present disclosure, the Cas protein is Cas13a protein, Cas13b protein, Cas13c protein or Cas13d protein. In some embodiments of the present disclosure, the Cas protein is a Cas13a protein, Cas13b protein or Cas13c protein in which the catalytic center RxxxxH motif of two HEPN domains carries a mutation, and the cleavage activity of these Cas proteins is completely or partially lost. In some embodiments, the Cas protein is a CasRx protein. In some embodiments, the Cas protein is a dCasRx in which both HEPN domains carry mutations (R239A, H244A of HEPN-1 and R858A, H863A of HEPN-2).

[0376] In some embodiments of the present disclosure, the Cas protein comprises the sequence shown in SEQ ID NO: 1.

[0377] In some embodiments of the present disclosure, the guide sequence is located at the 3' end or 5' end of the direct repeat sequence. In some embodiments of the present disclosure, the guide sequence is located at the 3' end of the direct repeat sequence. In some embodiments of the present disclosure, the guide sequence is located at the 5' end of the direct repeat sequence.

[0378] In some embodiments of the present disclosure, the direct repeat sequence comprises the sequence shown in SEQ ID NO: 2. In some embodiments of the present disclosure, the direct repeat sequence consists of the sequence shown in SEQ ID NO: 2.

[0379] target RNA

[0380] The gene editing systems and compositions disclosed herein can be used to target one or more target RNA molecules, such as target RNA molecules present in a biological sample. In some embodiments, the target RNA is pre-mRNA or mRNA (mature mRNA).

[0381] In some embodiments of the present disclosure, the target RNA is CTGF RNA, MITF RNA, or SRD5A2 RNA, or a fragment thereof. In some embodiments of the present 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 the present disclosure, the target RNA is CTGF mRNA, MITF mRNA, or SRD5A2 mRNA, or a fragment thereof. In some embodiments of the present disclosure, the target RNA is CTGF pre-mRNA, MITF pre-mRNA, or SRD5A2 pre-mRNA, or a fragment thereof. In some embodiments of the present disclosure, the target RNA is CTGF mRNA, MITF mRNA, or SRD5A2 mRNA. In some embodiments of the present disclosure, the target RNA is mammalian CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of the present disclosure, the target RNA is human CTGF RNA, MITF RNA, or SRD5A2 RNA. In some embodiments of the present disclosure, the target RNA is human CTGF mRNA, MITF mRNA, or SRD5A2 mRNA.

[0382] In some embodiments of the present disclosure, the target RNA sequence is a sequence as shown in SEQ ID NO: 14, 22 or 35.

[0383] In some embodiments of the present disclosure, the target RNA sequence is nucleotides 494 to 785 of the sequence shown in SEQ ID NO: 14, nucleotides 404 to 606 of the sequence shown in SEQ ID NO: 22, or nucleotides 484 to 820 of the sequence shown in SEQ ID NO: 35.

[0384] In some embodiments of the present disclosure, the target RNA sequence is a nucleotide sequence from nucleotide 494, 604, 678 or 761 to nucleotide 518, 628, 702 or 785 of the sequence shown in SEQ ID NO: 14 (human CCN2 mRNA, NCBI NM_001901.4).

[0385] In some embodiments of the present disclosure, the target RNA sequence is a nucleotide sequence from nucleotide 404, 429, 453, 479, 509, 546 or 582 to nucleotide 428, 453, 477, 503, 533, 570 or 606 of the sequence shown in SEQ ID NO: 22 (human MITF mRNA, NCBI NM_001354607.2).

[0386] In some embodiments of the present disclosure, the target RNA sequence is a nucleotide sequence from nucleotide 484, 524, 547, 585, 635, 721, 755 or 796 to nucleotide 508, 548, 571, 609, 659, 745, 779 or 820 of the sequence shown in SEQ ID NO: 35 (human SRD5 A2 mRNA, NCBI XM_011533072.3).

[0387] When referring to the target nucleic acid, CTGF mRNA, MITF mRNA or SRD5A2 mRNA transcript variants (NCBI accession numbers NM_001901.4, NM_001354607.2, XM_011533072.3) herein, it is not intended to limit the gRNA disclosed herein to targeting only this nucleic acid molecule; those skilled in the art should know that the gRNA disclosed herein can be used to target other nucleic acid molecules, such as human CTGF pre-mRNA, MITF pre-mRNA or SRD5A2 pre-mRNA, other transcript variants of CTGF mRNA, MITF mRNA or SRD5A2 mRNA, and the like.

[0388] In some embodiments of the present disclosure, the gene editing system described herein can be used to reduce the expression level of CTGF RNA, MITF RNA, or SRD5A2 RNA; for example, using an RNA-guided nuclease to contact and cleave CTGF RNA, MITF RNA, or SRD5A2 RNA under the guidance of a gRNA. In some embodiments, the gene editing system described herein can be used to reduce the expression level of CTGF RNA, MITF RNA, or SRD5A2 RNA in cells.

[0389] In some embodiments of the present disclosure, the target RNA is AR RNA. In some embodiments of the present disclosure, the target RNA is human AR RNA. In some embodiments of the present disclosure, the target RNA is human mRNA.

[0390] In some embodiments of the present disclosure, the target RNA is AR RNA or a fragment thereof.

[0391] In some embodiments of the present disclosure, the target RNA is AR mRNA or a fragment thereof.

[0392] In some embodiments of the present disclosure, the target RNA is AR pre-mRNA or a fragment thereof.

[0393] In some embodiments of the present disclosure, the target RNA is selected from the sequence shown in any one of SEQ ID NOs: 394-396.

[0394] In some embodiments of the present disclosure, the gene editing system described herein can be used to reduce the expression level of AR RNA; for example, using an RNA-guided nuclease to contact and cleave AR RNA under the guidance of gRNA. In some embodiments, the gene editing system described herein can be used to reduce the expression level of AR RNA in cells.

[0395] Aptamer / aptamer sequence

[0396] In some embodiments, the guide polynucleotide further comprises an aptamer sequence. In some embodiments, the aptamer sequence is inserted into a loop of the guide polynucleotide. In some embodiments, the aptamer sequence is attached to the end of the guide polynucleotide.

[0397] In some embodiments, the aptamer sequence comprises an MS2 aptamer sequence, a PP7 aptamer sequence, or a Qβ aptamer sequence.

[0398] Adaptor protein

[0399] In some embodiments, the gene editing system further comprises a fusion protein comprising an adaptor protein and a fusion domain, or a nucleic acid encoding the fusion protein, wherein the adaptor protein is capable of binding to an aptamer sequence.

[0400] In some embodiments, 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, the fusion domain comprises a cytosine deaminase domain, an adenosine deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, or an affinity or reporter tag or domain.

[0401] RNA-guided nucleases

[0402] When referring to RNA-guided nucleases in this disclosure, it can refer to the RNA-guided nuclease itself in a narrow sense, or it can refer to a fusion protein obtained by covalently linking or fusing the RNA-guided nuclease with other domains.

[0403] In some embodiments of the present disclosure, the RNA-guided nuclease is optionally selected from the group consisting of 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, Csn1 , 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, Systems comprising Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, Cas13f, TnpB, IscB, IsrB, Fancor; or fragments thereof (non-limiting examples include nucleic acid binding domain fragments).

[0404] In some embodiments of the present disclosure, the RNA-guided nuclease is selected from Cas9, Cas12, Cas13, TnpB, IscB, IsrB, Fancor nuclease; or fragments thereof, including but not limited to nucleic acid binding domain fragments.

[0405] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas protein.

[0406] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas13 protein.

[0407] In some embodiments of the present disclosure, the RNA-guided nuclease is selected from SpCas9, SaCas9, Nme2Cas9, Nme3Cas9, CjCas9, NmCas9, FnCas9, PpnCas9, FrCas9, SauCas9, SauriCas9, ScaCas9, St1Cas9, BlatCas9, CdiCas9, GeoCas9, fragments thereof, and mutants or fragments of mutants thereof.

[0408] In some embodiments of the present disclosure, the RNA-guided nuclease is selected from AsCpf1, enAsCas12a (addgene plasmid #196724), dFnCas12a (addgene plasmid #136379), ErCas12a, LbCas12a D832A, LbCas12a H759A, LbCas12a E795L, FnCas12a3, FnCas1 2a D917A, AsCas12a R1226A, AsCas12a D908A, AsCas12a E174R / S542R, AsCas12a(S542R / K548V / N552R), PrCas12a, PxCas12a, PcCas12a, PdCas12a, Mb2Cas12a, Mb3Cas12a, MlCas12 a, CMaCas12a, 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 Vogelbac teria), CasY.4 (from Parcubacteria), CasY.5 (from Komeilibacteria), CasY.6 (from Kerfeldbacteria), PlmCasX, DpbCasX, Un1Cas12f, CnCas12f1, enRhCas12f1, AsCas12f1, SpaCas12f1, Cas12g1 (addgene plasmid#120879), Cas12h(SEQ ID NO:1), Cas12i1(addgene plasmid#171670), Cas12i2(addgene plasmid#188275), Cas12i1(addgene plasmid#188275) of WO2021113522A1plasmid#120882)、Cas12i2(addgene plasmid#120883) and CN111757889B single-stranded Cas12f.4 / Cas12f.5 / Cas1 2f.6 Cas12i Processor, dSiCas12i(D1049A), SiCas12i, Si2Cas12i, WiCa s12i , Wi2Cas12i , Wi3Cas12i , SaCas12i , Sa2Cas12i , Sa3Cas12i , WaCas12i , Wa2Cas12i , xCas12i , hfCas12Max , Cas12i - Max ( addgene plasmid#188276); plasmid#188498); plasmid#181787)、Cas12k-TnsC(addgene plasmid#181789), Cas12l, MmCas12m, MmCas12mΔZF(H549A,C552A), d Cas12m-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-1and TnpB-2) 、TnpB Alicyclobacillus_macrosporagiidus_strain_DSM_17980 、TnpB Haloactinospora_alba_strain_DSM_45015 、TnpBLipingzhangella_halophila_strain_DSM_102030, TnpB Meiothermus_Silvanus_DSM_9946, TnpB QNFX01000004, ISDra2 TnpB (PDB:8H1J), KraIscB-1, AwaIscB, OgeuIscB, GtFz1 (from Guillardia theta), SpuFz1 (from Spizello myces punctatus), NlovFz2 (from Percolozoa Naegleria lovaniensis), MmeFz2 (from Mercenaria mercenaria), fragments thereof, and mutants or mutant fragments thereof.

[0409] In some embodiments of the present disclosure, the RNA-guided nuclease is selected from wild-type RNA-guided nucleases (including but not limited to CasRx, C13-2, etc.), variants thereof (including but not limited to mutants with complete loss of cleavage activity, mutants with partial loss of cleavage activity, mutants with increased cleavage activity, mutants with reduced off-target / bypass effects), or functional fragments or fusion proteins thereof.

[0410] In some embodiments of the present disclosure, the RNA-guided nuclease comprises any one or more of the following fused domains: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0411] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a subcellular localization signal.

[0412] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a subcellular localization signal and a deaminase domain.

[0413] In some embodiments of the present disclosure, the subcellular localization signal is selected from a nuclear localization signal and a nuclear export signal.

[0414] In some embodiments, the RNA-guided nuclease is fused to at least one subcellular localization signal. Exemplary subcellular localization signals include organelle localization signals, such as a nuclear localization signal (NLS), a nuclear export signal (NES), or a mitochondrial localization signal.

[0415] In some embodiments, the RNA-guided nuclease is fused to at least one heterologous NLS. In some embodiments, the RNA-guided nuclease is fused to at least two NLSs. In some embodiments, the RNA-guided nuclease is fused to at least three NLSs. In some embodiments, the RNA-guided nuclease is fused to at least one N-terminal NLS and at least one C-terminal NLS. In some embodiments, the RNA-guided nuclease is fused to at least two C-terminal NLSs. In some embodiments, the RNA-guided nuclease is fused to at least two N-terminal NLSs.

[0416] In some embodiments, the NLS is independently selected from 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), PPK KARED (SEQ ID NO: 406), POPKKKPL (SEQ ID NO: 407), SALIKKKKKMAP (SEQ ID NO: 408), DRLRR (SEQ ID NO: 410). 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).

[0417] In some embodiments, the RNA-guided nuclease is fused to a heterologous NES. In some embodiments, the RNA-guided nuclease is fused to at least two NESs. In some embodiments, the RNA-guided nuclease is fused to at least three NESs. In some embodiments, the RNA-guided nuclease is fused to at least one N-terminal NES and at least one C-terminal NES. In some embodiments, the RNA-guided nuclease is fused to at least two C-terminal NESs. In some embodiments, the RNA-guided nuclease is fused to at least two N-terminal NESs.

[0418] In some embodiments, the NES is independently selected from an adenovirus type 5 E1B NES, an HIV Rev NES, a MAPK NES, or a PTK2 NES.

[0419] In some embodiments, the RNA-guided nuclease is fused to an NLS and an NES, with a cleavable linker present between the NLS and the NES. In some embodiments, the NES promotes production of delivery particles (e.g., virus-like particles) comprising the RNA-guided nuclease in a production cell line. In some embodiments, cleavage of the linker in a target cell can expose the NLS and promote nuclear localization of the RNA-guided nuclease in the target cell. Exemplary fusion domains (e.g., fused heterologous protein domains) include domains that can cleave RNA (e.g., PIN endonuclease domains, NYN domains, SMR domains from SOT1, or RNase domains from staphylococcal nuclease), domains that can affect RNA stability (e.g., tristetraprolin (TTP) or domains from UPF1, EXOSC5, and STAU1), domains that can edit nucleotides or ribonucleotides (e.g., cytidine deaminases, PPR proteins, adenosine deaminases, ADAR family proteins, or APOBEC family proteins), domains that can activate translation (e.g., eIF4E and other translation initiation factors, yeast poly(A) binding protein, or domains of GLD2), domains that can inhibit translation (e.g., Pumilio or FBF). These proteins may be PUF proteins, deadenylases, CAF1, Argonaute proteins), domains that can methylate RNA (e.g., domains from m6A methyltransferase factors such as METTL14, METTL3, or WTAP), domains that can demethylate RNA (e.g., human alkylation repair homolog 5), domains that can affect splicing (e.g., the RS-rich domain of SRSF1, the Gly-rich domain of hnRNP A1, the alanine-rich motif of RBM4, or the proline-rich motif of DAZAP1), domains that can enable affinity purification or immunoprecipitation (e.g., FLAG, HA, biotin, or HALO tags), and domains that can enable proximity-based protein labeling and recognition (e.g., biotin ligases such as BirA or peroxidases such as APEX2 to biotinylate target DNA-interacting proteins).

[0420] In some embodiments of the present disclosure, the deaminase domain includes an adenosine deaminase domain. In some embodiments, the nuclease activity is maintained, partially inactivated or completely inactivated RNA-guided nuclease (non-limiting examples include the Cas13 protein with a mutant HEPN domain or the catalytically inactivated Cas13 protein) is covalently linked or fused to the adenosine deaminase domain to guide the A-to-I deaminase activity of RNA transcripts in mammalian cells. Cox et al., Science 358 (6366): 1019-1027 (2017) describes an adenosine deaminase domain for targeting A-to-I RNA editing based on ADAR2 engineering, which is incorporated herein by reference in its entirety. In some other embodiments, the adenosine deaminase domain is covalently linked or fused to a linker protein that is capable of binding to an aptamer sequence inserted into or attached to the guidance polynucleotide, thereby allowing the adenosine deaminase domain to be non-covalently linked to the RNA-guided nuclease complexed with the guidance polynucleotide.

[0421] In some embodiments of the present disclosure, the deaminase domain includes a cytosine deaminase domain. In some embodiments, the nuclease activity is maintained, partially inactivated or completely inactivated RNA-guided nuclease (non-limiting examples include the Cas13 protein with a mutant HEPN domain or the Cas13 protein with catalytic inactivation) is covalently linked or fused to a cytosine deaminase domain to guide the C-to-U deaminase activity of RNA transcripts in mammalian cells. Abudayyeh et al., Science 365(6451): 382-386 (2019) describes a cytosine deaminase domain for targeting C-to-U RNA editing evolved from ADAR2, which is incorporated herein by reference in its entirety. In other embodiments, the CDase domain is covalently linked or fused to an adaptor protein that is capable of binding an aptamer sequence inserted into or appended to the guide polynucleotide, thereby allowing non-covalent attachment of the CDase domain to the RNA-guided nuclease complexed with the guide polynucleotide.

[0422] In some embodiments, the fusion domain comprises a splicing factor domain. In some embodiments, the Cas13 protein with a mutant HEPN domain or a catalytically inactive Cas13 protein is covalently linked or fused to a splicing factor domain to guide the alternative splicing of the target RNA in mammalian cells. Non-limiting examples of splicing factor domains include the RS-rich domain of SRSF1, the Gly-rich domain of hnRNPA1, the alanine-rich motif of RBM4, or the proline-rich motif of DAZAP1. In other embodiments, the splicing factor domain is covalently linked or fused to a linker protein that is capable of binding to an aptamer sequence inserted into or attached to the guide RNA, thereby allowing the splicing factor domain to be non-covalently linked to the Cas13 protein compounded with the guide RNA.

[0423] In some embodiments of the present disclosure, the RNA-guided nuclease is covalently linked or fused to a translation activation domain. In some embodiments, the nuclease (non-limiting examples such as the Cas13 protein with a mutation HEPN domain or the Cas13 protein with catalytic inactivation) guided by the RNA of nuclease activity maintenance, partial inactivation or complete inactivation is covalently linked or fused to a translation activation domain to activate or increase the expression of the target RNA. Non-limiting examples of translation activation domains include eIF4E and other translation initiation factors, yeast poly (A) binding protein or the domain of GLD2. In some other embodiments, the translation activation domain is covalently linked or fused to a linker protein that can bind to an aptamer sequence inserted into or attached to the guidance polynucleotide, thereby allowing the translation activation domain to be non-covalently linked to the nuclease guided by the RNA compounded with the guidance polynucleotide.

[0424] In some embodiments of the present disclosure, the RNA-guided nuclease is covalently linked or fused to a translation inhibition domain. In some embodiments, the nuclease activity is maintained, partially inactivated, or completely inactivated RNA-guided nuclease (non-limiting examples include the Cas13 protein with a mutant HEPN domain or the catalytically inactivated Cas13 protein) is covalently linked or fused to a translation inhibition domain to inhibit or reduce the expression of the target RNA. Non-limiting examples of translation inhibition domains include Pumilio or FBF PUF proteins, deadenylases, CAF1, Argonaute proteins. In other embodiments, the translation inhibition domain is covalently linked or fused to a linker protein that is capable of binding to an aptamer sequence inserted into or attached to the guidance polynucleotide, thereby allowing the translation inhibition domain to be non-covalently linked to the RNA-guided nuclease complexed with the guidance polynucleotide.

[0425] In some embodiments of the present disclosure, the RNA-guided nuclease is covalently linked to or fused with an RNA methylation domain. In some embodiments, the nuclease (non-limiting examples such as the Cas13 protein with a mutant HEPN domain or the catalytically inactivated Cas13 protein) of RNA-guided nuclease activity retention, partial inactivation or complete inactivation is covalently linked to or fused with an RNA methylation domain for methylation of the target RNA. Non-limiting examples of RNA methylation domains include m6A domains, such as METTL14, METTL3 or WTAP. In some other embodiments, the RNA methylation domain is covalently linked to or fused with an adapter protein that can bind to an aptamer sequence inserted into or attached to the guidance polynucleotide, thereby allowing the RNA methylation domain to be non-covalently linked to the RNA-guided nuclease complexed with the guidance polynucleotide.

[0426] In some embodiments of the present disclosure, the RNA-guided nuclease is covalently linked to or fused to an RNA demethylation domain. In some embodiments, the nuclease activity is maintained, partially inactivated, or completely inactivated. The nuclease guided by RNA (non-limiting examples such as the Cas13 protein with a mutant HEPN domain or the catalytically inactivated Cas13 protein) is covalently linked to or fused to an RNA demethylation domain for demethylation of the target RNA. Non-limiting examples of RNA demethylation domains include human alkylation repair homolog 5 or ALKBH5. In some other embodiments, the RNA demethylation domain is covalently linked to or fused to an adapter protein that can bind to an aptamer sequence inserted into or attached to the guidance polynucleotide, thereby allowing the RNA demethylation domain to be non-covalently linked to the RNA-guided nuclease complexed with the guidance polynucleotide.

[0427] In some embodiments of the present disclosure, the RNA-guided nuclease is covalently linked or fused to a ribonuclease domain. In some embodiments, a nuclease (non-limiting example, such as the Cas13 protein with a mutant HEPN domain or the Cas13 protein with catalytic inactivation) guided by RNA with retained, partially inactivated, or completely inactivated nuclease activity is covalently linked or fused to a ribonuclease domain to cut the target RNA. Non-limiting examples of ribonuclease domains include a PIN endonuclease domain, a NYN domain, an SMR domain from SOT1, or an RNase domain from staphylococcal nuclease.

[0428] In some embodiments of the present disclosure, the RNA-guided nuclease is covalently linked or fused to an affinity tag, an affinity domain, a reporter tag or a reporter domain. In some embodiments, the RNA-guided nuclease is covalently linked or fused to a reporter domain, such as a fluorescent protein. Non-limiting examples of reporter domains include GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP. In some embodiments, the RNA-guided nuclease is covalently linked or fused to an affinity tag such as a purification tag. Non-limiting examples of affinity tags include 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.

[0429] In some embodiments of the present disclosure, the affinity tag and affinity domain are used interchangeably, and the reporter tag and reporter domain are used interchangeably.

[0430] In some embodiments of the present disclosure, the RNA-guided nuclease is covalently linked to the fusion domain with or without a linker sequence; that is, the RNA-guided nuclease is directly covalently linked to the fusion domain (without a linker sequence) or covalently linked via a linker sequence. Typically, the linker sequence consists of 1-100, 1-50, 1-30, 1-20, 1-10, or 1-5 amino acids.

[0431] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with the RNA-guided nuclease and directing sequence-specific binding of the complex to the target RNA.

[0432] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with the RNA-guided nuclease and directing the complex to sequence-specifically bind to and cleave the target RNA.

[0433] Cas proteins

[0434] In some embodiments of the present 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, Cas12k protein.

[0435] In some embodiments of the present disclosure, the Cas protein is selected from SpCas9, SaCas9, Nme2Cas9, Nme3Cas9, CjCas9, NmCas9, FnCas9, PpnCas9, FrCas9, SauCas9, SauriCas9, ScaCas9, St1Cas9, BlatCas9, CdiCas9, GeoCas9, fragments thereof, and mutants or mutant fragments thereof. The Cas12 protein is selected from AsCpf1, LbCas12a, AkCas12b, AacCas12b, PlmCasX, DpbCasX, Cas12i1, Cas12i2, Cas12i3, -2.

[0436] In some embodiments of the present disclosure, the Cas protein is selected from 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, MlCas12 a, CMaCas12a, 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 (addgeneplasmid#120882)、Cas12i2(addgene plasmid#120883) and CN111757889B single-stranded Cas12f.4 / Cas12f.5 / Cas1 2f.6 Cas12i Processor, dSiCas12i(D1049A), SiCas12i, Si2Cas12i, WiCa s12i , Wi2Cas12i , Wi3Cas12i , SaCas12i , Sa2Cas12i , Sa3Cas12i , WaCas12i , Wa2Cas12i , xCas12i , hfCas12Max , Cas12i - Max ( addgene plasmid#188276); plasmid#188498); plasmid#181787)、Cas12k-TnsC(addgene plasmid#181789), Cas12l, MmCas12m, MmCas12mΔZF(H549A,C552A), d Cas12m-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-1and TnpB-2) 、TnpB Alicyclobacillus_macrosporagiidus_strain_DSM_17980 、TnpB Haloactinospora_alba_strain_DSM_45015 、TnpBLipingzhangella_halophila_strain_DSM_102030, TnpB Meiothermus_Silvanus_DSM_9946, TnpB QNFX01000004, ISDra2 TnpB (PDB:8H1J), KraIscB-1, AwaIscB, OgeuIscB, GtFz1 (from Guillardia theta), SpuFz1 (from Spizellomyces punctatus), NlovFz2 (from Percolozoa Naegleria lovaniensis), MmeFz2 (from Mercenaria mercenaria), fragments thereof, and mutants or mutant fragments thereof.

[0437] In some embodiments of the present disclosure, the Cas13 protein is a Cas13a protein, a Cas13b protein, a Cas13c protein or a Cas13d protein. In some embodiments of the present disclosure, the Cas protein is a Cas13a protein, a Cas13b protein, a Cas13c protein or a Cas13d protein in which both HEPN domains carry mutations, and the cleavage activity of these Cas proteins is completely or partially lost. 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, Cas13bt3, CcaCas13b, MisCas13b , Hgm4Cas13b, Pba4Cas13b, Bba2Cas13b, CasRx, dCasRx(R239A / H244A / R858A / H863A), CasRx_N2V8(A134V, A140V, A141V, A143V), RspCas13d, C13-2.

[0438] In some embodiments of the present disclosure, the Cas13 protein is a Cas13d protein.

[0439] In some embodiments of the present disclosure, 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 CasRx or C13-2 protein.

[0440] In some embodiments of the present disclosure, the Cas13 protein is a CasRx protein. In some embodiments, the Cas13 protein is a dCasRx in which both HEPN domains carry mutations (R239A, H244A of HEPN-1 and R858A, H863A of HEPN-2).

[0441] In some embodiments of the present disclosure, the Cas13 protein comprises the sequence shown in SEQ ID NO:1.

[0442] 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 to the sequence shown in SEQ ID NO: 1.

[0443] In some embodiments of the present disclosure, the Cas13 protein is a C13-2 protein. In some embodiments, the Cas13 protein is a dead C13-2 protein.

[0444] In some embodiments, the Cas13 protein comprises 1, 2, 3, 4, 5 or 6 mutations at positions corresponding to amino acid residues R210, H215, R750, H755, R785 and / or H790 of the reference protein set forth in SEQ ID NO: 1 (C13-2). In some embodiments, the Cas13 protein is mutated to A (alanine) at positions corresponding to amino acid residues R210, H215, R750, H755, R785 and / or H790 of the reference protein set forth in SEQ ID NO: 1.

[0445] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210 and H215 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R750 and H755 of the reference protein set forth in SEQ ID NO: 1. In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R785 and H790 of the reference protein set forth in SEQ ID NO: 1.

[0446] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210, H215, R750, and H755 of the reference protein set forth in SEQ ID NO: 1.

[0447] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R750, H755, R785, and H790 of the reference protein set forth in SEQ ID NO: 1.

[0448] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210, H215, R785 and / or H790 of the reference protein set forth in SEQ ID NO: 1.

[0449] In some embodiments, the Cas13 protein comprises mutations at positions corresponding to amino acid residues R210, H215, R750, H755, R785, and H790 of the reference protein set forth in SEQ ID NO: 1.

[0450] In some embodiments, 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, the corresponding positions of R210, H215, R750, H755, R785, and H790 are all mutated to A.

[0451] In some embodiments, 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.

[0452] In some embodiments, the Cas13 protein is obtained by introducing 1, 2, 3, 4, 5 or 6 mutations at positions R210, H215, R750, H755, R785 and / or H790 of the sequence shown in SEQ ID NO: 1. In some embodiments, the Cas13 protein is obtained by mutating positions R210, H215, R750, H755, R785 and / or H790 of the sequence shown in SEQ ID NO: 1 to A (alanine).

[0453] In some embodiments, the Cas13 protein is obtained by mutating R210, H215, R785, and H790 of the sequence shown in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by mutating R210, H215, R750, and H755 of the sequence shown in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by mutating R750, H755, R785, and H790 of the sequence shown in SEQ ID NO: 1 to A. In some embodiments, the Cas13 protein is obtained by mutating R210, H215, R750, H755, R785, and H790 of the sequence shown in SEQ ID NO: 1 to A.

[0454] In some embodiments, the Cas13 protein comprises any one or more mutations at positions corresponding to the following amino acid residues of the reference protein as compared to 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+N6 8. N394+N68, N87+N68, R308+N265, N394+N265, N87+N265, R308+N68+N265, N87+N68+N265, T7, A16, S260, A263, M266, N274, F288, M 302, N303, L304, V305, I311, D313, H324, P326, H327, N332, N346, T353, T360, E365, A373, M380, S382, K395, Y396, D402, D411, S418.

[0455] In some embodiments, the Cas13 protein is obtained by introducing any one or more mutations at the following positions of 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, R32 8. N332, R341, N346, R358, N372, N383, N390, N394, R47+R290, R47+R314, R290+R314, R47+R29 0+R314, R308+N68, N394+N68, N87+N68, R308+N265, N394+N265, N87+N265, R308+N68+N265, N 87+N68+N265, T7, A16, S260, A263, M266, N274, F288, M302, N303, L304, V305, I311, D313, H32 4. P326, H327, N332, N346, T353, T360, E365, A373, M380, S382, K395, Y396, D402, D411, S418.

[0456] In some embodiments, the C13-2 protein, similar proteins of the C13-2 protein, mutants of the C13-2 protein, and their functional fragments or fusion proteins can form a complex with the direct repeat sequence described in the present disclosure comprising the sequence shown in SEQ ID NO: 2 or 3, and then specifically target the target RNA described in the present disclosure under the guidance of the guide sequence.

[0457] In some embodiments of the present disclosure, the Cas protein comprises any one or more of the following fusion domains: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0458] In some embodiments of the present disclosure, the Cas protein comprises a subcellular localization signal.

[0459] In some embodiments of the present disclosure, the Cas protein comprises a subcellular localization signal and a deaminase domain.

[0460] In some embodiments of the present disclosure, the subcellular localization signal is selected from a nuclear localization signal and a nuclear export signal.

[0461] In some embodiments of the present disclosure, the Cas protein is covalently linked to the fusion domain with or without a linker sequence; that is, the Cas protein is directly covalently linked to the fusion domain (without a linker sequence) or covalently linked via a linker sequence. Typically, the linker sequence consists of 1-100, 1-50, 1-30, 1-20, 1-10, or 1-5 amino acids.

[0462] In some embodiments of the present disclosure, the guide RNA is capable of forming a CRISPR complex with the Cas protein and guiding the sequence-specific binding of the CRISPR complex to the target RNA.

[0463] In some embodiments of the present disclosure, the guide RNA is capable of forming a CRISPR complex with the Cas protein and guiding the CRISPR complex to sequence-specifically bind to and cleave the target RNA.

[0464] Nucleotide sequence encoding guide RNA or RNA-guided nuclease

[0465] In some embodiments, the nucleotide sequence encoding the RNA-guided nuclease is a plasmid. In some embodiments, the nucleotide sequence encoding the RNA-guided nuclease is part of a viral vector genome, such as the DNA genome of an AAV vector flanked by ITRs. In some embodiments, the nucleotide sequence encoding the RNA-guided nuclease is an mRNA.

[0466] In some embodiments of the present disclosure, the nucleotide sequence encoding the guide RNA or RNA-guided nuclease is codon-optimized.

[0467] In some embodiments of the present disclosure, the nucleotide sequence encoding the guide RNA is DNA. In some embodiments, the sequence of the DNA is codon optimized.

[0468] In some embodiments of the present disclosure, the nucleotide sequence encoding the RNA-guided nuclease is DNA or mRNA. In some embodiments, the sequence of the DNA is codon-optimized. In some embodiments, the sequence of the mRNA is codon-optimized.

[0469] In some embodiments of the present disclosure, codon optimization is performed for expression in a desired cell type. In some embodiments, codon optimization is performed for expression in a eukaryotic cell environment. In some embodiments, codon optimization is performed for expression in a mammalian cell environment. In some embodiments, codon optimization is performed for expression in a human cell environment.

[0470] Generally, codon optimization refers to the modification of nucleic acid sequences to enhance expression in the host cell of interest by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the original sequence with a codon that is more frequently used in the genes of the host cell and simultaneously maintains the original amino acid sequence. Calculation programs or algorithms for codon optimization are also available, such as those available using the following tools or algorithms: ExpOptimizer, Codon OptimWiz, NGTM Codon, Codon optimization, Synthetic Gene Designer, and DNAWorks, etc.

[0471] carrier

[0472] Vectors can comprise any type of nucleotides, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, can be partially obtained from natural sources, and can comprise natural, non-natural or altered nucleotides. Suitable vectors include those designed for expression, such as plasmids and viruses.

[0473] In some embodiments, recombinant vectors contain regulatory sequences, such as transcriptional and translational start and stop codons, that are specific for the type of host cell into which the vector is to be introduced (eg, bacteria, fungi, plants, or animals, as appropriate).

[0474] In some embodiments, the recombinant vector optionally includes a gene carrier element (nucleic acid), such as a selectable marker region, a lactose operon, 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 replication origin, a Col E1 replication origin, a loxP site, or a Cre recombinase coding region.

[0475] In some embodiments, the coding sequence in the vector is codon-optimized for expression in prokaryotic cells (eg, bacteria) or eukaryotic cells (eg, mammalian cells, human cells).

[0476] Regulatory sequences

[0477] In some embodiments of the present disclosure, the regulatory sequence comprises 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.

[0478] In some embodiments of the present disclosure, the regulatory sequence is a U6 promoter or an eye-specific promoter.

[0479] In some embodiments of the present disclosure, the regulatory sequence is a CBh promoter. In some embodiments, the CBh promoter comprises a CMV enhancer sequence. In some embodiments, the CBh promoter comprises a chicken β-actin promoter. In some embodiments, the CBh promoter comprises a hybrid intron.

[0480] In some embodiments of the present disclosure, the regulatory sequence comprises an HRE enhancer element (hypoxia response element). In some embodiments, the regulatory sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 HRE enhancer elements in series. In some embodiments, the regulatory sequence comprises 2, 3, 4, 5, 6, 7, 8, or 9 HRE enhancer elements in series.

[0481] In some embodiments of the present disclosure, the regulatory sequence comprises an NRS element (neuron restrictive silencer) from the human synapsin gene (human synapsin gene). In some embodiments, the regulatory sequence comprises an NRS element and an HRE enhancer element. In some embodiments, the regulatory sequence comprises an NRS element and an HRE enhancer element in series. To enhance the regulatory effect, the NRS element and the HRE enhancer element in series can be repeated multiple times. In some embodiments, the regulatory sequence comprises an NRS element and an HRE enhancer element in series that are repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times or at least 6 times. In some embodiments, the regulatory sequence comprises an NRS element and an HRE enhancer element in series that are repeated 2 times, 3 times, 4 times, 5 times or 6 times.

[0482] promoter

[0483] 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., retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter (optionally with CMV enhancer), SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, or EF1α promoter), or a pol III promoter and a pol II promoter.

[0484] In some embodiments of the present disclosure, the promoter is a constitutive promoter, which is continuously active and not regulated by external signals or molecules. Suitable constitutive promoters include, but are not limited to, CMV, RSV, SV40, EF1α, CAG, and β-actin promoters. In some embodiments, the promoter is an inducible promoter regulated by external signals or molecules (e.g., transcription factors).

[0485] In some embodiments of the present disclosure, the promoter is a tissue-specific promoter, which can be used to drive the tissue-specific expression of Cas13 protein. Suitable muscle-specific promoters include but are not limited to CK8, MHCK7, myoglobin promoter (Mb), desmin (Desmin) promoter, muscle creatine kinase promoter (MCK) and variants thereof, and SPc5-12 synthetic promoters. Suitable immune cell-specific promoters include but are not limited to B29 promoter (B cell), CD14 promoter (monocyte), CD43 promoter (leukocyte and platelet), CD68 (macrophage) and SV40 / CD43 promoter (leukocyte and platelet). Suitable blood cell-specific promoters include but are not limited to CD43 promoter (leukocyte and platelet), CD45 promoter (hematopoietic cell), INF-β (hematopoietic cell), WASP promoter (hematopoietic cell), SV40 / CD43 promoter (leukocyte and platelet), and SV40 / CD45 promoter (hematopoietic cell). Suitable pancreas-specific promoters include but are not limited to elastase-1 promoter. Suitable endothelial cell-specific promoters include, but are not limited to, the Fit-1 promoter and the ICAM-2 promoter. Suitable neuronal tissue / cell-specific promoters include, but are not limited to, the GFAP promoter (astroglial cells), the SYN1 promoter (neurons), and the NSE / RU5' promoter (mature neurons). Neuronal tissue / cell-specific promoters can be the GFAP promoter and the SYN1 promoter. Suitable kidney-specific promoters include, but are not limited to, the NphsI promoter (podocytes). Suitable bone-specific promoters include, but are not limited to, the OG-2 promoter (osteoblasts, odontoblasts). Suitable lung-specific promoters include, but are not limited to, the SP-B promoter (lungs). Suitable liver-specific promoters include, but are not limited to, the SV40 / Alb promoter. Suitable heart-specific promoters include, but are not limited to, α-MHC.

[0486] In some embodiments of the present disclosure, the promoter is an eye-specific promoter.

[0487] In some embodiments of the present disclosure, the eye-specific promoter is selected from the group consisting of: a retinoschisis protein promoter, a K12 promoter, a rhodopsin promoter, a rod-specific promoter, a cone-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.).

[0488] In some embodiments of the present disclosure, the promoter is a chicken β-actin (CB) promoter. The chicken β-actin promoter can be a short chicken β-actin promoter or a long chicken β-actin promoter. In some embodiments, the promoter (e.g., chicken β-actin promoter) comprises an enhancer sequence, such as a cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence can be a short CMV enhancer sequence or a long CMV enhancer sequence. In some embodiments, the promoter comprises a long CMV enhancer sequence and a long chicken β-actin promoter. In some embodiments, the promoter comprises a short CMV enhancer sequence and a short chicken β-actin promoter. However, those skilled in the art will appreciate that a short CMV enhancer can be used together with a long CB promoter, and a long CMV enhancer can be used together with a short CB promoter. In some embodiments of the present disclosure, the promoter is a CBh promoter.

[0489] enhancer

[0490] In some embodiments of the present disclosure, the enhancer is selected from the group consisting of WPRE, CMV enhancer, SV40 enhancer, and an intronic sequence between exons 2 and 3 of rabbit β-globin.

[0491] In some embodiments of the present disclosure, the enhancer is located upstream of the promoter element; however, it can also be located downstream or within the coding sequence regulated by the promoter and maintain function. Therefore, the enhancer or a portion thereof can be present in the RNA sequence transcribed from the coding sequence.

[0492] In some embodiments of the present disclosure, the enhancer may be located within 100, 200, 300, 400, 500 or more base pairs upstream or downstream of the coding sequence regulated by the promoter.

[0493] In some embodiments of the disclosure, an enhancer increases expression of a coding sequence above that provided by a promoter.

[0494] Vector system

[0495] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA-guided nuclease or the guide RNA is codon-optimized for expression in eukaryotic cells.

[0496] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA-guided nuclease or the guide RNA is codon-optimized for expression in mammalian cells.

[0497] In some embodiments of the disclosure, the polynucleotide sequence encoding the RNA-guided nuclease or the guide RNA is codon-optimized for expression in human cells.

[0498] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA-guided nuclease or the guide RNA is codon-optimized for expression in prokaryotic cells.

[0499] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA-guided nuclease or the guide RNA is codon-optimized for expression in bacterial cells.

[0500] In some embodiments of the present disclosure, the nucleic acid molecule encoding the RNA-guided nuclease or the guide RNA is a plasmid. In some embodiments, the nucleic acid molecule encoding the RNA-guided nuclease or the guide RNA is part of a viral vector genome, such as, for example, a DNA genome of an AAV vector flanked by ITRs. In some embodiments, the nucleic acid molecule encoding the RNA-guided nuclease or the guide RNA is an mRNA.

[0501] Adeno-associated virus vector (AAV vector)

[0502] Delivery of the CRISPR-Cas system by 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 the present disclosure, the AAV vector comprises an ssDNA genome comprising an RNA-guided nuclease and a coding sequence for a guide RNA flanked by ITRs.

[0503] In some embodiments of the present disclosure, the guide RNA or gene editing system described herein is packaged in an AAV vector, for example, packaged into an 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 capsid.

[0504] In some embodiments of the present disclosure, the guide RNA or gene editing system described herein is packaged into an AAV2, AAV5, AAV6, AAV8, AAV9, or AAV PHP.eB capsid.

[0505] In some embodiments of the present disclosure, the AAV vector described herein is optionally 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.

[0506] In some embodiments of the present disclosure, the AAV vector described herein is selected from: AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 8, AAV2 / 9, AAV2 / PHP.eB.

[0507] In some embodiments, the gene editing system described herein is packaged in an AAV vector comprising an engineered capsid with tissue tropism, such as an engineered ocular tissue-tropic capsid.

[0508] lipid nanoparticles

[0509] Gillmore et al., N.Engl.J.Med., 385:493-502 (2021) describes the LNP delivery of the CRISPR-Cas system, which is incorporated herein by reference in its entirety. In some embodiments, in addition to RNA payload (Cas13 mRNA and guide RNA), lipid nanoparticles (LNPs) also include four components: cations or ionizable lipids, cholesterol, helper lipids, and PEG-lipids. In some embodiments, the cations 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, the PEG-lipid includes PEG-2000-C-DMG, PEG-2000-DMG, or ALC-0159. In some embodiments, the helper lipid includes DSPC. The components of LNPs are described in Paunovska et al., Nature Reviews Genetics 23:265-280 (2022), which is incorporated herein by reference in its entirety.

[0510] Lentiviral vectors

[0511] In some embodiments of the present disclosure, the lentiviral vector is pseudotyped with a homologous or heterologous envelope protein, such as VSV-G. In some embodiments, the mRNA encoding the RNA-guided nuclease is linked to an aptamer sequence.

[0512] RNP complex (ribonucleoprotein complex)

[0513] In some embodiments of the present disclosure, the RNP complex (ribonucleoprotein complex) can be delivered to eukaryotic cells, mammalian cells or human cells by microinjection or electroporation. In some embodiments, the ribonucleoprotein complex can be packaged in virus-like particles and delivered to mammals or human subjects in vivo.

[0514] virus-like particles

[0515] In some embodiments of the present disclosure, engineered virus-like particles (VLPs) are pseudotyped with homologous or heterologous envelope proteins such as VSV-G. In some embodiments, the RNA-guided nuclease is fused to a gag protein (e.g., MLVgag) via a cleavable linker, wherein the cleavage of the linker in the target cell exposes an NLS between the linker and the RNA-guided nuclease. In some embodiments, the fusion protein comprises (e.g., from 5' to 3') a gag protein (e.g., MLVgag), one or more NESs, a cleavable linker, one or more NLSs, and an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is fused to a first dimerization domain capable of dimerizing or heterodimerizing with a second dimerization domain fused to a membrane protein, wherein the presence of a ligand promotes the dimerization and enriches the RNA-guided nuclease or fusion protein into the VLP.

[0516] cell

[0517] The cell of the present disclosure can be an isolated cell. The cell of the present disclosure (for example, it can be used to produce a cell-free system) can be eukaryotic or prokaryotic. The example of such cells includes but is not limited to bacteria, archaebacteria, plants, fungi, yeast, insects and mammalian cells, such as lactobacillus, lactococcus, bacillus (such as bacillus subtilis), Escherichia (such as Escherichia coli), Clostridium, Saccharomyces or Pichia (such as saccharomyces cerevisiae or Pichia pastoris), Kluyveromyces lactis, Salmonella typhimurium, Drosophila cells, Caenorhabditis elegans cells, African clawed frog cells, SF9 cells, C129 cells, 293 cells, Neurospora and immortalized mammalian cell lines (for example, Hela cells, bone marrow cell lines and lymphoid cell lines).

[0518] In some embodiments, the cell is a prokaryotic cell, such as a bacterial cell, such as escherichia coli. In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell or a human cell. In some embodiments, the cell is a primary eukaryotic cell, a stem cell, a tumor / cancer cell, a circulating tumor cell (CTC), a blood cell (for example, T cell, B cell, NK cell, Tregs etc.), a hematopoietic stem cell, a specialized immune cell (such as tumor infiltrating lymphocytes or tumor suppressor lymphocytes), a stromal cell (such as cancer associated fibroblasts etc.) in a tumor microenvironment. In some embodiments, the cell is the brain or neuronal cell (for example, neuron, astrocyte, microglia, retinal ganglion cell, rod / cone cell etc.) of a central or peripheral nervous system.

[0519] Diseases or conditions associated with target RNA

[0520] In some embodiments of the present disclosure, the disease or condition associated with the target RNA refers to a disease or condition caused by abnormally high expression of the target RNA, and the target RNA is CTGF RNA, MITF RNA, SRD5A2 RNA or AR RNA.

[0521] The gene editing system disclosed herein that targets CTGF RNA, MITF RNA, SRD5A2 RNA, or AR RNA can effectively knock down CTGF RNA, MITF RNA, SRD5A2 RNA, or AR RNA, and thus can be used to prevent, treat, or diagnose these diseases.

[0522] In some embodiments, the pharmaceutical composition is delivered to a human subject in vivo. The pharmaceutical composition can be delivered by any effective route, and a therapeutically effective amount of the pharmaceutical composition can be delivered to a subject in need thereof. Exemplary routes of administration include, but are not limited to, intravenous infusion, intravenous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, subcutaneous injection, intradermal injection, intraventricular injection, intravascular injection, intracerebellar injection, intraocular injection, subretinal injection, intravitreal injection, intracameral injection, intratympanic injection, intranasal administration, and inhalation.

[0523] In some embodiments of the present disclosure, a therapeutically effective amount of the gene editing system or pharmaceutical composition described herein is delivered to a subject in need using a suitable delivery method, which can achieve scar repair, skin whitening, reduction or elimination of chloasma, prevention or treatment of melanoma, and / or can downregulate androgen receptor expression, ultimately for the prevention or treatment of androgenic alopecia.

[0524] In some embodiments of the present disclosure, the use of the inhibitors, guide RNAs, nucleic acids, vectors, vector systems, adeno-associated viral vectors, lipid nanoparticles, lentiviral vectors, ribonucleoprotein complexes, virus-like particles, eukaryotic cells, and / or pharmaceutical compositions described herein can downregulate MITF expression, thereby reducing melanin expression, reducing or eliminating chloasma, achieving skin whitening effects, and / or treating melanoma. The document Yi X, et al. MIT F-siRNA formulation is a safe and effective therapy for human melasma [J]. Molecular Therapy, 2011, 19(2): 362-371. describes the use of siRNA targeting MITF for the treatment of chloasma, which is incorporated herein by reference in its entirety.

[0525] In some embodiments of the present disclosure, the use of the inhibitors, guide RNAs, nucleic acids, vectors, vector systems, adeno-associated viral vectors, lipid nanoparticles, lentiviral vectors, ribonucleoprotein complexes, virus-like particles, eukaryotic cells, and / or pharmaceutical compositions described herein can downregulate SRD5A2 expression and prevent or treat hair loss caused by male pattern baldness. The document 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 plant extracts that can downregulate SRD5A2 expression and are used for AGA, the entire text of which is incorporated herein by reference.

[0526] cosmetic

[0527] In some embodiments of the present disclosure, the cosmetic can be used to repair scars, whiten skin, reduce or eliminate melasma, prevent or treat melanoma, and / or prevent or treat androgenic alopecia.

[0528] In some embodiments of the present disclosure, the use of inhibitors, guide RNAs, nucleic acids, vectors, vector systems, adeno-associated viral vectors, lipid nanoparticles, lentiviral vectors, ribonucleoprotein complexes, virus-like particles, eukaryotic cells and / or pharmaceutical compositions as described herein can downregulate CTGF expression, prevent excessive collagen expression in wound repair, prevent excessive wound fibrosis from forming scars, and ultimately achieve the purpose of scar repair and / or skin beautification. The document 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 that siRNA targeting CTGF reduces scar contraction during skin wound healing, and the entire text is incorporated herein by reference.

[0529] In some embodiments of the present disclosure, the use of the inhibitors, guide RNAs, nucleic acids, vectors, vector systems, adeno-associated viral vectors, lipid nanoparticles, lentiviral vectors, ribonucleoprotein complexes, virus-like particles, eukaryotic cells, and / or pharmaceutical compositions described herein can downregulate MITF expression, thereby reducing melanin expression, reducing or eliminating chloasma, achieving skin whitening effects, and / or treating melanoma. The document Yi X, et al. MIT F-siRNA formulation is a safe and effective therapy for human melasma [J]. Molecular Therapy, 2011, 19(2): 362-371. describes the use of siRNA targeting MITF for the treatment of chloasma, which is incorporated herein by reference in its entirety.

[0530] In some embodiments of the present disclosure, the use of the inhibitors, guide RNAs, nucleic acids, vectors, vector systems, adeno-associated viral vectors, lipid nanoparticles, lentiviral vectors, ribonucleoprotein complexes, virus-like particles, eukaryotic cells, and / or pharmaceutical compositions described herein can downregulate SRD5A2 expression and prevent or treat hair loss caused by male pattern baldness. The document Khantham C, et al. Antioxidation, Anti-Inflammation, and Regulation of SRD5A Gene Expression of Oryza sativa cv. Bue Bang 3CMU Husk and Bran Extracts as Androgenetic Alopecia Molecular Treatment Substances. Plants. 2022; 11(3): 330. https: / / doi.org / 10.3390 / plants11030330 describes plant extracts that can downregulate SRD5A2 expression and are used for AGA, which is incorporated herein by reference in its entirety.

[0531] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0532] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0533] Example

[0534] In the following examples, in order to verify the effect of targeting each target RNA, the applicant's recently discovered highly active Cas13 protein, namely C13-2 (also known as CasRfg.4), was used.

[0535] Amino acid sequence of C13-2 protein (SEQ ID NO: 1):

[0536] The DR sequence corresponding to C13-2 (SEQ ID NO: 2) is:

[0537] The applicant screened and obtained another DR with good effect, named DR-hf2 (SEQ ID NO: 3), the sequence of which is:

[0538] Example 1. Verification of endogenous CTGF gene editing efficiency

[0539] 1. Construction of an editing vector targeting the endogenous gene CTGF

[0540] The C13-2-BsaI plasmid (SEQ ID NO: 4) with a universal crRNA backbone expression cassette was synthesized at an outsourcing service company.

[0541] gRNA was designed against human CCN2 mRNA (NCBI NM_001901.4, SEQ ID NO: 14), as shown in Table 1 and Figure 1 .

[0542] Table 1. Designed gRNA guide sequences

[0543] The sense and antisense DNA sequences were synthesized. The sense strand was constructed by appending AGAC to the 5' end of the guide sequence shown in Table 1, while the antisense strand was constructed by appending AAA to the 5' end of the reverse complement of the guide sequence. Primer annealing was used to obtain fragments targeting the CTGF RNA target site.

[0544] The primer annealing reaction system is shown in Table 2 below. Incubate at 95°C in a PCR instrument for 5 minutes, then immediately remove and incubate on ice for 5 minutes to allow the primers to anneal to each other to form double-stranded DNA with sticky ends.

[0545] Table 2. Primer annealing reaction system

[0546] The synthesized C13-2-BsaI plasmid was digested with the endonuclease Bsa I, and the annealed product and the purified and recovered backbone were ligated by T4. After transformation into Escherichia coli, positive clones were selected and the target plasmid was extracted, i.e., the verification vector (which can express C13-2 protein and gRNA targeting CTGF RNA) was used for subsequent experiments.

[0547] 2. Transfect 293T cells with the vector to be verified

[0548] The 293T cell line highly expressing CTGF (293T-CTGF cells) was used.

[0549] Cell line construction method: A vector, Lv-CTGF-T2a-GFP (SEQ ID NO: 15), was constructed to overexpress the CTGF and EGFP genes. CTGF and EGFP were separated by a 2A peptide. The Lv-CTGF-T2a-GFP plasmid was packaged into a lentivirus and then transduced into 293T cells to establish a cell line stably overexpressing the CTGF gene.

[0550] 293T-CTGF cells were transfected with validation vectors carrying different target sites. The negative control group was transfected with C13-2-BsaI vector.

[0551] Transfection was performed in 24-well plates according to the instructions of Lipofectamine 2000 (Thermo).

[0552] 3. qPCR detection

[0553] 72 hours after transfection, RNA was extracted from 293T-CTGF cells using the SteadyPure Universal RNA Extraction Kit AG21017, and RNA concentration was measured using an ultra-micro-spectrophotometer. RNA products were reverse transcribed using the Evo M-MLV Mix Kit with gDNA Clean for qPCR Reverse Transcription Kit and detected using the SYBR Green Premix Pro Taq HS qPCR Kit. A separate 293T-NC control group was established: qPCR was used to measure CTGF RNA levels in normal 293T cells (which did not overexpress CTGF and were not transfected with the gene-editing plasmids described above).

[0554] The primers used in qPCR are as follows:

[0555] Detection of CTGF (primer my): GCGTGTGCACCGCCAAAGAT (SEQ ID NO: 16),

[0556] AACGTCCATGCTGCACAGGG (SEQ ID NO:17);

[0557] Detection of CTGF (primer 11): CAGCATGGACGTTCGTCTG (SEQ ID NO: 18),

[0558] AACCACGGTTTGGTCCTTGG (SEQ ID NO: 19);

[0559] Detection of internal reference GAPDH: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 20),

[0560] GAAGGGGTCATTGATGGCAAC (SEQ ID NO: 21).

[0561] according to Green Premix Pro Taq HS qPCR Kit Instructions: Configure the reaction system and use QuantStudio TM 5Real-Time PCR System was used for detection.

[0562] This experiment used the relative quantification method, 2 -△△Ct The target RNA level can be calculated by the following method:

[0563] ΔCt=Ct(CTGF)-Ct(GAPDH)

[0564] △△Ct=△Ct(sample to be verified, such as C13-2-CTGF-g2)-△Ct(C13-2-BsaI)

[0565] 2 -△△Ct =2^(-△△Ct)

[0566] Calculate the 2 of CTGF according to the above calculation method -△△Ct Repeat the test multiple times and take the average value of the results.

[0567] As shown in Figures 2 and 3. Figure 2 shows the detection results using CTGF-my primers. Figure 3 shows the detection results using CTGF-11 primers.

[0568] qPCR results showed that Cas13 protein combined with g6, g7, g8, and g10 gRNAs efficiently edited CTGF RNA, effectively downregulating CTGF expression with statistically significant differences (P < 0.0001). The editing efficiency was g8 > g10 > g6 > g7. Combinations of Cas13 protein with g2, g3, and g4 gRNAs had very low editing efficiency or failed to successfully knock down CTGF RNA.

[0569] Example 2. Verification of endogenous MITF gene editing efficiency

[0570] The editing efficiency was tested in a manner substantially similar to that of Example 1.

[0571] 1. Construction of an editing vector targeting the endogenous gene MITF

[0572] gRNA was designed against human MITF mRNA (NCBI NM_001354607.2, SEQ ID NO: 22), as shown in Table 3 and Figure 4.

[0573] Table 3. Guide sequences of designed gRNAs targeting MITF

[0574] The same method as in Example 1 was used to obtain a fragment targeting the MITF RNA target site by primer annealing.

[0575] After the C13-2-BsaI plasmid was digested with the endonuclease Bsa I, the annealed product and the purified and recovered backbone were ligated by T4. After transformation into Escherichia coli, positive clones were selected and the target plasmid was extracted, i.e., the verification vector (which can express C13-2 protein and gRNA targeting MITF RNA) was used for subsequent experiments.

[0576] 2. Transfect 293T cells with the vector to be verified

[0577] 293T cells were transfected with validation vectors carrying different target sites. The negative control group was transfected with C13-2-BsaI vector.

[0578] Transfection was performed in 24-well plates according to the instructions of Lipofectamine 2000 (Thermo).

[0579] 3. qPCR detection

[0580] 72 hours after transfection, qPCR detection was performed in the same manner as in Example 1. A 293T blank control group was also set up: qPCR was used to detect the MITF RNA level in untreated 293T cells (not transfected with plasmid).

[0581] The primers used in qPCR are as follows:

[0582] Detection of MITF: GCCTCCAAGCCTCCGATAAG (SEQ ID NO: 33),

[0583] GCACTCTCTGTTGCATGAACT(SEQ ID NO:34);

[0584] Detection of internal reference GAPDH: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 20),

[0585] GAAGGGGTCATTGATGGCAAC (SEQ ID NO: 21).

[0586] Use 2 -△△Ct The level of the edited target RNA was calculated using the same method. The experiment was repeated multiple times and the results were averaged. (See Figure 5).

[0587] qPCR results showed that the Cas13 protein in combination with g1, g2, g3, g4, g5, g6, and g7 gRNAs had a high editing efficiency for MITF RNA and could effectively downregulate MITF expression, with statistically significant differences (P < 0.0001). The editing efficiency of the Cas13 protein in combination with g2, g3, g4, g6, and g7 gRNAs exceeded 90%, while the editing efficiency in combination with g1 and g5 gRNAs was 80% and 71%, respectively. The editing efficiency in combination with g8, g9, and g10 gRNAs was less than 35%.

[0588] Example 3. Verification of endogenous SRD5A2 gene editing efficiency

[0589] The editing efficiency was tested in a manner substantially similar to that of Example 1.

[0590] 1. Construction of editing vectors and control vectors targeting the endogenous gene CTGF

[0591] gRNA was designed for human SRD5A2 mRNA (NCBI XM_011533072.3, SEQ ID NO: 35), as shown in Table 4 and Figure 6.

[0592] Table 4. Designed gRNAs targeting SRD5A2

[0593] The same method as in Example 1 was used to obtain a fragment targeting the SRD5A2 RNA target site by primer annealing.

[0594] The C13-2-BsaI plasmid was digested with the endonuclease Bsa I, and the annealed product and the purified and recovered backbone were ligated by T4. After transformation into Escherichia coli, positive clones were selected and the target plasmid was extracted, i.e., the verification vector (which can express C13-2 protein and gRNA targeting SRD5A2 RNA) was used for subsequent experiments.

[0595] Control vectors were also constructed to express shRNA-1 and shRNA-2 targeting SRD5A2 RNA, as shown in Table 5 .

[0596] Table 5. Designed shRNA

[0597] The primer annealing method was used to obtain the shRNA fragment targeting the SRD5A2 target site, and the primers are shown in Table 6.

[0598] Table 6. Primers corresponding to shRNA vectors

[0599] The backbone of the shRNA expression vector, pAAV-CMV-EGFP (SEQ ID NO: 58), was synthesized at an outsourcing service company. The backbone was double-digested with the endonucleases Bsa I and Not I. The annealed product and the purified backbone recovered after digestion were ligated by T4 ligation. After transformation into Escherichia coli, positive clones were selected and the target plasmid, which can express shRNA-1 and shRNA-2, was extracted and used for subsequent experiments.

[0600] 2. Transfect 293T cells with the vector to be verified

[0601] Construction of a 293T cell line overexpressing SRD5A2 (293T-SRD5A2 cells): A vector, Lv-SRD5A2-T2a-GFP (SEQ ID NO: 65), was constructed to overexpress the SRD5A2 and EGFP genes. SRD5A2 and EGFP were separated by a 2A peptide. The Lv-SRD5A2-T2a-GFP plasmid was packaged into lentivirus and then transduced into 293T cells to establish a 293T-SRD5A2 cell line stably overexpressing the SRD5A2 gene.

[0602] 293T-SRD5A2 cells were transfected with validation vectors carrying different target sites and shRNA control vectors, respectively. The negative control group was transfected with C13-2-BsaI vector.

[0603] Transfection was performed in 24-well plates according to the instructions of Lipofectamine 2000.

[0604] 3. qPCR detection

[0605] 72 hours after transfection, qPCR detection was performed in the same manner as in Example 1. A 293T-NC control group was also set up: qPCR detection of SRD5A2 RNA levels in untreated 293T cells (not overexpressing SRD5A2 and not transfected with gene editing plasmids and shRNA plasmids) was performed.

[0606] The primers used in qPCR are as follows:

[0607] Detection of SRD5A2 (primer 2, P2): ACTGCTCAATCGAGGGAGG (SEQ ID NO: 66),

[0608] CACCCAAGCTAAACCGTATGTC (SEQ ID NO: 67);

[0609] Detection of SRD5A2 (primer 3, P3): CGGTTTAGCTTGGGTGTCTTC (SEQ ID NO: 68),

[0610] CCGAGGAAATTGGCTCCAGAA (SEQ ID NO: 69);

[0611] Detection of internal reference GAPDH: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 20),

[0612] GAAGGGGTCATTGATGGCAAC (SEQ ID NO: 21).

[0613] Use 2 -△△Ct The level of the edited target RNA was calculated using the PCR method. C13-2-BsaI was used as the negative control group and was calculated as 1.00. The experiment was repeated multiple times and the results were averaged. As shown in Figures 7 and 8, Figure 7 shows the results of the detection using primer 2 (P2). Figure 8 shows the results of the detection using primer 3 (P3).

[0614] In Figure 7, all gRNA groups effectively downregulated target RNA expression compared to the negative control group, with statistically significant differences (P < 0.01). The editing efficiency (> 90%) of the h1, h7, h9, and h11 gRNA groups was higher than that of shRNA-1 and shRNA-2. The h9 and h11 gRNA groups showed statistically significant differences compared to shRNA-1 (P < 0.05).

[0615] In Figure 8, the h3, h5, h8, h9, and h19 gRNA groups all effectively downregulated target RNA expression compared to the negative control group, with statistically significant differences (P<0.01). The editing efficiency of the h3, h5, h8, and h9 gRNA groups was higher than that of shRNA-1. The editing efficiency of the h19 gRNA group was 95%, higher than that of shRNA-1 and shRNA-2.

[0616] Example 4. Verification of the editing efficiency of the gRNA of the present invention and other Cas13 targeting CTGF

[0617] The editing efficiency test is performed in substantially the same manner as in the above embodiment.

[0618] The CasRx-BsaI plasmid was synthesized outsourced, with its sequence shown as SEQ ID NO:70. CasRx-CTGF gRNA plasmids were constructed by annealing primers related to the guide sequence, followed by BsaI digestion and T4 ligation. These plasmids express CasRx and a gRNA targeting CTGF. Transfection was performed in a 293T cell line that overexpresses CTGF, and knockdown efficiency was assessed by qPCR.

[0619] The results are shown in Figures 9 and 10 . CasRx also has high editing efficiency when combined with CTGF-g6, g7, g8, and g10 gRNAs.

[0620] Example 5. Verification of the editing efficiency of the gRNA of the present invention and other Cas13 targeting MITF

[0621] The editing efficiency test is performed in substantially the same manner as in the above embodiment.

[0622] CasRx-MITF gRNA plasmids were constructed by annealing primers related to the guide sequence, digesting the CasRx-BsaI plasmid with BsaI, and ligating with T4 enzyme to express CasRx and a gRNA targeting MITF. Transfection was performed in 293T cells, and knockdown efficiency was assessed by qPCR 72 hours later.

[0623] The results are shown in Figure 11 . CasRx also had high editing efficiency when combined with MITF-g3, g4, g6, and g7 gRNAs.

[0624] Example 6. Off-target detection analysis of C13-2 combined with gRNA editing of MITF

[0625] The cells transfected 48 hours after the experiment in Example 5 were taken, total RNA was extracted, and the samples were sent for RNAseq sequencing (n=3 samples per group). The library type was LncRNA chain-specific library, the sequencing data volume was 16G, and the sequencing strategy was PE150.

[0626] RNAseq analysis principle:

[0627] 1. Use fastqc and multiqc to control the data quality, and use fastp to remove low-quality reads.

[0628] 2. Align to human rRNA sequence for removal, and use Hisat2 alignment software to align reads except rRNA sequence to hg38 reference genome.

[0629] 3. After alignment, Kallisto software was used to quantify gene expression levels, and then sleuth software was used to analyze expression differences (vs. 293T-NC). Genes with |b|>0.5, qval<0.05, and mean_obs>1 were considered differentially expressed genes.

[0630] 4. Align the sgRNA sequence to the reference cDNA using EMBOSS water software. Transcripts with a number of aligned bases >= 18, a number of mismatched bases <= 6, and a minimum number of consecutive paired bases >= 8 were considered predicted off-target transcripts, and the corresponding genes were considered off-target genes.

[0631] 5. Take the intersection of significantly downregulated genes and predicted off-target genes to obtain the off-target gene set.

[0632] RNASeq result analysis:

[0633] Using the unedited 293T-NC as a benchmark, the expression levels of the MITF gene after editing in different experimental groups were analyzed. The results are shown in Table 7 below.

[0634] Table 7. Expression levels of the MITF gene after editing

[0635] The off-target gene analysis results are shown in Table 8 below.

[0636] Table 8. Off-target gene analysis results

[0637] The experimental results showed that MITF-g3, MITF-g4, and MITF-g7 gRNAs only edited the MITF target site with no off-target effects. MITF-g6 gRNA had one off-target gene.

[0638] Example 7. Verification of the editing efficiency of the gRNA of the present invention and other Cas13 targeting SRD5A2

[0639] The editing efficiency test is performed in substantially the same manner as in the above embodiment.

[0640] CasRx-SRD5A2 gRNA plasmids were constructed by annealing primers related to the guide sequence, digesting the CasRx-BsaI plasmid with BsaI, and ligating with T4 enzyme. These plasmids express CasRx and a gRNA targeting SRD5A2. Transfection was performed in a 293T cell line overexpressing SRD5A2, and knockdown efficiency was assessed by qPCR 72 hours later.

[0641] The results are shown in Figure 12 . CasRx also had high editing efficiency when combined with SRD5A2-h1, h7, h9, and h11 gRNAs.

[0642] Example 8. Verification of endogenous AR gene editing efficiency

[0643] Using a combination of the Cas13 tool C13-2 protein and gRNA (crRNA), human AR mRNA (NCBI Reference Sequence: NM_000044.6, SEQ ID NO: 394) can be targeted and edited, thereby downregulating AR expression and achieving downregulation of androgen receptor expression, ultimately for the treatment of androgenic alopecia.

[0644] (1) Construction of a validation vector targeting the endogenous gene AR

[0645] The C13-2-BsaI plasmid (SEQ ID NO: 4) with a universal crRNA backbone expression cassette was synthesized at an outsourcing company.

[0646] The sense and antisense DNA sequences were synthesized. The sense strand was constructed by adding agac to the 5' end of the guide sequence shown in Table 9, and the antisense strand was constructed by adding aaaa to the 5' end of the reverse complement of the guide sequence. Primer annealing was used to obtain fragments targeting the AR RNA target site.

[0647] The primer annealing reaction system is shown in the table below. Incubate at 95°C in a PCR instrument for 5 minutes, then immediately remove and incubate on ice for 5 minutes to allow the primers to anneal to each other to form double-stranded DNA with sticky ends.

[0648] Table 10. Primer annealing reaction system

[0649] The C13-2-BsaI plasmid was digested with BsaI endonuclease, and the backbone was purified and recovered. The annealed product was ligated with T4 ligase to obtain the verification vector plasmid (CMV-C13-2-U6-gRNA). After transformation into E. coli, positive clones were selected and plasmids were extracted for subsequent experiments.

[0650] (2) Transfection of 293T cells with the vector to be verified

[0651] 293T cells were transfected with validation vectors carrying different target site sequences and a control vector (C13-2-BsaI).

[0652] The 24-well plates were transfected according to the instructions of Lipofectamine 2000 (Thermo).

[0653] (3) qPCR detection

[0654] 72 hours after transfection, RNA was extracted from cells using the SteadyPure Universal RNA Extraction Kit AG21017, and RNA concentration was determined using an ultra-micro-spectrophotometer. RNA products were reverse transcribed using the Evo M-MLV Mix Kit with gDNA Clean for qPCR AG11728 Reverse Transcription Kit, and the reverse transcription products were detected using the SYBR Green Premix Pro Taq HS qPCR Kit (Low Rox Plus).

[0655] The primers used in qPCR are as follows:

[0656] Detection of AR (primer 4): GACGACCAGATGGCTGTCATT (SEQ ID NO: 380)

[0657] GGGCGAAGTAGAGCATCCT(SEQ ID NO:381)

[0658] Detection AR (primer 5): CCAGGGACCATGTTTTGCC (SEQ ID NO: 382)

[0659] CGAAGACGACAAGATGGACAA(SEQ ID NO:383)

[0660] Detection internal reference GAPDH: CCATGGGGAAGGTGAAGGTC (SEQ ID NO: 384)

[0661] GAAGGGGTCATTGATGGCAAC (SEQ ID NO:385).

[0662] according to Green Premix Pro Taq HS qPCR Kit (Rox Plus) instructions for preparing the reaction system and using QuantStudio TM 5Real-Time PCR System was used for detection.

[0663] This experiment used the relative quantification method, 2 -△△Ct The target RNA level can be calculated by the following method:

[0664] ΔCt=Ct(AR)-Ct(GAPDH)

[0665] △△Ct=△Ct(vector to be verified, such as C13-2-AR-h1)-△Ct(control C13-2-BsaI)

[0666] 2 -△△Ct =2^(-△△Ct)

[0667] The 2 of the edited AR RNA was calculated according to the above calculation method. -△△Ct The values ​​are shown in Table 11, Table 12, and Figure 13 below.

[0668] Table 11. qPCR detection of edited AR RNA levels (primer 4)

[0669] *NC refers to the blank control of 293T cells without plasmid transfection

[0670] Table 12. qPCR detection of edited AR RNA levels (primer 5)

[0671] *NC refers to the blank control of 293T cells without plasmid transfection

[0672] qPCR results showed that C13-2 combined with gRNA could downregulate AR RNA expression levels.

[0673] Example 9. Editing effect test of different Cas13

[0674] (1) Construction of a validation vector targeting the endogenous gene AR

[0675] Different control groups were set up to compare the editing effects of C13-2+gRNA, CasRx+gRNA, and shRNA on AR RNA.

[0676] The CasRx-BsaI plasmid (SEQ ID NO: 70), the control vector shRNA-asiAR72 plasmid (SEQ ID NO: 387), and the shRNA-ARstart plasmid (SEQ ID NO: 388) with a universal crRNA backbone expression cassette were synthesized by an outsourcing company.

[0677] The shRNA-asiAR72 plasmid is used to express shRNA with the target sequence: GTTCACTTTTGACCTGCTAAT (SEQ ID NO: 390), located in exon 8 of the AR gene.

[0678] The shRNA-ARstart plasmid is used to express shRNA with the target sequence: GACCTACCGAGGAGCTTTC (SEQ ID NO: 391), located in exon 1 of the AR gene.

[0679] The CasRx+gRNA vector (CMV-CasRx-U6-gRNA) was constructed using the method of Example 9. The sense and antisense strands of the DNA sequences were synthesized. The sense strand was aaac added to the 5'-end of the guide sequence shown in Table 9, and the antisense strand was aaaa added to the 5'-end of the reverse complementary sequence of the guide sequence. A fragment targeting the AR RNA target site was obtained using primer annealing. It was then ligated with the Bsa I digested fragment of the CasRx-BsaI plasmid using T4 ligase, and positive clones were selected after transformation into E. coli, and the plasmid was extracted for subsequent experiments.

[0680] (2) Transfection of 293T cells with the vector to be verified

[0681] 293T cells were transfected with the vectors carrying different target sites to be verified and the control vector. 293T cells without plasmid transfection served as blank controls (NC group).

[0682] The 24-well plates were transfected according to the instructions of Lipofectamine 2000 (Thermo).

[0683] (3) qPCR detection

[0684] 72 hours after transfection, cells were subjected to qPCR detection in the same manner as in Example 8. The relative quantification method, i.e., 2 -△△Ct The target RNA levels were calculated.

[0685] Table 13, Table 14, and Figure 14 show the comparative test results with shRNA (all experiments were repeated 3 times, and the results were averaged). AR-h2, AR-13, and AR-h8 gRNAs had better knockdown efficiency on AR RNA than shRNA.

[0686] Table 13. Comparative test results of qPCR detection and shRNA (primer 4)

[0687] Table 14. Comparative test results of qPCR detection and shRNA (primer 5)

[0688] Table 15, Table 16 and Figure 15 show the test results in the same batch experiments with CasRx (all repeated 3 times, the results are averaged). C13-2 and CasRx can knock down AR RNA after being combined with different gRNAs, and the editing efficiency of C13-2 is slightly better than that of CasRx.

[0689] Table 15. qPCR and CasRx test results in the same batch of experiments (primer 4)

[0690] Table 16. qPCR and CasRx test results in the same batch of experiments (primer 5)

[0691] (4) Protein level detection (WB)

[0692] 72 hours after transfection, cells were trypsinized, centrifuged, washed with PBS, and lysed on ice for 30 minutes in 100 μL of cold RIPA lysis buffer. 5× loading buffer was added, the cells were boiled in a boiling water bath for 5 minutes, and centrifuged at maximum speed for 1 minute. The supernatant was then subjected to protein electrophoresis. After electrophoresis, the cells were transferred to a PVDF membrane for Western blotting. GAPDH antibody (CellSignal, #5174) was used as an internal control, and AR antibody (Abcam, ab133273) was used to detect target gene protein expression. A goat anti-rabbit secondary antibody (Sigma-Aldrich, A0545) was used.

[0693] The WB results are shown in Figure 16. As can be seen from the figure, compared with the C13-2-BsaI and CasRx-BsaI control groups, the C13-2 and CasRx experimental groups can significantly downregulate the expression of AR protein.

[0694] Example 10. Off-target detection

[0695] Control vector construction

[0696] Because 293T cells do not contain the EGFP sequence, a C13-2-GFP vector targeting EGFP was constructed as a negative control using the method of Example 8. The spacer targeting GFP is tgccgttcttctgcttgtcggccatgatat (SEQ ID NO: 393).

[0697] RNAseq sequencing

[0698] The method of Example 8 was used to construct a verification vector expressing C13-2 and AR-h2, AR-h4, AR-h8 or AR-h9 gRNA, which was used as the experimental group; 293T cells were transfected together with the negative control group. After 48 hours, total RNA samples were extracted for RNAseq sequencing (n = 3 samples per group). The library type was LncRNA chain-specific library, the sequencing data volume was 16G, and the sequencing strategy was PE150.

[0699] Principles of RNAseq Analysis

[0700] Fastqc and multiqc were used to perform data quality control, and fastp was used to remove low-quality reads.

[0701] Sequences aligned to human rRNA were removed and aligned to the hg38 reference genome using Hisat2 alignment software.

[0702] After alignment, Kallisto software was used to quantify the gene expression levels, and then sleuth software was used to perform expression difference analysis. Genes with |b|>0.5, qval<0.05, and mean_obs>2 were considered differentially expressed genes (DEGs).

[0703] The guide sequence of gRNA was aligned to the reference cDNA using EMBOSS water software, and transcripts with an aligned base number ≥18, a mismatched base number ≤6, and a minimum number of consecutive paired bases ≥8 were considered predicted off-target transcripts, and the corresponding genes were considered predicted off-target genes.

[0704] The off-target gene set was obtained by taking the intersection of the significantly down-regulated genes in DEGs and the predicted off-target genes and excluding the AR gene.

[0705] RNASeq result analysis

[0706] The analysis results are shown in Table 17 below.

[0707] Table 17 Off-target detection analysis results

[0708] RNAseq sequencing results showed that no guide sequence-related off-targets were detected after C13-2 combined with AR-h2, AR-h4, AR-h8, and AR-h9 gRNA editing.

[0709] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, 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 knocks down the level of CTGF RNA, MITF RNA or SRD5A2 RNA, or inhibits the translation of CTGF RNA, MITF RNA or SRD5A2 RNA.

2. A gene editing system guide RNA, characterized in that It comprises a guide sequence that hybridizes to a target RNA, wherein the target RNA is CTGF RNA, MITF RNA, or SRD5A2 RNA; Optionally, the target RNA is CTGF pre-mRNA, MITF pre-mRNA or SRD5A2 pre-mRNA, or CTGF mRNA, MITF mRNA or SRD5A2 mRNA; Optionally, the target RNA is mammalian CTGF RNA, MITF RNA or SRD5A2 RNA.

3. A pharmaceutical composition, characterized in that It comprises the inhibitor according to claim 1 or the guide RNA according to claim 2; Optionally, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

4. Use of the inhibitor according to claim 1 or the guide RNA according to claim 2 in the preparation of a medicament for diagnosing, treating or preventing a disease or condition associated with a target RNA; Optionally, the disease or disorder associated with the target RNA refers to a disease or disorder caused by abnormally high expression of the target RNA; Optionally, the target RNA is CTGF pre-mRNA, MITF pre-mRNA or SRD5A2 pre-mRNA, 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 a sequence shown in SEQ ID NO: 14, 22 or 35; Optionally, the target RNA sequence is nucleotides 494 to 785 of the sequence shown in SEQ ID NO:14, nucleotides 404 to 606 of the sequence shown in SEQ ID NO:22, or nucleotides 484 to 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 preparation of cosmetics.

6. A cosmetic comprising the inhibitor according to claim 1 or the guide RNA according to claim 2.

7. Use of a gene editing system in the preparation of a drug for diagnosing, treating or preventing androgenic alopecia, 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 in that: The gene editing system: Knock down AR RNA levels or inhibit AR RNA translation.

9. A gene editing system guide RNA, characterized in that It contains a guide sequence that hybridizes to the AR RNA.

10. A pharmaceutical composition, characterized in that It comprises the gene editing system according to claim 2.

Images