Cas13 protein, CRISPR-Cas system and application of CRISPR-Cas system

Abstract

The invention relates to a Cas13 protein, a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system and application of the CRISPR-Cas system. The amino acid sequences of the Cas13 protein are shown as SEQ ID NO.1-SEQ ID NO.9, and the Cas13 protein is respectively named as Cas13a10, Cas13a11, Cas13b3, Cas13b4, Cas13b5, Cas13T9, Cas13T15, Cas13T16 and Cas13Z2; the 9 Cas13 proteins have low homology with reported Cas13 proteins, show RNA nuclease activity, and have great application prospects in gene editing.

Description

Cas13 protein, CRISPR-Cas system and its applications Technical Field

[0001] The present invention relates to Cas13 protein, CRISPR-Cas system and applications thereof, and belongs to the field of biotechnology. Background Art

[0002] The CRISPR (Clustered regularly interspaced short palindromic repeats) system was developed by bacteria and archaea to defend against invading phage DNA. CRISPR systems include two families: type 1 systems are further divided into types I, III, and IV, and type 2 systems are divided into types II, V, and VI. These six system types are further divided into 19 subtypes. Many prokaryotes contain multiple CRISPR-Cas systems, suggesting that they are compatible and may share components.

[0003] The CRISPR / Cas13 system is a CRISPR system that can target RNA for cleavage. The system is composed of the Cas13 protein and CRISPR RNA (guide RNA) to form an RNA-targeting effector complex guided by the guide RNA. Currently, four subtypes of the Cas13 family have been identified, including Cas13a, Cas13b, Cas13c, and Cas13d.

[0004] Compared to RNAi and CRISPRi technologies, the CRISPR / Cas13 system can achieve higher RNA degradation rates while minimizing the likelihood of off-target effects. However, the Cas13 proteins currently discovered still have defects, such as large size and low cleavage activity, which can affect the CRISPR / Cas13 system's ability to edit target RNA. CRISPR / Cas13 systems from different microbial sources exhibit functional and structural diversity. Therefore, there is a need to continue exploring more Cas13 proteins to construct CRISPR-Cas13 systems with better performance.

[0005] Summary of the Invention

[0006] The present disclosure provides a new Cas13 protein and CRSPR-Cas13 system to solve the above problems.

[0007] In one aspect of the present disclosure, a Cas13 protein is provided, wherein the Cas13 protein is the following protein (a) or (b):

[0008] (a) a protein comprising the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9;

[0009] (b) A protein in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (a) and which substantially retains the biological function of the sequence from which it is derived.

[0010] In some preferred embodiments, the amino acid sequence of the protein in (b) has at least 85% sequence identity with that in (a), and substantially retains the biological function of the sequence from which it is derived.

[0011] In some preferred embodiments, the amino acid sequence of the protein in (b) has at least 95% sequence identity with that in (a), and substantially retains the biological function of the sequence from which it is derived.

[0012] In a second aspect of the present disclosure, a fusion protein is provided, comprising the aforementioned Cas13 protein and a heterologous functional domain.

[0013] In some preferred embodiments, the heterologous functional domain includes at least one of a deaminase, a reporter protein, a detection marker, a localization signal, a protein targeting domain, a DNA binding domain, an epitope tag, a transcription activation domain, a transcription repression domain, a nuclease, a methylation active enzyme, a demethylase, a transcription release factor, an RNA cleavage polypeptide, a nucleic acid ligase, a histone modification active factor, or a nucleic acid binding active factor.

[0014] In some preferred embodiments, the heterologous functional domain is fused to the N-terminus or C-terminus of the Cas13 protein, or the heterologous functional domain is fused inside the Cas13 protein.

[0015] In some preferred embodiments, the localization signal is a nuclear localization signal or a nuclear export signal.

[0016] In a third aspect of the present disclosure, a CRISPR-Cas system is provided, comprising the Cas13 protein of the first aspect and a guide RNA, or the CRISPR-Cas system comprises the fusion protein of the second aspect and a guide RNA; the guide RNA can bind to the Cas13 protein to form a complex and guide the complex to contact the target RNA.

[0017] In some preferred embodiments, the guide RNA comprises a spacer sequence and a direct repeat sequence; the spacer sequence is 15 to 60 nucleotides in size; and the direct repeat sequence is 15 to 40 nucleotides in size.

[0018] In some preferred embodiments, the spacer sequence is between 25-35 nucleotides.

[0019] In some preferred embodiments, the spacer sequence is about 30 nucleotides.

[0020] In some preferred embodiments, there are at least 15 nucleotides of complementarity between the spacer sequence and the target RNA.

[0021] In the fourth aspect of the present disclosure, a polynucleotide is provided, which encodes the Cas13 protein of the first aspect, or the polynucleotide encodes the fusion protein of the second aspect, or the polynucleotide encodes the CRISPR-Cas system of the third aspect.

[0022] In a fifth aspect of the present disclosure, a vector is provided, wherein the vector carries the polynucleotide described in the fourth aspect.

[0023] In some preferred embodiments, the polynucleotide carried by the vector is linked to a regulatory element.

[0024] In some preferred embodiments, the regulatory element comprises at least one of a promoter, an enhancer, a polyadenylation signal, a terminator, a protein degradation signal, a transposon, a leader sequence, a polyadenylation sequence, or a marker gene.

[0025] In some preferred embodiments, the vector comprises at least one of a viral vector or a non-viral vector.

[0026] In some preferred embodiments, the viral vector comprises at least one of a retroviral vector, a phage vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a hybrid viral vector, a baculoviral vector, a herpes simplex viral vector, or a lentiviral vector.

[0027] In some preferred embodiments, the non-viral vector comprises a plasmid vector.

[0028] In the sixth aspect of the present disclosure, a delivery system is provided, which carries the Cas13 protein of the first aspect, the fusion protein of the second aspect, the CRISPR-Cas system of the third aspect, the polynucleotide of the fourth aspect, or the vector of the fifth aspect.

[0029] In some preferred embodiments, the delivery vector of the delivery system includes at least one of a liposome vector, a chitosan vector, a polymer vector, a nanoparticle vector, an exosome vector, an exosome vector, a microvesicle vector, a viral vector, a gene gun, or an electroporation device.

[0030] In the seventh aspect of the present disclosure, a host cell is provided, which carries the Cas13 protein of the first aspect, the fusion protein of the second aspect, the CRISPR-Cas system of the third aspect, the polynucleotide of the fourth aspect, or the vector of the fifth aspect.

[0031] In an eighth aspect of the present disclosure, a model is provided, which is a plant model or an animal model; the plant model or animal model comprises the host cell described in the seventh aspect.

[0032] In the ninth aspect of the present disclosure, a method for gene editing is provided, comprising contacting the target RNA with the Cas13 protein of the first aspect, the fusion protein of the second aspect, the CRISPR-Cas system of the third aspect, the polynucleotide of the fourth aspect, the vector of the fifth aspect, or the host cell of the seventh aspect.

[0033] In some preferred embodiments, the guide RNA binds to the Cas13 protein to form a complex, and guides the complex to contact the target RNA to perform gene editing on the target RNA.

[0034] In the tenth aspect of the present disclosure, a gene editing product is provided, wherein the gene editing product is obtained by gene editing the target RNA using the method described in the ninth aspect.

[0035] In the eleventh aspect of the present disclosure, the use of the Cas13 protein described in the first aspect, the fusion protein described in the second aspect, the CRISPR-Cas system described in the third aspect, the polynucleotide described in the fourth aspect, the vector described in the fifth aspect, the delivery system described in the sixth aspect, the host cell described in the seventh aspect, or the method described in the ninth aspect in gene editing is provided.

[0036] In the twelfth aspect of the present disclosure, a kit is provided, comprising the Cas13 protein described in the first aspect, the fusion protein described in the second aspect, the CRISPR-Cas system described in the third aspect, the polynucleotide described in the fourth aspect, the vector described in the fifth aspect, the delivery system described in the sixth aspect, or the host cell described in the seventh aspect.

[0037] In the thirteenth aspect of the present disclosure, a nucleic acid detection method is provided, wherein the method comprises performing nucleic acid detection on a sample to be tested using the kit described in the twelfth aspect.

[0038] In the fourteenth aspect of the present disclosure, provided is the use of the Cas13 protein described in the first aspect, the fusion protein described in the second aspect, the CRISPR-Cas system described in the third aspect, the polynucleotide described in the fourth aspect, the vector described in the fifth aspect, the delivery system described in the sixth aspect, the host cell described in the seventh aspect, the kit described in the twelfth aspect, or the nucleic acid detection method described in the thirteenth aspect in nucleic acid detection.

[0039] In the fifteenth aspect of the present invention, provided is the use of the Cas13 protein of the first aspect, the fusion protein of the second aspect, the CRISPR-Cas system of the third aspect, the polynucleotide of the fourth aspect, the vector of the fifth aspect, the delivery system of the sixth aspect, or the host cell of the seventh aspect in the preparation of a medicament for treating a disorder or disease of a subject in need thereof.

[0040] In the sixteenth aspect of the present invention, a method for treating a condition or disease in a subject in need is provided, the method comprising: administering to the subject a composition comprising the CRISPR-Cas system of the third aspect of the present invention or a polynucleotide encoding the system; wherein the spacer sequence is complementary to at least 15 nucleotides in a target RNA associated with the disease or condition; wherein the Cas13 protein binds to the guide RNA to form a complex, and the complex binds to the target RNA, and the Cas13 protein edits (e.g., cuts) the target RNA to achieve the purpose of treating the condition or disease of the subject.

[0041] In some preferred embodiments, the condition or disease is cancer, an infectious disease, or other disease.

[0042] In some embodiments, the cancer is Wilms' tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, a skin cancer, a breast cancer, a colon cancer, a rectal cancer, a prostate cancer, a liver cancer, a kidney cancer, a pancreatic cancer, a lung cancer, a biliary tract cancer, a cervical cancer, an endometrial cancer, an esophageal cancer, a gastric cancer, a head and neck cancer, a medullary thyroid cancer, an ovarian cancer, a glioma, a lymphoma, a leukemia, a myeloma, a Hodgkin lymphoma, a non-Hodgkin lymphoma, or a bladder cancer.

[0043] In some preferred embodiments, the leukemia includes acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia or chronic myeloid leukemia.

[0044] In some embodiments, the additional disease comprises a neurological disease, an otological disease, a muscle disorder, or an ophthalmic disease.

[0045] In some preferred embodiments, the nervous system disease includes: the neurodevelopmental disorder "Angelman syndrome" or amyotrophic lateral sclerosis.

[0046] In some preferred embodiments, the otological disease includes dominant hereditary deafness.

[0047] In some preferred embodiments, the muscle abnormality comprises spinal muscular atrophy.

[0048] In some preferred embodiments, the ophthalmic disease includes: congenital amaurosis type 2.

[0049] In some embodiments, the method is an in vitro method, an in vivo method, or an ex vivo method.

[0050] The technical solution of the present invention has the following advantages:

[0051] The Cas13 proteins provided by the present invention, including Cas13a_10, Cas13a_11, Cas13b_3, Cas13b_4, Cas13b_5, Cas13T_9, Cas13T_15, Cas13T_16, and Cas13Z_2, have low homology with the reported Cas13 proteins and exhibit RNA nuclease editing activity on target RNA. Therefore, Cas13a_10, Cas13a_11, Cas13b_3, Cas13b_4, Cas13b_5, Cas13T_9, Cas13T_15, Cas13T_16, and Cas13Z_2 all have great application prospects in gene editing.

[0052] The Cas protein involved in the present invention has side-cutting activity and can be used for RNA detection. For example, the Cas13a_10, Cas13a_11, Cas13b_3, Cas13b_4, Cas13b_5, Cas13T_9, Cas13T_15, Cas13T_16, and Cas13Z_2 proteins of the present invention show nonspecific / incidental RNase activity (side-cutting activity), which can be used to detect the presence of specific RNA, and can achieve highly sensitive and convenient nucleic acid detection. For example, it can be widely used in on-site detection of various medical, biological, and environmental samples, and has a very broad application prospect.

[0053] In addition, Cas13a_10 has only 519 amino acids, Cas13a_11 has only 726 amino acids, Cas13T_15 has 543 amino acids, Cas13T_16 has only 845 amino acids, and Cas13Z_2 has 849 amino acids. The smaller Cas13 protein is conducive to encapsulation, packaging and delivery during gene editing. For example, only AAV vector is required for delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG1 shows a schematic diagram of the genomic locus structure of the CRISPR-Cas system.

[0055] Figure 2 shows the secondary structure predicted by the direct repeat (DR) sequence associated with Cas13a_10, Cas13a_11, Cas13b_3, Cas13b_4, and Cas13b_5, wherein, since the DR sequences of Cas13a_10 and Cas13a_11 are the same, the DR sequences of the two are named DR-Cas13a_10 / 11.

[0056] Figure 3 shows the predicted secondary structures of the direct repeat (DR) sequences associated with Cas13T_9, Cas13T_15, Cas13T_16, and Cas13Z_2.

[0057] Figure 4 shows a phylogenetic tree of Cas13a_10, Cas13a_11, Cas13b_3, Cas13b_4, Cas13b_5, Cas13T_9, Cas13T_15, Cas13T_16, Cas13Z_2 and previously identified Cas13 proteins.

[0058] Figure 5 shows the conserved domain structures of Cas13a_10, Cas13a_11, Cas13b_3, Cas13b_4, Cas13b_5, Cas13T_15, Cas13T_16, and Cas13Z_2, showing the relative positions of the RXXXXH motifs.

[0059] Figure 6 shows the predicted three-dimensional protein structures of Cas13a_10, Cas13a_11, Cas13b_3, Cas13b_4, Cas13b_5, and Cas13T_9.

[0060] Figure 7 shows the predicted three-dimensional protein structures of Cas13T_15, Cas13T_16, and Cas13 Z_2.

[0061] Figure 8 shows the relationship between the editing activity of target RNA by guide RNA and Cas13a_10, Cas13a_11, Cas13b_3, and Cas13b_5 using different structures (i.e., the reporter gene mCherry expression level of human HEK293T cells after transfection is knocked down). In the figure, the ordinate is the relative mean fluorescence intensity, and the abscissa is different crRNAs.

[0062] Figure 9 shows the relationship between the editing activity of target RNA using guide RNAs of different structures and Cas13b_4, Cas13T_9, and Cas13T_15 (i.e., the knockdown of reporter gene mCherry expression in transfected human HEK293T cells). In the figure, the ordinate is the relative mean fluorescence intensity, and the abscissa is different crRNAs.

[0063] Figure 10 shows the relationship between the editing activity of target RNA using guide RNAs with different structures and Cas13T_16 and Cas13Z_2 (i.e., the knockdown of reporter gene mCherry expression in transfected human HEK293T cells). In the figure, the ordinate is the relative mean fluorescence intensity, and the abscissa is the different crRNAs.

[0064] Figure 11 shows the relationship between the side-cutting activity of different spacer sequences and Cas13b_3, Cas13 b_4, and Cas13T_9 on the GFP reporter gene (i.e., the GFP knockdown efficiency of transfected human HEK293T cells represents the non-specific nuclease activity of the above-mentioned Cas proteins). In the figure, the ordinate is the relative mean fluorescence intensity, and the abscissa is different crRNAs.

[0065] Figure 12 shows the relationship between the side-cutting activity of different spacer sequences and Cas13T_15 and Cas13T_16 on the GFP reporter gene (i.e., the GFP knockdown efficiency of transfected human HEK293T cells represents the non-specific nuclease activity of the above-mentioned Cas proteins). In the figure, the ordinate is the relative mean fluorescence intensity, and the abscissa is different crRNA.

[0066] FIG13 shows the plasmid map of U6-BbSI-PUC57 used to construct the guide RNA recombinant plasmid.

[0067] Figure 14 shows a comparison of the cleavage activities of Cas13T_9, Cas13Z_2, Cas13T_15, Cas13T_16, Cas13b_3 and Cas13d at specific sites.

[0068] Figure 15 shows the knockdown effect of Cas13T_15 on the mRNA expression of various endogenous genes. DETAILED DESCRIPTION

[0069] Embodiments of the present invention relate to Cas13 proteins.

[0070] Cas13 protein is a class of proteins that have the ability to cut single-stranded RNA. The present invention relates to 9 novel Cas13 proteins, including:

[0071] A protein having an amino acid sequence as shown in any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9, and a functionally active variant thereof, i.e., a protein or polypeptide having an amino acid sequence that is not completely identical to any one of SEQ ID NOs.1-9 but having the same activity or substantially the same activity, for example, the activity calculated according to the activity characterized in the examples is 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1.0 times, 1.2 times, 1.5 times, 1.8 times, 2 times, etc., of any one of SEQ ID NOs.1-9.

[0072] It is well known to those skilled in the art that Cas13 protein can undergo amino acid changes, such as substitutions, deletions or additions, and the resulting Cas13 protein can maintain its function or activity.

[0073] The "substitution" mentioned above refers to the replacement of an amino acid residue at a certain position in an amino acid sequence by another amino acid residue; wherein, the "substitution" may be a conservative amino acid substitution.

[0074] "Conservative modification," "conservative substitution," or "conservative replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main-chain conformation, and rigidity, etc.), such that changes can frequently be made without altering the biological activity of the protein.

[0075] Those skilled in the art will recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). In addition, substitutions with structurally or functionally similar amino acids are unlikely to disrupt biological activity. Exemplary conservative substitutions are set forth in "Exemplary Conservative Amino Acid Substitutions" (see Table 1).

[0076] Table 1 Exemplary conservative amino acid substitutions

[0077] Embodiments of the present invention relate to fusion proteins comprising the Cas13 protein of the present invention. Preferred embodiments of the present invention relate to fusion proteins comprising the Cas13 protein of the present invention and a heterologous functional domain.

[0078] In some embodiments of the present invention, the heterologous functional domain of the fusion protein includes at least one of a deaminase, a reporter protein, a detection label, a localization signal, a protein targeting domain, a DNA binding domain, an epitope tag, a transcription activation domain, a transcription repression domain, a nuclease, a methylation active enzyme, a demethylase, a transcription release factor, an RNA cleavage polypeptide, a nucleic acid ligase, a histone modification active factor, or a nucleic acid binding active factor.

[0079] The fusion protein of the present invention obtained by fusing Cas13 of the present invention with a heterologous functional domain can achieve specific detection, mutation, or therapeutic purposes by combining two or more protein functional activities, for example, as described below.

[0080] In some embodiments, the fusion protein comprising Cas13 of the present invention can improve the signal-to-noise ratio by using a negative feedback regulation mechanism with the help of a nuclear localization sequence (NLS), a zinc finger protein, and a KRAB domain.

[0081] In some embodiments, a fusion protein comprising Cas13 of the present invention can achieve RNA methylation at a specific site and / or achieve RNA demethylation at a specific site.

[0082] In some embodiments, the fusion protein comprising Cas13 of the present invention can target pathogenic tau pre-mRNA, change the alternative splicing mode, and achieve significant therapeutic effects.

[0083] Embodiments of the present invention relate to CRISPR-Cas systems comprising the Cas13 protein of the present invention.

[0084] The CRISPR-Cas system of the present invention comprises the Cas13 protein of the present invention and a guide RNA, or comprises the fusion protein of the present invention and a guide RNA.

[0085] In some embodiments, the guide RNA of the present invention can bind to the Cas13 protein to form a complex and guide the complex to contact the target RNA.

[0086] The guide RNA of the present invention comprises a spacer sequence and a direct repeat sequence; the spacer sequence has a size of 15 to 60 nucleotides, for example, 20 to 50 nucleotides, 30 to 40 nucleotides or 30 nucleotides; the direct repeat (DR) sequence has a size of 15 to 40 nucleotides, for example, 20 to 35 nucleotides, 25 to 30 nucleotides or 30 nucleotides.

[0087] In some embodiments, the guide RNA is chemically modified, for example, by incorporation of a 2'-O-methyl, 2'-O-methyl 3'-phosphorothioate, or 2'-O-methyl 3'-thioPACE at one or more terminal nucleotides. These chemically modified guide RNAs are more stable and have increased activity compared to unmodified guide RNAs.

[0088] In some embodiments, there are at least 15, e.g., 15, 16, 18, 19, 20, 21-30 nucleotides of complementarity between the spacer sequence and the target RNA.

[0089] The CRISPR-Cas system of the present invention can be used for nucleic acid detection. In some embodiments of the present invention, after the CRISPR-Cas13 system is combined with crRNA and substrate RNA to form a ternary complex, the Cas13 protein is activated. Now, the Cas13 protein can not only specifically cut the substrate RNA, but also cut any single-stranded RNA free in the environment. This phenomenon is referred to as non-specific nuclease activity, incidental activity or collateral cleavage activity. Utilizing this characteristic, the system of the present invention can detect RNA, for example, within 10-15 minutes, the detection sensitivity reaches femtomolar level. Compared with traditional nucleic acid detection gold standard qPCR, the detection speed of the present invention is faster and less dependent on equipment.

[0090] The CRISPR-Cas system of the present invention can be used for RNA imaging. In some embodiments of the present invention, the Cas13 protein is mutated to obtain a protein variant that loses the cleavage enzyme activity but retains the binding enzyme activity, thereby being able to target the target RNA molecule.

[0091] The CRISPR-Cas system of the present invention can be used for transcriptome regulation. In some embodiments of the present invention, various effector proteins are fused to the Cas13 protein or its variants that have lost their cleavage enzyme activity but retained their binding enzyme activity, enabling various RNA-targeting regulatory actions. For example, methylation regulation can be used to treat genetic diseases caused by single-base mutations.

[0092] By fusing a specific mutant of the Cas13 protein of the present invention (e.g., a mutant that has lost some or all of its nuclease activity) with a deaminase (e.g., ADAR2DD, etc.), an RNA base editor can be constructed.

[0093] In some embodiments, the CRISPR-Cas system of the present invention can achieve therapeutic purposes by targeting overexpressed RNA or toxic RNA, or mutant RNA (such as splicing defects or truncations, whose overexpression can cause disease). Alternatively, the CRISPR-Cas system of the present invention can also be used for treatment by targeting trans-acting mutant sequences of RNA-dependent functions that cause disease. For example, mutations in the SNHG5 gene are associated with various cancers (e.g., prostate cancer), and by targeting the RNA expressed therein, related diseases can be treated.

[0094] Embodiments of the present invention relate to polynucleotides.

[0095] The polynucleotide of the present invention encodes the Cas13 protein of the present invention, or the polynucleotide encodes the fusion protein of the present invention, or the polynucleotide encodes the CRISPR-Cas system of the present invention.

[0096] In some embodiments, the polynucleotides are DNA molecules. In other embodiments, the polynucleotides are RNA molecules (such as mRNA molecules encoding the Cas13 protein, its derivatives or its functional fragments). In some embodiments, the RNA molecules (such as mRNA molecules) are capped, polyadenylated, or replaced by 2'-O-methyl analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, 2'-fluoro analogs, 5-methylcytosine nucleosides, pseudouridine, or any combination thereof, including but not limited to 2-aminopurine, 5-bromo-uridine, pseudouridine (Ψ), N1-methylpseudouridine (melΨ), 5-methoxyuridine (5moU), inosine, 7-methylguanosine, etc.

[0097] In some embodiments, the polynucleotide shares at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or 100% of the nucleotide sequence with the nucleotide sequence disclosed herein.

[0098] In some embodiments, the amino acid sequence encoded by the polynucleotide has at least 85% (eg, 85%, 95%, 99%) sequence identity to the amino acid sequence shown in any one of SEQ ID NOs. 1-9.

[0099] Embodiments of the present invention relate to vectors, which can deliver or introduce the Cas13 protein (including its homologue) and / or guide RNA involved in the present invention into host cells, and in some specific cells (such as prokaryotic cells), these components (such as in prokaryotic cells) can also be replicated. When operably connected to appropriate regulatory elements, the vector can replicate. The aforementioned vector includes a variety of vectors, and common viral vectors and non-viral vectors are available. One type of vector is a viral vector, in which viral-derived DNA or RNA sequences are present in vectors for packaging viruses (such as retroviral vectors, phage vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia virus vectors, hybrid viral vectors, baculovirus vectors, herpes simplex virus vectors or slow virus vectors). Viral vectors often include polynucleotides carried by viruses for transfection into host cells. In addition, non-viral vectors are common plasmids, which are circular double-stranded DNA loops that can, for example, be inserted into other DNA segments by standard molecular cloning techniques. Some plasmid vectors (such as bacterial vectors and additional mammalian vectors with bacterial replication origins) can replicate autonomously in the host cells into which they are introduced.

[0100] The vector of the present invention carries the polynucleotides described above, for example, encoding the Cas13 protein of the present invention, or encoding a fusion protein of the aforementioned Cas13 protein, or encoding a polynucleotide of the CRISPR-Cas system of the present invention.

[0101] The polynucleotide further comprises a nucleotide sequence encoding a direct repeat (DR) sequence as described in SEQ ID NO. 20-27.

[0102] In some embodiments of the present invention, the polynucleotide carried by the vector is linked to a regulatory element.

[0103] In some embodiments of the invention, the regulatory element comprises at least one of a promoter, an internal ribosome entry site (IRES), an enhancer, a polyadenylation signal, a terminator, a protein degradation signal, a transposon, a leader sequence, a polyadenylation sequence, or a marker gene.

[0104] Promoters include tissue-specific promoters that can direct expression primarily in tissues of interest such as muscle, neurons, bones, skin, blood, or specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Specifically, promoters can include U6 promoters, H1 promoters, retroviral Rous sarcoma virus LTR promoters (optionally with RSV enhancers), cytomegalovirus (CMV) promoters (optionally with CMV enhancers), SV40 promoters, dihydrofolate reductase promoters, β-actin promoters, phosphoglycerol kinase (PGK) promoters, and EF1α promoters.

[0105] Enhancer elements include CMV enhancers, RSV enhancers, SV40 enhancers, and the like. When designing an expression vector, factors such as the host cell to be transformed, the expected expression level, and the like may be considered. The vector can be introduced into a host cell to produce transcripts, proteins, or peptides encoded by the nucleic acids described herein, including fusion proteins or peptides (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, and the like).

[0106] Embodiments of the present invention relate to delivery systems. The delivery systems of the present invention are used to deliver the proteins, nucleic acids, systems or vectors of the present invention into the body for detection and treatment.

[0107] In some embodiments of the present invention, the delivery system carries the Cas13 protein of the present invention, or the fusion protein of the present invention, or the CRISPR-Cas system of the present invention, or the polynucleotide of the present invention, or the vector of the present invention.

[0108] In some embodiments of the present invention, the delivery vector of the delivery system includes at least one of a liposome vector, a chitosan vector, a polymer vector, a nanoparticle vector, an exosome vector, an exosome vector, a microvesicle vector, a viral vector, a gene gun, or an electroporation device.

[0109] In addition, when the delivery object is some cells (such as plant cells), a method of delivery such as cell penetrating peptides (CPP) will also be adopted. For example, in one embodiment, Cas protein and / or at least one guide RNA is coupled to one or more CPPs, thereby effectively transporting the CPP coupled with Cas protein and / or guide RNA into plant cells (such as protoplasts). CPP has a short peptide of less than 35 amino acids, which is derived from a protein or derived from a chimeric sequence and can transport biomolecules across the cell membrane in a non-receptor-dependent manner. CPP can be a cationic peptide, a peptide with a hydrophobic sequence, an amphipathic peptide, a peptide rich in proline and antimicrobial sequences, and a chimeric or dichotomous peptide. CPP can penetrate biological membranes and therefore trigger different biomolecules to move across the cell membrane into the cytoplasm, and can improve their intracellular pathways and therefore promote the interaction between biomolecules and targets.

[0110] Exemplary CPPs include Tat (a nuclear transcription activator protein required for viral replication by HIV type 1), penetratin, Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin β3 signal peptide sequence, polyarginine peptide Arg sequence, guanine-rich molecule transporter, sweet arrow peptide, etc.

[0111] Embodiments of the present invention relate to host cells carrying the Cas13 protein of the present invention, the fusion protein of the present invention, the CRISPR-Cas system of the present invention, the polynucleotide of the present invention, or the vector of the present invention.

[0112] In some embodiments, the host cell is an in vitro, in vivo or in vitro cell, or a cell line or progeny, and the cell or cell line or progeny comprises: the Cas13 protein, fusion protein, CRISPR-Cas system, polynucleotide, vector, delivery system.

[0113] In some embodiments, the cell is a prokaryotic cell, eg, E. coli.

[0114] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a yeast cell, a plant cell or a mammalian cell (including humans and non-human mammals). In some embodiments, the cell is a non-human mammalian cell, such as a cell of a non-human primate, cattle, sheep, pig, dog, monkey, rabbit, rodent (such as rat or mouse). In some embodiments, the cell is a non-mammalian eukaryotic cell, such as a cell of a bird (such as chicken), fish or crustacean. In some embodiments, the cell is a plant cell, such as a cell that a monocot or dicot has or a cell of a cultivated plant or food crop such as corn, sorghum, soybean, wheat, oat or rice. In some embodiments, the cell is a stem cell or stem cell line. In some cases, the host cell of the present invention comprises a modification of a gene or genome that does not exist in its wild type.

[0115] In some embodiments, the host cell is selected from the group consisting of Escherichia coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, yeast cells, or a combination thereof.

[0116] Embodiments of the present invention relate to models.

[0117] In some embodiments of the present invention, the model comprises the host cell described in the present invention.

[0118] Embodiments of the present invention relate to methods of gene editing.

[0119] In some embodiments of the invention, the method comprises contacting the target RNA with a Cas13 protein of the invention, a fusion protein of the invention, a CRISPR-Cas system of the invention, a polynucleotide of the invention, or a vector of the invention. In some embodiments of the invention, the contacting can be edited by the Cas13 protein.

[0120] In some embodiments of the present invention, in the method, the guide RNA binds to the Cas13 protein to form a complex, and guides the complex to contact the target RNA to perform gene editing on the target RNA.

[0121] Embodiments of the present invention relate to gene editing products. In some embodiments of the present invention, the gene editing product is obtained by gene editing a target RNA using the gene editing method of the present invention.

[0122] Therefore, embodiments of the present invention relate to applications in gene editing.

[0123] Embodiments of the present invention relate to kits.

[0124] In some embodiments of the present invention, the kit comprises the Cas13 protein of the present invention, the fusion protein of the present invention, the CRISPR-Cas system of the present invention, the polynucleotide of the present invention, the vector of the present invention, the delivery system of the present invention, or the host cell of the present invention. In some embodiments, the kit also includes instructions for using the kit, such as instructions in more than one language. The kit may also include one or more reagents for use in the process of utilizing one or more of the above components. The reagents may be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. The above reagents may be provided in a form (e.g., in concentrated or lyophilized form) requiring the addition of one or more other components before use; the buffer may be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. The buffer may have a suitable pH value, for example, it may be alkaline. In some embodiments, the pH of the buffer is between about 7-10.

[0125] Embodiments of the present invention relate to nucleic acid detection methods. In some embodiments of the present invention, the nucleic acid detection methods of the present invention are performed using a kit of the present invention, or a kit comprising multiple components thereof, such as a Cas13 protein, a fusion protein, a CRISPR-Cas system, a polynucleotide, a vector, a delivery system, or a host cell, to perform nucleic acid detection on a sample to be tested.

[0126] In some embodiments, the CRISPR-Cas system of the present invention can be used for DNA and / or RNA detection.For example, as shown in the embodiments, when the interval (spacer) sequence is about 30 nucleotides, when the specific RNase activity activated by its guide RNA dependence, Cas13b_3 of the present invention, Cas13b_4, Cas13T_9, Cas13T_15, Cas13T_16 show side cutting activity (non-specific cutting activity).Therefore, the Cas13 protein involved in the present invention, by guide RNA (crRNA) is reprogrammed to carry out sample nucleic acid detection, realizes specific RNA detection.

[0127] For example, the CRISPR-Cas system of the present invention can adopt the SHERLOCK method (the method can be seen, for example, in Gootenberg et al., Nucleic acid detection with CRISPR-Cas13a / C2c2, Science, 356(6336):438-442 (2017)), which is incorporated herein by reference in its entirety.

[0128] Embodiments of the present invention relate to applications in nucleic acid detection, or embodiments of the present invention relate to applications in preparing drugs for treating disorders or diseases.

[0129] In some embodiments of the application of the present invention, the above-mentioned nucleic acid detection is performed using the kit of the present invention, or a kit comprising one or more components, such as Cas13 protein, fusion protein, CRISPR-Cas system, polynucleotide, vector, delivery system or host cell.

[0130] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the invention belongs. As used herein, the following terms have the meanings ascribed to them below, unless otherwise indicated.

[0131] "Polynucleotide" and "nucleic acid" refer to a polymeric form of nucleotides (e.g., RNA or DNA) of any length. The term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0132] A "DNA sequence encoding a specific RNA" refers to a DNA nucleotide sequence that is transcribed into RNA.

[0133] "Regulatory element" or "control element" refers to transcriptional and translational regulatory sequences such as promoters, enhancers, terminators, and the like.

[0134] A "promoter (also known as a promoter sequence)" is a DNA regulatory region that can bind RNA polymerase and initiate transcription of downstream (3' direction) coding or non-coding sequences. Generally speaking, various promoters, including inducible promoters, can be used to drive expression of various vectors of the present invention.

[0135] "Codon optimization" refers to the modification of a nucleic acid sequence to enable better expression of the aforementioned nucleic acid sequence in a host cell. Generally speaking, this is achieved by replacing at least one codon (for example, one or more, such as 10, 20 or more) in the original nucleic acid sequence with a codon that is more frequently or most frequently used in the host cell gene, while maintaining the expressed amino acid sequence.

[0136] "Target RNA" can be any target RNA molecule, which can be a naturally occurring RNA molecule or an engineered RNA molecule (e.g., mRNA, tRNA, ribosomal RNA (rRNA), microRNA (miRNA), interfering RNA (siRNA), ribozyme, satellite RNA, or viral RNA, etc.). The target RNA can be a target RNA associated with a condition or disease (e.g., an infectious disease or cancer). Complete complementarity between the target RNA and the guide RNA is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR-Cas complex.

[0137] "Naturally occurring" (also referred to as unmodified, unmodified, wild-type (wt)) refers to a nucleic acid, polypeptide, cell, or organism that occurs in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature is naturally occurring.

[0138] A "fusion protein" is a hybrid polypeptide comprising protein domains from at least two different proteins. One protein can be located at the amino-terminal (N-terminal) portion or the carboxyl-terminal (C-terminal) portion of the fusion protein, thereby forming an amino-terminal fusion protein or a carboxyl-terminal fusion protein, respectively.

[0139] A "CRISPR-Cas system" (also referred to as a "CRISPR system") generally comprises transcripts or other elements associated with the expression of a CRISPR-associated ("Cas") gene, or that are capable of directing the activity of the Cas gene. In some embodiments, components of a CRISPR system may comprise nucleic acids (e.g., vectors) encoding one or more components of the system, components in protein form, or a combination thereof.

[0140] "Cas protein" refers to a CRISPR-associated protein (Cas) (also referred to as a "CRISPR-associated protein," "CRISPR effector," "effector," "Cas protein," "Cas enzyme," or "CRISPR enzyme") that performs enzymatic activity and / or binds to a target site on a nucleic acid specified by an RNA guide. In some embodiments, a Cas protein has endonuclease, nickase, exonuclease, transposase, and / or excision activity; in other embodiments, the Cas protein may be nuclease-inactivated.

[0141] "Guide RNA (also known as guide RNA, gRNA or crRNA)" refers to any RNA molecule that facilitates targeting the Cas13 protein of the present invention to a target nucleic acid (such as DNA and / or RNA).

[0142] In the CRISPR-Cas system, the degree of complementarity between the guide RNA and its corresponding target nucleic acid may not be 100%, for example, about 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%. To reduce off-target interactions, for example, to reduce the interaction between the guide RNA and a target nucleic acid with low complementarity, mutations can be introduced into the CRISPR-Cas system so that the CRISPR-Cas system can distinguish between target nucleic acids with greater than 80%, 85%, 90%, 95%, 99% complementarity and off-target sequences. In some specific embodiments, the degree of hybridization / complementarity between the guide RNA and the corresponding target nucleic acid is from 80% to 95% (for example, there is about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% complementarity). Thus, in some embodiments, the degree of complementarity between a guide RNA and its corresponding target sequence is greater than 93%, 93.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9%. In some embodiments, the degree of complementarity is 100%.

[0143] It is known in the art that complete complementarity is not required on the basis that there is sufficient complementarity to work, and therefore, if necessary, the cutting efficiency can be adjusted by introducing mismatches (e.g., one or more mismatches between the spacer sequence and the target nucleic acid, such as mismatches of 1 or 2 nucleotides (including the position of the mismatch along the spacer sequence / target sequence)). If the mismatch (e.g., double mismatch) is located closer to the center (i.e., not at the 3' or 5' end), the cutting efficiency is more affected. Therefore, by selecting the position of the mismatch along the spacer sequence, the cutting efficiency can be adjusted. For example, if a cutting rate of less than 100% of the target is desired (e.g., in a cell population), 1 or 2 mismatches between the spacer sequence and the target sequence can be introduced into the spacer sequence.

[0144] "Vector" refers to a polynucleotide composition used to transfer, deliver or introduce nucleic acid into a host cell. Suitable vectors include plasmid vectors, phage vectors, viral vectors (e.g., retroviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, AAV vectors, lentiviral vectors, baculoviral vectors), and the like.

[0145] A "delivery system" comprises a delivery vehicle comprising one or more liposomes, nanoparticles, exosomes, microvesicles, viral vectors, or a gene gun.

[0146] In some embodiments, the vector (e.g., a viral vector or a non-viral vector, such as a lentiviral vector or a plasmid) can be delivered to the target tissue by, for example, intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration. The above-mentioned delivery can be carried out via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dose to be delivered herein can vary to a large extent according to a variety of factors, including but not limited to vector selection, target cells, organisms, tissues, the general condition of the subject to be treated, the degree of transformation / modification sought, the route of administration, the mode of administration, and the type of transformation / modification sought.

[0147] "Functional fragment" means that the protein or polypeptide sequence includes fewer amino acids than the original sequence of the protein or polypeptide, but the remaining amino acid sequence still has a certain proportion (such as 10%, 20%, 30%, 40%, 50% or 60-99%, 100%) of functional activity relative to the functional activity of the original reference sequence (for example, the protein or polypeptide can be modified by substitution, insertion, deletion and / or addition of one or more amino acids while retaining a certain proportion of enzymatic activity).

[0148] "Identity" refers to the sequence matching between two polypeptides or two nucleic acids. When the positions in the two sequences being compared are occupied by the same base or amino acid monomer subunit (for example, if the position in each of the two DNA molecules is occupied by adenine, or the position in each of the two polypeptides is occupied by lysine), each molecule is identical at that position. "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 being compared and multiplied by 100. For example, if 6 out of 10 positions of the two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have a total of 50% identity (3 out of a total of 6 positions match). Typically, comparison is performed when two sequences are aligned to produce maximum identity.

[0149] "Hybridization" or "complementary" or "substantially complementary" refers to a nucleic acid (e.g., RNA, DNA) comprising a nucleotide sequence that enables the nucleic acid to non-covalently bind (i.e., form Watson-Crick base pairs and / or G / U base pairs), "anneal," or "hybridize" with another nucleic acid in a sequence-specific, antiparallel manner (i.e., nucleic acid-specific binding to complementary nucleic acid) under appropriate in vitro and / or in vivo temperature and solution ionic strength conditions. Hybridization requires that the two nucleic acids contain complementary sequences, but mismatches between bases are possible. Typically, hybridizable nucleic acids are 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more) in length.

[0150] "Direct repeat (DR) sequence" (also known as DR sequence) refers to the DNA coding sequence in the CRISPR locus, or the RNA encoded by it in the guide RNA. When described at the RNA level, each T in the DNA coding sequence should be understood to represent a U.

[0151] The direct repeat sequence comprises a nucleotide sequence that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the nucleotide sequence set forth in any one of SEQ ID NOs. 20-27.

[0152] "Host cell" includes in vitro, ex vivo or in vivo cells or cell lines or their progeny, which contain: the Cas13 protein, fusion protein, CRISPR-Cas composition, recombinant vector, delivery vector described in the present invention.

[0153] In some embodiments, the CRISPR-Cas system provided by the present invention can be used for prokaryotic cells or eukaryotic cells. Exemplary, prokaryotic cells include Escherichia coli. Eukaryotic cells, including but not limited to plant cells, fungal cells (e.g., yeast cells, exemplary, such as Pichia pastoris, Kluyveromyces, Kluyveromyces lactis, etc.), mammalian cells, reptile cells, insect cells, bird cells, fish cells, parasite cells, arthropod cells, invertebrate cells, vertebrate cells, rodent cells, mouse cells, rat cells, primate cells, non-human primate cells or human cells. In some embodiments, eukaryotic cells are in vitro cultures. In some embodiments, eukaryotic cells are in vivo. In some embodiments, eukaryotic cells are isolated (in vitro).

[0154] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0155] If no specific experimental steps or conditions are specified in the following examples, the experiments were carried out according to the conventional experimental steps or conditions described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional reagents.

[0156] Example 1 Identification of novel Cas13 proteins

[0157] The inventors identified 9 new Cas13 proteins through metagenome analysis, de-redundancy (removal of identical protein sequences), and protein cluster analysis. The analysis found that they belong to four Cas13 protein families: Cas13a, Cas13b, Cas13T, and Cas13Z families (wherein, Cas13T and Cas13Z families do not belong to the previously identified Cas13 proteins and are different, belonging to a new Cas13 family): including Cas13a_10 (SEQ ID NO: 1), Cas13a_11 (SEQ ID NO: 2), Cas13b_3 (SEQ ID NO: 3), Cas13b_4 (SEQ ID NO: 4), Cas13b_5 (SEQ ID NO: 5), Cas13T_9 (SEQ ID NO: 6), Cas13T_15 (SEQ ID NO: 7), Cas13T_16 (SEQ ID NO: 8), and Cas13Z_2 (SEQ ID NO: 9). NO: 9), through analysis, it was found that the genomic locus structure of the above four Cas13 families is shown in Figure 1. The various components in the locus are not in proportion. As shown in the figure, the pointed bar marked with "Cas12a / b / T / Z" represents the Cas coding sequence, the rectangle represents the DR sequence, and the diamond represents the spacer sequence. Upon comparison, it was found that these 9 Cas13 proteins have low similarity with the previously identified Cas13 proteins.

[0158] Through sequence analysis, the direct repeat (DR) sequence associated with the above-mentioned Cas13 protein was found, and the DNA sequence encoding the direct repeat (DR) sequence is shown in Table 2.

[0159] Table 2 Coding sequences of direct repeat (DR) sequences associated with different Cas13 proteins

[0160] The secondary structure of the direct repeat (DR) sequences associated with Cas13a_10, Cas13a_11, Cas13b_3, Cas13b_4, Cas13b_5, Cas13T_9, Cas13T_15, Cas13T_16, and Cas13Z_2 was analyzed using RNA secondary structure prediction software RNAfold. The structural analysis results are shown in Figures 2 and 3. As can be seen from Figures 2 and 3, all DR sequences have relatively conserved secondary structures.

[0161] The DR sequences of the two proteins in the Cas13a family, Cas13a_10 and Cas13a_11, are exactly the same. From their secondary structure, it can be seen that they form a stem composed of 3 base pairs (5'-GCA-3' and its complementary base), followed by two smaller protrusions, followed by a stem composed of 5 base pairs (5'-CGCCC-3' and its complementary base), and the end is a ring structure formed by 7 bases (5'-UUCACUU-3').

[0162] Among the three proteins in the Cas13b family, the secondary structures of the DR sequences of Cas13b_3 and Cas13b_4 are very similar. They both form a stem composed of 5 base pairs (5'-GUUGU-3' and its complementary bases), followed by a protrusion structure (the base types and base numbers of the protrusion structure are different. For Cas13b_3, the protrusion structure formed by the associated DR sequence at this position consists of 3+3 bases; and for Cas13b_4, the protrusion formed by its associated DR sequence at this position consists of 4+4 bases), followed by a stem structure (the base types and base numbers of the stem structure are different. For Cas13b_3, the stem structure formed by the associated DR sequence at this position consists of 5'-UGCCUUG-3' and its complementary bases; and for Cas1 For Cas13b_3, the stem formed by its associated DR sequence at this position consists of 5'-GCCCUUG-3' and complementary bases), followed by a ring structure (the type and number of bases in the ring structure are different. For Cas13b_3, the protrusion structure formed by the associated DR sequence at this position consists of 6 bases; and for Cas13b_4, the protrusion formed by its associated DR sequence at this position consists of 4 bases); and the secondary structure of the DR sequence associated with Cas13b_5 is significantly different from that of Cas13b_3 and Cas13b_4; as can be seen from Figure 2, it has 3 stem structures consisting of 5 base pairs, 3 base pairs, and 6 base pairs, respectively, there is a protrusion structure between the 1st and 2nd stems, and between the 2nd and 3rd stems, and there is also a ring structure consisting of 6 bases at the end.

[0163] In the Cas13T family, the DR sequence associated with Cas13T_9, Cas13T_15, and Cas13T_16 has a similar secondary structure, including a stem structure composed of 5 base pairs (the structure of this part of Cas13T_9 and Cas13T_16 is exactly the same, both consisting of 5'-GUUGU-3' and its complementary base; the base types of the base pairs of the stem structure of Cas13T_15 are different), followed by a protrusion structure composed of multiple bases (among them, the protrusion part of the DR secondary structure associated with Cas13T_9 and Cas13T_15 is composed of 5+5 bases, while the protrusion structure of the DR secondary structure associated with Cas13T_16 is composed of 3+3 bases, and the base types are also different), followed by another It is a stem structure (the stem structure of the DR secondary structure associated with Cas13T_9 and Cas13T_15 is exactly the same, both consisting of 4 base pairs 5'-CCUU-3' and its complementary bases, and the stem structure of the DR secondary structure associated with Cas13T_16 consists of 7 base pairs 5'-UGUCUUU-3' and its complementary bases), and the end is a ring structure (the ring structure of the DR secondary structure associated with Cas13T_9 and Cas13T_15 consists of 8 bases, with one base difference, and the rest of the bases are the same; while the ring structure of the DR secondary structure associated with Cas13T_16 consists of 5 bases, and the base types are significantly different from those of Cas13T_9 and Cas13T_15).

[0164] From the secondary structure of the DR sequence associated with Cas13Z_2, it can be seen that it includes a stem structure composed of 5'-GC-3' and its complementary bases, followed by a small protrusion structure, then a stem structure composed of 5'-GU-3' and its complementary bases, followed by a protrusion structure composed of 5+5 bases, followed by a stem structure composed of 5'-CCUU-3' and its complementary bases, and the end is a ring structure composed of 8 bases.

[0165] The above-mentioned Cas13 protein was subjected to multiple sequence alignment with the previously identified Cas13 family protein using sequence comparison software, and an evolutionary tree was constructed. The construction results are shown in Figure 4. As can be seen from Figure 4, Cas13a_10 and Cas13a_11 are located on two relatively close branches of the phylogenetic tree, Cas13b_3, Cas13b_4, and Cas13b_5 are relatively close on the phylogenetic tree, and Cas13b_3 and Cas13b_5 are closer. The relationship between Cas13T_9, Cas13T_15, and Cas13T_16 in the phylogenetic tree is relatively close, and the relationship between Cas13Z_2 and other Cas13 proteins in the phylogenetic tree is relatively far.

[0166] The inventors also used the publicly available Cas protein database to perform functional annotation and multiple sequence comparison on each of the above-mentioned Cas13s, analyzed the conserved regions of the 9 Cas13 protein sequences, and identified the conserved RXXXXH motif positions of these 9 Cas13 proteins. The identification results are shown in Figure 5.

[0167] As shown in Figure 5, Cas13a_10, Cas13b_5, Cas13T_15, and Cas13T_16 all contain only one RXXXXH motif, of which the RXXXXH motifs of Cas13a_10, Cas13T_15, and Cas13T_16 are all closer to the C-terminus, while the RXXXXH motif of Cas13b_5 is closer to the N-terminus. Cas13a_11, Cas13b_3, Cas13b_4, and Cas13Z_2 all contain two RXXXXH motifs, of which the two RXXXXH motifs of Cas13a_11 are closer to the C-terminus, and the two RXXXXH motifs of Cas13b_3, Cas13b_4, and Cas13Z_2 are closer to both ends (N-terminus and C-terminus).

[0168] Specifically, one RXXXXH motif of Cas13a_10 is located at positions 490-495, two RXXXXH motifs of Cas13a_11 are located at positions 453-458 and 581-586, respectively, two RXXXXH motifs of Cas13b_3 are located at positions 125-130 and 1079-1084, and two RXXXXH motifs of Cas13b_4 are located at positions 148-153 and 1176-1181, respectively. One RXXXXH motif of Cas13b_5 is located at positions 128-133. One RXXXXH motif of Cas13T_15 is located at positions 480-486. One RXXXXH motif of Cas13T_16 is located at positions 800-805. Two RXXXXH motifs of Cas13Z_2 are located at positions 143-148 and 813-818, respectively. No obvious RXXXXH motif was found in the Cas13T_9 protein. The RXXXXH motif is the potential catalytic active site of the Cas13 protein. Mutating this domain may produce a protein that inactivates the nuclease activity of the Cas13 protein while essentially maintaining the ability to bind to the guide RNA and the target RNA.

[0169] The applicant also used protein structure prediction software to predict the three-dimensional structure of the above-mentioned Cas13 protein. Specifically, see Figures 6 and 7. The two RXXXXH motifs of Cas13b_3 and Cas13b_4 are very close to the N-terminus and C-terminus in the protein sequence, but are very close in the three-dimensional structure (see Figure 6). The two RXXXXH motifs of Cas13Z_2 are relatively close to the N-terminus and C-terminus, but appear relatively close in the three-dimensional structure (see Figure 7).

[0170] Example 2 Activity Verification of Cas13 Protein

[0171] 1. In order to verify whether the nine Cas13 proteins Cas13a_10, Cas13a_11, Cas13b_3, Cas13b_4, Cas13b_5, Cas13T_9, Cas13T_15, Cas13T_16, and Cas13Z_2 have RNA nuclease activity, this example provides a cleavage activity verification experiment of these nine Cas13 proteins. The specific experimental process is as follows:

[0172] (1) The codon-optimized (to be more suitable for expression in human cells) polynucleotides encoding Cas13a_10, Cas13a_11, Cas13b_3, Cas13b_4, Cas13b_5, Cas13T_9, Cas13T_15, Cas13T_16, and Cas13Z_2 (SEQ ID NO.10-18) were cloned into the p23_puro_NPLS_Cas13_msfGFP_NLS_3xFlag_C plasmid carrying the green fluorescent protein (GFP) gene (SEQ ID NO.28) (the nucleotide sequence of the p23_puro_NPLS_Cas13_msfGFP_NLS_3xFlag_C plasmid is shown in SEQ ID As shown in NO.29, the restriction site is NheI:XbaI), and a Cas13 protein recombinant plasmid is obtained (in the Cas13 protein recombinant plasmid, the polynucleotide of Cas13 protein replaces the sequence at positions 3472 to 6369 in the p23_puro_NPLS_Cas13_msfGFP_NLS_3xFlag_C plasmid).

[0173] (2) Take the mCherry reporter gene recombinant plasmid (the nucleotide sequence of the mCherry reporter gene recombinant plasmid is shown in SEQ ID NO.30).

[0174] (3) spacer_1 to spacer_10 (SEQ ID NO. 33-42) are spacer sequences targeting different positions of mCherry (SEQ ID NO. 31), and NT-spacer (SEQ ID NO. 43) is a spacer sequence that does not target mCherry. For specific sequence design, see Table 3.

[0175] Table 3 Spacer sequences targeting mCherry and those not targeting mCherry

[0176] In theory, the guide RNA precursor in the CRISPR / Cas13 system can produce two types of guide RNA (i.e. crRNA) structures during maturation: direct repeat (DR) sequence+spacer sequence (i.e. 5'DR, DR sequence is connected to the 5' end of the spacer sequence) and spacer sequence+direct repeat (DR) sequence (i.e. 3'DR, DR sequence is connected to the 3' end of the spacer sequence). Since the DR sequences of Cas13a_10 and Cas13a_11 are exactly the same, the DR sequences of the two are named DR-Cas13a_10 / 11. For Cas13a_10, the inventors constructed a guide RNA of 3'DR structure. In Figure 8, the guide RNA is named Cas13a_10-crRNA_1; for Cas13a_11, the inventors constructed a guide RNA of 5'DR structure. In Figure 8, it is named Cas13a_11-crRNA_1. Similarly, DR-Cas13b_3, DR-Cas13b_4, DR-Cas13b_5, DR-Cas13T_9, DR-Cas13T_15, DR-Cas13T_16, and DR-Cas13Z_2 were respectively connected to the 3' or 5' end of spacer_1 to spacer_10 to construct guide RNAs targeting the reporter gene mCherry; and the DR sequences of the aforementioned Cas proteins were respectively connected to the 3' or 5' end of NT-spacer to construct guide RNAs that did not target the reporter gene mCherry.

[0177] (4) The coding sequences of the guide RNA targeting the reporter gene mCherry and the guide RNA not targeting the reporter gene mCherry were cloned into the U6-BbSI-PUC57 plasmid (purchased from Shanghai Shenggong Company, the plasmid map of the U6-BbSI-PUC57 plasmid is shown in Figure 13, and the plasmid nucleotide sequence is shown in SEQ ID NO.32) after enzyme digestion (enzyme digestion site is BbSI) to obtain a guide RNA recombinant plasmid; wherein, the guide RNA targeting mCherry and the guide RNA not targeting mCherry are both composed of a direct repeat (DR) sequence and a spacer sequence. The structures of the guide RNA targeting mCherry and the guide RNA not targeting mCherry used for the 9 Cas13 proteins are shown in Table 4.

[0178] Table 4 Guide RNA

[0179] (5) Human HEK293T cells (purchased from ATCC) were cultured at 1×10 4 After inoculation at an inoculum size of 100 μg / well in a 48-well plate, the Cas13 protein recombinant plasmid, guide RNA recombinant plasmid (containing a guide RNA targeting the reporter gene mCherry) and mCherry reporter gene recombinant plasmid were co-transfected into human HEK293T cells using LIPOFECTAMINE3000 and P3000TM reagents (purchased from ThermoFisher, L3000015) to obtain transfected human HEK293T cells in the experimental group. The Cas13 protein recombinant plasmid, the guide RNA recombinant plasmid (containing a guide RNA that does not target the reporter gene mCherry) and the mCherry reporter gene recombinant plasmid were co-transfected into human HEK293T cells to obtain transfected human HEK293T cells for the negative control group; human HEK293T cells that were not transfected with any plasmid were used as the blank control group, and the transfected human HEK293T cells of the experimental group, the transfected human HEK293T cells of the negative control group and the blank control group were cultured in a cell culture incubator at 37°C and 5% (v / v) CO2.

[0180] (6) After 48 hours of culture, microscopic observation revealed that the growth and morphology of the transfected human HEK293T cells in the experimental group were similar to those of the negative control group and the blank control group. The transfected human HEK293T cells were observed under a fluorescence microscope and finally detected and quantitatively analyzed by flow cytometry. The analysis results are shown in Figures 8-10. Flow cytometry (FCM) analysis revealed that in the negative control group, the use of guide RNA that did not target the reporter gene mCherry failed to knock down the expression of the mCherry fluorescence signal; compared with the negative control group, the mCherry fluorescence signal intensity of the transfected human HEK293T cells in the experimental group was significantly reduced, indicating that all nine Cas13 proteins can effectively knock down the mCherry mRNA level using the guide RNA targeting mCherry, thereby reducing the expression of the mCherry protein. When using a 3'DR structured guide RNA (Cas13a_10-crRNA9), Cas13a_10 had a significant knockdown effect on mCherry expression, with the fluorescence signal intensity significantly reduced by about 80% (when using crRNA_9). When using a guide RNA with a specific structure, Cas13T_15, Cas13T_16, and Cas13Z_2 all had a good knockdown effect on mCherry expression, with a significant reduction in fluorescence signal intensity. Cas13a_11, Cas13b_3, Cas13b_4, Cas13b_5, Cas13T_9, and Cas13T_16 had a knockdown effect on mCherry expression, significantly reducing the fluorescence signal intensity. By comparison, it was found that when the spacer sequences (crRNA) at different positions of the mCherry plasmid were used, the degree of reduction in fluorescence signal intensity was different. This indicates that the spacer sequences targeting different positions of the target RNA have an impact on the editing effect of the Cas13 protein. For example, Cas13b_4 has a clear preference for guide RNAs with two structures (3'DR and 5'DR). Among them, when Cas13b_4 is guided by the crRNA of 3'DR, the knockdown effect of mCherry corresponding to some spacer sequences is relatively better.

[0181] 2. The inventors also conducted comparative experiments on Cas13d with Cas13b_3, Cas13T_15, Cas13T_16, Cas13b_4, and Cas13T_9 for specific crRNAs, as follows:

[0182] (1) Construction of Cas13d recombinant expression plasmid

[0183] The nucleotide sequence encoding Cas13d was optimized for mammalian codons, and the nucleotide sequence was shown in SEQ ID NO. 56. The Cas13d recombinant plasmid was constructed in the same manner as in step 1 (1) of Example 2 (in the Cas13d protein recombinant plasmid, the polynucleotide encoding the Cas13d protein replaced the sequence at positions 3472 to 6369 in the p23_puro_NPLS_Cas13_msfGFP_NLS_3xFlag_C plasmid).

[0184] (2) Take the mCherry reporter gene recombinant plasmid (the nucleotide sequence of the mCherry reporter gene recombinant plasmid is shown in SEQ ID NO.30).

[0185] (3) Designing guide RNA recombinant plasmids for comparison, taking Cas13d known in the art to have Cas13 activity as a comparison object, verifying the cleavage activity of some of the Cas13 proteins involved in the present invention, the DR sequence used by Cas13d is shown in SEQ ID NO.57. Two groups of experiments were designed as needed, wherein,

[0186] The spacer sequence used in the first group of experiments was spacer_1 (SEQ ID NO.33), the guide RNA (crRNA) used for Cas13d was Cas13d-DR-spacer_1 (SEQ ID NO.58), and the crRNAs used for Cas13T_9 and Cas13Z_2 were DR-Cas13T_9-spacer_1 (SEQ ID NO.59) and DR-Cas13Z_2-spacer_1 (SEQ ID NO.60), respectively.

[0187] The spacer sequence used in the second group of experiments was spacer_6 (SEQ ID NO.38), the crRNA used for Cas13d was Cas13d-DR-spacer_6 (SEQ ID NO.61), and the crRNA used for Cas13b_3, Cas13T_15, and Cas13T_16 were DR-Cas13b_3-spacer_6 (SEQ ID NO.62), DR-Cas13T_15-spacer_6 (SEQ ID NO.63), and DR-Cas13T_16-spacer_6 (SEQ ID NO.19), respectively.

[0188] In addition, for each Cas13 protein in the first and second groups, a non-targeted control group was set up. The spacer sequences used were all NT-spacer (SEQ ID NO: 43), the DR sequences were all DR sequences corresponding to each Cas13 protein, and the crRNA sequence structures were all DR-NT-spacer (5'DR).

[0189] Using the same approach as in step 1 of Example 2, the Cas13 protein involved in each group of experiments and the corresponding crRNA were cloned and constructed into p23_puro_NPLS_Cas13_msfGFP_NLS_3x Flag_C plasmid and U6-BbSI-PUC57 plasmid, and then transfected into human HEK293T cells (purchased from ATCC) with mCherry reporter gene recombinant plasmids in the same manner for cutting activity detection. It was found by flow cytometry analysis that at one site (spacer_1 targeting position), the cutting activity of Cas13Z_2 was higher than that of Cas13d, and the cutting activity of Cas13T_9 was comparable to that of Cas13d; at another site (spacer_6 targeting sequence position), the cutting efficiency of Cas13T_15 and Cas13T_16 was higher than that of Cas13d, and the cutting activity of Cas13b_3 was slightly lower than that of Cas13d (see Figure 14).

[0190] Example 3 Verification of the Cas13 protein's cleavage activity (i.e., non-specific cleavage activity)

[0191] In order to verify the above-mentioned Cas13 protein's side-cutting activity, the applicant also conducted a verification experiment on the side-cutting activity of some Cas13 proteins. The experimental process is as follows:

[0192] (1) The same guide RNA targeting mCherry as in Example 2 was used, and the experiment of Example 2 was repeated. The Cas13 protein recombinant plasmid containing the nucleotide sequence encoding Cas13b_3, Cas13b_4, Cas13T_9, Cas13T_15, and Cas13T_16 was co-transfected into human HEK293T cells with the mCherry reporter gene recombinant plasmid and the guide RNA recombinant plasmid (containing a guide RNA that does not target the reporter gene mCherry).

[0193] (2) Flow cytometry was used to analyze the knockdown efficiency of the GFP reporter gene in cells 48 hours after transfection. The knockdown efficiency of GFP represents the nonspecific cleavage activity of Cas13. The experimental results are shown in Figures 11 and 12.

[0194] As can be seen from Figures 11-12, Cas13b_3, Cas13b_4, Cas13T_9, Cas13T_15, and Cas13T_16 all have nonspecific cleavage activity, and the guide RNA corresponding to different spacer sequences also has an impact on the level of lateral cutting activity. For example, in Figure 11, when using crRNA_1, crRNA_3, crRNA_8, and crRNA_10 in guide RNA, Cas13T_9 has a nonspecific cleavage activity higher than other guide RNAs; and for Cas13b_4, using the crRNA of 3'DR and 5'DR structures, there are also differences in the level of lateral cutting activity; As shown in Figure 12, when using crRNA_4, Cas13T_15 has a higher lateral cutting activity, and the lateral cutting activity of the above-mentioned Cas13 protein can be used for DNA and / or RNA molecular detection.

[0195] Example 4 Verification of endogenous gene cleavage activity in eukaryotic cells

[0196] In order to verify the cleavage activity of the eukaryotic endogenous genes of the Cas13 protein described in the present invention, the applicant selected multiple eukaryotic endogenous genes: ANXA4, B4GALNT1, EZH2, PPIB and KRAS as target genes, and determined them in combination with the qPCR relative quantification method.

[0197] The applicant selected Cas13T_15 as the verification object and designed spacer sequences targeting endogenous genes ANXA4, B4GALNT1, EZH2, PPIB and KRAS mRNA, as shown in Table 5 below:

[0198] Table 5 Spacer sequences of human endogenous genes ANXA4, B4GALNT1, EZH2, PPIB and KRAS mRNA

[0199] DR-Cas13T_15 was connected to the 5' end of each spacer sequence in Table 5 (i.e., a guide RNA in the form of 5'DR was used) to obtain each guide RNA targeting endogenous gene mRNA, and an NT guide RNA that did not target any endogenous gene RNA was set as a control. The coding sequence of each guide RNA was cloned into the U6-BbSI-PUC57 plasmid in the same manner as in Example 2 to obtain guide RNA recombinant plasmids targeting different endogenous gene mRNAs.

[0200] The Cas13T_15 recombinant plasmid obtained in Example 1 and the above-mentioned guide RNA recombinant plasmids targeting different endogenous gene mRNAs, NT control guide RNA were transfected (transfection method was the same as in Example 2) into HEK293 cells (purchased from ATCC) to obtain experimental and control groups, and then the expression of endogenous gene mRNA was determined by RT-PCR to evaluate the knockdown efficiency, which was compared with the control cells transfected with Cas13T_15 recombinant plasmid / NT control guide RNA recombinant plasmid. After 48 h of culture in a cell culture incubator at 37 ° C, 5% (v / v) CO2, the cells were collected, RNA was extracted, and reverse transcribed into cDNA, and qPCR relative quantitative analysis was performed using GAPDH as the internal reference gene to compare the reduction in mRNA expression of each endogenous gene relative to the control group.

[0201] The quantitative analysis results are shown in Figure 15, which show that Cas13T_15 knocks down the mRNA expression of various endogenous genes to varying degrees. When using specific guide RNAs, the expression of ANXA4 mRNA, KRAS mRNA, and B4GALNT1 mRNA was knocked down by over 60%, and the mRNA expression of EZH2 and PPIB was also significantly knocked down, which also proves that Cas13T_15 can perform targeted cleavage in eukaryotic cells.

[0202] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A Cas13 protein, characterized in that The Cas13 protein is the following protein (a) or (b): (a) a protein comprising an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9; (b) A protein in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (a) and which substantially retains the biological function of the sequence from which it is derived.

2. The Cas13 protein according to claim 1, wherein In (b), the amino acid sequence of the protein has at least 85% sequence identity with that of (a), and substantially retains the biological function of the sequence from which it is derived.

3. The Cas13 protein as claimed in claim 2, characterized in that In (b), the amino acid sequence of the protein has at least 95% sequence identity with that of (a), and substantially retains the biological function of the sequence from which it is derived.

4. A fusion protein, characterized in that The fusion protein comprises the Cas13 protein according to any one of claims 1 to 3 and a heterologous functional domain.

5. The fusion protein according to claim 4, characterized in that The heterologous functional domain includes at least one of a deaminase, a reporter protein, a detection marker, a localization signal, a protein targeting domain, a DNA binding domain, an epitope tag, a transcription activation domain, a transcription inhibition domain, a nuclease, a methylation active enzyme, a demethylase, a transcription release factor, an RNA cleavage polypeptide, a nucleic acid ligase, a histone modification active factor or a nucleic acid binding active factor.

6. The fusion protein according to claim 5, characterized in that The heterologous functional domain is fused to the N-terminus or C-terminus of the Cas13 protein, or the heterologous functional domain is fused inside the Cas13 protein.

7. The fusion protein according to claim 5, characterized in that The localization signal is a nuclear localization signal or a nuclear export signal.

8. A CRISPR-Cas system, characterized in that The CRISPR-Cas system comprises the Cas13 protein and a guide RNA as described in any one of claims 1 to 3, or the CRISPR-Cas system comprises the fusion protein and a guide RNA as described in any one of claims 4 to 7; the guide RNA can bind to the Cas13 protein to form a complex and guide the complex to contact the target RNA.

9. The CRISPR-Cas system of claim 8, wherein: The guide RNA comprises a spacer sequence and a direct repeat sequence; the spacer sequence has a size of 15 to 60 nucleotides; and the direct repeat sequence has a size of 15 to 40 nucleotides.

10. The CRISPR-Cas system of claim 9, wherein: There are at least 15 nucleotides of complementarity between the spacer sequence and the target RNA.

11. A polynucleotide, characterized in that The polynucleotide encodes the Cas13 protein according to any one of claims 1 to 3, or the polynucleotide encodes the fusion protein according to any one of claims 4 to 7, or the polynucleotide encodes the CRISPR-Cas system according to any one of claims 8 to 10.

12. A carrier, characterized in that The vector carries the polynucleotide according to claim 11.

13. The vector according to claim 12, characterized in that The polynucleotide carried by the vector is connected to the regulatory elements.

14. The vector according to claim 13, characterized in that The regulatory element comprises at least one of a promoter, an enhancer, a polyadenylation signal, a terminator, a protein degradation signal, a transposon, a leader sequence, a polyadenylation sequence or a marker gene.

15. The carrier according to any one of claims 12 to 14, characterized in that The vector comprises at least one of a viral vector and a non-viral vector.

16. The vector according to claim 15, characterized in that The viral vector includes at least one of a retroviral vector, a phage vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a hybrid viral vector, a baculoviral vector, a herpes simplex viral vector or a lentiviral vector.

17. The vector according to claim 15, characterized in that The non-viral vector includes a plasmid vector.

18. A delivery system, characterized in that The delivery system carries the Cas13 protein described in any one of claims 1 to 3, the fusion protein described in any one of claims 4 to 7, the CRISPR-Cas system described in any one of claims 8 to 10, the polynucleotide described in claim 11, or the vector described in any one of claims 12 to 17.

19. The delivery system of claim 18, wherein The delivery vector of the delivery system includes at least one of a liposome vector, a chitosan vector, a polymer vector, a nanoparticle vector, an exosome vector, an exosome vector, a microvesicle vector, a virus vector, a gene gun, or an electroporation device.

20. A host cell, characterized in that The host cell carries the Cas13 protein described in any one of claims 1 to 3, the fusion protein described in any one of claims 4 to 7, the CRISPR-Cas system described in any one of claims 8 to 10, the polynucleotide described in claim 11, or the vector described in any one of claims 12 to 17.

21. A model, characterized in that The model is a plant model or an animal model; the plant model or the animal model comprises the host cell according to claim 20.

22. A method of gene editing, characterized in that: The method comprises contacting the target RNA with the Cas13 protein described in any one of claims 1 to 3, the fusion protein described in any one of claims 4 to 7, the CRISPR-Cas system described in any one of claims 8 to 10, the polynucleotide described in claim 11, the vector described in any one of claims 12 to 17, or the host cell described in claim 20.

23. The method of claim 22, wherein: The guide RNA binds to the Cas13 protein to form a complex, and guides the complex to contact the target RNA to perform gene editing on the target RNA.

24. A gene editing product, characterized in that: The gene editing product is obtained by gene editing the target RNA using the method described in claim 22 or 23.

25. Use of the Cas13 protein described in any one of claims 1 to 3, the fusion protein described in any one of claims 4 to 7, the CRISPR-Cas system described in any one of claims 8 to 10, the polynucleotide described in claim 11, the vector described in any one of claims 12 to 17, the delivery system described in claim 18 or 19, the host cell described in claim 20, or the method described in any one of claims 22 to 23 in gene editing.

26. A kit, characterized in that The kit comprises the Cas13 protein according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 7, the CRISPR-Cas system according to any one of claims 8 to 10, the polynucleotide according to claim 11, the vector according to any one of claims 12 to 17, the delivery system according to claim 18 or 19, or the host cell according to claim 20.

27. A method for nucleic acid detection, characterized in that: The method comprises using the kit described in claim 26 to perform nucleic acid detection on the sample to be tested.

28. Use of the Cas13 protein described in any one of claims 1 to 3, or the fusion protein described in any one of claims 4 to 7, or the CRISPR-Cas system described in any one of claims 8 to 10, or the polynucleotide described in claim 11, or the vector described in any one of claims 12 to 17, or the delivery system described in claim 18 or 19, or the host cell described in claim 20, or the kit described in claim 26, or the method described in claim 27 in nucleic acid detection.

29. Use of the Cas13 protein described in any one of claims 1 to 3, or the fusion protein described in any one of claims 4 to 7, or the CRISPR-Cas system described in any one of claims 8 to 10, or the polynucleotide described in claim 11, or the vector described in any one of claims 12 to 17, or the delivery system described in claim 18 or 19, or the host cell described in claim 20 in the preparation of a medicament for treating a disorder or disease in a subject in need thereof.

30. A method of treating a condition or disease in a subject in need thereof, the method comprising: Administering a composition to the subject, the composition comprising the CRISPR-Cas system of any one of claims 8 to 10 or a polynucleotide encoding the system; wherein the spacer sequence is complementary to at least 15 nucleotides in a target RNA associated with a disease or condition; wherein the Cas13 protein binds to the guide RNA to form a complex, and the complex binds to the target RNA, and the Cas13 protein edits the target RNA to achieve the purpose of treating the condition or disease of the subject; Preferably, the condition or disease is cancer, an infectious disease or other disease; Preferably, the cancer is Wilms tumor, Ewing sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma or bladder cancer; Preferably, the leukemia includes acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia or chronic myeloid leukemia; Preferably, the other diseases include nervous system diseases, ear diseases, muscle abnormalities or eye diseases; Preferably, the nervous system diseases include: neurodevelopmental disorders such as Angelman syndrome or ALS; Preferably, the ear diseases include: dominant hereditary deafness; Preferably, the muscle abnormalities include: spinal muscular atrophy; Preferably, the ophthalmic diseases include: congenital amaurosis type 2.

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