WO2022266849A1 - Screening of novel crispr-cas13 protein and use thereof - Google Patents

Abstract

Screening of a novel CRISPR-Cas13 protein and the use thereof. A novel Cas13 family protein, a method for screening the novel Cas13 family protein, and corresponding RNA detection systems, RNA editing systems and other systems, and the use thereof. Disclosed are the Cas13 protein and related RNA detection systems, RNA editing systems and other systems. The novel Cas13 protein contains more extended HEPN domains. By means of the method for rapidly searching the Cas13 protein, various novel Cas13 proteins are obtained, which have broad application prospects and a huge market value.

Description

Screening and application of novel CRISPR-Cas13 proteins technical field

This disclosure relates to the fields of biotechnology and medicine. More specifically, the present disclosure relates to novel Cas13 family proteins, methods for screening novel Cas13 family proteins, and corresponding RNA detection and editing systems and applications thereof. In particular, the present disclosure relates to Cas13 proteins and related RNA detection systems.

Background technique

The CRISPR-Cas system is an adaptive immune system of prokaryotes (mainly bacteria and archaea), which can defend against the invasion of foreign viruses (such as phages). According to different targets, they can be divided into two categories, one is the CRISPR-Cas system targeting DNA, such as CRISPR-Cas9; the other is the CRISPR-Cas system targeting RNA, such as the Cas13 family, including Cas13a, Cas13b, Cas13c, Cas13d, Cas13X, Cas13Y, etc. Compared with the CRISPR-Cas system for DNA editing, the CRISPR-Cas13 system mainly uses the HEPN (higher eukaryotes and prokaryotes nucleotide) domain to cut and defend against foreign invading nucleic acids, so the role of CRISPR-Cas targeting RNA is milder and more direct , enabling the regulation of RNA transcripts without altering the genome, improving the safety of gene editing. In addition, previous studies have found that the currently known Cas13 protein/system will undergo conformational changes when it is activated by sgRNA-guided target sequence recognition, showing specific RNase activity as well as non-specific RNase activity (called bystander RNase activity). enzyme cleavage activity), and the bystander cleavage property will indiscriminately cleave RNA molecules adjacent to the target RNA, especially Cas13a and Cas13b, which show very strong bystander RNase cleavage activity. Using this feature, researchers have applied it to the detection of RNA viruses, disease treatment and other fields. For example, in 2017, Zhang Feng et al. used Cas13a protein combined with RPA isothermal amplification technology and reverse transcription technology to develop a method that can detect trace amounts of New method SHERLOCK for DNA and RNA detection. In 2018, Jennifer and others developed a new method DETECTR that can detect nucleic acids. They combined Cas12a protein and LAMP isothermal amplification technology, and its sensitivity can realize the detection of aM level samples.

However, sgRNAs of different Cas13 proteins will have strong targeting sequence preference (Protospacer flanking site, PFS) characteristics (similar to the PAM of the Cas9 system) when targeting the target RNA region, which will limit its application to a certain extent range, because sometimes the nucleic acid that needs to be targeted will greatly reduce or even fail to activate the RNase activity of the Cas13 protein if the PFS does not exist. Therefore, it is urgent to find Cas13 proteins suitable for a variety of different PFS properties from nature to increase the application of Cas13 proteins in the field of detection, clinical diagnosis and treatment, etc.

Contents of the invention

Aiming at the deficiencies and actual needs of the existing technology for screening novel CRISPR-Cas proteins, the present disclosure provides a method for quickly finding novel CRISPR-Cas13 orthologous proteins containing more expanded HEPN domains (at least 2) And the RNA editing activity of the candidate protein was verified from the level of biological information analysis (for example, sequence alignment, protein structure prediction, etc.) and experimental level. These proteins are potentially applied to the regulation, editing, and detection of RNA levels, and have broad academic and commercial value.

The technical problem solved in this disclosure is how to quickly find a candidate CRISPR-Cas13 protein and its system with more novel RNA enzymatic cleavage active domains (extended HEPN domain); secondly, to verify the candidate CRISPR-Cas13 protein and its system activity; and finally obtained a variety of novel Cas13 proteins.

The disclosure achieves the following technical effects:

(1) Developed an analysis method for rapid screening of novel Cas13 family proteins, which can perform CRIPSR array system analysis and screening of related effector proteins on newly updated prokaryotic microbial DNA sequences and metagenomic sequences;

(2) The screened Cas13 family members can expand the application range of CRISPR-Cas13, and can enhance the sensitivity of multiple or single virus detection by integrating Cas13 proteins with different PFS characteristics. At the same time, through the packaging of adeno-associated virus and other delivery vectors, the diagnosis and treatment of related diseases can also be realized, such as the diagnosis and treatment of nerve-related degenerative diseases. In the field of plants, research on breeding and adversity stress can be carried out. In the field of microorganisms, related engineering bacteria can be transformed. Wait;

(3) In the screening process of this method, in addition to using the known HEPN domain of the Cas13 protein to screen, the conserved domain with RNA cleavage activity in other types of proteins will also be included, thereby providing a new screening method. The possibility of Cas13 protein, and due to the identification of these new functional domains in these new Cas13 proteins, provides new ideas and possibilities for further modifying Cas13 proteins.

In one aspect of the disclosure, a Cas13 protein is provided.

In a preferred embodiment, the Cas13 protein comprises an amino acid sequence as described in any one of SEQ ID NO: 1-204, or SEQ ID NO: 1 with conservative amino acid substitutions of one or more residues - the amino acid sequence of any one of 198.

In a preferred embodiment, the RNA cleavage activity of the Cas13 protein is retained.

In a preferred embodiment, the HEPN domain or the RNA cleavage domain of the Cas13 protein is further modified or transformed to reduce or eliminate its RNA cleavage activity, and become dCas13 with reduced or eliminated RNA cleavage activity.

In a preferred embodiment, the Cas13 protein is fused with one or more heterologous functional domains.

In a preferred embodiment, the fusion is at the N-terminal, C-terminal or internal of the Cas13 protein.

In a preferred embodiment, said one or more heterologous functional domains have the following activities: deaminases such as cytidine deaminase and deoxyadenosine deaminase, methylase, demethylase enzyme, transcriptional activation, transcriptional repression, nuclease, single-stranded RNA cleavage, double-stranded RNA cleavage, single-stranded DNA cleavage, double-stranded DNA cleavage, DNA or RNA ligase, reporter protein, detection protein, localization signal, or any combination. In another aspect of the present disclosure, a nucleic acid molecule comprising the nucleotide sequence encoding the above-mentioned Cas13 protein is provided.

In a preferred embodiment, the nucleic acid molecule is codon-optimized for expression in a particular host cell.

In a preferred embodiment, the host cell is a prokaryotic or eukaryotic cell, preferably a human cell.

In a preferred embodiment, the nucleic acid molecule comprises a promoter operatively linked to the nucleotide sequence encoding Cas13, which is a constitutive promoter, an inducible promoter, a tissue-specific promoter, a chimeric promoter or development-specific promoters.

In another aspect of the present disclosure, an expression vector is provided, which comprises the above-mentioned nucleic acid molecule, and expresses the above-mentioned amino acid sequence or nucleotide sequence in the form of DNA, RNA, or protein.

In a preferred embodiment, the expression vector is adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes simplex virus, oncolytic virus.

In another aspect of the present disclosure, a delivery system is provided, comprising (1) the above-mentioned expression vector, or the above-mentioned Cas13 protein; and (2) a delivery vector.

In a preferred embodiment, the delivery vehicle is a nanoparticle, liposome, exosome, microvesicle or gene gun.

In another aspect of the present disclosure, a CRISPR-Cas system is provided, which comprises: (1) the above-mentioned Cas13 protein or nucleic acid molecule, or a derivative or a functional fragment thereof; (2) a CRISPR-Cas system for targeting target RNA gRNA sequence.

In a preferred embodiment, wherein the gRNA sequence comprises a direct repeat (DR) sequence and a sequence targeting a spacer region of the target RNA portion.

In a preferred embodiment, wherein the DR sequence is the sequence shown in Table 1; wherein the spacer sequence is 15-60 nucleotides, preferably 25-50 nucleotides, more preferably 30 cores glycosides.

In a preferred embodiment, the DR sequence may be a derivative corresponding to any of the following, wherein the derivative (i) has one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) nucleotide additions, deletions, or substitutions; (ii) any one of the sequences shown in Table 1 has at least 20 %, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 97% sequence identity; (iii) any one of the sequences shown in Table 1 under stringent conditions, or hybridize to any one of (i) and (ii); or (iv) is the complement of any one of (i)-(iii), provided that the derivative is not any of the sequences shown in Table 1 One, and said derivative encodes, or is itself an RNA, said RNA substantially maintaining the same secondary structure as any RNA encoded by SEQ ID NO: 199-397.

In a preferred embodiment, the CRISPR-Cas system further comprises: (3) target RNA.

In a preferred embodiment, the CRISPR-Cas system causes degradation, cleavage or sequence change of the target RNA sequence.

In a preferred embodiment, the target RNA is mRNA or ncRNA, including non-coding RNA selected from lncRNA, miRNA, misc_RNA, Mt_rRNA, Mt_tRNA, rRNA, scaRNA, scRNA, snoRNA, snRNA, sRNA.

In another aspect of the present disclosure, there is provided a cell comprising the above-mentioned Cas13 protein, nucleic acid molecule, expression vector, delivery system or CRISPR-Cas system.

In a preferred embodiment, the cells are prokaryotic or eukaryotic cells, preferably human cells.

In another aspect of the present disclosure, there is provided a method for degrading or cutting target RNA in the target cell, modifying the sequence of the target RNA in the target cell, which includes using the above-mentioned Cas13 protein, nucleic acid molecule, expression vector, delivery vector or CRISPR-Cas system.

In a preferred embodiment, the target cells are prokaryotic cells or eukaryotic cells, preferably human cells.

In a preferred embodiment, the target cells are ex vivo cells, in vitro cells or in vivo cells.

In another aspect of the present disclosure, use of the above-mentioned Cas13 protein, nucleic acid molecule, or CRISPR-Cas system for detecting nucleic acid molecules is provided.

In a preferred embodiment, the detected target is RNA or DNA, wherein the RNA or DNA is RNA or DNA in prokaryotic microorganisms or eukaryotic organisms.

In another aspect of the present disclosure, the prokaryotic microorganism is a DNA virus or nucleic acid thereof, an RNA virus or nucleic acid thereof.

In another aspect of the present disclosure, the eukaryotes include animals and plants, preferably humans; the RNA or DNA in vivo includes RNA or DNA in cells or body fluids.

In another aspect of the present disclosure, the bodily fluid includes blood, urine, or lymph fluid.

Description of drawings

Figure 1 shows the prediction results of the RNA secondary structure of the DR sequence of the candidate Cas13 protein DZ109. Among them, DZ109a represents DR1, and DZ109b represents DR2 (see Table 1 for the sequence).

Figure 2A shows the results of verifying the RNase activity of the candidate protein DZ109 at the cell level. Experimental results of detection of candidate protease cleavage activities in mammalian cell lines: the above picture shows the plasmid containing DZ109 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing mCherry protein after co-transfection of 293T cell line for 24 hours, observed under 10X microscope The obtained fluorescence results. It can be found that compared with the negative control group, the RNase activity of the candidate protein DZ109 is very strong, and the side-cutting activity is also very strong, and the corresponding green light and red light are greatly reduced. Among them, White light represents the cell results under white light field; Green light represents the result graph under green fluorescence; Red light represents the result graph under red fluorescence; Negative-R1 is the negative control group; among them, PS394～PS396 are different regions containing targeted mcherry The number of the sgRNA used is DZ109a. R1 and R2 represent two different experimental replicates.

Figure 2B shows the results of verifying the RNase activity of the candidate protein DZ109 at the cell level. Experimental results of detection of candidate protease cleavage activities in mammalian cell lines: the above picture shows the plasmid containing DZ109 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing mCherry protein after co-transfection of 293T cell line for 24 hours, observed under 10X microscope The obtained fluorescence results. It can be found that compared with the negative control group, when the candidate protein DZ109 has only ps397 sgRNA when the DZ109b sequence is used as DR, the RNase activity and side cut activity of DZ109 are also very strong, and the corresponding green light and red light are greatly reduced. In the presence of other sgRNAs, there was no effect. Among them, White light represents the cell results under white light field; Green light represents the result graph under green fluorescence; Red light represents the result graph under red fluorescence; Negative-R1 is the negative control group; among them, PS397～PS399 are different regions containing targeted mcherry The number of the sgRNA used, the DR used is DZ109b. R1 and R2 represent two different experimental replicates.

Figure 2C shows the results of flow cytometric analysis of the RNase activity of DZ109 at the cell level. The results of flow cytometry assays for detecting the cleavage activity of candidate proteases in mammalian cell lines: the figure above shows the 293T cell line after co-transfection of the plasmid containing DZ109 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing mCherry protein for 48 hours. The result graph of formula analysis. It can be found that compared with the negative control group, the RNase activity of the candidate protein DZ109 is very strong, and the main group of red and green double yang has a significant shift, corresponding to the red light being greatly knocked down. The negative control is the control group that only expresses mcherry protein (red light) and DZ109 protein (green light); PS394-PS396 is the experimental group containing sgRNA targeting different regions of mcherry, and the DR used is DZ109a. R1 and R2 represent two different experimental replicates.

Figure 2D shows the results of flow cytometric analysis of the RNase activity of DZ109 at the cell level. The results of flow cytometry assays for detecting the cleavage activity of candidate proteases in mammalian cell lines: the figure above shows the 293T cell line after co-transfection of the plasmid containing DZ109 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing mCherry protein for 48 hours. The result graph of formula analysis. It can be found that compared with the negative control group, when the candidate protein DZ109 uses PS397 sgRNA, the RNase activity is very strong, and the main group of red and green double positives has a very obvious shift, and the corresponding red light and green light are greatly knocked down. The negative control is the control group that only expresses mcherry protein (red light) and DZ109 protein (green light); PS397-PS399 is the experimental group containing sgRNA targeting different regions of mcherry, and the DR used is DZ109b. R1 and R2 represent two different experimental replicates.

Figure 3 shows the results of the Cas13d[xdz9] positive control. Experimental results of detection of candidate protease cleavage activity in mammalian cell lines: the above picture shows the plasmid containing Cas13d protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing mCherry protein after co-transfection of 293T cell line for 24 hours, observed under a 10X microscope The obtained fluorescence results. Among them, White light represents the cell results under white light field; Green light represents the result graph under green fluorescence; Red light represents the result graph under red fluorescence; px262 is the negative control group and px261 is the experimental group.

Figure 4 shows the schematic diagram of the candidate protein DZ109, in which Figure 4A shows the position information of the expanded HEPN; Figure 4B shows the adjacent schematic diagram of the candidate protein and CRISPR array.

detailed description

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

As used in the specification, nouns modified by quantifiers may mean one or more. As used in the claims, when used in conjunction with the word "comprising/comprising", nouns modified by a quantifier may mean one or more than one.

The use of the term "or/or" in the claims is used to mean "and/or" unless expressly stated to mean only the alternative or the alternatives are mutually exclusive, although this disclosure supports referring only to the alternative and "and /or" is limited. "Another" as used herein may mean at least a second or more.

Throughout this application, the term "about" is used to indicate that a value includes error in the device, inherent variation in the methods used to determine the value, or inherent variation among study subjects. Such inherent variation may be a variation of ±10% of the labeled value.

Throughout the application, unless otherwise stated, nucleotide sequences are listed in a 5' to 3' orientation and amino acid sequences are listed in an N-terminal to C-terminal orientation.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating certain preferred embodiments of the invention, are given by way of illustration only, since many changes can be made from the detailed description within the spirit and scope of the invention. and modifications will become apparent to those skilled in the art.

definition

NCBI (https://www.ncbi.nlm.nih.gov/) refers to the National Center for Biological Information of the United States, which is a public database facing the world. Those skilled in the art can use the nucleic acid database provided by this database to download prokaryotic Genome, proteome-related databases, etc., can also use the blast alignment software provided by the data for sequence alignment analysis.

IMG (https://img.jgi.doe.gov/) refers to the integrated database of microbial genomes, which is a representative of the new generation of genome databases. and analysis services, storing sequencing data into the IMG/M database. The data can download the data of pure culture bacterial sequencing genome, metagenome, metagenomic assembly genome, and single cell sequencing genome.

CRISPR (cluster regularly interspaced short palindromic repeats) is a prokaryote, mainly referring to a string of DNA sequences in bacteria and archaea, including direct repeat (DR) regions and non-repeating spacer (spacer) regions. In addition to the CRISPR array, the CRIPSR system also includes related Cas proteins. Together, they make up the immune system that keeps bacteria below the invasion of foreign viruses.

The Cas13 family is the currently known CRIPSR enzyme family capable of targeting RNA, and its members include the Cas13a, Cas13b, Cas13c, Cas13d, Cas13X and Cas13Y families. Unlike the activity of CRISPR/Cas9 to cut DNA, CRISPR/Cas13 can be used to cut specific RNA sequences in bacterial cells.

Collateral cleavage is also called Bystander cleavage activity. In CRISPR-cas13family, it usually refers to the non-specific enzyme cleavage activity of CRISPR-Cas system, that is, the process of CRISPR-cas13 protein binding to sgRNA to target region CRISPR-cas13 The conformational change of the protein becomes a non-specific RNase, which can not only cleave the target nucleic acid, but also cleave adjacent nucleic acid molecules. As reported, Cas13a, Cas13b, etc. all show very strong bystander paralysis RNase activity.

HEPN domain is the abbreviation of higher eukaryotes and prokaryotes nucleotide domain, which is an important domain of Cas13 protein in the CRISPR-Cas13 enzyme system to cut and resist foreign invading nucleic acids.

The ABE system is the abbreviation of Adenine base editors, that is, the purine base conversion technology, which can realize the single base change from A/T to G/C. The most commonly used enzyme is adar enzyme (adenosine deaminases acting on RNA, an adenosine deaminase acting on RNA). It is mainly through the deamination of adenine to inosine, which will be regarded as G when reading the code in DNA or RNA, so as to realize the mutation from A/T to G/C. Since cells are insensitive to inosine excision repair, this mutation maintains high product purity.

CBE system is the abbreviation of Cytidine base editor, that is, pyrimidine base conversion technology. Currently, there are BE1, BE2 and BE3 tools, among which BE3 has the highest efficiency, so it is widely used in the fields of gene therapy, animal model making and functional gene screening.

Eukaryotic cells such as mammalian cells, including human cells (human primary cells or established human cell lines). The cells may be non-human mammalian cells, e.g., from non-human primates (e.g. monkeys), cows/bulls/cattle, sheep, goats, pigs, horses, dogs, cats, rodents (e.g. rabbits, small, rats, hamsters) etc. The cells are from fish (eg, salmon), birds (eg, birds, including chickens, ducks, geese), reptiles, shellfish (eg, oysters, clams, lobsters, shrimps), insects, worms, yeast, and the like. The cells may be from a plant, such as a monocot or a dicot. The plants may be food crops such as barley, cassava, cotton, peanuts, corn, millet, oil palm fruit, potatoes, beans, rapeseed or canola, rice, rye, sorghum, soybeans, sugar cane, sugar Beets, sunflowers and wheat. The plants may be cereals (eg barley, corn, millet, rice, rye, sorghum and wheat). The plants may be tubers (eg cassava and potatoes). In some embodiments, the plant may be a sugar crop (eg, sugar beet and sugar cane). The plant may be an oleaginous crop (such as soybean, peanut, rapeseed or canola, sunflower and oil palm fruit). The plant may be a fiber crop (eg cotton). The plant may be a tree, such as a peach or nectarine tree, an apple tree, a pear tree, an apricot tree, a walnut tree, a pistachio tree, a citrus tree (such as an orange, grapefruit or lemon tree), a grass, a vegetable, a fruit or algae. The plant may be a plant of the genus Solanum; a plant of the genus Brassica; a plant of the genus Lactuca; a plant of the genus Spinacia; a plant of the genus Capsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli , cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.

CRISPR system

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas13 (CRISPR-associated protein 13)-mediated RNA editing is becoming a promising tool for disease diagnosis and treatment, plant breeding, etc.

CRISPR is a DNA locus comprising short repeats of base sequences. Each repeat is followed by a short segment of "spacer DNA" from previous exposure to the virus. CRISPR is found in approximately 40% of sequenced eubacterial genomes and 90% of sequenced archaea. CRISPR is usually associated with a Cas gene that encodes a protein associated with CRISPR. The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPR spacers recognize and silence these exogenous genetic elements (eg RNAi) in eukaryotic organisms.

CRISPR repeats are 24 to 48 base pairs in size. They usually show some double symmetry, which means that secondary structures such as hairpins are formed, but are not true palindromic structures. Repeats are separated by spaces of similar length. Some CRISPR spacer sequences matched exactly to sequences from plasmids and bacteriophages, although some spacers matched the genomes of prokaryotes. New spacers can be added rapidly in response to phage infection.

guide RNA (gRNA). As an RNA-guiding protein, Cas13 requires short RNAs to guide the recognition of RNA targets.

nuclease

Cas nuclease. CRISPR-associated (Cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families have been described. Among these protein families, Cas1 appears to be ubiquitous in different CRISPR/Cas systems. Specific combinations of Cas genes and repeat structures have been used to define eight CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with encoding repeat-associated mysterious proteins (repeat-associated mysterious proteins). protein, RAMP) related to another gene module. More than one CRISPR isoform can exist in a single genome. The sporadic distribution of CRISPR/Cas isoforms suggests that this system underwent horizontal gene transfer during microbial evolution.

The foreign DNA is apparently processed into small elements (about 30 base pairs in length) by the protein encoded by the Cas gene, which is then somehow inserted into the CRISPR locus close to the leader sequence. RNAs from CRISPR loci are constitutively expressed and processed by Cas proteins into small RNAs composed of individually exogenously derived sequence elements with flanking repeats. RNA guides other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Evidence for functional diversity among CRISPR isoforms. The Cse (Cas subtype Ecoli) protein (referred to as CasA-E in E. coli) forms the functional complex Cascade, which processes CRISPR RNA transcripts into Cascade-retaining spacer-repeat units. In other prokaryotes, Cas6 processes CRISPR transcripts. Interestingly, Cascade and Cas3, but not Cas1 and Cas2, are required for CRISPR-based phage inactivation in E. coli. The Cmr (Cas RAMP module) protein found in Pyrococcus furiosus and some other prokaryotes forms a functional complex with small CRISPR RNAs that recognize and cleave complementary target RNAs. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

Example

Example 1: Novel Cas13 protein de novo screening

We also performed a de novo search for other CRISPR-Cas13 family members. To put it simply, the analysis system includes 2 large blocks, the identification of a part of the CRISPR array region. We first download the sequences of all bacteria, archaeal genomes and metagenomics from NCBI and IMG as of February 2021, and use the CRISPR array identification software ( Such as Pilercr) to identify the CRISPR array region; the other part is to search for Cas-related proteins near the upstream and downstream of this region, that is, to take 6 proteins adjacent to the upstream and downstream of this region, a total of 12 proteins for target domain analysis. See Table 2 for the amino acid sequence numbers, expanded HEPN domains and coordinates of the final candidate proteins.

Among them, the HEPN domain of this screening system, in addition to including the RxxxxxH (R4xH) features found in the past members of the Cas13 family (we denote it as the early HEPN domain), has also been expanded, including other domains with RNA cleavage activity RxxxxxH (R5xH ) and RxxxxxxH (R6xH) (we denote RxxxxxH (R5xH) and RxxxxxxH (R6xH) and RxxxxxH (R4xH) collectively as the extended HEPN domain). Wherein the conservative amino acid adjacent to R is preferably N, Q, H or D, such as combinations such as R[NDQH]xxxH, R[NDQH]xxxxH, R[NDQH]xxxxxH; wherein R4xH is preferably R[NQH]xxxH, and R5xH and R6xH are Preferred are R[NDQH]xxxxH and R[NQDH]xxxxxH; wherein x represents any amino acid, and N, D, Q and H in square brackets are preferentially considered conservative amino acids.

Example 2: Functional verification of novel candidate Cas13 protein

Functional verification will be carried out after the candidate protein is screened. In short, we first send the nucleic acid sequence, DR sequence and target spacer sequence of the candidate protein to the company for synthesis, and then introduce them into the expression plasmid to construct the corresponding plasmid. Then, the plasmid was amplified and cultured in DH5a E. coli competent cells through plasmid transformation, and then the transfection test of the human 293T (can express red light) cell line was carried out after the plasmid was extracted. At the same time, we also designed the corresponding negative and A positive control group was used to further confirm the cleavage activity of the candidate protein. After 48 hours of co-transfection of the plasmids, experiments such as flow cytometry analysis were performed to finally determine the RNA cleavage activity of the candidate protein.

According to the above research strategy, we randomly selected DZ109 for verification. As shown in Figures 2 to 3, the fluorescence results and flow cytometry results show that DZ109 has strong RNase activity.

Embodiment 3: RNA nucleic acid detection function of novel candidate Cas13 protein

In view of the very strong non-specific bystander RNase activity of the candidate Cas13 protein, it is potentially applied to the detection of RNA, such as RNA viruses and tumor signal RNA molecules. To put it simply, by constructing a CRISPR-cas system capable of cleaving the target detection nucleic acid (for example, it can exist in the form of a detection test paper, or a delivery vector coating, etc.), including the candidate CRISPR-Cas13 protein, sgRNA (targeted detection virus RNA) and reporter detection molecules (such as RNA fluorescent reporter molecules), and then when the system binds to the target RNA, it can exert the bystander RNase activity of the candidate cas13 protein and continue to cut the reporter detection molecule, so that the signal molecule can send a signal, such as sending fluorescence. These signals can be received by detection instruments and converted into electrical signals before being read out, so that the detection of target nucleic acids can be achieved. For example, further integration of machine learning algorithm models can further quantify and predict target nucleic acids. Therefore, it can be widely used in virus detection, such as the detection of new coronavirus; it can also be widely used in the non-invasive diagnosis of diseases (such as tumors), such as liquid biopsy.

Example 4: Verification of the base editing function of the novel candidate Cas13 protein

There are two main systems currently used for single base editing, one is the ABE system and the other is the CBE system. To put it simply, the cleavage domain (extended HEPN domain) of the candidate Cas13 protein is mutated to obtain a candidate dCas13 protein that only binds RNA but has no cleavage activity, and then fuses the adar enzyme sequence to construct the ABE single base editing system. Plasmids, and then design sgRNAs for site-directed base mutation processing on specific sequences, such as transcripts of the TP53 gene, and construct corresponding plasmid vectors. Then, the co-transfected cell line was obtained by co-transfecting the human 293T cell line and sorting by flow cytometry 48 hours later. Then the extraction of RNA transcripts and library construction were performed. Then enter deep seq sequencing. After the sequencing is completed, the mutation status of the TP53 gene transcript can be analyzed by bioinformatics methods to obtain the single-base editing efficiency analysis of the corresponding ABE system. In this way, the optimal single base editing system for the target region can be constructed by continuously optimizing sgRNA.

Example 5: Homology Analysis of Candidate Cas13 Proteins and Known Cas13 Proteins

Based on the principle that the higher the coverage of the unknown protein on the known protein and the larger the proportion of similarity, the closer the homology between the unknown protein and the known protein. After screening the candidate proteins, we first download the relevant protein sequences of Cas13a, b, c, d, x(e), y(f) from the NCBI database and patent literature, and then merge them with our data to construct a local blastp index file, and then compare the candidate protein sequence to the local blastp index library for protein sequence alignment analysis. For the part whose identity between proteins is less than 20% or cannot be compared to the local index library, we will uniformly mark it as 20%; similarly, for the coverage (coverage) is less than 5% or cannot be compared to the local index Libraries are marked at 1%. The homology level between the novel Cas13 protein identified by the method of the present invention and the known Cas13 protein of each family is extremely low. For example, DZ109, DZ110, DZ140, DZ159, DZ163, DZ183, DZ264, DZ280, etc. have less than 20% homology with currently known Cas13 types.

The DR sequences of candidate Cas13 proteins are shown in Table 1 below.

Table 1. DR sequences of candidate Cas13 proteins

SEQ ID No. DR-ID DR-SEQ 199 DZ109a GTTGTGTATGCCCTATATTTGTAGGGTTGAAACAAC 200 DZ109b GTTGTTTCAACCCTACAAATAGGGCATACACAAC

201 DZ110 GGTGTGAATGCCCTTGTTTTGAAGGGTTAATACATC 202 DZ111 GCTGGATTCATCTGTATTTTTGCAGGTATTCACAGC 203 DZ112 GCTGTGTTTACTTACCAAAATGCAAGTGTTACCAGC 204 DZ113 GTTGTAACTGCCTTTGTTTTGAAAGGTAAAAACAAC 205 DZ114 GTTGTAACTGCTATTACTTTGAATAGAGAAAAACAAC 206 DZ119 GTTGTGATTGCTCTAATTTTAGGGCGCCAACAAC 207 DZ120 GCTGGAGCAGCCCTCGATTTGCTGGGTAATCACAGC 208 DZ121 GCTGGAGCAGCCCTCGATTTGCAGGGTTATCACAGC 209 DZ123 GAGATAGACCCTTGTTAACTCGTAAGGTTCTGTGACT 210 DZ124 GAACTATAATCCTGTAACTGAACAGGATTCTGAAA 211 DZ126 TGTTTCTACCTTTTCAAAACAAAGGCTTGCACACC 212 DZ127 GTGTCAGTCCGCCGTGAAACAGGCGGTCATGCAGAAC 213 DZ128 GCTCTACAGCCCTCTGAAACATGAGGGTTCTGAAAC 214 DZ129 GCACAAGATAACCCGAAAACAGTAGGGTTCTAAAAC 215 DZ130 GATTGAAAGGATTGTAAAATTTACAAGGTCTTAAAAC 216 DZ131 GTACTAATGCCCTACAGAAATGCAGGGTTCTAAAAC 217 DZ132 GAACTACTACCTCAATGAATGTTGGGGTTCAGAAACT 218 DZ133 GTGGAGAACCCGATATAGTGGGTACTAGAG 219 DZ134 ACTTAAAATCCCCCTGTAAATGCGGGGGTTCTAAAAC 220 DZ135 GGTGGAAAAGCCTTTGATTTGAAAGGTAAAAGCACC 221 DZ136 GATTGAAAGCTATGCGAATTTGCACAGTCTTAAAAC 222 DZ137 CAAGTAAACCCCTACCAACTGGTAGGGGTCTGAAAC 223 DZ138 GGTGTAGTGCTCCCTTATTTGGAGCTCATCTCCGGC 224 DZ139 GAACTACACCCGTGCAAAAATGCAGGGGTCTAAAAC 225 DZ140 GCTGTAAATGCCTTTCAAAATGAGGCCTTTGCCAGC 226 DZ141 GTACAATAGCCCGTTGAAATAGATGGGTTCTAAGACT 227 DZ142 CTTGAAGATGACCTCCAATTAAGGGAGGACTGCACT 228 DZ143 GTTGTGGGAGCCTTTATTTTGTAAGGTATAAACAAC

229 DZ144 GGTTCAGTCGCCTGTGAAAAGAGGCGTCGTCCTCAAC 230 DZ145 GTTGTGAATGCCTTAATTTTGGAAGGTGAGAACAAC 231 DZ146 GTTGTAAAAGCCTTTAGTTTGTAAGGTAAAAACAAC 232 DZ147 GTTGTGGATGCCTTAACTTTGAAAGGTGAAAACAAC 233 DZ148 GTTCCAGACCCCGTGTTTTTCAACGGGTTGTTGTTC 234 DZ149 GTTGTGGAAGCCTGACTTTTATTAGGTAAGCACAAC 235 DZ150 GTTGTAAATGCCTTATATTTGCAAGGTGAAAACAAC 236 DZ151 GTTGTAAATGCCTTATATTTGCAAGGTGAAAACAAC 237 DZ152 GTTGTAAATACCCTATATTTGAAGGGTAATAACAA 238 DZ153 GTTGTGTTTCCACTTCAAATATAGGGTATTCACAAC 239 DZ154 AGCTGGTAACACTAGTATTTTTGCTGGTAATCACAGC 240 DZ155 GTTGTAGAAGCCCCTTATTTGAAGGGGTA 241 DZ156 GCTGTAGAAGCCTTCGTTTTGGGAGGTAAGTACAGC 242 DZ157 GTTGTAGCAACCTATATTTTGTTAGGAAAAGACAAC 243 DZ158 GAAGCCCCTATTTTGTGGGGTAGTTACAGC 244 DZ159 TGCTGTAACTACCTGCTGTTTAAGAGGTACATACAGT 245 DZ160 GTTGTAGAGGCCCTCGATTTGCAGGGTAGGTACAAC 246 DZ161 GTTGTGAAAGCCTTATATTTGAAGGGTAAATACAAC 247 DZ162 TTGTAATTGCTACCCAAAATGCAGCATTCAACAAC 248 DZ163 GGTGTGAATGCCCTTGTTTTGAAGGGTTAATACATCT 249 DZ164 GCTGTGGAAGCCTTGACTTTGAAAGGTAGTTACAGC 250 DZ165 GGTGGAAAAGCCTTTGATTTGAAAGGTAAAAGCACC 251 DZ166 GCTGTTGCAGCCTGCTATTTGGTGGGTATTTACAGC 252 DZ167 GTTGTGTTTACCTTTCAAATTAAGGGCAGCCACAAC 253 DZ168 GTAGAAATGAAGACAAAGCGATAGAGTGCTTAATAAC 254 DZ169 GTTGTAACTGCTCTTAGTTTG 255 DZ170 GTTGTCGAAAGCCTTATTTTGAAGGGCTATTACAAC 256 DZ171 GCTGTTTTTACCTTTCAAATTCAAGGCATTCACAGC

257 DZ172 GCTGTAGAAGCCGGCACTTTGGCTGGTAATTACAGT 258 DZ173 GTTGCATCTGCCTTTCTATTTGAGAGGCACAAACAAC 259 DZ174 GTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATC 260 DZ175 GTTGTTGAAGACCAAATTTTGAATGGTTATAACAAC 261 DZ176 TTGTTTCTTCCTACTAAAAGTGCAGGTCTTTACAAC 262 DZ177 TTGTAACTGCCTCTTATTTTGAAGGGTAAAAACAAC 263 DZ178 TGCTGTAATTACCCCTTCAAAAAGAAGGCTTCCACAGC 264 DZ179 GTTGTAAGTGCTCTTAATTTGAAGGGTAAACACAAC 265 DZ180 GGTGTGGAAGCCTTCTTTTTGAAAGG 266 DZ181 ACTGTAGATGATCCCAAAAGTGAAGGGAACT 267 DZ182 GTTGTGTTTACCTTTCAAATTAAGGGCAGCCACAAC 268 DZ183 TTAGTTGTAACTGCCCTTATTTTGAAGGGTAAACACAACTT 269 DZ184 GTTGTCTCTTCCCTCTCAAATGAGGGCTTTTACAAC 270 DZ185 GTTGTAACTGCTCTTAGTTTGAAGGGTAAAAACAAC 271 DZ186 GTTGTAACTGCTCTTAATTAGAAGGGTAAAAACAAC 272 DZ187 GTTGTAACAAGCCTAAGTTTGAAAGGTAAAAACAAC 273 DZ188 GGTGTAATCAGTTTCTTTTTGAAAGCTATTAACACCA 274 DZ189 TTGTAACTGCCTCTTATTTTGAA 275 DZ191 CTATGTTGGGACATACCTGTTTTTGAAAGGTATTTACAAC 276 DZ192 TTGAGTTGTTACAGCCTTTGTTTTGAAAGGTATTTACAAC 277 DZ193 AAGTTGTCATACCCACTCATATGTGGGCTTCTGCAAC 278 DZ194 GCTGTAACTACATCCCCAAACGGGAGCCTCTACAGC 279 DZ195 GTTGTAGCTGCCCTTAATTTGAAGGGTAAAAACAAC 280 DZ196 GCTGTAAGTACCTTTCAAAAATCAAGGCTTCAACAGC 281 DZ197 GTTGTAACTGCCCCTTATTTTGATGGGTAAAAACAAC 282 DZ198 TGATATAGACCACCCCAATATCAAAGGGGACTAAAAC 283 DZ199 GCTGTAGAGGCCCTGCGTTTGGCAGGTAGGTACAGC 284 DZ200 GTTGCAGAAGCCCACATGTGAGTGGGTATGACAAC

285 DZ201 GTTGTAACTGCCCTTGATTTGAAGGGTAAAA 286 DZ202 TATTTTGAAGGGTATAAACAAC 287 DZ203 GTTGTAACCGTGCTTGATTTTGAAGCGCAATTCCAAC 288 DZ204 AGTATTATCTAACCCCTAAATAACGGGGGGCTATAACT 289 DZ205 GGTGTGTCCGCCTTTGATTTGAAAGGTATGTGCACC 290 DZ206 GCTGTAACTGCCTTATTTTTGAGAGGTAAATACAGC 291 DZ207 GTTGTAAATGCCCTTGTTTGAAGGGTAAAAACAAC 292 DZ208 GTTGTGAATGCTCTTAGTTTGTGGAGTAAAGACAAC 293 DZ209 GTCGAAGAAGCCTCCAGTTTGAGGGGTGAGTTTGAC 294 DZ210 GTTGTAAGTGCCCCTTAGTTTGAAAGGTAGAAACAAC 295 DZ211 GTTGTAACTGCCCTTATTTTAAAGGGTACAAACAAC 296 DZ212 GTTGTAACTGCCCTCGGTTTGGGGGGTGAACACAAC 297 DZ213 GTTGTAACTGCTCTTGTTTTGAAGGGTAAACACAAC 298 DZ215 GCTGTTATTACCTTTTCAAATCAAAGGCATACACAGC 299 DZ216 GTTGTAAATGCCTTATATTTGTAAGGTGAAAACAAC 300 DZ217 GTTGTAGCTGCCCCTTATTTTGAAGGGTAAACACAAC 301 DZ218 AGGGTTTTGAAGCCCTCTATGCTGAGAGGGTTGTAAAC 302 DZ219 TTGTTGTAGCTGCTCTTTGTTTGGAGGGTAAAAACAAC 303 DZ220 GATATAGACCACCCCAATATCGAAGGGGACTAAAAC 304 DZ221 GTTGGGACGTATCACAATTTGAAAGGTACTCACAAC 305 DZ222 GCTGTGTATGCCTTTGATTTGAAAGGTAATAACAGC 306 DZ223 GTTGTTACAGCCCTATATTTGAAGGGTACTCACAAC 307 DZ224 ATTACAACCCCTATATTCACGGGGACTATAAC 308 DZ225 GTTGTTACTGCCCCTTATTTTGAAGGGTAAACACAAC 309 DZ226 GTTGTAGCTGCCCTTTATTTTGAAAGGTAAAAACAAC 310 DZ227 GCTGTAGAATCCCTTATTTTGAAGGGTAGGAACAGC 311 DZ228 GTTGTAACTGCCCCTTATTTTGAAGGGTATAAACAACA 312 DZ229 GGTGTAGTGCTCCCTCATTTGGAGCTCATCTTTAGC

313 DZ230 GTTGTAACTGCCCCTTATTTTGAAGGGTAAACACAAC 314 DZ231 GATATAGACTACCCCAATATCGAAGGGGACTAAAAC 315 DZ232 GAGAAACATCACCCCCAAATGGAGGGGGACTGCACC 316 DZ233 GTTGTAAGAACCTTGCAAATATAAGGCATTTACAAC 317 DZ234 GTTGTTACAGTCCTTAATTTGAAGGGTAAACACAAC 318 DZ235 GTTGTTATTACCTCTCCAAACTAAGAGCCTTTACAAC 319 DZ236 GTTGTAAAGGCTCTTAGTTTGGAGGGTAATAACAAC 320 DZ237 GTTGTAAATACCCTATATTTGAAGGGTAATAACAAC 321 DZ238 GTTGTAGATGCCTTGATTTTGCAGGGTAAACACAAC 322 DZ239 GTTGGGACGTATCACAATTTGAAGGGTACTCACAAC 323 DZ240 GATATAGACCACCCCAATATCGAAGGGGACTAAAAC 324 DZ241 GTCACAATCCCTGAACGCATGGGAACTGAAAC 325 DZ242 GTTGTTTTTACCTACAAAATGAGGCCAGCTACAAC 326 DZ243 GTTGTAAATACTCTATATTTGAAGGGT 327 DZ244 GCTGTGGAAGCCTTTCGTTTGAATGGTAATTACAGC 328 DZ245 GATATAGACCACCCCAATATCGAAGGGGACTAAAAC 329 DZ246 GTTGTAGATGCCCCGATTTTGCAGGGTAAACACAAC 330 DZ247 GGTGTGAATGATCCCAAAATGAAAGGGAACTACAAC 331 DZ248 GATATAGATAACCCCCAAAAACGAAGGGGTCTAAAAC 332 DZ249 GTTGTCATACCTCTCATGTGAGGGCGTTAGCAAC 333 DZ250 GATATAGATAACCCCCAAAAACGAAGGGGACTAAAAC 334 DZ251 GGTGTGGAAGCCCTCTGTTTGAAGGGTAGATACACC 335 DZ252 GTTGTAACTGCCCTCAGTTTGAAGGGTAAAAACAAC 336 DZ253 GTTGTAGAAGCCTGTATTTGAGCAGGTATGACAAC 337 DZ254 GATATAGATAACCCCCAAAAACGAAGGGGACTAAAAC 338 DZ255 GTTGTGAAAGGCCTCAGTTTGATGGGTACTAACAAC 339 DZ256 GTCACAACTCCCATGTAGGCGGAGACTGCAAC 340 DZ257 GTTTTAGTCCCCTTTGATATTGGGGTGGTCTATATC

341 DZ258 ATTACAATCCCCATATACAGGGGAACTGAAAC 342 DZ259 GGGGGTGATCTCCAAC 343 DZ260 ACTGTAGATAATCCCAATAGTGAAGGGAACTACAAC 344 DZ261 TTTGTGGGGTAGAAACAAC 345 DZ262 GGTGTGAAAGCCATCTTTTTGTATGGTAGGGACACC 346 DZ263 GGTGCAGTCGCCCTTCAGTGGGCGTGGTCGGTGCAAC 347 DZ264 TATGTTGTAACTGCCCCTTATTTTGAAGGGTAAACACAAC 348 DZ265 GTTTGAATACCACCCCCACATGACGGGGGACTGCAAC 349 DZ266 ACTGTAGACTATCCCAATAGTGAAGGGAACTACAAC 350 DZ267 GCTGTTTAAGCCCTTCGTTTGAAGGGTATTGACAGC 351 DZ268 GTTGTAGAAGCCGTTCATTTGGAATGGTATGACAAC 352 DZ269 GGTGCAGTCGCCCTTCATTTGGGCGTGGTCTGCAGG 353 DZ270 CCCTGCACACCACGCCCAAGTTGAGGGCGACTGCACCT 354 DZ271 GTTGGATAAGCCTGCTATTTGCAAGGTGAAGACAACA 355 DZ272 GTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATC 356 DZ273 GTTGTATCCCCTTTCAAATTGGGGTTATTCCACATC 357 DZ274 GGTTCGGAACCACGCCCAATCACGGGCGACTGCACC 358 DZ275 CGTAACAATCCCCGTGTGTAGGGGAACTGCAACTT 359 DZ276 GGTGCAGTCGCCCTCCATTCGGGCGTGATGTGCAGG 360 DZ277 GAATGAAACTATCGTCACATAGCGACGAACTACACC 361 DZ278 GGTGTAGTCGCCTCTTAATTAGGCGTCATGTCC 362 DZ279 GATTTAGATAACCCCAATAATGAAGGGGACTAAAAC 363 DZ280 GTTGAATTAACCTTTCAAAATAGGGCTACTTCAAC 364 DZ281 GTTTCAGTTCCCCTGCCTACGGGGATTGTTAC 365 DZ282 GTCACAACTCCCGAGAG 366 DZ283 GTTGTCATACCCATCCAAACGACAGGCTTCTACAACA 367 DZ284 GTTGTAATACCATTCTCAAATGATGGCTTCGGCAAC 368 DZ285 GGTGTGAACGCGCTTGTTTTGAAGCGTAAATACACC

369 DZ286 GTTTCAGTTCCCCTGTATGCGGGGATTGTTAC 370 DZ287 TTGTCATACCCCTCCTGACGAGAGGCTTCTACAAC 371 DZ288 GAAGGAGATCACCGCCACATGACGGCGGACTGCACC 372 DZ289 GTTGTCATACCCCTCCAAACGAGAGGCTTCTACAAC 373 DZ290 GCCTCACATCACCGCCAAAACGACGGCGGACTACACC 374 DZ292 GTTGAAATTATCCCCACATAAAGGGGAACTAAGAC 375 DZ293 GTTTTGAGAATAGCCCGACATAGAGGGCAATAGAC 376 DZ294 GTAGAAATGAGTACAAAGCGATAGAGAGCTTAATAAC 377 DZ296 GCTATATATCACCCCACTATGTAAGGGGACTAGAAC 378 DZ297 ATTTAAGATGACTGCTTCTTTAACAGCAGACTGAACC 379 DZ298 GGTCCAGTCCGCTGTGAAAGGAGCGGTCATCCAGAAC 380 DZ299 GTGAATACAGCTCGATATAGTGAGCAATAAGATT 381 DZ300 GTTGTAATACCATTCTCAAATGATGGCTTCGGCAAC 382 DZ301 CTGTTCAGGAGGACCGCTCATTTCACAGCGGACTGACCC 383 DZ302 GAATACAGCTCGATATAGCGAGTAATAAC 384 DZ303 TGTGAAAGTAGCCCGATATAGAGGGCAATAA 385 DZ304 GTGAATACAGCTCGATATAGTGAGCAATAAG 386 DZ305 GTTTCAGCATGACCCCCTTCTTTCACAGGGGACTGAAC 387 DZ306 GTTAAAAGAAAACAGCCCGACATAGCGGGCAATAAC 388 DZ307 ACTTATTGCTCACTATATCGAGCTTTTCTCAC 389 DZ308 GATATAGACCACCCCAATATCGAAGGGGACTAAAAC 390 DZ309 TGTTCTGCATGACCGCCTGTTTCACGGCGGACTGACAC 391 DZ310 GATATAGACCACCCCAATATCAAAGGGGACTAAAAC 392 DZ311 GATATAGACCACCCCAATATCGAAGGGGACTAAAAC 393 DZ312 GTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATC 394 DZ313 GGTGCAGGCCGCTGTGAAAGAAGCGGTCATGCGGAAC 395 DZ314 GTGAAAGTAGCCCGATATAGAGGGCAATAAC 396 DZ315 GATATAGACCACCCCAATATCAAAGGGGACTAAAACT

397 DZ316 GTTGTAGAAGCCTATCGTTTGGATAGGTATGACAAC

See Table 2 for the amino acid sequence number, expanded HEPN domain and coordinates of the final candidate Cas13 protein.

Table 2. Summary table of candidate Cas13 proteins

Claims (32)

Cas13 protein, it comprises as described in any one of SEQ ID NO:1 to 198 aminoacid sequence, or has any one of SEQ ID NO:1 to 198 of the conservative amino acid substitution of one or more residues amino acid sequence. According to the Cas13 protein described in claim 1, its RNA cutting activity is retained. According to the Cas13 protein according to claim 1, its HEPN domain or RNA cleavage domain is further modified or transformed to reduce or eliminate its RNA cleavage activity, and become dCas13 with reduced or eliminated RNA cleavage activity. The Cas13 protein according to any one of claims 1 to 3, wherein the Cas13 protein is fused with one or more heterologous functional domains, wherein the fusion is at the N-terminal and C-terminal of the Cas13 protein or inside. The Cas13 protein according to claim 4, wherein said one or more heterologous functional domains have the following activities: deaminases such as cytidine deaminase and deoxyadenosine deaminase, methylase, Demethylase, transcriptional activation, transcriptional repression, nuclease, single-stranded RNA cleavage, double-stranded RNA cleavage, single-stranded DNA cleavage, double-stranded DNA cleavage, DNA or RNA ligase, reporter protein, detection protein, localization signal, or any combination thereof. A nucleic acid molecule comprising a nucleotide sequence encoding the Cas13 protein according to any one of claims 1 to 5. The nucleic acid molecule according to claim 6, which is codon-optimized for expression in a specific host cell. The nucleic acid molecule according to claim 7, wherein said host cell is a prokaryotic or eukaryotic cell, preferably a human cell. The nucleic acid molecule according to any one of claims 6 to 8, comprising a promoter effectively linked to the nucleotide sequence encoding Cas13, which is a constitutive promoter, an inducible promoter, a tissue-specific promoter, Chimeric or developmental specific promoters. An expression vector, comprising the nucleic acid molecule of any one of claims 6 to 9, expressing the amino acid sequence of claim 1 or the nucleotide sequence of any one of claims 6 to 9 in the form of DNA, RNA or protein. The expression vector according to claim 10, which is adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes simplex virus, oncolytic virus. A delivery system comprising (1) the expression vector according to claim 10, or the Cas13 protein according to any one of claims 6 to 11; and (2) a delivery vector. The delivery system according to claim 12, wherein the delivery vehicle is a nanoparticle, liposome, exosome, microvesicle or gene gun. CRISPR-Cas system, it comprises: (1) Cas13 protein according to any one of claims 1 to 5 or its derivative or functional fragment, or the nucleic acid molecule according to any one of claims 6 to 9; ( 2) The gRNA sequence used to target the target RNA. According to the CRISPR-Cas system according to claim 14, the functional fragment of the Cas13 protein shall refer to any one amino acid (such as 1, 2, 3, 4, 5, 6, 7) containing one or more SEQ ID NO: 1 to 204 , 8, 9 or 10 residues), additions, deletions and/or substitutions (eg conservative substitutions). According to the CRISPR-Cas system according to claim 15, the derivative of the Cas13 protein should at least have at least ≥70% amino acid sequence identity with any protein fragment in SEQ ID NO: 1 to 198 (such as 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agreement). The CRISPR-Cas system according to claim 14, wherein the gRNA sequence comprises a direct repeat (DR) sequence and a sequence targeting a spacer region of the target RNA portion. The CRISPR-Cas system according to claim 17, wherein the DR sequence is the sequence shown in Table 1; wherein the spacer sequence is 15-60 nucleotides, preferably 25-50 nucleotides, more Preferably 30 nucleotides. The CRISPR-Cas system according to claim 18, wherein the DR sequence can be a derivative corresponding to any of the following, wherein the derivative (i) has, compared with any of the sequences shown in Table 1, One or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) additions, deletions, or substitutions of nucleotides; (ii) with any of the sequences shown in Table 1 One has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 97% sequence identity; (iii) under stringent conditions as shown in Table 1 Any of the sequences, or hybridize to any of (i) and (ii); or (iv) is the complement of any of (i)-(iii), provided that the derivative is not shown in Table 1 Any one of the sequences, and said derivative encodes an RNA, or itself is an RNA, said RNA substantially maintains the same secondary structure as any RNA encoded by SEQ ID NO: 199-397. The CRISPR-Cas system according to claim 14, further comprising: (3) target RNA. The CRISPR-Cas system according to claim 14, which causes degradation, cleavage or sequence change of the target RNA sequence. The CRISPR-Cas system according to claim 20, wherein the target RNA is mRNA or ncRNA, including non-coding RNA selected from lncRNA, miRNA, misc_RNA, Mt_rRNA, Mt_tRNA, rRNA, scaRNA, scRNA, snoRNA, snRNA, sRNA . A cell comprising the Cas13 protein described in any one of claims 1 to 5, the nucleic acid molecule described in any one of claims 6 to 9, the expression vector described in claim 10 or 11, the delivery described in claim 12 or 13 system, or the CRISPR-Cas system described in any one of claims 14 to 22. The cell according to claim 23, which is a prokaryotic cell or a eukaryotic cell, preferably a human cell. The method for degrading or cutting the target RNA in the target cell, modifying the sequence of the target RNA in the target cell, which includes using the Cas13 protein described in any one of claims 1 to 5, the nucleic acid molecule described in any one of claims 6 to 9 , the expression vector of claim 10 or 11, the delivery system of claim 12 or 13, or the CRISPR-Cas system of any one of claims 14 to 22. The method according to claim 25, the target cells are prokaryotic cells or eukaryotic cells, preferably human cells. The method according to claim 25, wherein the target cells are ex vivo cells, in vitro cells or in vivo cells. The Cas13 protein described in any one of claims 1 to 5, the nucleic acid molecule described in any one of claims 6 to 9, or the CRISPR-Cas system described in any one of claims 14 to 22 is used to detect nucleic acid molecules use. The use according to claim 28, wherein the detected target is RNA or DNA, wherein the RNA or DNA is RNA or DNA in prokaryotic microorganisms or eukaryotic organisms. The use according to claim 29, wherein the prokaryotic microorganism is a DNA virus or a nucleic acid thereof, an RNA virus or a nucleic acid thereof. The use according to claim 29, wherein the eukaryotic organisms include animals and plants, preferably humans; the RNA or DNA in the body includes RNA or DNA in cells or body fluids. The use according to claim 31, wherein said body fluid comprises body fluids such as blood, urine or lymph.

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