Development of RNA-targeted gene editing tools

Abstract

The invention discloses a screening method and application of a compact Cas13 protein. The present disclosure relates to the fields of biotechnology and medicine. More specifically, the present disclosure relates to a new Cas13 family protein, a method for screening the new Cas13 family protein, and a corresponding RNA editing system and application thereof. The present disclosure relates, inter alia, to Cas13 proteins and related RNA editing systems. The molecular weight of the novel Cas13 protein is very low, the CRISPR-Cas protein which is guided by guide RNA and has RNase activity is almost pushed to the limit, and the novel Cas13 protein contains more expanded HEPN structural domains. The screening method for rapidly searching the CRISPR-Cas13 protein which is ultralow in molecular weight, depends on guide of guide RNA and has RNase activity is provided for the first time, multiple new Cas13 proteins and new families thereof are obtained, and the CRISPR-Cas13 protein has wide application prospects and huge market value.

Description

Development of RNA-targeted gene editing tools

[0001] This application claims priority to application No. CN202210246868.4, filed on March 14, 2022, entitled “Development of RNA-Targeted Gene Editing Tools,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present disclosure relates to the fields of biotechnology and medicine. More specifically, the present disclosure relates to new Cas13 family proteins, methods for screening new Cas13 family proteins, and corresponding RNA editing systems and applications thereof. The present disclosure particularly relates to low molecular weight Cas13 proteins and related RNA editing systems. Background Art

[0003] The CRISPR-Cas system, considered a key component of a new generation of genome engineering tools, functions as an adaptive immune mechanism in microorganisms such as bacteria and archaea, protecting them from viruses and other foreign nucleic acids. The CRISPR-Cas immune response primarily involves three phases: adaptation, expression and processing, and interference. Similar to other defense mechanisms, the CRISPR-Cas system evolved in the context of constant competition with mobile genetic elements, resulting in extreme diversity in Cas protein sequences and CRISPR-Cas loci architecture.

[0004] Since 2011, CRISPR-Cas systems have been divided into two categories based on their genetic composition, locus structure, and sequence similarity clustering. The first category is an effector module composed of multiple Cas proteins, some of which form crRNA-binding complexes that mediate pre-crRNA processing and interference through additional Cas proteins. The second category consists of a single Cas effector protein with a multifunctional domain binding region that can bind to crRNA and participate in all activities required for interference. In some variants, it also includes participation in pre-crRNA maturation. The second category is mainly divided into three subtypes: type II (such as Cas9), type V (such as Cas12a), and type VI (such as Cas13d). Type II and type V subtypes mainly target DNA, while type VI effector Cas proteins mainly target RNA.

[0005] Currently, a variety of CRISPR-Cas-based gene editing tools have been developed based on the second type of CRISPR-Cas system, including CRISPRa, CRISPRi, and single-base editing technologies. However, gene delivery into cells requires a delivery vehicle. Commonly used delivery vehicles include retroviruses, adenoviruses, and adeno-associated viruses. These tools have limited carrying capacity. For example, adeno-associated virus (AAV) cannot accommodate DNA larger than 4.7kb, making it difficult to package large CRISPR-Cas-related tools.

[0006] In 2020, researchers discovered a Casφ (also classified as a subfamily of Cas12j) protein in a large bacterial and viral phage. This protein, with a molecular weight only half that of the Cas9 and Cas12a genome-editing enzymes, can cut DNA in eukaryotic cells. Zhang Feng's team recently discovered the ancestral protein IscB (approximately 400 amino acids) and the TnpB family, both of which are DNA-editing enzymes. Currently, the smallest known Cas13 effector proteins capable of editing RNA, such as Cas13bt and Cas13X, are over 700 amino acids.

[0007] Previous research strategies were mainly based on the sequence conservation of the Cas1 protein to determine the adjacent Cas proteins, but this approach will miss some single effector proteins that do not have the Cas1 protein. Based on the coexistence of CRISPR-array and Cas protein, scholars have been prompted to start directly from predicting the CRISPR array and then look for adjacent CRISPR-Cas associated proteins. However, due to the limitations of the current algorithms for predicting CRISPR arrays, no algorithm has been classified as the gold standard. In addition, the identification of candidate proteins mainly relies on DNA and protein sequence alignment, which can easily ignore the influence of protein spatial folding. Therefore, there is an urgent need to develop new methods for screening single effector proteins related to the CRISPR-Cas13 system with smaller molecular weight and new cas13 proteins with smaller molecular weight.

[0008] Summary of the Invention

[0009] To address the shortcomings and practical needs of existing technologies for screening novel CRISPR-Cas proteins, this disclosure provides a method for rapidly identifying novel guide RNAs containing a relatively large number of extended HEPN domains (at least one) that guide CRISPR-Cas13 proteins with RNase activity. The method also verifies the RNase activity of candidate proteins through both bioinformatics analysis (e.g., sequence alignment, protein structure prediction, etc.) and experimental validation. These proteins have potential applications in RNA regulation, editing, and detection, and possess broad academic and commercial value.

[0010] The technical problem solved by the present disclosure is how to quickly find candidate CRISPR-Cas13 proteins and their systems with more novel RNA enzymatic activity domains (expanded HEPN domains); secondly, verify the activity of candidate CRISPR-Cas13 proteins and their systems; and finally obtain a variety of novel Cas13 proteins.

[0011] In a first aspect of the present disclosure, a Cas13 protein is provided, comprising an amino acid sequence as described in any one of SEQ ID NOs: 1 to 78, or a protein having at least 70%, 80%, 85%, 90%, or 95% homology to a sequence as described in any one of SEQ ID NOs: 1 to 78; preferably, the protein comprises an amino acid sequence as described in any one of SEQ ID NOs: 1-34, 37, 38, 41, 42, 43, 45, 46, 47, 49, 52, 54, 55, 58, 61, 62, 64, 65, or 68-71, or a protein having at least 70%, 80%, 85%, 90%, or 95% homology to a sequence as described in any one of SEQ ID NOs: 1 to 78; NO: 1-34,37,38,41,42,43,45,46,47,49,52,54,55,58,61,62,64,65, or 68-71 having at least 70%, 80%, 85%, 90%, or 95% homology to any one of the sequences described in NO: 1-34,37,38,41,42,43,45,46,47,49,52,54,55,58,61,62,64,65, or 68-71; More preferably, the protein comprises an amino acid sequence as described in any one of SEQ ID NO: 1, 3, 6, 17, 19, 21, 27, 31, 33, 55, 68, 69, 71, or comprises a protein having at least 80%, 85%, 90%, or 95% homology to any one of the sequences described in SEQ ID NO: 1, 3, 6, 17, 19, 21, 27, 31, 33, 55, 68, 69, 71.

[0012] In a preferred embodiment, the cas13 protein described in the first aspect of the present invention, the protein having at least 80%, 85%, 90%, or 95% homology is a protein having conservative amino acid additions, deletions, or substitutions of one or more residues; preferably, it is a protein having conservative amino acid additions, deletions, or substitutions of 1-10 residues.

[0013] In a second aspect of the present disclosure, a Cas13 protein is provided, wherein the HEPN domain of the protein comprises at least one RXXXXXXH and / or RXXXXXXH structure, wherein X is an optional amino acid; preferably, the HEPN domain comprises 1-9 RXXXXXH and / or RXXXXXXH structures; more preferably, the HEPN domain comprises 2, 3, 4, or 5 HEPN domains.

[0014] In a preferred embodiment, in the cas13 protein provided in the second aspect, the amino acid X adjacent to R is preferably N, Q, H or D.

[0015] In a preferred embodiment, the HEPN structure of the cas13 protein described in the second aspect contains the HEPN structure of the protein described in Table 2.

[0016] In a preferred embodiment, the RNA cleavage activity of the cas13 protein described in the first aspect or the second aspect of the present invention is retained.

[0017] In a preferred embodiment, the Cas13 protein according to any one of the first aspect or the second aspect of the present invention, the HEPN domain of the Cas13 protein has a mutation of at least one nucleotide.

[0018] In a preferred embodiment, the Cas13 protein described in any one of the first aspect or the second aspect of the present invention is fused with one or more heterologous functional domains, wherein the fusion is at the N-terminus, C-terminus or inside the Cas13 protein; preferably, the heterologous functional domain has the following activities: deaminase such as cytidine deaminase and deoxyadenosine deaminase, methylase, demethylase, transcription activation, transcription 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.

[0019] In a preferred embodiment, the HEPN domain of the Cas13 protein according to any one of the first or second aspects of the present invention is the same as the HEPN domain of any one of SEQ ID NOs: 1 to 78.

[0020] In a preferred embodiment, at least one HEPN domain of the cas13 protein according to any one of the first or second aspects of the invention comprises a RXXXXH, RXXXXXH, and / or RXXXXXXH structure, wherein X is an optional amino acid, preferably, the amino acid adjacent to R is preferably N, Q, H or D.

[0021] In a preferred embodiment, the HEPN domain of the aforementioned cas13 protein comprises at least one RXXXXXXH and / or RXXXXXXH structure; preferably, the HEPN domain comprises 1-9 RXXXXXH and / or RXXXXXXH structures; more preferably, the HEPN domain comprises 2, 3, 4, or 5 HEPN domains.

[0022] In the third aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleotide sequence encoding the Cas13 protein described in any one of the first and second aspects of the present invention.

[0023] In a preferred embodiment, the nucleic acid molecule is a nucleic acid that has been codon-optimized for a specific host cell; the host cell is preferably a prokaryotic cell or a eukaryotic cell, more preferably a eukaryotic cell, and more preferably a cell of human origin.

[0024] In a preferred embodiment, any of the aforementioned nucleic acid molecules comprises a promoter operably linked to a nucleotide sequence encoding Cas13, wherein the promoter is a constitutive promoter, an inducible promoter, a tissue-specific promoter, a chimeric promoter, or a development-specific promoter.

[0025] In a fourth aspect of the present invention, a CRISPR-Cas system is provided, comprising: (1) the Cas13 protein or its derivative or functional fragment according to any one of the first or second aspects of the present invention, or the nucleic acid molecule according to any one of the third aspects of the present invention; and (2) a gRNA targeting a target nucleic acid;

[0026] Preferably, the gRNA sequence comprises a direct repeat (DR) sequence and a spacer sequence with the target nucleic acid;

[0027] More preferably, the DR sequence comprises a nucleic acid as shown in any one of SEQ ID NOs: 79-234 or a nucleic acid comprising a derivative sequence of a sequence as shown in any one of SEQ ID NOs: 79-234;

[0028] The derived sequence is:

[0029] (i) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) nucleotide additions, deletions, or substitutions compared to any of the sequences shown in Table 1;

[0030] (ii) having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 97% sequence identity to any one of the sequences shown in Table 1;

[0031] (iii) hybridizes under stringent conditions to any one of the sequences shown in Table 1, or to any one of (i) and (ii); or

[0032] (iv) is the complement of any one of (i) to (iii), provided that the derivative is not any of the sequences shown in Table 1, and the derivative encodes an RNA, or is itself an RNA, and the RNA retains substantially the same secondary structure as any RNA encoded by SEQ ID NOs: 79-234.

[0033] In a preferred embodiment, in any of the aforementioned CRISPR-Cas systems, the spacer sequence is 15-60 nucleotides, preferably 25-50 nucleotides, and more preferably 30 nucleotides.

[0034] In a preferred embodiment, the target nucleic acid of any of the aforementioned CRISPR-Cas systems is a target RNA; preferably, the target RNA is mRNA or ncRNA, including a non-coding RNA selected from 1ncRNA, miRNA, misc_RNA, Mt_rRNA, Mt_tRNA, rRNA, scaRNA, scRNA, snoRNA, snRNA, and sRNA.

[0035] In the fifth aspect of the present invention, a vector is provided, which comprises the nucleic acid molecule of any one of the third aspect and is capable of expressing the cas13 protein of any one of the first aspect or the second aspect of the present invention or the nucleic acid molecule of any one of the third aspect of the present invention; preferably, the vector is selected from viral vectors, lipid nanoparticles (LNP), liposomes, cationic polymers (such as PEI), nanoparticles, exosomes, microvesicles, and gene guns; more preferably, the vector is selected from viral vectors, and more preferably, the viral vector is selected from adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes simplex virus, and oncolytic virus.

[0036] In a sixth aspect of the present invention, a delivery system is provided, comprising (1) the vector described in any one of the fifth aspect, or the nucleic acid molecule described in any one of the third aspect, and (2) a delivery vector.

[0037] In a preferred embodiment, the delivery vector of the delivery system described in this aspect is a nanoparticle, a liposome, an exosome, a microvesicle or a gene gun.

[0038] In the seventh aspect of the present invention, a cell is provided, which comprises the Cas13 protein described in any one of the first or second aspects of the present invention, the nucleic acid molecule described in any one of the third aspect of the present invention, the expression vector described in the fifth aspect of the present invention, the delivery system described in the sixth aspect of the present invention, or the CRISPR-Cas system described in any one of the fourth aspect of the present invention.

[0039] In a preferred embodiment, the cell described in any one of the present invention is a prokaryotic cell or a eukaryotic cell, preferably a human cell.

[0040] In the seventh aspect of the present invention, a method for degrading or cutting a target RNA in a target cell, or modifying the sequence of a target RNA in a target cell is provided, comprising using the Cas13 protein described in any one of the first or second aspects of the present invention, the nucleic acid molecule described in any one of the third aspects of the present invention, the expression vector described in the fifth aspect of the present invention, the delivery system described in the sixth aspect of the present invention, or the CRISPR-Cas system described in any one of the fourth aspects of the present invention.

[0041] In a preferred embodiment, the target cell described in any one of the aspects is a prokaryotic cell or a eukaryotic cell, preferably a human cell.

[0042] In a preferred embodiment, the target cell described in any one of the aspects is an in vitro cell, an in vitro cell or an in vivo cell.

[0043] In a seventh aspect of the present invention, a method for screening Cas13 proteins is provided, wherein the Cas13 proteins comprising at least one RXXXXXXH and / or RXXXXXXH structure in the HEPN structure are screened, where X is an optional amino acid; preferably, the HEPN domain comprises 1-9 RXXXXXH and / or RXXXXXXH structures; more preferably, the HEPN domain comprises 2, 3, 4, or 5 HEPN domains.

[0044] In a preferred embodiment, in any of the methods described in this aspect, the HEPN structure of the screened Cas13 protein contains the HEPN structure of the protein described in Table 2, or contains a HEPN structure with at least 80%, 85%, 90%, or 95% similarity to the HEPN structure of the protein described in Table 2.

[0045] In a preferred embodiment, in any of the methods described herein, the screening method comprises:

[0046] 1) Download bacterial genome and / or metagenome sequences and identify CRISPR array regions;

[0047] 2) Analyze the proteins upstream and downstream of the CRISPR array region, and select proteins whose HEPN structure contains at least one RXXXXXH and / or RXXXXXXH structure as candidate Cas13 proteins;

[0048] Preferably, the HEPN structure further contains at least one RXXXXH structure.

[0049] In a preferred embodiment, in the method described in any one of the aspects of the present invention, six proteins upstream and downstream of the CRISPR array region adjacent to the CRISPR array region are analyzed.

[0050] In a preferred embodiment, in any of the methods described herein, the amino acid X adjacent to R in the HEPN structure is preferably N, Q, H or D.

[0051] In a preferred embodiment, in any of the methods described in this aspect, the preference for candidate proteins (PFS) is screened; further, by screening the preference for candidate proteins, better usage functions of the candidate proteins are obtained.

[0052] The present disclosure achieves the following technical effects:

[0053] (1) Developed a method for rapid screening of novel Cas13 family proteins, which can be used to analyze newly updated prokaryotic microbial DNA sequences and metagenomic sequences using the CRIPSR array system and screen for related effector proteins;

[0054] (2) Screening low-molecular-weight Cas13 family members to expand the application scope of CRISPR-Cas13. Since the low molecular weight of the candidate Cas13 protein can be better packaged through delivery vectors such as adeno-associated viruses, it can achieve the diagnosis and treatment of related diseases, such as the diagnosis and treatment of neurodegenerative diseases. In the field of plants, it can be used to carry out research on breeding and adverse stress, and in the field of microorganisms, it can be used to transform related engineered bacteria.

[0055] (3) In the screening process, in addition to using the known HEPN domain of Cas13 protein for screening, this method also includes conserved domains with RNA cleavage activity in other types of proteins, thereby providing the possibility of screening new Cas13 proteins. Moreover, the identification of these new functional domains in these new Cas13 proteins provides new ideas and possibilities for further modification of Cas13 proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 shows the RNase activity results of protein DZ4. DZ4's enzymatic activity in 293T cells was detected by flow cytometry. 293T cells were co-transfected with a plasmid containing DZ4 protein (containing the corresponding sgRNA targeting mCherry) and a plasmid containing mCherry protein. Flow cytometry analysis was performed 48 hours later. It was found that the candidate protein DZ4 had strong RNase activity compared to the negative control group, which only expressed mCherry protein (red light) and DZ4 protein (green light); the experimental group (also labeled AP459) also contained one of the sgRNAs targeting different regions of mCherry.

[0057] Figure 2 shows the RNase activity results of the candidate protein DZ28. Flow cytometry analysis results for detecting candidate protease cleavage activity in mammalian cell lines: The figure shows the flow cytometry results 48 hours after co-transfection of a plasmid containing the DZ28 protein (containing the corresponding sgRNA targeting mCherry) with a plasmid containing the mCherry protein into a 293T cell line. It can be seen that compared with the negative control group, the candidate protein DZ28 has strong RNase activity, and the corresponding red light is significantly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ28 protein (green light); among them, AP393 is an experimental group containing one of the sgRNAs targeting different regions of mCherry.

[0058] Figure 3 shows the results of RNase activity of protein DZ29. The results of flow cytometry experiments to detect candidate protease cleavage activity in mammalian cell lines: The figure shows the results of flow cytometry analysis 48 hours after the plasmid containing DZ29 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein DZ29 has strong RNase activity, and the corresponding red light is significantly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ29 protein (green light); among them, the control group AP405 and the control group AP407 are experimental groups containing sgRNAs targeting different regions of mCherry.

[0059] Figure 4 shows the flow cytometry results of the candidate Cas13 protein DZ30 at the cell level to verify its RNase activity. The results of the flow cytometry experiment to detect the candidate protease cleavage activity in the mammalian cell line: The figure shows the results of the flow cytometry analysis 48 hours after the plasmid containing the DZ30 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing the mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein DZ30 has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ30 protein (green light); among them, AP411 and AP413 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0060] Figure 5 shows the flow cytometry results of the candidate Cas13 protein DZ31 at the cell level to verify its RNase activity. The results of the flow cytometry experiment to detect the candidate protease cleavage activity in the mammalian cell line: The figure shows the results of the flow cytometry analysis 48 hours after the plasmid containing the DZ31 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing the mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein DZ31 has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ31 protein (green light); among them, AP417 and AP419 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0061] Figure 6 shows the flow cytometry results of the candidate Cas13 protein DZ32 at the cell level to verify its RNase activity. The results of the flow cytometry experiment to detect the candidate protease cleavage activity in the mammalian cell line: The figure shows the results of the flow cytometry analysis 48 hours after the plasmid containing the DZ32 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing the mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein DZ32 has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ32 protein (green light); among them, AP421 and AP423 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0062] Figure 7 shows the flow cytometry results of the candidate Cas13 protein DZ33 at the cell level to verify its RNase activity. The results of the flow cytometry experiment to detect the candidate protease cleavage activity in the mammalian cell line: The figure shows the results of the flow cytometry analysis 48 hours after the plasmid containing the DZ33 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing the mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein DZ33 has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ33 protein (green light); among them, AP427 and AP429 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0063] Figure 8 shows the flow cytometry results of the candidate Cas13 protein DZ35 at the cell level to verify its RNase activity. The results of the flow cytometry experiment to detect the candidate protease cleavage activity in the mammalian cell line: The figure shows the results of the flow cytometry analysis 48 hours after the plasmid containing the DZ35 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing the mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein DZ35 has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ35 protein (green light); among them, AP441 and AP443 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0064] Figure 9 shows the flow cytometry results of the candidate Cas13 protein DZ36 at the cell level to verify its RNase activity. The results of the flow cytometry experiment to detect the candidate protease cleavage activity in the mammalian cell line: The figure shows the results of the flow cytometry analysis 48 hours after the plasmid containing the DZ36 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing the mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein DZ36 has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ36 protein (green light); among them, AP25 and AP27 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0065] Figure 10 shows the flow cytometry analysis results of the candidate Cas13 protein DZ37 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ37 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ37 can target mCherry RNA and has strong RNase activity, and the corresponding red light is significantly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (red light) and DZ37 protein (green light); among them, AP33 and AP35 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0066] Figure 11 shows the flow cytometry analysis results of the candidate Cas13 protein DZ38 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ38 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ38 can target mCherry RNA and has strong RNase activity, and the corresponding red light is significantly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ38 protein (green light); among them, AP38 and AP47 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0067] Figure 12 shows the flow cytometry analysis results of the candidate Cas13 protein DZ39 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ39 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ39 targets mCherry RNA and has a certain RNase activity, and the corresponding red fluorescence is slightly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ39 protein (green light); among them, AP39 and AP43 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0068] Figure 13 shows the flow cytometry analysis results of the candidate Cas13 protein DZ40 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ40 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ40 can target mCherry RNA and has a certain RNase activity, and the corresponding red light is knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ40 protein (green light); among them, AP49 and AP53 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0069] Figure 14 shows the flow cytometry analysis results of the candidate Cas13 protein DZ44 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ44 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ44 can target mCherry RNA and has a certain RNase activity, and the corresponding red light is knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ44 protein (green light); among them, AP59 and AP55 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0070] Figure 15 shows the flow cytometry analysis results of the candidate Cas13 protein DZ45 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ45 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ45 can target mCherry RNA and has strong RNase activity, and the corresponding red light is significantly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ45 protein (green light); among them, AP63 and AP65 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0071] Figure 16 shows the flow cytometry analysis results of the candidate Cas13 protein DZ46 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ46 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ46 can target mCherry RNA and has strong RNase activity, and the corresponding red light is significantly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ46 protein (green light); among them, AP69 and AP71 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0072] Figure 17 shows the flow cytometry analysis results of the candidate Cas13 protein DZ47 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ47 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ47 can target mCherry RNA and has strong RNase activity, and the corresponding red light is significantly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ47 protein (green light); among them, AP91 and AP93 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0073] Figure 18 shows the flow cytometry analysis results of the candidate Cas13 protein DZ50 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ50 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ50 can target mCherry RNA and has a certain RNase activity, and the corresponding red light is knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ50 protein (green light); among them, AP121 and AP125 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0074] Figure 19 shows the flow cytometry analysis results of the candidate Cas13 protein DZ51 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ51 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ51 can target mCherry RNA and has strong RNase activity, and the corresponding red light is significantly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ51 protein (green light); among them, AP127 and AP131 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0075] Figure 20 shows the flow cytometry analysis results of the candidate Cas13 protein DZ52 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ52 protein (containing the corresponding gRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ52 can target mCherry RNA and has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ52 protein (green light); among them, AP133 and AP135 are experimental groups containing two gRNAs targeting different regions of mCherry.

[0076] Figure 21 shows the flow cytometry analysis results of the candidate Cas13 protein DZ54 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ54 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ54 can target mCherry RNA and has a certain RNase activity, corresponding to the knockdown of red light. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ54 protein (green light); among them, AP153 is an experimental group containing gRNA targeting mCherry.

[0077] Figure 22 shows the flow cytometry analysis results of the candidate Cas13 protein DZ55 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ55 protein (containing the corresponding sgRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ55 can target mCherry RNA and has a certain RNase activity, and the corresponding red light is knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ55 protein (green light); among them, AP157 is an experimental group containing gRNA targeting mCherry.

[0078] Figure 23 shows the flow cytometry analysis results of the candidate Cas13 protein DZ57 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ57 protein (containing the corresponding gRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ57 can target mCherry RNA and has a certain RNase activity, and the corresponding red light is knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ57 protein (green light); among them, AP169 and AP171 are experimental groups containing two gRNAs targeting different regions of mCherry.

[0079] Figure 24 shows the flow cytometry analysis results of the candidate Cas13 protein DZ62RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ62 protein (containing the corresponding gRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ62 can target mCherry RNA, has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ62 protein (green light); among them, AP187 and AP191 are experimental groups containing two gRNAs targeting different regions of mCherry.

[0080] Figure 25 shows the flow cytometry analysis results of the candidate Cas13 protein DZ63 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ63 protein (containing the corresponding gRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ63 can target mCherry RNA and has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ63 protein (green light); among them, AP193 and AP197 are experimental groups containing two gRNAs targeting different regions of mCherry.

[0081] Figure 26 shows the flow cytometry analysis results of the candidate Cas13 protein DZ65 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ65 protein (containing the corresponding gRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ65 can target mCherry RNA and has strong RNase activity, and the corresponding red light is significantly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ65 protein (green light); among them, AP201 and AP203 are experimental groups containing two gRNAs targeting different regions of mCherry.

[0082] Figure 27 shows the flow cytometry analysis results of the candidate Cas13 protein DZ68 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ68 protein (containing the corresponding gRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ68 can target mCherry RNA and has strong RNase activity, and the corresponding red light is significantly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ68 protein (green light); among them, AP217 and AP219 are experimental groups containing two gRNAs targeting different regions of mCherry.

[0083] Figure 28 shows the flow cytometry analysis results of the candidate Cas13 protein DZ86 RNase activity. The flow cytometry results of the candidate protease cleavage activity were detected in a mammalian cell line (HEK293T). The plasmid containing the DZ86 protein (containing the corresponding gRNA targeting mCherry) was co-transfected with the plasmid containing the mCherry protein into the 293T cell line for 48 hours and then flow cytometry was performed. It can be found that compared with the negative control group, the candidate protein DZ86 can target mCherry RNA and has strong RNase activity, and the corresponding red light is significantly knocked down. The negative control is a control group (without gRNA) that only contains the expression of mCherry protein (FB132) and DZ86 protein (green light); among them, AP711 and AP713 are experimental groups containing two gRNAs targeting different regions of mCherry.

[0084] Figure 29 shows the flow cytometry results of the candidate Cas13 protein DZ90 at the cell level to verify its RNase activity. The results of the flow cytometry experiment for detecting the candidate protease cleavage activity in the mammalian cell line: The figure shows the results of the flow cytometry analysis 48 hours after the plasmid containing the DZ90 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing the mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein DZ90 has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ90 protein (green light); among them, AP313 and AP317 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0085] Figure 30 shows the flow cytometry results of the candidate Cas13 protein DZ91 at the cell level to verify its RNase activity. The results of the flow cytometry experiment for detecting the candidate protease cleavage activity in the mammalian cell line: The figure shows the results of the flow cytometry analysis after the plasmid containing the DZ91 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing the mCherry protein were co-transfected into the 293T cell line for 48 hours. It can be found that compared with the negative control group, the candidate protein DZ91 has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ91 protein (green light); among them, AP319 and AP323 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0086] Figure 31 shows the flow cytometry results of the candidate Cas13 protein DZ93 at the cell level to verify its RNase activity. The results of the flow cytometry experiment for detecting the candidate protease cleavage activity in the mammalian cell line: The figure shows the results of the flow cytometry analysis 48 hours after the plasmid containing the DZ93 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing the mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein DZ93 has strong RNase activity, and the corresponding red light is knocked down to a certain extent. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ93 protein (green light); among them, AP151 is an experimental group containing one of the sgRNAs targeting different regions of mCherry.

[0087] Figure 32 shows the flow cytometry results of the candidate Cas13 protein DZ96 at the cell level to verify its RNase activity. The results of the flow cytometry experiment for detecting the candidate protease cleavage activity in the mammalian cell line: The figure shows the results of the flow cytometry analysis 48 hours after the plasmid containing the DZ96 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing the mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein DZ96 has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ96 protein (green light); among them, AP349 and AP353 are experimental groups containing two sgRNAs targeting different regions of mCherry.

[0088] Figure 33 shows the flow cytometry results of the candidate Cas13 protein DZ98 at the cell level to verify its RNase activity. The results of the flow cytometry experiment for detecting the candidate protease cleavage activity in the mammalian cell line: The figure shows the results of the flow cytometry analysis 48 hours after the plasmid containing the DZ98 protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing the mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein DZ98 has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and DZ98 protein (green light); among them, AP361 is an experimental group containing one of the sgRNAs targeting different regions of mCherry.

[0089] Figure 34 shows the flow cytometry results of the positive control Cas13d protein to verify its RNase activity at the cellular level. Flow cytometry experimental results for detecting candidate protease cleavage activity in mammalian cell lines: The figure shows the results of flow cytometry analysis 48 hours after the plasmid containing Cas13d protein (containing the corresponding sgRNA targeting mCherry) and the plasmid containing mCherry protein were co-transfected into the 293T cell line. It can be found that compared with the negative control group, the candidate protein Cas13d has strong RNase activity, and the corresponding red light is greatly knocked down. The negative control is a control group containing only the expression of mCherry protein (red light) and Cas13d protein (green light); among them, px261 is an experimental group containing one of the sgRNAs targeting different regions of mCherry.

[0090] Figure 35 shows the qPCR results of knocking down the endogenous gene STAT3 by the candidate protein DZ4. It can be found that the randomly designed sgRNA targeting the STAT3 gene can knock down the endogenous gene to a certain extent when transiently transfected with the DZ4 protein.

[0091] Figures 36A and 36B show the qPCR results of knockdown of the endogenous genes STAT3 and EZH2 by the candidate protein DZ29. It can be seen that randomly designed sgRNAs targeting STAT3 and EZH2 genes, when transiently transfected with the DZ29 protein, can knock down the endogenous genes to a certain extent. Different DRs have a certain influence on DZ29 knockdown of endogenous genes.

[0092] Figure 37 shows the qPCR results of knocking down the endogenous gene EZH2 by the candidate protein DZ32. It can be found that the randomly designed sgRNA targeting the EZH2 gene can be transiently transfected together with the DZ32 protein to knock down the endogenous gene to a certain extent.

[0093] Figure 38 shows the qPCR results of knocking down the endogenous genes STAT3 and EZH2 by the candidate protein DZ47. It can be found that randomly designed sgRNAs targeting the STAT3 and EZH2 genes can knock down the endogenous genes to a certain extent when transiently transfected with the DZ47 protein.

[0094] Figure 39 shows the qPCR results of knocking down the endogenous genes STAT3 and EZH2 by the candidate protein DZ51. It can be found that randomly designed sgRNAs targeting the STAT3 and EZH2 genes can be transiently transfected together with the DZ51 protein to knock down the endogenous genes to a certain extent.

[0095] Figure 40 shows the qPCR results of knocking down the endogenous genes STAT3 and EZH2 by the candidate protein DZ54. It can be found that randomly designed sgRNAs targeting the STAT3 and EZH2 genes can be transiently transfected together with the DZ54 protein to knock down the endogenous genes to a certain extent.

[0096] Figure 41 shows the qPCR results of knocking down the endogenous genes STAT3 and EZH2 by the candidate protein DZ68. It can be found that randomly designed sgRNAs targeting the STAT3 and EZH2 genes can be transiently transfected together with the DZ68 protein to knock down the endogenous genes to a certain extent.

[0097] Figures 42A and 42B show the qPCR results of knockdown of the endogenous genes STAT3 and EZH2 by the candidate protein DZ93. It can be seen that randomly designed sgRNAs targeting STAT3 and EZH2, when transiently transfected with DZ93 protein, can knock down the endogenous genes to a certain extent. Different DRs have a certain influence on DZ93 knockdown of endogenous genes.

[0098] Figure 43 shows the qPCR results of knocking down the endogenous gene STAT3 by the candidate protein DZ98. It can be found that the randomly designed sgRNA targeting the STAT3 gene can be transiently transfected together with the DZ98 protein to knock down the endogenous gene to a certain extent.

[0099] Figure 44 shows the qPCR results of candidate proteins knocking down the endogenous gene STAT3 in 293T cells. The boxed portion shows the efficiency of some proteins, which have the potential to knock down STAT3 RNase relative to the control group.

[0100] Figures 45A and 45B show the qPCR results of knocking down the endogenous genes STAT3 and EZH2 by the candidate protein DZ806. It can be found that transient transfection of randomly designed sgRNAs targeting the STAT3 and EZH2 genes together with the DZ806 protein can knock down the endogenous genes to a certain extent.

[0101] Figures 46A and 46B show the qPCR results of knocking down the endogenous genes STAT3 and EZH2 by the candidate protein DZ821. It can be found that transient transfection of randomly designed sgRNAs targeting the STAT3 and EZH2 genes together with the DZ821 protein can knock down the endogenous genes to a certain extent.

[0102] Figures 47A and 47B show the qPCR results of knocking down the endogenous genes STAT3 and EZH2 by the candidate protein DZ822. It can be found that transient transfection of randomly designed sgRNAs targeting the STAT3 and EZH2 genes together with the DZ822 protein can knock down the endogenous genes to a certain extent.

[0103] Figures 48A and 48B show the qPCR results of knocking down the endogenous genes STAT3 and EZH2 by the candidate protein DZ825. It can be found that transient transfection of randomly designed sgRNAs targeting the STAT3 and EZH2 genes together with the DZ825 protein can knock down the endogenous genes to a certain extent.

[0104] Figure 49A shows the results of a preferential motif analysis experiment to screen DZ796 for RNA targeting. It reveals a strong 5' base preference. The first base of the 5' segment from the target sequence is either G or C, while the first base of the 3' segment is G. The 3' PFS is not distinct. The overall PFS is 5'[C / G]-targetSeq-NNNNN[G]-3'.

[0105] Figure 49B shows the results of a preferential motif analysis experiment to screen DZ806 for RNA targeting, revealing strong base preferences at both the 5' and 3' ends. The third base from the 5' end of the target sequence has a strong T preference, while the first and second bases are primarily G or A-preferred. The first base from the 3' end exhibits a T preference. The overall PFS is 5'-T[G / A][G / A]-targetSeq-TN[G / A]-3'.

[0106] Figure 49C shows the results of a preferential motif analysis experiment to screen for DZ821's RNA targeting, revealing strong base preferences at both the 5' and 3' ends. The fifth base from the 5' and 3' ends of the target sequence has a strong preference for T, with an overall PFS of 5'-TNNN[C / G]-targetSeq-NNNNT-3'.

[0107] Figure 49D shows the results of the DZ822 preferential motif analysis for RNA targeting. It can be seen that it has a strong base preference at the 5' end. The third base from the 5' end of the target sequence has a strong preference for T, while the 3' end has a weaker PFS, with a preference for G or C at the third base from the target sequence. The overall PFS is 5'-TN[G / C / A]-targetSeq-[G / A][C / G][G / C]-3'

[0108] Figure 49E shows the results of a preferential motif analysis experiment to screen DZ824 for RNA targeting. It shows a strong 5' base preference. The third base from the 5' end of the target sequence has a strong preference for T, while the 3' end has a weaker PFS and the second base from the target sequence has a weaker preference for G. The overall PFS is 5'-N[C / G][C / G][C / T]-targetSeq-NG[C / A]-3'.

[0109] Figure 49F shows the results of an experimental motif analysis to screen for DZ825's RNA targeting preference. It reveals strong base preferences at both the 5' and 3' ends of the target sequence. The first base in the 5' end of the target sequence is a C, while the first base in the 3' end is a G.

[0110] Figure 50A shows an endogenous gene knockdown (KD) experiment validating the DZ825 PFS. Two 293T endogenous genes, STAT3 and EZH2, were selected. The first spacer in each gene experimental group was designed when the PFS was unknown, and the next three spacers were newly designed based on the PFS motif. As can be seen in the figure, some of the newly designed sgRNAs demonstrated significant knockdown efficacy in the 293T cell line knockdown experiments.

[0111] Figure 50B shows an endogenous gene knockdown experiment validating the DZ822 PFS. Three 293T endogenous genes were selected: STAT3, EGFR, and HRAS. The first experimental group in each gene contained a spacer designed when the PFS was unknown, while subsequent groups contained newly designed spacers based on the PFS motif. As can be seen in the figure, some of the newly designed sgRNAs, such as KRAS, demonstrated significant knockdown efficacy in the 293T cell line knockdown experiments.

[0112] Figure 50C shows a knockdown (KD) experiment for endogenous genes in 293T cells targeting DZ806 PFS. The selected endogenous genes include STAT3, EZH2, EGFR, HRAS, RAF1, NF2, SMARCA4, NFKB1, PPARG, KRAS, PTBP1, and NRAS. The first group of each knockdown gene experimental group is a spacer designed when the PFS is unknown, and the subsequent groups are newly designed spacers based on the PFS motif. As can be seen from the figure, in the knockdown experiment of the 293T cell line, some of the newly designed sgRNAs can show good knockdown effects, such as NF2 and SMARCA4.

[0113] Figure 50D shows the optimal knockdown effect of the original protein numbered DZ806 on endogenous genes in 293T cells. The highest knockdown efficiency of the tested genes was for the KD EGFR gene, exceeding 50%.

[0114] Figure 51 shows the evolutionary relationship between candidate CRISPR-Cas13s with guide RNA and potential RNase activity and known Cas13 protein family members. It reveals that our candidate proteins are potentially divided into two new families: Cas13m1 and Cas13m2, for example, DZ30 and DZ32; and DZ47 and DZ29 of the Cas13m2 family. DETAILED DESCRIPTION

[0115] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the present invention. Where specific conditions are not specified in the examples, the conditions were followed as recommended by the manufacturer.

[0116] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Unless otherwise indicated, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology and pharmacology known to those skilled in the art are employed in the practice of the present invention.

[0117] It should be noted that all headings and subheadings used in this application are for convenience only and should not be construed as limiting the invention in any way.

[0118] Unless otherwise stated, the use of exemplary terms (eg, "such as") provided herein is illustrative only and does not limit the scope of the invention.

[0119] In this document, "a" or "an" or "the" may refer to one or more than one. Unless otherwise specified in this specification, terms presented in the singular form also include plural cases.

[0120] In this document, a noun without a quantifier may mean one or more than one. As used in the claims, when used in conjunction with the word "comprises", a noun without a quantifier may mean one or more than one.

[0121] In the text, regardless of whether the content in this text adopts only alternatives or adopts and / or, unless otherwise stated or the alternatives are completely independent, the term "or / or" is used to mean "and / or".

[0122] Throughout the text, "another" can mean at least a second or more.

[0123] In the text, the term "about" is used to indicate an error in a value. Such an error may be a variation of ±10% from the stated value.

[0124] In the text, unless otherwise indicated, nucleotide sequences are listed in 5' to 3' direction and amino acid sequences are listed in N-terminal to C-terminal direction.

[0125] definition

[0126] NCBI (https: / / www.ncbi.nlm.nih.gov / ) refers to the National Center for Bioinformation in the United States. It is a public database for the whole world. Technicians in this field can use the nucleic acid database provided by this database to download prokaryotic genomes, proteome-related databases, etc., and can also use the blast comparison software provided by this database to perform sequence comparison analysis.

[0127] IMG (https: / / img.jgi.doe.gov / ) stands for the Integrated Microbial Genome Database and represents a new generation of genome databases. It not only fully integrates the content of existing databases but also provides enhanced data upload, annotation, and analysis services, storing sequencing data in the IMG / M database. This database allows downloading of pure culture bacterial sequenced genomes, metagenomes, metagenome-assembled genomes, and single-cell sequenced genomes.

[0128] The term "CRISPR" (cluster regularly interspaced short palindromic repeats) refers to a DNA sequence in the genome of prokaryotes, including a direct repeat (DR) region and a non-repetitive spacer region.

[0129] The term "CRISPR array" refers to the region containing the repeat sequence and the spacer sequence.

[0130] The term "CRISPR-Cas system" refers to a system comprising a CRISPR array and associated Cas proteins.

[0131] The Cas13 family is a family of CRISPR enzymes that can target RNA, and its members include Cas13a, Cas13b, Cas13c, Cas13d, Cas13X, and Cas13Y families. Unlike CRISPR / Cas9, which only cuts DNA, CRISPR / Cas13 can be used to cut specific RNA sequences in bacterial cells.

[0132] The term "HEPN domain" is the abbreviation of higher eukaryotes and prokaryotes nucleotide domain, which is an important domain of the Cas13 protein in the CRISPR-Cas13 enzyme system that cuts and resists foreign invading nucleic acids.

[0133] The term "ABE system" is short for Adenine base editors, which refers to purine base conversion technology that can achieve single-base changes 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 mainly achieves the mutation of A / T to G / C by deaminating adenine to inosine, which is read as G when reading in DNA or RNA. Because cells are insensitive to inosine excision repair, this mutation can maintain a high product purity.

[0134] The term "CBE system" is the abbreviation of Cytidine base editor, which is a pyrimidine base conversion technology. Currently, there are BE1, BE2 and BE3 tools, among which BE3 has the highest efficiency and is therefore widely used in gene therapy, animal model preparation, and functional gene screening.

[0135] The term "eukaryotic cell" includes, for example, mammalian cells, including human cells (primary human cells or established human cell lines). The cell can be a non-human mammalian cell, for example, from a non-human primate (e.g., monkey), cow / bull / cattle, sheep, goat, pig, horse, dog, cat, rodent (e.g., rabbit, mouse, rat, hamster), etc. The cell is from fish (e.g., salmon), bird (e.g., poultry, including chicken, duck, goose), reptile, shellfish (e.g., oyster, clam, lobster, shrimp), insect, worm, yeast, etc. The cell can be from a plant, such as a monocot or dicot. The plant can be a food crop, such as barley, cassava, cotton, peanut, corn, millet, oil palm fruit, potato, bean, rapeseed or canola, rice, rye, sorghum, soybean, sugarcane, sugar beet, sunflower, and wheat. The plant can be a cereal (e.g., barley, corn, millet, rice, rye, sorghum, and wheat). The plant may be a tuber (e.g., cassava and potato). In some embodiments, the plant may be a sugar crop (e.g., sugar beet and sugar cane). The plant may be an oil crop (e.g., soybean, peanut, rapeseed or canola, sunflower, and oil palm fruit). The plant may be a fiber crop (e.g., 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 (e.g., an orange, grapefruit, or lemon tree), a grass, a vegetable, a fruit, or an algae. The plant may be a nightshade plant; a Brassica plant; a Lactuca plant; a Spinacia plant; a Capsicum plant; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberries, blueberries, raspberries, blackberries, grapes, coffee, cocoa, etc.

[0136] The term "host cell" in this application includes any cell expressing the Cas13 protein described in this application, or transducing a nucleic acid molecule containing the Cas13 protein, or a CRISPR-Cas system, or a delivery system, including prokaryotic cells and eukaryotic cells.

[0137] CRISPR system

[0138] 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, and other aspects.

[0139] CRISPR is a DNA locus containing short repetitions of base sequences. Each repetition is followed by a short segment of "spacer DNA" from previous exposure to viruses. CRISPR is found in about 40% of sequenced eubacterial genomes and 90% of sequenced archaea. CRISPR is typically associated with Cas genes that encode proteins 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. The CRISPR spacer recognizes and silences these exogenous genetic elements (such as RNAi) in eukaryotic organisms.

[0140] CRISPR repeats range in size from 24 to 48 base pairs. They typically display some two-fold symmetry, meaning they form secondary structures such as hairpins, but not true palindromes. The repeats are separated by spacers of similar length. Some CRISPR spacer sequences exactly match sequences from plasmids and phages, while some spacers match prokaryotic genomes. New spacers can be rapidly added in response to phage infection.

[0141] The "guide RNA (gRNA)" is a sequence in the guide RNA that is complementary (partially complementary or fully complementary) and / or hybridizes with the target sequence in the target nucleic acid, thereby guiding the CRISPR-Cas complex (such as the CRISPR-Cas13 complex) to the target nucleic acid sequence and specifically binding to it.

[0142] Nuclease

[0143] In this application, "Cas nuclease" and "cas13 protein" can be used interchangeably. CRISPR-related (Cas) genes are generally 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 sequence structures have been used to define eight CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with additional gene modules encoding repeat-associated mysterious proteins (RAMPs). More than one CRISPR subtype may exist in a single genome. The sporadic distribution of CRISPR / Cas subtypes suggests that the system has undergone horizontal gene transfer during microbial evolution.

[0144] The exogenous DNA is apparently processed into small elements (about 30 base pairs in length) by proteins encoded by the Cas genes, which are then somehow inserted into the CRISPR locus near the leader sequence. RNA from the CRISPR locus is constitutively expressed and is processed by the Cas protein into small RNAs consisting of separate exogenous sequence elements with flanking repeats. RNA guides other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Evidence suggests functional diversity between CRISPR subtypes. Cse (Cas subtype Ecoli) protein (called CasA-E in Escherichia coli (E. coli)) forms a functional complex Cascade, which processes CRISPR RNA transcripts into spacer-repeat sequence units that retain Cascade. In other prokaryotes, Cas6 processes CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but does not require Cas1 and Cas2. The Cmr (Cas RAMP module) protein, found in Pyrococcus furiosus and some other prokaryotes, forms a functional complex with a small CRISPR RNA that recognizes and cleaves complementary target RNA. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

[0145] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present disclosure only and are not intended to limit the scope of the present disclosure. The experimental procedures in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

[0146] Unless otherwise specified, the materials and reagents used in the examples of this disclosure are commercially available products.

[0147] Example

[0148] Example 1: Screening of novel Cas13 proteins

[0149] It is generally believed in the art that the HEPN domain necessary for Cas13 function refers to the sequence of RxxxxH (R4xH). Therefore, in the process of screening potential Cas13, at least two R4xH domains are usually used as the first step screening criteria. However, the applicant found that RxxxxxH (R5xH) or RxxxxxxH (R6xH) can actually also function as the HEPN domain of Cas13. Therefore, the inventors used R4xH, R5xH and R6xH (hereinafter referred to as the extended HEPN domain) as inclusion criteria during the screening process, thereby discovering a class of cas13 proteins with new HEPN domains (R5xH and R6xH) that were not found by traditional screening methods. The inventors also found that the molecular weight of these cas13 proteins containing R5xH and R6xH type HEPN domains is much smaller than that of known cas13. This means that the R5xH and R6xH domains are likely to be the characteristic structures of a class of smaller molecular weight Cas13 proteins, thereby providing a method for screening more small cas13 proteins.

[0150] We first downloaded the sequences of all bacteria, archaeal genomes and metagenomes from NCBI and IMG as of July 2021, and used CRISPR array identification software (such as Pilercr) to identify the CRISPR array region; we took 6 proteins upstream and downstream of the CRISPR array region adjacent to the CRISPR array region for target domain analysis, and obtained 78 candidate proteins. The expanded HEPN domains and coordinates of the candidate proteins are shown in Table 2.

[0151] The HEPN domain of the candidate cas13 protein includes RxxxxH (R4xH) features, RxxxxxH (R5xH), and RxxxxxxH (R6xH) features, where x represents any amino acid. The conserved amino acid adjacent to R is preferably N, Q, H or D, such as R[NDQH]xxxH, R[NDQH]xxxxH, R[NDQH]xxxxxH and other combinations; wherein R4xH, R5xH and R6xH are preferably R[NDQH]xxxH, R[NDQH]xxxxH and R[NQDH]xxxxxH, respectively.

[0152] Example 2: Verification of RNA cleavage activity

[0153] The nucleic acid sequence, DR sequence and target spacer sequence of the candidate protein were synthesized and then introduced into the expression plasmid to construct the corresponding plasmid. The plasmid was transformed into DH-5a Escherichia coli competent cells for plasmid amplification and culture. After the plasmid was extracted, a transfection test of the human 293T (capable of expressing red light) cell line was performed. A negative control group and a positive control group were designed. The negative control contained only the expressed mCherry protein (denoted as FB132), and the positive control was the cas13d protein. Flow cytometric analysis and other tests were performed 48 hours after co-transfection of the plasmid to finally determine the RNA enzymatic activity of the candidate protein.

[0154] The results are shown in Figures 1 to 33. The results from DZ4, DZ28, DZ29, DZ30, DZ31, DZ32, DZ33, DZ35, DZ36, DZ37, DZ38, DZ39, DZ40, DZ44, DZ45, DZ46, DZ47, DZ50, DZ51, DZ52, DZ54, DZ55, DZ57, DZ62, DZ63, DZ65, DZ68, DZ86, DZ90, DZ91, DZ93, DZ96, DZ98 and the positive control protein Cas13d show that the Cas13 protein we screened can well knock down mCherry (red light) protein in transient mammalian cell lines, while the negative control group cannot be cut, showing red fluorescence highlight; indicating that our candidate protein can play the role of RNase in mammals, etc.

[0155] Example 3: Functional verification of knockdown of 293T endogenous genes

[0156] In order to further verify the ability of the candidate protein to cut endogenous genes, we further screened some proteins DZ4, DZ29, DZ32, DZ47, DZ51, DZ54, DZ68, DZ93, DZ98, etc. that had high RNase activity for cutting mCherry from Example 2, and performed experimental verification of knocking down endogenous genes (STAT3, EZH2). We randomly designed sgRNA for the two endogenous genes of 293T. The cutting results are shown in Figures 35-43. The results show that all proteins have cutting function, although there are also some differences between the experimental group and the control group. This may be related to the PFS of the protein. Previous studies have reported that Cas13 proteins, such as Cas13a, Cas13b, etc., have a strong preference (PFS) when targeting RNA sequences. The results from qPCR fully demonstrate the feasibility of our method for screening candidate guide RNA-guided CRISPR-Cas proteins with Rase activity.

[0157] We used a portion of the screened guide RNA-guided CRISPR-Cas proteins with potential RNase activity (including dz806, dz825, dz822, dz821, etc.) to directly perform knockdown experiments on the endogenous genes STAT3 and EZH2. The results are shown in Figures 44 to 48.

[0158] It was found that although the PFS of the proteins was unknown, some candidate proteins showed a certain effect of knocking down the endogenous gene STAT3, including DZ784, DZ787, DZ788, DZ791, DZ793, DZ794, DZ796, DZ797, DZ798, DZ800, DZ803, DZ805, DZ810, DZ813, DZ814, DZ816, DZ817, DZ821, and DZ824. Subsequently, DZ806, DZ810, DZ821, DZ822, and DZ825 were further subjected to knockdown experiments on the endogenous gene EZH2, and the results are shown in Figures 45 to 48. It was found that multiple repeated experiments showed that they had a certain knockdown effect on the endogenous genes EZH2 and STAT3.

[0159] Example 4: PFS function screening of candidate Cas13 proteins

[0160] The candidate proteins screened can be preferentially screened using techniques known to those skilled in the art, which may improve the cleavage efficiency of the screened enzymes.

[0161] To further explore the bias of candidate protein-targeting RNAs, we designed an assay to identify protein PFS. The assay involves first designing a library plasmid containing either a 5′-6N(NNNNNN)-spacer(target sequence)-resistance gene or a spacer(target sequence)-NNNNNN(6N)-3′-resistance gene (collectively referred to as the 6N library plasmid) before a resistance gene (e.g., ampicillin). A guide RNA plasmid targeting the target sequence is also designed. E. coli cells are transfected with the 6N library plasmid, along with the candidate protein plasmid and a guide RNA targeting the target region. A negative control is established, in which the candidate protein plasmid is co-transfected with a guide RNA plasmid targeting a region other than the target (i.e., non-target). All surviving E. coli cells are then extracted and sequenced using deep-seq. Using bioinformatics methods, differences in 5′ or 3′ sequence preferences between the experimental and control groups are analyzed to calculate the PFS of the corresponding proteins.

[0162] According to this method, we tested proteins numbered DZ796, DZ806, DZ821, DZ822, DZ824 and DZ825. As shown in Figure 49, their potential PFS were 5′-[C / G]-targetSeq-NNNNN[G]-3′ (Figure 49A), 5′-T[G / A][G / A]-targetSeq-TN[G / A]-3′ (Figure 49B), 5′-TNNN[C / G]-targetSeq-NNNNT-3′ (Figure 49C), 5′-TN[G / C / A]-targetSeq-[G / A][C / G][G / C]-3′ (Figure 49D), 5′-N[C / G][C / G][C / T]-targetSeq-NG[C / A]-3′ (Figure 49E) and 5′-C-tareget-G-3′ (Figure 49F).

[0163] We then used PFSs identified by proteins DZ825, DZ822, and DZ806 to knock down endogenous genes in a mammalian cell line (293T), as shown in Figure 50. Figure 50A shows the results of knockdown (KD) experiments using protein DZ825 for the endogenous genes STAT3 and EZH2 in the 293T cell line. For each gene, the first experimental group consisted of a randomly designed spacer with an unknown PFS, followed by three newly designed spacers based on the PFS motif. As can be seen in the figure, some of the newly designed sgRNAs still demonstrated good KD effects in the 293T cell line KD experiments. Figure 50B shows the results of experiments using protein DZ822 for the three endogenous genes STAT3, EGFR, and HRAS in the 293T cell line. For each gene, the first experimental group consisted of a randomly designed spacer with an unknown PFS, followed by newly designed spacers based on the PFS motif. As can be seen in the figure, some newly designed sgRNAs, such as KRAS, demonstrated good KD effects in KD experiments in 293T cell lines. Figure 50C shows the results of experiments targeting endogenous genes in 293T cells, designated DZ806, for their PFS-specific KD motifs. The selected endogenous genes included STAT3, EZH2, EGFR, HRAS, RAF1, NF2, SMARCA4, NFKB1, PPARG, KRAS, PTBP1, and NRAS. The first spacer in each KD gene experimental group was designed when the PFS was unknown, while subsequent groups contained newly designed spacers based on the PFS motif. As can be seen in the figure, some newly designed sgRNAs, such as NF2 and SMARCA4, demonstrated good KD effects in KD experiments in 293T cell lines.

[0164] Example 5: Verification of base editing function

[0165] By mutating the cleavage domain (expanded HEPN domain) of the candidate Cas13 protein, a candidate dCas13 protein that only binds to RNA but has no cleavage activity is obtained, and then the adar enzyme sequence is fused to construct a plasmid for the ABE single-base editing system. Then, the sgRNA for site-directed base mutation of a specific sequence, such as the transcript of the TP53 gene, is designed and the corresponding plasmid vector is constructed. Then, the human 293T cell line is co-transfected, and flow cytometry is performed 48 hours later to obtain the co-transfected cell line. RNA transcripts are then extracted and a library is constructed. Then deep seq sequencing is performed. After sequencing, the mutation of the TP53 gene transcript is analyzed by bioinformatics methods to obtain the single-base editing efficiency analysis of the corresponding ABE system. Thus, the optimal single-base editing system for the target region can be constructed by continuously optimizing sgRNA.

[0166] Example 6: Homology analysis of candidate Cas13 proteins and known Cas13 proteins

[0167] It is based on the principle that the higher the coverage of the unknown protein in the known protein and the greater the similarity ratio, the closer the homology of the unknown protein to the known protein. After screening the candidate proteins, we first download the relevant protein sequences of Cas13a, b, c, d, x (e), y (f), bt from the NCBI database and patent literature, and then merge them with our data to build a local blastp index file, and then align the candidate protein sequence to the local blastp index library for protein sequence alignment analysis. For the parts with a similarity (identity) between proteins less than 20% or that cannot be aligned to the local index library, we uniformly mark them as 20%; similarly, for coverage (coverage) less than 5% or that cannot be aligned to the local index library, it is marked as 1%. The vast majority of the new Cas13 proteins identified by the method of the present invention have extremely low similarity to the homology levels of the known Cas13 proteins of each family. The numbers are DZ28; DZ29; DZ30; DZ31; DZ32; DZ33; DZ35; DZ36; DZ37; DZ40; DZ44; DZ45; DZ46; 54; DZ55; DZ57; DZ63; DZ65; DZ68; DZ86; DZ91; DZ98; DZ784; DZ785; DZ786; DZ787; DZ788; DZ789; DZ793; DZ795; DZ The homologies of the following proteins are less than 20% with the currently known Cas13 types: 797; DZ798; DZ799; DZ801; DZ803; DZ804; DZ805; DZ806; DZ807; DZ809; DZ10; DZ812; DZ813; DZ814; DZ815; DZ816; DZ817; DZ819; DZ820; DZ821; DZ822; DZ825; DZ826; DZ827; DZ829; DZ831; DZ844, etc. The proteins labeled DZ4, DZ38, DZ843, DZ62, DZ93, DZ794, DZ796, DZ824, and DZ828 have a similarity of 20% to 50% with known Cas13 family proteins, while the remaining proteins have a similarity of 50% to 80% with known proteins.

[0168] As shown in Figure 51, further phylogenetic analysis revealed that our candidate guide RNA-guided CRISPR-Cas13 proteins with RNase activity have independent branches, potentially forming two relatively large new families of compact Cas13 proteins, tentatively designated as the Cas13 m1 family and the Cas13 m2 family. The Cas13 m1 family includes proteins such as DZ30 and DZ32, while the Cas13 m2 family includes proteins such as DZ47 and DZ29.

[0169] The DR sequences of candidate Cas13 proteins are shown in Table 1 below.

[0170] Table 1. DR sequences of candidate Cas13 proteins

[0171] Table 2 Sequence numbers and characteristics of candidate Cas13 proteins

[0172] See Table 3 below for plasmid construction primers for sgRNA that knocks down endogenous genes in the 293T cell line expressing the candidate Cas13 protein.

[0173] Table 3. sgRNA primers for targeted knockdown of endogenous genes

Claims

1. Cas13 protein, characterized in that The protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1 to 78, or comprises a protein having at least 70%, 80%, 85%, 90%, or 95% homology to a sequence as set forth in any one of SEQ ID NOs: 1 to 78; Preferably, the protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-34, 37, 38, 41, 42, 43, 45, 46, 47, 49, 52, 54, 55, 58, 61, 62, 64, 65, or 68-71, or a protein having at least 70%, 80%, 85%, 90%, or 95% homology to a sequence as set forth in any one of SEQ ID NOs: 1-34, 37, 38, 41, 42, 43, 45, 46, 47, 49, 52, 54, 55, 58, 61, 62, 64, 65, or 68-71; More preferably, the protein comprises an amino acid sequence as described in any one of SEQ ID NOs: 1, 3, 6, 17, 19, 21, 27, 31, 33, 55, 68, 69, 71, or a protein having at least 80%, 85%, 90%, or 95% homology to the sequence described in any one of SEQ ID NOs: 1, 3, 6, 17, 19, 21, 27, 31, 33, 55, 68, 69, 71.

2. Cas13 protein, characterized in that The HEPN domain of the protein comprises at least one RXXXXXH and / or RXXXXXXH structure, wherein X is an optional amino acid; preferably, the HEPN domain comprises 1-9 RXXXXXH and / or RXXXXXXH structures; more preferably, the HEPN domain comprises 2, 3, 4, or 5 HEPN domains; In a preferred embodiment, the amino acid X adjacent to R is preferably N, Q, H or D.

3. The Cas13 protein according to claim 1, wherein The protein having at least 80%, 85%, 90%, or 95% homology is a protein having conservative amino acid additions, deletions, or substitutions of one or more residues; Preferably, the protein has conservative amino acid additions, deletions, or substitutions of 1-10 residues.

4. The Cas13 protein according to claim 1 or 2, characterized in that Its RNA cleavage activity is retained.

5. The Cas13 protein according to claim 1 or 2, characterized in that The HEPN domain of the Cas13 protein has a mutation of at least one nucleotide.

6. The Cas13 protein according to any one of claims 1 to 5, characterized in that The Cas13 protein is fused to one or more heterologous functional domains, wherein the fusion is at the N-terminus, C-terminus or inside the Cas13 protein; Preferably, the heterologous functional domain has the following activities: deaminase 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.

7. The cas13 protein according to claim 1 or 2, characterized in that The HEPN domain of the protein is identical to the HEPN domain of any one of SEQ ID NOs: 1 to 78.

8. The cas13 protein according to claim 1 or 2, characterized in that At least one of the HEPN domains of the protein comprises a RXXXXH, RXXXXXH, and / or RXXXXXXH structure, wherein X is an optional amino acid, Preferably, the amino acid adjacent to R is N, Q, H or D.

9. The Cas13 protein according to claim 8, characterized in that The HEPN domain of the protein comprises at least one RXXXXXH and / or RXXXXXXH structure; Preferably, the HEPN domain comprises 1-9 RXXXXXH and / or RXXXXXXH structures; More preferably, the HEPN domain comprises 2, 3, 4, or 5 HEPN domains.

10. The Cas13 protein according to claim 2, characterized in that The HEPN structure of the cas13 protein contains the HEPN structure of the protein described in Table 2.

11. A nucleic acid molecule comprising a nucleotide sequence encoding the Cas13 protein according to any one of claims 1 to 10.

12. The nucleic acid molecule according to claim 11, characterized in that The nucleic acid molecule is a nucleic acid that has been codon-optimized for a specific host cell; The host cell is preferably a prokaryotic cell or a eukaryotic cell, more preferably a eukaryotic cell, and even more preferably a cell of human origin.

13. The nucleic acid molecule according to claim 11 or 12, characterized in that The nucleic acid molecule comprises a promoter operably linked to a nucleotide sequence encoding Cas13, wherein the promoter is a constitutive promoter, an inducible promoter, a tissue-specific promoter, a chimeric promoter, or a development-specific promoter.

14. CRISPR-Cas system, characterized in that The system comprises: (1) the Cas13 protein or its derivative or functional fragment according to any one of claims 1 to 10, or the nucleic acid molecule according to any one of claims 11 to 13; (2) a gRNA targeting a target nucleic acid; Preferably, the gRNA sequence comprises a direct repeat (DR) sequence and a spacer sequence with the target nucleic acid; More preferably, the DR sequence comprises a nucleic acid as shown in any one of SEQ ID NOs: 79-234 or a nucleic acid comprising a derivative sequence of a sequence as shown in any one of SEQ ID NOs: 79-234; The derived sequence is: (i) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) nucleotide additions, deletions, or substitutions compared to any of the sequences shown in Table 1; (ii) having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 97% sequence identity to any one of the sequences shown in Table 1; (iii) hybridizes under stringent conditions to any one of the sequences shown in Table 1, or to any one of (i) and (ii); or (iv) is the complement of any one of (i) to (iii), provided that the derivative is not any of the sequences shown in Table 1, and the derivative encodes an RNA, or is itself an RNA, and the RNA retains substantially the same secondary structure as any RNA encoded by SEQ ID NOs: 79-234.

15. The CRISPR-Cas system according to claim 14, wherein The spacer sequence is 15-60 nucleotides, preferably 25-50 nucleotides, more preferably 30 nucleotides.

16. The CRISPR-Cas system according to claim 15, wherein the target nucleic acid acted by the system is a target RNA; Preferably, 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.

17. A carrier, characterized in that The vector comprises the nucleic acid molecule of any one of claims 11-13, and is capable of expressing the cas13 protein of any one of claims 1-10 or the nucleic acid molecule of any one of claims 11-13; Preferably, the vector is selected from viral vectors, lipid nanoparticles (LNP), liposomes, cationic polymers (such as PEI), nanoparticles, exosomes, liposomes, microvesicles, and gene guns; More preferably, the vector is selected from viral vectors, More preferably, the viral vector is selected from adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes simplex virus, and oncolytic virus.

18. A delivery system comprising (1) the vector of claim 17, or the nucleic acid molecule of any one of claims 11 to 13, and (2) a delivery vehicle.

19. The delivery system according to claim 18, wherein the delivery vehicle is a nanoparticle, a liposome, an exosome, a microvesicle or a gene gun.

20. A cell comprising the Cas13 protein of any one of claims 1 to 10, the nucleic acid molecule of any one of claims 11 to 13, the vector of claim 17, the delivery system of claim 18 or 19, or the CRISPR-Cas system of any one of claims 14-16.

21. The cell according to claim 20, which is a prokaryotic cell or a eukaryotic cell, preferably a human cell.

22. A method for degrading or cutting a target RNA in a target cell, or modifying the sequence of a target RNA in a target cell, comprising using a Cas13 protein according to any one of claims 1 to 10, a nucleic acid molecule according to any one of claims 11 to 13, a vector according to claim 17, a delivery system according to claim 18 or 19, or a CRISPR-Cas system according to any one of claims 14 to 16.

23. The method according to claim 22, wherein the target cell is a prokaryotic cell or a eukaryotic cell, preferably a human cell.

24. The method of claim 22, wherein the target cell is an ex vivo cell, an in vitro cell, or an in vivo cell.

25. A method for screening cas13 protein, characterized in that The screening protein HEPN domain comprises at least one Cas13 protein of RXXXXXH and / or RXXXXXXH structure, where X is an optional amino acid; preferably, the HEPN domain comprises 1-9 RXXXXXH and / or RXXXXXXH structures; more preferably, the HEPN domain comprises 2, 3, 4, or 5 HEPN domains.

26. The method according to claim 25, characterized in that The HEPN structure of the screened cas13 protein contains the HEPN structure of the protein described in Table 2, or contains a HEPN structure having at least 80%, 85%, 90%, or 95% similarity to the HEPN structure of the protein described in Table 2.

27. The method according to claim 25 or 26, characterized in that The screening method comprises: 1) Download bacterial genome and / or metagenome sequences and identify CRISPR array regions; 2) Analyze the proteins upstream and downstream of the CRISPR array region, and select proteins whose HEPN structure contains at least one RXXXXXH and / or RXXXXXXH structure as candidate Cas13 proteins; Preferably, the HEPN structure further contains at least one RXXXXH structure.

28. The method according to claim 27, characterized in that Six proteins upstream and downstream of the CRISPR array region adjacent to the CRISPR array region were analyzed.

29. The method according to any one of claims 25 to 27, characterized in that: The bias of screening candidate proteins.

30. The method according to claims 25-27, characterized in that The amino acid X adjacent to R is preferably N, Q, H or D.

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