Novel crispr / cas13 system and use thereof

Abstract

The present specification discloses a novel CRISPR / Cas13 system. The novel CRISPR / Cas13 system has both target-specific nucleic acid cleavage activity and ancillary cleavage activity. The novel / Cas13 system has both target-specific nucleic acid cleavage activity and ancillary cleavage activity. The inventors of the present specification have specified a novel CRISPR / Cas13 system from a known database, and have demonstrated the function thereof through experimentation, thereby completing the invention.

Description

Novel CRISPR / CAS13 system and its uses

[0001] This specification covers the technology of the CRISPR / Cas13 system, which belongs to the Type VI CRISPR / Cas system of Class 2.

[0002]

[0003] CRISPR / Cas13 system

[0004] Overview of the CRISPR / Cas13 System

[0005] This specification discloses a novel CRISPR / Cas13 system. The CRISPR / Cas13 system is a type of CRISPR / Cas system. The CRISPR / Cas13 system comprises an effector protein (Cas13 protein) and a guide RNA. The CRISPR / Cas13 system recognizes and cleaves a single-stranded RNA having a sequence complementary to the spacer sequence of the guide RNA and a protospacer flanking sequence (PFS). Some CRISPR / Cas13 systems, after cleaving the single-stranded RNA, activate a secondary cleavage activity to randomly cleave surrounding single-stranded RNA.

[0006] Below, the structure and function of the CRISPR / Cas13 system are described in more detail.

[0007] CRISPR / Cas13 system classification

[0008] The CRISPR / Cas13 system belongs to the Type VI CRISPR / Cas system of Class 2. The CRISPR / Cas13 system is further divided into several subtypes, including Cas13a, Cas13b, Cas13c, Cas13d, Cas13x, and Cas13y. These subtypes are also referred to as IV-a, IV-b, IV-c, IV-d, IV-x, and IV-y, respectively. The CRISPR / Cas13 systems belonging to each subtype show slight differences in the size of the Cas13 protein, the structure of the guide RNA, etc.

[0009] CRISPR / Cas13 system configuration and function

[0010] The CRISPR / Cas13 system comprises the Cas13 protein and its corresponding guide RNA. The Cas13 protein and guide RNA function as a complex. The protein-RNA complex can bind to a target nucleic acid (by recognizing the target nucleic acid) and cleave it. Furthermore, some protein-RNA complexes have secondary cleavage activity, cleaving any surrounding nucleic acids after cleaving the target nucleic acid. The Cas13 protein-guide RNA complex is also referred to as a ribonucleoprotein (RNP). The Cas13 protein recognizes the Protospacer Flanking Sequence (PFS) and can cleave nucleic acids. The guide RNA can guide the Cas13 protein-guide RNA complex to the target nucleic acid by binding to the target nucleic acid containing the target sequence.

[0011] The above CRISPR / Cas13 system is known to function by targeting single-stranded RNA as a target nucleic acid.

[0012] Structure of the Cas13 protein

[0013] The Cas13 protein can be broadly divided into the REC (RECognition) lobe and the NUC (NUClease) lobe. The NUC lobe contains two HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains. The REC lobe is an important part of the Cas13 protein that recognizes the guide RNA and forms a complex. The NUC lobe cleaves nucleic acids. The structure of the Cas13 protein has already been reported by several researchers. For example, it is described in detail in Xue et. al (Engineering CRISPR / Cas13 System against RNA Viruses: From Diagnostics to Therapeutics. Bioengineering (Basel), 2022 Jul; 9(7): 291).

[0014] Structure of guide RNA

[0015] Unlike the guide RNA of the CRISPR / Cas9 system, the guide RNA of the CRISPR / Cas13 system consists of a single RNA molecule, called crRNA. This guide RNA contains direct repeats and a spacer.

[0016] The direct repeats and spacers have different linking structures depending on the subtype of the CRISPR / Cas13 system. For example, the guide RNAs of the Cas13a and Cas13d subtypes have direct repeats and spacers sequentially linked from the 5' end to the 3' end. In contrast, the guide RNA of the Cas13b subtype has spacers and direct repeats sequentially linked from the 5' end to the 3' end.

[0017] Target-specific nucleic acid cleavage activity and PFS (Protospacer Flanking Sequence) of the CRISPR / Cas13 system

[0018] The CRISPR / Cas13 system can target-specifically cleave nucleic acids. Two conditions are necessary for target-specific nucleic acid cleavage activity to occur. First, the nucleic acid must contain a base sequence of a certain length that the Cas13 protein can recognize. Second, a sequence capable of complementarily binding to a spacer contained in the guide RNA must be present around the base sequence of a certain length. 1) The Cas13 protein recognizes the base sequence of a certain length, and 2) the spacer complementarily binds to the region surrounding the base sequence of a certain length, and the CRISPR / Cas13 system cleaves the nucleic acid. The base sequence of a certain length recognized by the Cas13 protein is called the protospacer flanking sequence (PFS). This corresponds to the protospacer adjacent motif (PAM) of CRISPR / Cas9.

[0019] The PFS is determined by the type of Cas13 protein. Knowing the PFS of the Cas13 protein can be used to design a CRISPR / Cas13 system that targets nucleic acids surrounding the PFS.

[0020] Among Cas13 subtypes, the CRISPR / Cas13d system is known to not require a PFS. The CRISPR / Cas13d system can cleave a target nucleic acid without any other conditions (regardless of the presence of a PFS sequence), as long as the guide RNA spacer can bind complementarily.

[0021] Collateral cleavage activity of the CRISPR / Cas13 system

[0022] Some CRISPR / Cas13 systems possess a collateral cleavage function in addition to their target-specific nucleic acid cleavage activity. When a CRISPR / Cas13 system recognizes a target nucleic acid, the collateral cleavage function is activated. A CRISPR / Cas13 system with this collateral cleavage function randomly cleaves surrounding single-stranded nucleic acids. By applying this collateral cleavage function, the CRISPR / Cas13 system can be used to detect the presence of a target nucleic acid in a sample.

[0023]

[0024] This specification discloses a novel, previously unreported CRISPR / Cas13 system. Each component of the novel CRISPR / Cas13 system is disclosed. The function of the novel CRISPR / Cas13 system is elucidated. The potential applications of the novel CRISPR / Cas13 system are also elucidated.

[0025]

[0026] The inventors of this specification have identified components of a novel CRISPR / Cas13 system from microbial genetic sequence information disclosed in a public database. Specifically, they have identified a guide RNA comprising the Cas13 protein of SEQ ID NO: 1 and a direct repeat sequence of SEQ ID NO: 2. Based on the identified information, the inventors have constructed a novel CRISPR / Cas13 system. Furthermore, they have experimentally confirmed that the novel CRISPR / Cas13 system can cleave nucleic acids target-specifically. Furthermore, they have confirmed that the novel CRISPR / Cas13 system belongs to the Cas13d subtype and can operate without protospacer flanking sequence (PFS) limitations. Furthermore, they have experimentally confirmed that the novel CRISPR / Cas13 system has ancillary cleavage functions.

[0027] The inventors discovered that the novel CRISPR / Cas13 system can be used for off-target nucleic acid cleavage or target nucleic acid detection. The inventors specifically proposed and demonstrated methods for cleaving off-target nucleic acids and detecting target nucleic acids.

[0028]

[0029] The novel CRISPR / Cas13 system described in this specification can target-specifically cleave single-stranded nucleic acids. Because the novel CRISPR / Cas13 system possesses secondary cleavage activity, it can randomly cleave surrounding nucleic acids after recognizing the target nucleic acid. Applying this characteristic, the novel CRISPR / Cas13 system can be used for target nucleic acid detection.

[0030]

[0031] Figure 1 shows the results of measuring fluorescence signal intensity over time according to Experimental Example 2.1. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents reaction time in minutes. Example 2.1 represents the results for Example 2.1 according to Table 3, and Control represents the results when only Cas13 protein was added.

[0032] Figure 2 shows the results of measuring fluorescence signal intensity over time according to Experimental Example 2.1. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents reaction time in minutes. Example 2.2 shows the results for Example 2.2 according to Table 3, and Control shows the results when only Cas13 protein was added.

[0033] Figure 3 shows the results of measuring fluorescence signal intensity over time according to Experimental Example 2.1. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents reaction time in minutes. Example 2.3 shows the results for Example 2.3 according to Table 3, and Control shows the results when only Cas13 protein was added.

[0034] Figure 4 shows the results of measuring fluorescence signal intensity over time according to Experimental Example 2.1. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents reaction time in minutes. Example 2.4 shows the results for Example 2.4 according to Table 3, and Control shows the results when only Cas13 protein was added.

[0035] Figure 5 shows the results of measuring fluorescence signal intensity over time according to Experimental Example 2.1. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents reaction time in minutes. Example 2.5 shows the results for Example 2.5 according to Table 3, and Control shows the results when only Cas13 protein was added.

[0036] Figure 6 shows the results of measuring fluorescence signal intensity over time according to Experimental Example 2.1. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents reaction time in minutes. Example 2.6 shows the results for Example 2.6 according to Table 3, and Control shows the results when only Cas13 protein was added.

[0037] Figure 7 shows the results of measuring fluorescence signal intensity by concentration according to Experimental Example 2.2. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents reaction time in minutes. Examples 2.2.1 to 2.2.5 each represent examples of the corresponding labels disclosed in Table 4, and the Control represents the results obtained by adding only Cas13 protein.

[0038] Figure 8 shows the results of measuring fluorescence signal intensity by concentration according to Experimental Example 2.2. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents reaction time in minutes. Examples 2.5.1 to 2.5.5 each represent examples of the corresponding labels disclosed in Table 4, and the Control represents the results obtained by adding only Cas13 protein.

[0039] Figure 9 shows the results of measuring fluorescence signal intensity over time according to Experimental Example 3.1. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents reaction time in minutes. Example 3.1 represents Example 3.1 according to Table 4, and Control represents the results obtained by adding only Cas13 protein.

[0040] Figure 10 shows the results of measuring fluorescence signal intensity over time according to Experimental Example 3.1. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents reaction time in minutes. Example 3.2 represents Example 3.2 according to Table 4, and Control represents the results obtained by adding only Cas13 protein.

[0041] Figure 11 shows the results of measuring fluorescence signal intensity over time according to Experimental Example 3.1. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents reaction time in minutes. Example 3.3 represents Example 3.3 according to Table 4, and Control represents the results obtained by adding only Cas13 protein.

[0042] Figure 12 shows the results of measuring fluorescence signal intensity by concentration according to Experimental Example 3.2. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents the reaction time in minutes. Examples 3.3.1 to 3.3.5 each represent examples of the corresponding labels disclosed in Table 6, and the Control represents the results obtained by adding only Cas13 protein.

[0043] Figure 13 shows the results of measuring fluorescence signal intensity by concentration according to Experimental Example 3.2. The vertical axis of the chart represents relative fluorescence intensity, and the horizontal axis represents the reaction time in minutes. Examples 3.4.1 to 3.4.5 each represent examples of the corresponding labels disclosed in Table 6, and the Control represents the results obtained by adding only Cas13 protein.

[0044]

[0045] Hereinafter, the best mode for carrying out the invention is exemplified. This includes some, but not all, implementations of the invention disclosed herein. The embodiments described in this paragraph are merely exemplary, and the implementations described in this paragraph should not be construed as the "best mode for carrying out the invention." Those skilled in the art will likely envision numerous variations and more desirable implementations of the examples described in this paragraph, and such variations should also be considered to be included within the best mode for carrying out the invention.

[0046] This specification provides a novel CRISPR / Cas13 composition comprising: a Cas13 protein comprising a nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid encoding said Cas13 protein; and a programmable guide RNA, or a nucleic acid encoding said programmable guide RNA, wherein said programmable guide RNA has a structure in which a direct repeat portion and a guide domain are linked in a 5'-to-3'-end direction, said direct repeat portion interacting with said Cas13 protein to form a complex, said direct repeat portion comprising an RNA sequence of SEQ ID NO: 2, and said guide domain can complementarily bind to a predetermined target nucleic acid.

[0047] In one embodiment, the nucleic acid encoding the above Cas13 protein may be DNA or mRNA, and the nucleic acid encoding the above programmable guide RNA may be DNA.

[0048] In one embodiment, the novel CRISPR / Cas13 composition comprises the Cas13 protein and the programmable guide RNA, wherein the Cas13 protein and the programmable guide RNA can form a protein-RNA complex.

[0049] In one embodiment, the novel CRISPR / Cas13 composition may comprise a nucleic acid encoding the Cas13 protein and a nucleic acid encoding the programmable guide RNA.

[0050] In one embodiment, the nucleic acid encoding the above Cas13 protein and the nucleic acid encoding the above programmable guide RNA can be contained in a single molecule of a vector.

[0051] In one embodiment, the nucleic acid encoding the above Cas13 protein and the nucleic acid encoding the above programmable guide RNA may each be contained in two or more vector molecules.

[0052] In one embodiment, the vector may be a viral vector, a non-viral vector, or a suitable combination thereof.

[0053] This specification provides a method for cleaving a non-target RNA using a novel CRISPR / Cas13 system comprising: mixing the novel CRISPR / Cas13 composition with a sample, wherein the sample comprises a target RNA and a non-target RNA, and a guide domain of a programmable guide RNA of the novel CRISPR / Cas13 composition is capable of complementarily binding to the target RNA.

[0054] This specification provides a method for detecting a target RNA using a novel CRISPR / Cas13 system, comprising: (a) mixing the novel CRISPR / Cas13 composition, a sample, and a detection reagent, wherein a guide domain of a programmable guide RNA of the novel CRISPR / Cas13 composition can complementarily bind to the target RNA, the detection reagent comprises a reporter, the reporter comprises a probe nucleic acid, and when the probe nucleic acid is cleaved, the reporter generates a detectable signal, wherein when the sample comprises the target RNA, a protein-RNA complex of the novel CRISPR / Cas13 composition complementarily binds to the target RNA, and a secondary cleavage function of the protein-RNA complex is activated, thereby cleaving the probe nucleic acid; and (b) measuring the detectable signal in the mixture, wherein information about the target RNA can be obtained by measuring the detectable signal.

[0055] In one embodiment, the above process (b) may be a process of measuring whether a detectable signal occurs; the intensity of the occurring signal; the change in signal intensity over time; or any combination of the above.

[0056] In one embodiment, the information about the above target RNA may be whether the above target RNA is present, how much of the above target RNA is contained in the sample, or all of the above.

[0057] In one embodiment, the detectable signal may be a fluorescent signal.

[0058] This specification provides a method for providing COVID-19 diagnostic information, comprising: (a) mixing a novel CRISPR / Cas13 complex, a sample, and a detection reagent, wherein the sample is a sample collected from a patient suspected of being infected with COVID-19, and wherein the novel CRISPR / Cas13 complex comprises: a Cas13 protein of SEQ ID NO: 1; and a guide RNA, wherein the guide RNA has a structure in which a direct repeat of SEQ ID NO: 2 is sequentially linked to a guide domain from a 5' end to a 3' end, and the guide domain comprises an RNA sequence selected from among SEQ ID NOs: 37 to 51, and SEQ ID NOs: 52 to 56, wherein the detection reagent comprises a reporter, the reporter comprises a probe nucleic acid, and when the probe nucleic acid is cleaved, the reporter generates a fluorescent signal; (b) measuring a fluorescent signal in the mixture; And, (c) a process of analyzing the above fluorescence signal to obtain information on whether the patient who provided the sample was infected with COVID-19.

[0059] In one embodiment, the above process (b) may be a process of measuring whether a fluorescence signal is generated; the intensity of the generated fluorescence signal; a change in the fluorescence signal over time; or any combination thereof.

[0060]

[0061] Hereinafter, the present invention will be described in more detail through specific implementations and examples with reference to the attached drawings. It should be noted that the attached drawings include some, but not all, implementations of the invention. The invention disclosed by this specification may be implemented in various ways and is not limited to the specific implementations described herein. These implementations should be considered as provided to satisfy the legal requirements applicable to this specification. Those skilled in the art will be able to think of many modifications and other implementations of the invention disclosed herein. Therefore, the invention disclosed herein is not limited to the specific implementations described herein, and it should be understood that modifications and other implementations thereof are also included within the scope of the claims.

[0062]

[0063] Definition of terms

[0064] approximately

[0065] The term "about" as used herein means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or 0% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0066] Amino acid sequence notation

[0067] Unless otherwise stated, when describing amino acid sequences in this specification, amino acid single-letter notation or three-letter notation is used, and is written in the N-terminal to C-terminal direction. For example, when written as VSKN, it means a peptide in which valine, serine, lysine, and asparagine are sequentially connected from the N-terminal to the C-terminal. Another example, when written as Thr-Leu-Lys, it means a peptide in which threonine, leucine, and lysine are sequentially connected from the N-terminal to the C-terminal. In the case of amino acids that cannot be expressed in the single-letter notation, other letters are used and additional explanations are provided.

[0068] The notation for each amino acid is as follows: Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine ​​(Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys, K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Ty, Y); and Valine (Val, V).

[0069] Nucleic acid sequence notation

[0070] The symbols A, T, C, G, and U used herein are to be interpreted as meanings understood by a person skilled in the art. They may be appropriately interpreted as bases, nucleosides, or nucleotides in DNA or RNA, depending on the context and technology. For example, when referring to a base, they may be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) themselves, respectively; when referring to a nucleoside, they may be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G), or uridine (U), respectively; and when referring to a nucleotide in a sequence, they should be interpreted to mean a nucleotide containing each of the above nucleosides.

[0071]

[0072] Discovering a new CRISPR / Cas13 system from the public database

[0073] The inventors of this specification discovered a novel CRISPR / Cas13 system from a publicly available database. They first identified the Cas13 protein and guide RNA from microbial genetic sequence information in the publicly available database. They then synthesized the identified Cas13 protein and guide RNA, and prepared a CRISPR / Cas13 complex. The inventors confirmed that the prepared CRISPR / Cas13 complex exhibited target-specific nucleic acid cleavage activity and ancillary cleavage function, thereby completing the invention.

[0074] Specifically, the inventors utilized genetic information published by the National Center for Biotechnology Information (NCBI). They decoded the metagenome sequencing data of bovine stomach published by NCBI to obtain genomic information on each microorganism contained in bovine stomach juice. Based on the sequence information of the known Cas13 protein, they searched for genes encoding the CRISPR / Cas13 system among the genes of microorganisms contained in the camel stomach. From the above genes, they identified the amino acid sequence of the Cas13 protein and the guide RNA sequence. Among the candidates for the CRISPR / Cas13 system obtained in this way, the inventors selected candidates with particularly excellent collateral cleavage function.

[0075]

[0076] Novel CRISPR / Cas13 system

[0077] Overview of the Novel CRISPR / Cas13 System

[0078] This specification discloses a novel CRISPR / Cas13 system. The novel CRISPR / Cas13 system was discovered from a known database. The novel CRISPR / Cas13 system can cleave a target nucleic acid. After recognizing the target nucleic acid, the novel CRISPR / Cas13 system can also cleave any surrounding nucleic acid. In other words, the novel CRISPR / Cas13 system has both target-specific nucleic acid cleavage activity and secondary cleavage activity. The novel CRISPR / Cas13 system belongs to the Cas13d subtype. The novel CRISPR / Cas13 system can recognize and cleave a target nucleic acid without PFS limitation. The inventors of this specification identified the novel CRISPR / Cas13 system from a known database and experimentally verified its function, thereby completing the invention.

[0079] Novel Cas13 protein - amino acid sequence

[0080] The novel CRISPR / Cas13 system comprises a novel Cas13 protein. This novel Cas13 protein is an ortholog of the Cas13 protein. The novel Cas13 protein was identified from a known database, but its function or use has not been previously elucidated. The novel Cas13 protein can form a complex with a programmable guide RNA, as described below. The novel Cas13 protein comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence similar thereto.

[0081] Novel Cas13 protein - PFS sequence

[0082] The above Cas13 protein can recognize and exhibit the above-described target-specific nucleic acid cleavage function and / or ancillary cleavage function when 1) the target nucleic acid contains a specific sequence, and 2) a sequence adjacent to the specific sequence can complementarily bind to a guide RNA. In this case, the specific sequence is called a protospacer flanking sequence (PFS). The PFS is a sequence adjacent to the 5'-terminal side, the 3'-terminal side, or both ends of a sequence (target sequence) that can complementarily bind to a guide RNA.

[0083] Novel Cas13 protein - PFS sequence, for the Cas13d subtype

[0084] CRISPR / Cas13 systems belonging to the Cas13d subtype are known to not require a phase-dependent synthetase (PFS). CRISPR / Cas13d systems function as long as the target sequence can complementarily bind to the guide RNA. The novel CRISPR / Cas13 system disclosed in this specification experimentally confirms that it belongs to the Cas13d subtype. Therefore, the novel CRISPR / Cas13 system can recognize target nucleic acids and function solely with programmable guide RNA, without the limitations of a phase-dependent synthetase (PFS).

[0085] Programmable Guide RNA - Guide RNA Structure

[0086] The novel CRISPR / Cas13 system comprises a programmable guide RNA. The programmable guide RNA comprises direct repeats and a guide domain. The direct repeats and the guide domain are sequentially linked from the 5'-end to the 3'-end. In other words, the programmable guide RNA comprises the following structure:

[0087] 5' - (direct repeat) - (guide domain) - 3'

[0088] Here, additional domains may be included between the direct repeat portion and the guide domain as needed.

[0089] Each domain is described in more detail below.

[0090] Programmable guide RNA - direct repeats

[0091] The direct repeat region forms a stem-loop structure. The direct repeat region allows the programmable guide RNA to interact with the novel Cas13 protein to form a complex. The nucleic acid sequence of the direct repeat region can vary depending on the Cas13 protein.

[0092] Programmable guide RNA - guide domain

[0093] The above guide domain corresponds to the guide RNA spacer described in the CRISPR / Cas13 system. The above guide domain is artificially designed based on a predetermined target sequence. The design of the above guide domain determines which nucleic acid the CRISPR / Cas13 system will target.

[0094] Function of the novel CRISPR / Cas13 system - Target RNA cleavage activity

[0095] The novel CRISPR / Cas13 system described above can cleave target RNA. When the novel CRISPR / Cas13 complex binds to the target RNA, it can be cleaved. A programmable guide RNA for the novel CRISPR / Cas13 system is designed based on the target RNA. The programmable guide RNA is designed to complementarily bind to the target RNA.

[0096] Functionality of the Novel CRISPR / Cas13 System - Collateral Cutting Activity

[0097] The novel CRISPR / Cas13 system possesses collateral cleavage activity. When the novel CRISPR / Cas13 system binds to a target RNA, the collateral cleavage function is activated. The novel CRISPR / Cas13 system with the collateral cleavage function activated randomly cleaves surrounding single-stranded RNA. The nucleic acid sequence of the single-stranded RNA cleaved by the collateral cleavage activity is unrelated to the sequence of the target RNA. Any nucleic acid sequence is cleaved by the novel CRISPR / Cas13 system with the collateral cleavage function activated.

[0098] Novel CRISPR / Cas13 complex

[0099] The novel CRISPR / Cas13 system may refer to a complex of the Cas13 protein and the programmable guide RNA. The complex may be referred to as a protein-RNA complex or a ribonucleoprotein (RNP).

[0100] Expression vectors for each component of the novel CRISPR / Cas13 system

[0101] The novel CRISPR / Cas13 system may refer to a vector capable of expressing the Cas13 protein and the programmable guide RNA. The expression vector is not limited in its composition or form as long as it can express each component of the novel CRISPR / Cas13 system. The expression vector comprises a nucleic acid encoding the Cas13 protein and a nucleic acid encoding the programmable guide RNA, and may include other components such as a promoter. The expression vector may be DNA, mRNA, or a suitable combination thereof.

[0102] Novel CRISPR / Cas13 compositions

[0103] The above novel CRISPR / Cas13 system may refer to a novel CRISPR / Cas13 composition.

[0104] The novel CRISPR / Cas13 composition comprises: the novel Cas13 protein or a nucleic acid encoding the novel Cas13 protein; and the programmable guide RNA or a nucleic acid encoding the programmable guide RNA.

[0105]

[0106] Uses of the new CRISPR / Cas13 system

[0107] The novel CRISPR / Cas13 system can bind to a predetermined target RNA based on the design of a programmable guide RNA (target RNA recognition function). The novel CRISPR / Cas13 system can cleave the predetermined target RNA (target-specific nucleic acid cleavage activity). After cleaving the predetermined target RNA, the novel CRISPR / Cas13 system can randomly cleave surrounding single-stranded RNAs regardless of sequence (collateral cleavage activity). By utilizing these properties, the CRISPR / Cas13 system can be used to cleave non-target RNAs having arbitrary nucleic acid sequences. Furthermore, the CRISPR / Cas13 system can be used to detect the presence of target RNA in a sample. These applications are described in more detail below.

[0108]

[0109] Non-target nucleic acid cleavage method using a novel CRISPR / Cas13 system

[0110] Overview of non-target nucleic acid cleavage methods

[0111] This specification discloses a method for non-target nucleic acid cleavage using a novel CRISPR / Cas13 system. This non-target nucleic acid cleavage method utilizes the property of the novel CRISPR / Cas13 system that, when it recognizes a target nucleic acid, a secondary cleavage function is activated to cleave any nucleic acid. This non-target nucleic acid cleavage method comprises a step of contacting a target nucleic acid and a non-target nucleic acid with the novel CRISPR / Cas13 system. The novel CRISPR / Cas13 system is described in the "Novel CRISPR / Cas13 System" section. The guide domain of the guide RNA of the CRISPR / Cas13 system is designed to target the target nucleic acid.

[0112] Below, the above method is explained in more detail.

[0113] Novel CRISPR / Cas13 system

[0114] To cleave the above non-target nucleic acid, the novel CRISPR / Cas13 system provided herein is used. The novel CRISPR / Cas13 system is as described in "Novel CRISPR / Cas13 System." The form in which the CRISPR / Cas13 system is used may vary depending on the purpose. For example, a novel CRISPR / Cas13 complex; an expression vector for each component of the novel CRISPR / Cas13 system; a novel CRISPR / Cas13 composition; or an appropriate combination thereof may be used.

[0115] Target nucleic acid

[0116] In the above non-target nucleic acid cleavage method, the target nucleic acid refers to a nucleic acid that the novel CRISPR / Cas13 system can recognize. Specifically, the target nucleic acid can hybridize with the guide domain of the programmable guide RNA of the novel CRISPR / Cas13 system. In other words, the target nucleic acid comprises a nucleic acid sequence complementary to the guide domain.

[0117] non-target nucleic acids

[0118] The above non-target nucleic acid is a single-stranded nucleic acid that can be cleaved by the Cas13 protein of the novel CRISPR / Cas13 system. Unlike the above target nucleic acid, the above non-target nucleic acid has no restrictions on its sequence or length.

[0119] Order of contacting target nucleic acid and non-target nucleic acid

[0120] The above off-target nucleic acid cleavage method utilizes the ancillary cleavage function of the novel CRISPR / Cas13 system. This ancillary cleavage function is activated when the novel CRISPR / Cas13 system recognizes the target nucleic acid. Therefore, the above off-target nucleic acid cleavage method is preferably performed in such a way that the novel CRISPR / Cas13 system first contacts the target nucleic acid, followed by the non-target nucleic acid. However, even if the non-target nucleic acid contacts first, as long as it is sufficiently adjacent to the novel CRISPR / Cas13 system, the non-target nucleic acid can be cleaved even if the target nucleic acid contacts later. Consequently, when performing the above off-target nucleic acid cleavage method, the order in which the novel CRISPR / Cas13 system contacts the target nucleic acid and the non-target nucleic acid is not particularly important. When performing the above off-target nucleic acid cleavage method, the order in which the novel CRISPR / Cas13 system contacts the target nucleic acid and the non-target nucleic acid can vary as needed. In some cases, the target nucleic acid and the non-target nucleic acid can be contacted simultaneously with the novel CRISPR / Cas13 system.

[0121]

[0122] A method for detecting target nucleic acids using a novel CRISPR / Cas13 system

[0123] Overview of Target Nucleic Acid Detection Methods

[0124] This specification discloses a method for detecting a target nucleic acid using a novel CRISPR / Cas13 system. The method utilizes the ancillary cleavage activity of the novel CRISPR / Cas13 system. The method comprises mixing the novel CRISPR / Cas13 system with a sample and a detection reagent; and measuring whether a predetermined signal is generated from the mixture. The sample is a sample likely to contain a target nucleic acid. The detection reagent includes a reporter that generates a specific signal under specific conditions. The reporter includes a probe nucleic acid. When the probe nucleic acid is cleaved, the reporter generates a detectable signal (e.g., fluorescence). If the target nucleic acid is present in the sample, the novel CRISPR / Cas13 system recognizes the target nucleic acid. When the novel CRISPR / Cas13 system recognizes the target nucleic acid, the ancillary cleavage function is activated to randomly cleave surrounding nucleic acids. This cleavage of the reporter probe nucleic acid results in the generation of a detectable signal. By measuring and analyzing the above detectable signal, it is possible to determine whether and how much of the above target nucleic acid is present in the sample.

[0125] Below, each component and each process used in this method is described in detail.

[0126] sample

[0127] The method for detecting target nucleic acids using the novel CRISPR / Cas13 system described above is performed using a sample. By performing the method described above, it is possible to determine whether the sample contains the target nucleic acid and the amount of target nucleic acid contained therein. In other words, the sample is the target nucleic acid to be detected. The sample can take various forms, as needed. For example, the sample can contain bodily fluids from a human or non-human animal. In another example, the sample can be an appropriately pretreated sample of bodily fluids from a human or non-human animal.

[0128] Novel CRISPR / Cas13 system

[0129] The novel CRISPR / Cas13 system is used to determine whether the sample above contains the target nucleic acid and how much of the target nucleic acid it contains. The novel CRISPR / Cas13 system is as described in “Novel CRISPR / Cas13 System.” The novel CRISPR / Cas13 system can be used in the form of a novel CRISPR / Cas13 complex, an expression vector for each component of the novel CRISPR / Cas13 system, a novel CRISPR / Cas13 composition, or a suitable combination thereof.

[0130] Detection reagent

[0131] When the sample above contains a target nucleic acid, a detection reagent is used to generate a measurable signal. The detection reagent above includes a reporter. The reporter includes a probe nucleic acid. The reporter emits a measurable signal when the probe nucleic acid is cleaved. The measurable signal can be, for example, a fluorescent signal. If the target nucleic acid is present in the sample above, the novel CRISPR / Cas13 system can bind to the target nucleic acid. When the novel CRISPR / Cas13 system binds to the target nucleic acid, a secondary cleavage function is activated. When the secondary cleavage function is activated, nucleic acids surrounding the novel CRISPR / Cas13 system are randomly cleaved. When the probe nucleic acid of the reporter is cleaved as a result, the reporter generates a measurable signal. In conclusion, when the sample, the novel CRISPR / Cas13 system, and the detection reagent are mixed, a measurable signal will be generated by the reporter if the target nucleic acid is present in the sample above.

[0132] The above detection reagent may further include additional substances. These additional substances can be selected as needed. For example, the additional substance may be a substance that promotes the collateral cleavage activity of the novel CRISPR / Cas13 system. In another example, the additional substance may be a substance that amplifies the measurable signal generated from the reporter.

[0133] measurable signal

[0134] The measurable signal is a signal that can be measured when performing a target nucleic acid detection method. This is determined by the type of reporter included in the detection reagent. As long as the purpose of the method can be achieved, the nature of the measurable signal is not important. The signal generated from the reporter can be diverse. For example, the signal can be fluorescence at a specific wavelength. By measuring whether the measurable signal is generated, how strong the signal is, and how the signal intensity changes, information about the target nucleic acid can be obtained. Information about the target nucleic acid can include, for example, whether the target nucleic acid is present in the sample and the amount thereof.

[0135] Process of mixing samples, CRISPR / Cas13 system, and detection reagents

[0136] As described above, when the target nucleic acid, the novel CRISPR / Cas13 system, and the reporter come into contact, a measurable signal is generated. Therefore, to detect the target nucleic acid, the sample, the novel CRISPR / Cas13 system, and the detection reagent must all be brought together in one location. This process can be expressed in various ways. For example, the sample, the novel CRISPR / Cas13 system, and the detection reagent can be brought together in one location by mixing, mixing, reacting, contacting, or any appropriate combination of these actions. This process must be performed under conditions where the novel CRISPR / Cas13 system recognizes the target nucleic acid, the secondary cleavage function is activated to cleave the probe nucleic acid of the reporter, a measurable signal is generated from the reporter, and the sequence of events measured is uninterrupted. As long as these conditions are met, the process is not limited in terms of method, environment, or location. For ease of explanation, this process will hereinafter be referred to as "mixing."

[0137] The order of mixing the sample, CRISPR / Cas13 system, and detection reagent

[0138] In the above target nucleic acid detection method, the order in which the sample, the novel CRISPR / Cas13 system, and the detection reagent are mixed is not particularly important. The sample, the novel CRISPR / Cas13 system, and the detection reagent can be mixed simultaneously or in any order. Based on the principle of the above target nucleic acid detection method, it may be preferable to mix the sample and the novel CRISPR / Cas13 system first, followed by the detection reagent. However, as long as mixing is performed within an appropriate time, the series of reactions described above can sufficiently occur. The order in which the sample, the novel CRISPR / Cas13 system, and the detection reagent are mixed does not significantly affect the effectiveness of the target nucleic acid detection method.

[0139] The process of measuring a detectable signal

[0140] The above target nucleic acid detection method involves measuring a detectable signal. As described above, if a target nucleic acid is present in the sample, a series of reactions occur, causing the reporter in the detection reagent to emit a detectable signal. The method for measuring this detectable signal is determined by considering factors such as the nature of the emitted signal and the pattern in which it is emitted. The method is not particularly limited, and any known method can be used.

[0141]

[0142] Possible embodiments of the invention

[0143] Cas13 protein

[0144] Example 1: Novel Cas13d protein

[0145] Cas13 protein of subtype VI-D (also called Cas13d protein).

[0146] Example 2, novel Cas13 protein

[0147] 실시예 1에 있어서, 상기 Cas13 단백질은 VSKNDNIKSKAKALGLKSTFQVGDEVVMTSFGKGNKAIVEKIVRGTDVQSVPTEPNFSAE IEGKKFDLVGRAHIQTKSDNPQYSKKRTGDDMIGAKAALEKRFFGGTFDDNIHIQLAYNV LDIEKILSVHINNIVYTLNNIRRKDNAEDDDFIGYMSTRNDYDTFIEPRKHNISEDAAKS IDKSRASFEEYLQPPVKDCLHYFGNTFFAPREIEKTYTDNRGFERKKKVTVNSLIDEKEI YYIFALLGGLRQFCTHDNKSGRNWLYSLEENGINSDAKAVLDKYYNSAVARIDESFVDNA SKTNFKLIFNAMDVSDEALQDNIAKAFYKFTVCKSFKNMGFSIKKLREQLLELPEYEGLK DKHYDSVRSKLYQIMDFVIYLSFKDEKFKKDNEEKINNIVNELRATLNEEDKGRVYASYA ERMKSELKPAIARLKSDIDKIKDSRVKEFELDASVKYRLSKVVESVRLKDRATYFTKLIY LTTLFLDGKEINDLLTTLIHQFENIASFIDVMNDRGIDCRFSDGYKLFESSKQIAFELRN VNSFARMTRTSKDDENATHMMYIDAAEILGTDYTEEQIEEHLNLEKKRMIPGTKKADMNF RNFIINNVIKSSRFNYLVRYSNPKKIRALADNEGVIRFVLGELPDAQIDRYTLLCGFNPD ADRQEKTDKLAKAITGLRFNDFENVKQGANTEGESQESIDKAQKQGLISLYLTVLYLLTK NLVYVNSRYFLAFHCLERDAQLLGSGAGHHEPYVALTQRFINEDKLNEHACEYLKTNIAN SDEYTIRIFRNNAAHLSAVRNANLYIDKLKEFKSYYEIYHFLSQENIYGKYCVDKKYVTA DENGSKTISVKISKDYCPQVYIDKSLEYFDKLNKYGTYCKDFTKALNSPFGYNLARYKNL SIEGLFDRNRPGDKGENTFED(서열번호 1)의 아미노산 서열을 포함함.

[0148] Example 3, including similar sequences

[0149] In Example 1, the Cas13 protein is about 50% or more, about 51% or more, about 52% or more, about 53% or more, about 54% or more, about 55% or more, about 56% or more, about 57% or more, about 58% or more, about 59% or more, about 60% or more, about 61% or more, about 62% or more, about 63% or more, about 64% or more, about 65% or more, about 66% or more, about 67% or more, about 68% or more, about 69% or more, about 70% or more, about 71% or more, about 72% or more, about 73% or more, about 74% or more, about 75% or more, about 76% or more, about 77% or more, about 78% or more, about 79% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 100% identical, same, matched, and / or equivalent amino acid sequence.

[0150] Example 4, Functional Limitation

[0151] In any one of Examples 1 to 3, the Cas13 protein has the function of cleaving a single-stranded nucleic acid.

[0152] Example 5, PFS only

[0153] In any one of Examples 1 to 4, the Cas13 protein recognizes a protospacer flanking sequence (PFS) selected from the following:

[0154] RNA sequences selected from 5'-N-3', 5'-NN-3', and 5'-NNN-3',

[0155] Here, N is independently selected from A, U, C, or G; and

[0156] DNA sequences selected from 5'-N-3', 5'-NN-3', and 5'-NNN-3',

[0157] Here, N is independently selected from A, T, C, or G.

[0158] Example 6, no PFS required

[0159] In any one of Examples 1 to 3 to 4, when the Cas13 protein exhibits the function of cleaving a single-stranded nucleic acid,

[0160] Not PFS aware, not needing to be PFS aware, and / or not dependent on PFS.

[0161] Example 7, including a modified domain

[0162] In any one of Examples 1 to 6, the Cas13 protein comprises a modified protein domain.

[0163] Example 8, Meaning of Transformation

[0164] In Example 7, the modified protein domain of the Cas13 protein means that one or more amino acids are substituted, deleted, and / or inserted compared to the unmodified protein domain.

[0165] Example 9, modified domain

[0166] In Example 7 or Example 8, the Cas13 protein comprises a modified HEPN domain.

[0167] Example 10, including additional domains

[0168] In any one of Examples 1 to 9, the Cas13 protein further comprises an additional protein domain.

[0169] Example 11, nucleic acid limitation

[0170] In any one of Examples 1 to 10, the single-stranded nucleic acid is single-stranded RNA or single-stranded DNA.

[0171]

[0172] programmable guide RNA

[0173] Example 12, Programmable Guide RNA

[0174] Programmable guide RNA containing:

[0175] direct repeats; and

[0176] A guide domain designed to target a predetermined target sequence;

[0177] Here, the programmable guide RNA has the direct repeat portion and the guide domain sequentially linked from the 5' end to the 3' end,

[0178] The above direct repeats can interact with the Cas13 protein of the VI-D subtype to form a complex.

[0179] Example 13, structural formula expression

[0180] In Example 12, the programmable guide RNA comprises:

[0181] 5'-[direct repeat]-[guide domain]-3',

[0182] Here, the direct repeat portion and the guide domain are as defined in Example 12.

[0183] Example 14, direct repeat sequence limitation

[0184] In any one of Examples 12 to 13, the direct repeat portion has an RNA sequence of GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACC (SEQ ID NO: 2).

[0185] Example 15, including a sequence similar to a direct repeat

[0186] In Example 12, the direct repeat portion is about 50% or more, about 51% or more, about 52% or more, about 53% or more, about 54% or more, about 55% or more, about 56% or more, about 57% or more, about 58% or more, about 59% or more, about 60% or more, about 61% or more, about 62% or more, about 63% or more, about 64% or more, about 65% or more, about 66% or more, about 67% or more, about 68% or more, about 69% or more, about 70% or more, about 71% or more, about 72% or more, about 73% or more, about 74% or more, about 75% or more, about 76% or more, about 77% or more, about 78% or more, about 79% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 100% identical, same, matched, and / or equivalent RNA sequence.

[0187] Example 16, Direct Repeat Function Limitation

[0188] In any one of Examples 12 to 15, the direct repeat portion can interact with the Cas13 protein of any one of Examples 1 to 11 to form a complex.

[0189] Example 17, Length limitation

[0190] In any one of Examples 12 to 16, the guide domain has a length selected from among 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, 30nt, 31nt, 32nt, 33nt, 34nt, 35nt, 36nt, 37nt, 38nt, 39nt, 40nt, 41nt, 42nt, 43nt, 44nt, 45nt, 46nt, 47nt, 48nt, 49nt, and 50nt, or has a length within two numerical ranges selected from the numerical ranges. (e.g., 17nt to 20nt).

[0191] Example 18, Target Meaning

[0192] In any one of Examples 12 to 17, the guide domain is capable of complementary binding and / or hybridizing with a nucleic acid of the predetermined target sequence.

[0193] Example 19, Complementary Sequence, % Limitation

[0194] In any one of Examples 12 to 18, the guide domain is about 50% or more, about 51% or more, about 52% or more, about 53% or more, about 54% or more, about 55% or more, about 56% or more, about 57% or more, about 58% or more, about 59% or more, about 60% or more, about 61% or more, about 62% or more, about 63% or more, about 64% or more, about 65% or more, about 66% or more, about 67% or more, about 68% or more, about 69% or more, about 70% or more, about 71% or more, about 72% or more, about 73% or more, about 74% or more, about 75% or more, about 76% or more, about 77% or more, about 78% or more, about 79% or more, about 80% or more, about Having a sequence that is at least 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% complementary.

[0195] Example 20, Complementary sequence, limited number of mismatches

[0196] In any one of Examples 12 to 19, the guide domain is a sequence complementary to the predetermined target sequence, but includes 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

[0197] Example 21, Target RNA limitation

[0198] In any one of Examples 12 to 20, the nucleic acid of the predetermined target sequence is RNA or DNA.

[0199]

[0200] Cas13 complex

[0201] Example 22, Cas13 complex

[0202] CRISPR / Cas13 complex containing:

[0203] The Cas13 protein of any one of Examples 1 to 11; and

[0204] The programmable guide RNA of any one of Examples 12 to 21,

[0205] Here, the direct repeats of the Cas13 protein and the programmable guide RNA interact to form a complex.

[0206] Example 23, PFS and target nucleic acid recognition

[0207] In Example 22, the CRISPR / Cas13 complex can bind to a target nucleic acid, and the sequence of the target nucleic acid includes the following sequence:

[0208] With a predetermined target sequence, the predetermined target sequence is about 50% or more, about 51% or more, about 52% or more, about 53% or more, about 54% or more, about 55% or more, about 56% or more, about 57% or more, about 58% or more, about 59% or more, about 60% or more, about 61% or more, about 62% or more, about 63% or more, about 64% or more, about 65% or more, about 66% or more, about 67% or more, about 68% or more, about 69% or more, about 70% or more, about 71% or more, about 72% or more, about 73% or more, about 74% or more, about 75% or more, about 76% or more, about 77% or more, about 78% or more, about 79% or more, about 80% or more, about At least 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% complementary sequences.

[0209] Example 24, including additional PFS

[0210] In Example 23, when the Cas13 protein of the CRISPR / Cas13 complex recognizes PFS, the sequence of the target nucleic acid further includes a Protospacer Flanking Sequence (PFS) recognized by the Cas13 protein.

[0211] Example 25, PFS only

[0212] In Example 24, the PFS is selected from the following:

[0213] RNA sequences selected from 5'-N-3', 5'-NN-3', and 5'-NNN-3',

[0214] Here, N is independently selected from A, U, C, or G; and

[0215] DNA sequences selected from 5'-N-3', 5'-NN-3', and 5'-NNN-3',

[0216] Here, N is independently selected from A, T, C, or G.

[0217] Example 26, PFS and target sequence position relationship

[0218] In any one of Examples 23 to 25, the PFS in the target nucleic acid is adjacent to the 3'-end or the 5'-end of the target sequence.

[0219] Example 27, PFS and distance to target sequence

[0220] In Example 26, the PFS and the target sequence are separated by a distance of 0 nt, 1 nt, 2 nt, or 3 nt.

[0221] Example 28, Cas13 complex, ancillary cleavage function

[0222] In any one of Examples 22 to 27, the CRISPR / Cas13 complex has a collateral cleavage function,

[0223] When the CRISPR / Cas13 complex binds to the target nucleic acid, the secondary cleavage function is activated to cleave any nucleic acid present in the vicinity of the CRISPR / Cas13 complex.

[0224] Example 29, Cas13 complex, target nucleic acid cleavage function

[0225] In any one of Examples 22 to 28, the CRISPR / Cas13 complex has a target specific cleavage function,

[0226] When the CRISPR / Cas13 complex binds to the target nucleic acid, the target nucleic acid is cut.

[0227] Example 30, Nucleic Acid Type Restriction

[0228] In any one of Examples 28 to 29, the target nucleic acid and any nucleic acid are RNA or DNA.

[0229]

[0230] A vector capable of expressing each component of the CRISPR / Cas13 system

[0231] Example 31, expression vector

[0232] A vector capable of expressing components of the CRISPR / Cas13 system, including:

[0233] A nucleic acid encoding the Cas13 protein of any one of Examples 1 to 11; and

[0234] A nucleic acid encoding a programmable guide RNA of any one of Examples 12 to 21.

[0235] Example 32, included in one vector

[0236] In Example 31, the nucleic acid encoding the Cas13 protein and the nucleic acid encoding the programmable guide RNA are contained in one molecule of nucleic acid.

[0237] Example 33, included in different vectors

[0238] In Example 31, the nucleic acid encoding the Cas13 protein and the nucleic acid encoding the programmable guide RNA are each contained in nucleic acids of different molecules.

[0239] Example 34, vector types

[0240] In any one of Examples 31 to 33, the vector is a non-viral vector and / or a viral vector.

[0241] Example 35, nonviral vector type

[0242] In Example 34, the non-viral vector is selected from among a plasmid, a phage, naked DNA, a DNA complex, mRNA, and a PCR amplicon.

[0243] Example 36, Type of Viral Vector

[0244] In Examples 34 to 35, the viral vector is selected from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus.

[0245] Example 37, Nucleic Acid Type Restriction

[0246] In any one of Examples 31 to 36, the nucleic acid encoding the Cas13 protein and the nucleic acid encoding the programmable guide RNA are each independently selected from among DNA and RNA.

[0247] Example 38, DNA encoding Cas13 protein

[0248] In Example 37, the nucleic acid encoding the Cas13 protein has the DNA sequence of (SEQ ID NO: 4).

[0249]

[0250] CRISPR / Cas13 composition

[0251] Example 39, CRISPR / Cas13 composition

[0252] A CRISPR / Cas13 composition comprising:

[0253] The Cas13 protein of any one of Examples 1 to 11, or a nucleic acid encoding the Cas13 protein; and

[0254] A programmable guide RNA of any one of Examples 12 to 21, or a nucleic acid encoding said programmable guide RNA.

[0255] Example 40, CRISPR / Cas13 complex

[0256] In Example 39, the CRISPR / Cas13 composition comprises the Cas13 protein and the programmable guide RNA, and the Cas13 protein and the programmable guide RNA combine to form a ribonucleoprotein.

[0257] Example 41, Complex Limitation

[0258] In Example 40, the CRISPR / Cas13 composition comprises a CRISPR / Cas13 complex of any one of Examples 22 to 30.

[0259] Example 42, CRISPR / Cas13 system component expression vector

[0260] In Example 39, the CRISPR / Cas13 composition comprises a nucleic acid encoding the Cas13 protein and a nucleic acid encoding the programmable guide RNA.

[0261] Example 43, Expression Vector Restriction

[0262] In Example 42, the CRISPR / Cas13 composition comprises an expression vector of each component of the CRISPR / Cas13 system of any one of Examples 31 to 38.

[0263] Example 44, protein-encoding nucleic acid and guide RNA

[0264] In Example 39, the CRISPR / Cas13 composition comprises a nucleic acid encoding the Cas13 protein and the programmable guide RNA.

[0265] Example 45, mRNA-limited

[0266] In Example 44, the nucleic acid encoding the Cas13 protein is mRNA.

[0267]

[0268] Composition for detecting target nucleic acid

[0269] Example 46, Composition for detecting target nucleic acid

[0270] Detection reagents including:

[0271] A reporter comprising a probe nucleic acid,

[0272] Here, when the probe nucleic acid is cleaved, the reporter generates a detectable signal.

[0273] Example 47, Limiting the detectable signal

[0274] In Example 46, the detectable signal is a fluorescence signal.

[0275]

[0276] Non-target nucleic acid cleavage method / use

[0277] Example 48, Non-target Nucleic Acid Cleavage Method

[0278] A method for cleaving a non-target nucleic acid using a CRISPR / Cas13 system, comprising:

[0279] A process of contacting a CRISPR / Cas13 complex of any one of Examples 22 to 30 with a target nucleic acid and a non-target nucleic acid,

[0280] Here, the target nucleic acid has 1) a predetermined target sequence, and 2) a PFS recognized by the Cas13 protein of the CRISPR / Cas13 complex,

[0281] The guide domain of the programmable guide RNA of the above CRISPR / Cas13 complex is designed to target the predetermined target sequence,

[0282] The above non-target nucleic acid has a different sequence from the target nucleic acid,

[0283] When the CRISPR / Cas13 complex comes into contact with the target nucleic acid, the secondary cleavage function is activated, thereby cleaving the non-target nucleic acid.

[0284] Example 49, Contact Sequence

[0285] In Example 48, the process of contacting the CRISPR / Cas13 complex with the target nucleic acid and the non-target nucleic acid is performed in a selected order among the following:

[0286] (a) contacting the CRISPR / Cas13 complex with the target nucleic acid, and then contacting it with the non-target nucleic acid;

[0287] (b) contacting the CRISPR / Cas13 complex with the non-target nucleic acid, and then contacting it with the target nucleic acid; and

[0288] (c) Contacting the CRISPR / Cas13 complex with the target nucleic acid and non-target nucleic acid simultaneously.

[0289] Example 50, Non-target nucleic acid cleavage use

[0290] Use of the CRISPR / Cas13 complex of any one of Examples 22 to 30, the component expression vector of the CRISPR / Cas13 system of any one of Examples 31 to 38, and / or the CRISPR / Cas13 composition of any one of Examples 39 to 45 in the non-target nucleic acid cleavage method of any one of Examples 48 to 49.

[0291]

[0292] Target nucleic acid detection method / use

[0293] Example 51, Method for detecting target nucleic acid

[0294] A method for detecting a target nucleic acid using a CRISPR / Cas13 system, comprising:

[0295] (a) a process of blending, mixing, reacting, and / or contacting a sample, a CRISPR / Cas13 complex of any one of Examples 22 to 30, and a detection reagent of any one of Examples 46 to 47;

[0296] Here, the target nucleic acid has 1) a predetermined target sequence, and 2) a PFS recognized by the Cas13 protein of the CRISPR / Cas13 complex,

[0297] The guide domain of the programmable guide RNA of the above CRISPR / Cas13 complex is designed to target the predetermined target sequence,

[0298] When the target nucleic acid is present in the sample, the target nucleic acid binds to the CRISPR / Cas13 complex and activates the secondary cleavage activity, whereby the probe nucleic acid included in the reporter can be cleaved by the Cas13 complex.

[0299] When the probe nucleic acid contained in the reporter of the above detection reagent is cleaved, the reporter generates a detectable signal; and

[0300] (b) a process of observing, measuring and / or detecting the detectable signal from the mixture;

[0301] Here, the presence or absence of the target nucleic acid and / or the amount present in the sample can be derived, found out, measured, determined, detected and / or analyzed from the detectable signal.

[0302] Example 52, Sample, Complex, and Detection Reagent Contact Sequence

[0303] In Example 51, the process of blending, mixing, reacting, and / or contacting the sample, the CRISPR / Cas13 complex of any one of Examples 22 to 30, and the detection reagent of any one of Examples 46 to 47 is performed in a selected order among the following:

[0304] (1) Blending, mixing, reacting, and / or contacting the sample and the CRISPR / Cas13 complex, and then blending, mixing, reacting, and / or contacting the detection reagent;

[0305] (2) After blending, mixing, reacting, and / or contacting the sample and the detection reagent, blending, mixing, reacting, and / or contacting the CRISPR / Cas13 complex;

[0306] (3) After blending, mixing, reacting, and / or contacting the CRISPR / Cas13 complex and the detection reagent, blending, mixing, reacting, and / or contacting the sample; and

[0307] (4) Blending, mixing, reacting, and / or contacting the sample, the CRISPR / Cas13 complex, and the detection reagent simultaneously.

[0308] Example 53, COVID-19 detection method

[0309] In any one of Examples 51 to 52, the method for detecting a target nucleic acid using the CRISPR / Cas13 system is a method for detecting whether the sample contains a nucleic acid of a COVID-19 virus.

[0310] Example 54, COVID-19 N-gene, E-gene detection method

[0311] 실시예 53에 있어서, 상기 표적 핵산은 ucugauaauggaccccaaaaucagcgaaaugcaccccgcauuacguuugguggacccucagauucaacuggcaguaaccagaauggagaacgcaguggggcgcgaucaaaacaacgucggccccaagguuuacccaauaauacugcgucuugguucaccgcucucacucaacauggcaaggaagaccuuaaauucccucgaggacaaggcguuccaauuaacaccaauagcaguccagaugaccaaauuggcuacuaccgaagagcuaccagacgaauucgugguggugacgguaaaaugaaagaucucaguccaagaugguauuucuacuaccuaggaacugggccagaagcuggacucccuauggugcuaacaaagacggcaucauaggguugcaacugagggagccuugaauacaccaaaagaucacauuggcacccgcaauccugcuaacaaugcugcaaucgugcuacaacuuccucaaggaacaacauugccaaaaggcuucuacgcagaagggagcagaggcggcagucaagccucuucucguuccucaucacguagucgcaacaguucaagaaauucaacuccaggcagcaguaggggaaccuccugcuagaauggcuggcaauggcggugaugcugcucuugcuuugcugcugcuugacagauugaaccagcuugagagcaaaaugucugguaaaggccaacaacaacaaggccaaacugucacuaagaaaucugcugcugaggcuucuaagaagccucggcaaaaacguacugccacuaaagcauacaauguaacacaagcuuucggcagacgugguccagaacaaacccaaggaaauuuuggggaccaggaacuaaucagacaaggaacugauuacaaacauuggccgcaaauugcacaauuugcccccagcgcuucagcguucuucggaaugucgcgcauuggcauggaagaaaaaa(서열번호 5)and / or uacucauucguuucggaagagagacagguacguuaauaguuaauagcguacuucuuuuuucuugcuuucgugguauucuugcuaguuacacuagccauccuuacugcgcuucgauugugu gcguacugcugcaauauuguuaacgugagucuuguaaaaccuucuuuuuacguuuacucucguguuaaaaaucugaauucuucuagaguuccugaucuucuggucuaaaaaa (SEQ ID NO: 6) Im.

[0312] Example 55, Target Sequence Limitation

[0313] In Example 54, the target sequence of the target nucleic acid is CGCAUUACGUUUGGUGGACC (SEQ ID NO: 57), UUCAACUGGCAGUAACCAGA (SEQ ID NO: 58), UUACAAACAUUGGCCGCAAA (SEQ ID NO: 59), GGGAGCCUUGAAUACACCAA (SEQ ID NO: 60), CACAUUGGCACCCGCAAUCC (SEQ ID NO: 61), AAUGCUGCAAUCGUGCUACA (SEQ ID NO: 62), GGGGAACUUCUCCUGCUAGA (SEQ ID NO: 63), CUUUGCUGCUGCUUGACAGA (SEQ ID NO: 64), CAGCUUGAGAGCAAAAUGUC (SEQ ID NO: 65), GAUUCAACUGGCAGUAACCA (SEQ ID NO: 66), CACAUUGGCACCCGCAAUCCU (SEQ ID NO: 67), AAUGCUGCAAUCGUGCUACAA (SEQ ID NO: 68), A nucleic acid sequence selected from among UUGGCCGCAAAUUGCACAAUUUG (SEQ ID NO: 69), AGGAACUGAUUACAAACAUUGGC (SEQ ID NO: 70), GGGACCAGGAACUAAUCAGACAA (SEQ ID NO: 71), CUAGCCAUCCUUACUGCG (SEQ ID NO: 72), CGGAAGAGACAGGUACGUUA (SEQ ID NO: 73), GUUACACUAGCCAUCCUUAC (SEQ ID NO: 74), UGUGCGUACUGCUGCAAUAUU (SEQ ID NO: 75), and CAAUAUUGUUAACGUGAGUCUU (SEQ ID NO: 76).

[0314] Example 56, Guide Domain Limited

[0315] In Example 54, the guide domain of the programmable guide RNA of the CRISPR / Cas13 complex is ggUccaccaaacgUaaUgcg (SEQ ID NO: 37), UCUGGUUACUGCCAGUUGAA (SEQ ID NO: 38), UUUgcggccaaUgUUUgUaa (SEQ ID NO: 39), UUGGUGUAUUCAAGGCUCCC (SEQ ID NO: 40), GGAUUGCGGGUGCCAAUGUG (SEQ ID NO: 41), UGUAGCACGAUUGCAGCAUU (SEQ ID NO: 42), UCUAGCAGGAGAAGUUCCCC (SEQ ID NO: 43), UCUGUCAAGCAGCAGCAAAG (SEQ ID NO: 44), GACAUUUUGCUCUCAAGCUG (SEQ ID NO: 45), UggUUacUgccagUUgaaUc (SEQ ID NO: 46), Having a nucleic acid sequence selected from among aggaUUgcgggUgccaaUgUg (SEQ ID NO: 47), UUgUagcacgaUUgcagcaUU (SEQ ID NO: 48), CAAAUUGUGCAAUUUGCGGCCAA (SEQ ID NO: 49), GCCAAUGUUUGUAAUCAGUUCCU (SEQ ID NO: 50), UUGUCUGAUUAGUUCCUGGUCCC (SEQ ID NO: 51), cgcagUaaggaUggcUag (SEQ ID NO: 52), UaacgUaccUgUcUcUUccg (SEQ ID NO: 53), gUaaggaUggcUagUgUaac (SEQ ID NO: 54), aaUaUUgcagcagUacgcaca (SEQ ID NO: 55), and aagacUcacgUUaacaaUaUg (SEQ ID NO: 56).

[0316] Example 57, Programmable Guide RNA Limited

[0317] In Example 54, the programmable guide RNA of the CRISPR / Cas13 complex is GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCUggUUacUgccagUUgaaUc (SEQ ID NO: 16), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCaggaUUgcgggUgccaaUgUg (SEQ ID NO: 17), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCUUgUagcacgaUUgcagcaUU (SEQ ID NO: 18), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCCAAAUUGUGCAAUUUGCGGCCAA (SEQ ID NO: 19), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCGCCAAUGUUUGUAAUCAGUUCCU (SEQ ID NO: 20), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCUUGUCUGAUUAGUUCCUGGUCCC (SEQ ID NO: 21), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCggUccaccaaacgUaaUgcg (SEQ ID NO: 22), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCUCUGGUUACUGCCAGUUGAA (SEQ ID NO: 23), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCUUUgcggccaaUgUUUgUaa (SEQ ID NO: 24), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCUUGGUGUAUUCAAGGCUCCC (SEQ ID NO: 25), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCGGAUUGCGGGUGCCAAUGUG (SEQ ID NO: 26), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCUGUAGCACGAUUGCAGCAUU (SEQ ID NO: 27), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCUCUAGCAGGAGAAGUUCCCC (SEQ ID NO: 28),GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCUCUGUCAAGCAGCAGCAAAG(SEQ ID NO: 29), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCGACAUUUUGCUCUCAAGCUG(SEQ ID NO: 30), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCUaacgUaccUgUcUcUUccg(SEQ ID NO: 32), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCgUaaggaUggcUagUgUaac(SEQ ID NO: 33), GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCaaUaUUgcagcagUacgcaca(SEQ ID NO: 34), Having a nucleic acid sequence selected from among GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCaagacUcacgUUaacaaUaUUg (SEQ ID NO: 35), and GAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACCcgcagUaaggaUggcUag (SEQ ID NO: 36).

[0318] Example 58, Use for detecting target nucleic acid

[0319] Use of the CRISPR / Cas13 complex of any one of Examples 22 to 30, the component expression vector of the CRISPR / Cas13 system of any one of Examples 31 to 38, and / or the CRISPR / Cas13 composition of any one of Examples 39 to 45 in the target nucleic acid detection method of any one of Examples 51 to 57.

[0320]

[0321] Experimental example

[0322] Hereinafter, the invention provided by this specification will be described in more detail through experimental examples and examples. These examples are intended solely to illustrate the subject matter disclosed by this specification, and it will be apparent to those skilled in the art that the scope of the subject matter disclosed by this specification is not limited by these examples.

[0323]

[0324] Experimental Example 1. Experimental Method and Materials

[0325] Experimental Example 1.1. Identification of a New CRISPR / Cas13 System Sequence from a Publication Database

[0326] We used the bovine metagenome data published in the NCBI (National Center for Biotechnology Information) database. Using the NGS data, we characterized the CRISPR / Cas13 system using the following method:

[0327] (1) Microbial genome information was derived by assembling sequence data using MEGAHIT v.1.2.9 (NGS assembly software). (Li D, Liu CM, Luo R, Sadakane K, Lam TW. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics. 2015 May 15;31(10):1674-6. doi: 10.1093 / bioinformatics / btv033. Epub 2015 Jan 20. PMID: 25609793).

[0328] (2) With reference to the known Cas13 sequence information (e.g., Cas13 derived from Lachnospiraceae bacterium), a group of Cas13 protein candidates included in the genome information of the microorganism was identified through homology analysis using the (BLAST) tool.

[0329] (3) In the same manner as (2), guide RNA candidates were identified for each Cas13 protein candidate.

[0330] Below, an experiment was conducted using the sequence information of the CRISPR / Cas13 system derived by the above method.

[0331] Each piece of information about the CRISPR / Cas13 system specified by the above method is as follows:

[0332] SubjectSequenceSEQ ID NOCas13 proteinVSKNDNIKSKAKALGLKSTFQVGDEVVMTSFGKGNKAIVEKIVRGTDVQSVPTEPNFSAEIEGKKFDLVGRAHIQTKSDNPQYSKKRTGDDMIGAKAALEKRFFGGTFDDNIHIQLAYNVLDIEKILSVHINNIVYTLNNIRRKDNAEDDDFIGYMSTRNDYDTFIEPRKHNISEDAAKSIDKSRASFEEYLQPPVKDCLHYFGNTFFAPREIEKTYTDNRGFERKKKVTVNSLIDEKEIYYIFALLGGLRQFCTHDNKSGRNWLYSLEENGINSDAKAVLDKYYNSAVARIDESFVDNASKTNFKLIFNAMDVSDEALQDNIAKAFYKFTVCKSFKNMGFSIKKLREQLLELPEYEGLKDKHYDSVRSKLYQIMDFVIYLSFKDEKFKKDNEEKINNIVNELRATLNEEDKGRVYASYAERMKSELKPAIARLKSDIDKIKDSRVKEFELDASVKYRLSKVVESVRLKDRATYFTKLIYLTTLFLDGKEINDLLTTLIHQFENIASFIDVMNDRGIDCRFSDGYKLFESSKQIAFELRNVNSFARMTRTSKDDENATHMMYIDAAEILGTDYTEEQIEEHLNLEKKRMIPGTKKADMNFRNFIINNVIKSSRFNYLVRYSNPKKIRALADNEGVIRFVLGELPDAQIDRYTLLCGFNPDADRQEKTDKLAKAITGLRFNDFENVKQGANTEGESQESIDKAQKQGLISLYLTVLYLLTKNLVYVNSRYFLAFHCLERDAQLLGSGAGHHEPYVALTQRFINEDKLNEHACEYLKTNIANSDEYTIRIFRNNAAHLSAVRNANLYIDKLKEFKSYYEIYHFLSQENIYGKYCVDKKYVTADENGSKTISVKISKDYCPQVYIDKSLEYFDKLNKYGTYCKDFTKALNSPFGYNLARYKNLSIEGLFDRNRPGDKGENTFED1DirectrepeatGAAACGUACUACACCCUUUUUGUAAGGGUCUGAAACC2

[0333] Experimental Example 1.2. Production of a Novel Cas13 Protein Expression Vector

[0334] For the amino acid sequence of the novel Cas13 protein discovered in Experimental Example 1.1, an E. coli codon-optimized nucleic acid sequence was specified, and this was cloned into a pET28 vector to produce a novel Cas13 protein expression vector.

[0335] Experimental Example 1.3. Production and Purification of a Novel Cas13 Protein

[0336] The novel Cas13 protein expression vector manufactured in Experimental Example 1.2 was induced into cells, and the novel Cas13 protein was manufactured in various E. coli strains (NiCo21(DE3), T7 Express lysY / Iq, NEB® Express Iq) under various growth conditions (medium, temperature, induction conditions, etc.), and the manufactured amount was tested through SDS-PAGE analysis.

[0337] Based on the above test results, optimized growth conditions and strains for flask-scale production were selected.

[0338] Specifically, the E. coli cells were transformed with the plasmid vector prepared in Experimental Example 1.2 according to the manufacturer's instructions. A single colony was grown overnight in Luria-Bertani broth containing 100 ug ml-1 ampicillin at 37°C. The cells were diluted 1:200 in 250 ml of the same medium and grown at 37°C until the OD600 reached 0.70 to 0.75. The culture was incubated at room temperature for 1 hour, and protein expression was induced with 0.5 mM isopropyl-bD-1-thiogalactopyranoside by incubation for 16-18 hours at 23°C with shaking (220 rpm).

[0339] After inducing E. coli to produce the novel Cas13 protein under the above optimized growth conditions, the cells were pelleted by centrifugation at ~4000 g for 20 min and stored in a refrigerator at -20°C.

[0340] Prior to purification of the novel Cas13 protein, frozen cells were resuspended in sonication buffer (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 5% glycerol, 0.2% Tween20, 1 mM DTT, 1 mM EDTA, 1x Halt Protease Inhibitor Cocktail (Fisher Scientific), 40 mM imidazole at 25°C) using 5 ml of buffer per 1 g of pellet.

[0341] The pellets suspended in the above sonication buffer were sonicated using a Qsonica Microtip Probe 420-A at 30% amp per 30 ml of sample, 5 sec ON / 15 sec OFF, on ice for 4 x 2 min (interval treatment was performed to prevent overheating).

[0342] The sample after the above sonication was centrifuged at 40,000 g for 1 hour to remove cell debris.

[0343] The supernatant from the above centrifugation results was filtered through a 0.2 μm PES syringe filter (Fisher Scientific) and loaded onto a Ni2+-charged HiTrap chelate HP column (Cytiva) equilibrated with 20 mM Tris-HCl pH 7.5 in 0.5 M NaCl, 40 mM imidazole buffer at 25°C.

[0344] The column was eluted with a linear gradient of imidazole with increasing concentration from 40 mM to 700 mM at 25°C, 0.5 M NaCl to 20 mM Tris-HCl, pH 7.5 to obtain the novel Cas13 protein.

[0345] Experimental Example 1.4. Production of Programmable Guide RNA

[0346] The programmable guide RNA (hereinafter also referred to as crRNA) of the novel CRISPR / Cas13 system specified in Experimental Example 1.1 was synthesized through in vitro transcription using TranscriptAid T7 High Yield Transcription kit (Thermo fisher) or HiScribe T7 Quick High Yield RNA Synthesis Kits (New England Biolabs).

[0347] For the above in vitro transcription, a fragment containing a T7 promoter at the proximal end of the crRNA coding sequence was generated by PCR using a primer as a template, or a T7 primer oligonucleotide was annealed to a single-stranded template oligonucleotide.

[0348] The manufactured crRNA was purified using the RNeasy MiniElute Cleanup Kit (Qiagen) or the Monarch RNA Cleanup Kit (50 μg) (New England Biolabs).

[0349] The primers used for the above in vitro transcription are as follows:

[0350] Primers used to synthesize programmable guide RNAs of the novel CRISPR / Cas13 system LabelSequenceSEQ ID NOcrRNA Forward primerGAAATTAATACGACTCACTATAGGAAACGTACTACACCCTTTTTGTAAGGGTCTGAAACC77crRNA Example 2.1 Reverse primerGATTCAACTGGCAGTAACCAGGTTTCAGACCCTTACAAAAAGGG78crRNA Example 2.2 Reverse primerCACATTGGCACCCGCAATCCTGGTTTCAGACCCTTACAAAAAGGG79crRNA Example 2.3 Reverse primerAATGCTGCAATCGTGCTACAAGGTTTCAGACCCTTACAAAAAGGG80crRNA Example 2.4 Reverse primerTTGGCCGCAAATTGCACAATTTGGGTTTCAGACCCTTACAAAAAGGG81crRNA Example 2.5 Reverse primerAGGAACTGATTACAAACATTGGCGGTTTCAGACCCTTACAAAAAGGG82crRNA Example 2.6 Reverse primerGGGACCAGGAACTAATCAGACAAGGTTTCAGACCCTTACAAAAAGGG83crRNA Example 3.1 Reverse primerCGGAAGAGACAGGTACGTTAGGTTTCAGACCCTTACAAAAAGGG84crRNA Example 3.2 Reverse primerGTTACACTAGCCATCCTTACGGTTTCAGACCCTTACAAAAAGGG85crRNA Example 3.3 Reverse primerTGTGCGTACTGCTGCAATATTGGTTTCAGACCCTTACAAAAAGGG86crRNA Example 3.4 Reverse primerCAATATTGTTAACGTGAGTCTTGGTTTCAGACCCTTACAAAAAGGG87

[0351] Experimental Example 1.5. Preparation of a Novel CRISPR / Cas13 Complex

[0352] A novel CRISPR / Cas13 complex was prepared by incubating 40 nM of Cas13 protein prepared by Experimental Example 1.3 and 40 nM of crRNA prepared by Experimental Example 1.4 in NEB r2.1 buffer containing 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 ug / mL BSA (pH=7.9) at room temperature for 15 minutes.

[0353] Experimental Example 1.6. Target nucleic acid detection method using a CRISPR / Cas13 complex.

[0354] A reaction solution was prepared by mixing 1x NEB 2.0 buffer, 0.6 U / uL of Rnase Inhibitor, 12.5 nM of Rnase alert (IDT reporter), target RNA at various concentrations (0.1 - 100 ng / uL) depending on the experimental example, and 100 uL of purified water containing no nuclease.

[0355] The novel CRISPR / Cas13 complex prepared by Experimental Example 1.5 was added to the above reaction solution.

[0356] The above mixture was loaded into 384-well (SPL) and incubated at 37°C for up to 1 hour in a fluorescence plate reader (TECAN, Infinite 200 Pro M Plex), and fluorescence measurements were performed every 3 minutes (λex: 535 nm; λem: 485 nm, gain: -135).

[0357]

[0358] Experimental Example 2. Target Nucleic Acid Detection Experiment 1 Using a Novel CRISPR / Cas13 Complex

[0359] Experimental Example 2.1. Target Nucleic Acid Detection Experiment Based on Guide Domain Sequence

[0360] To confirm whether the novel CRISPR / Cas13 complex manufactured according to Experimental Example 1.5 has ancillary cleavage activity and can be further utilized for target nucleic acid detection, a target nucleic acid detection experiment was conducted according to Experimental Example 1.6.

[0361] The target nucleic acid corresponds to the N-gene of COVID-19, and the sequence is as follows:

[0362] ucugauaauggaccccaaaaucagcgaaaugcaccccgcauuacguuugguggacccucagauucaacuggcaguaaccagaauggagaacgcaguggggcgcgaucaaaacaacgucggccccaagguuuacccaauaauacugcgucuugguucaccgcucucacucaacauggcaaggaagaccuuaaauucccucgaggacaaggcguuccaauuaacaccaauagcaguccagaugaccaaauuggcuacuaccgaagagcuaccagacgaauucgugguggugacgguaaaaugaaagaucucaguccaagaugguauuucuacuaccuaggaacugggccagaagcuggacucccuauggugcuaacaaagacggcaucauaggguugcaacugagggagccuugaauacaccaaaagaucacauuggcacccgcaauccugcuaacaaugcugcaaucgugcuacaacuuccucaaggaacaacauugccaaaaggcuucuacgcagaagggagcagaggcggcagucaagccucuucucguuccucaucacguagucgcaacaguucaagaaauucaacuccaggcagcaguaggggaaccuccugcuagaauggcuggcaauggcggugaugcugcucuugcuuugcugcugcuugacagauugaaccagcuugagagcaaaaugucugguaaaggccaacaacaacaaggccaaacugucacuaagaaaucugcugcugaggcuucuaagaagccucggcaaaaacguacugccacuaaagcauacaauguaacacaagcuuucggcagacgugguccagaacaaacccaaggaaauuuuggggaccaggaacuaaucagacaaggaacugauuacaaacauuggccgcaaauugcacaauuugcccccagcgcuucagcguucuucggaaugucgcgcauuggcauggaagaaaaaa(서열번호 5)

[0363] The compositions of each novel CRISPR / Cas13 complex used in Experimental Example 2.1 are shown in the following table:

[0364] As a negative control for each experiment, only Cas13 protein was added without programmable guide RNA.

[0365] Each component of the novel CRISPR / Cas13 complex used in Experimental Example 2 LabelCas13 protein (SEQ ID NO) Direct Repeat (SEQ ID NO) Guide domain PFS Example 2.1 12TGGTTACTGCCAGTTGAATCG Example 2.2AGGATTGCGGGTGCCAATGTGGE xample 2.3TTGTAGCACGATTGCAGCATTC Example 2.4CAAATTGTGCAATTTGCGGCCAACE xample 2.5GCCAATGTTTGTAATCAGTTCCTC Example 2.6TTGTCTGATTAGTTCCTGGTCCCG

[0366] The results of the above experiments are shown in Figures 1 to 6:

[0367] As a result of the experiment, it can be seen that the novel CRISPR / Cas13 complex 1) has guide domains of various sequences that all bind to target nucleic acids and activate the ancillary cleavage function, and 2) exhibits ancillary cleavage activity sufficient to be used for target nucleic acid detection.

[0368] Experimental Example 2.2. Target nucleic acid detection experiment according to concentration

[0369] To confirm whether the novel CRISPR / Cas13 complex manufactured according to Experimental Example 1.5 has ancillary cleavage activity and can be further utilized for target nucleic acid detection, a target nucleic acid detection experiment was conducted according to Experimental Example 1.6 using the N-gene of COVID-19 as the target nucleic acid.

[0370] The compositions of each novel CRISPR / Cas13 complex used in Experimental Example 2 are shown in the following table:

[0371] Each composition and concentration of the novel CRISPR / Cas13 complex used in Experimental Example 2.2 LabelCas13 protein (SEQ ID NO) Direct Repeat (SEQ ID NO) Guide domain PFS Concentration (ng / uL) Example 2.2.1 12 AGGATTGCGGGTGCCAATGTGG 0.05 Example 2.2.2 0.1 Example 2.2.3 0.25 Example 2.2.4 0.5 Example 2.2.5 1 Example 2.5.1 GCCAATGTTTGTAATCAGTTCCTC 0.05 Example 2.5.2 0.1 Example 2.5.3 0.25 Example 2.5.4 0.5 Example 2.5.5 1

[0372] As a negative control for each experiment, only Cas13 protein was added without programmable guide RNA.

[0373] The results of the above experiment are shown in Figures 7 and 8:

[0374] Experimental results show that the novel CRISPR / Cas13 complex 1) binds to target nucleic acids even at low concentrations and activates ancillary cleavage function, and 2) exhibits sufficient ancillary cleavage activity to be used for target nucleic acid detection.

[0375]

[0376] Experimental Example 3. Target Nucleic Acid Detection Experiment 2 Using a Novel CRISPR / Cas13 Complex

[0377] Experimental Example 3.1. Target Nucleic Acid Detection Experiment Based on Guide Domain Sequence

[0378] To confirm whether the novel CRISPR / Cas13 complex manufactured according to Experimental Example 1.5 has ancillary cleavage activity and can be further utilized for target nucleic acid detection, a target nucleic acid detection experiment was conducted according to Experimental Example 1.6.

[0379] The compositions of each novel CRISPR / Cas13 complex used in Experimental Example 3.1 are shown in the following table:

[0380] The target nucleic acid corresponds to the E-gene of COVID-19, and the sequence is as follows:

[0381] uacucauucguuucggaagagacagguacguuaauaguuaauagcguacuucuuuuuucuugcuuucgugguauucuugcuaguuacacuagccauccuuacugcgcuucgauugugu gcguacugcugcaauauuguuaacgugagucuuguaaaaccuucuuuuuacguuuacucucguguuaaaaaucugaauucuucuagaguuccugaucuucuggucuaaaaaa (SEQ ID NO: 6)

[0382] Each component of the novel CRISPR / Cas13 complex used in Experimental Example 3 LabelCas13 protein (SEQ ID NO) Direct Repeat (SEQ ID NO) Guide domain PFS Example 3.1 12 TAACGTACCTGTCTCTTCCGA Example 3.2 GTAAGGATGGCTAGTGTAACT Example 3.3 AATATTGCAGCAGTACGCACAGE example 3.4 AAGACTCACGTTAACAATATTGC

[0383] The results of the above experiments are shown in Figures 9 to 11:

[0384] Here, in the case of Example 3.4 above, the fluorescence signal intensity measurement range was exceeded immediately after the reaction, and a separate graph was not drawn.

[0385] As a result of the experiment, it can be seen that the novel CRISPR / Cas13 complex 1) activates the ancillary cleavage function by binding all guide domains of various sequences to the target nucleic acid, 2) exhibits ancillary cleavage activity sufficient to be used for target nucleic acid detection, and 3) functions only with the presence of a sequence complementary to the guide domain, without relying on the protospacer flanking sequence.

[0386] Experimental Example 3.2. Target nucleic acid detection experiment according to concentration

[0387] To confirm whether the novel CRISPR / Cas13 complex manufactured according to Experimental Example 1.5 has ancillary cleavage activity and can be further utilized for target nucleic acid detection, a target nucleic acid detection experiment was conducted according to Experimental Example 1.6 using the E-gene of COVID-19 as the target nucleic acid.

[0388] The compositions of each novel CRISPR / Cas13 complex used in Experimental Example 3.2 are shown in the following table:

[0389] Each composition and concentration of the novel CRISPR / Cas13 complex used in Experimental Example 2.2 LabelCas13 protein (SEQ ID NO) Direct Repeat (SEQ ID NO) Guide domain PFS Concentration (ng / uL) Example 3.3.1 12AATATTGCAGCAGTACGCACAG0.05Example 3.3.2 0.1Example 3.3.3 0.25Example 3.3.4 0.5Example 3.3.51Example 3.4.1AAGACTCACGTTAACAATATTGC0.05Example 3.4.2 0.1Example 3.4.3 0.25Example 3.4.4 0.5Example 3.4.51

[0390] As a negative control for each experiment, only Cas13 protein was added without programmable guide RNA.

[0391] The results of the above experiment are shown in Figures 12 and 13:

[0392] Here, for Example 3.4.5, the detection limit is exceeded after 4 minutes of reaction, for Example 3.4.4, after 8 minutes of reaction, and for Example 3.4.3, after 14 minutes of reaction, and the figure of 65000 is inserted to fill in the data points.

[0393] Experimental results show that the novel CRISPR / Cas13 complex 1) binds to target nucleic acids even at low concentrations and activates ancillary cleavage function, and 2) exhibits sufficient ancillary cleavage activity to be used for target nucleic acid detection.

[0394]

[0395] The invention disclosed in this specification relates to a novel CRISPR / Cas13 system. The novel CRISPR / Cas13 system can specifically cleave nucleic acids and, additionally, cleave any nucleic acid. The novel CRISPR / Cas13 system can be used for non-target nucleic acid cleavage or for target nucleic acid detection.

Claims

1. A novel CRISPR / Cas13 composition comprising: A Cas13 protein comprising a nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the Cas13 protein; and a programmable guide RNA, or a nucleic acid encoding said programmable guide RNA; The above programmable guide RNA has a structure in which the repeat portion and the guide domain are directly linked from the 5' end to the 3' end. The above direct repeat interacts with the above Cas13 protein to form a complex, The above direct repeat portion contains the RNA sequence of sequence number 2, The above guide domain can complementarily bind to a predetermined target nucleic acid.

2. In the novel CRISPR / Cas13 composition of paragraph 1, The nucleic acid encoding the above Cas13 protein is DNA or mRNA, The nucleic acid encoding the above programmable guide RNA is DNA.

3. In any one of the novel CRISPR / Cas13 compositions of paragraphs 1 and 2, The novel CRISPR / Cas13 composition comprises the Cas13 protein and the programmable guide RNA, The above Cas13 protein and the above programmable guide RNA form a protein-RNA complex.

4. In any one of the novel CRISPR / Cas13 compositions of paragraphs 1 and 2, The novel CRISPR / Cas13 composition comprises a nucleic acid encoding the Cas13 protein and a nucleic acid encoding the programmable guide RNA.

5. In the novel CRISPR / Cas13 composition of Article 4, The nucleic acid encoding the above Cas13 protein and the nucleic acid encoding the above programmable guide RNA are contained in one molecule of a vector.

6. In the novel CRISPR / Cas13 composition of paragraph 4, The nucleic acid encoding the above Cas13 protein and the nucleic acid encoding the above programmable guide RNA are each contained in two or more vector molecules.

7. In any one of the novel CRISPR / cas13 compositions of clauses 5 to 6, The above vector is a viral vector, a non-viral vector, or a suitable combination thereof.

8. A method for cleaving non-target RNA using a novel CRISPR / Cas13 system, comprising: The process of mixing the novel CRISPR / Cas13 composition of Article 3 with a sample; Here, the sample contains target RNA and non-target RNA, The guide domain of the programmable guide RNA of the novel CRISPR / Cas13 composition above can complementarily bind to the target RNA above.

9. A method for detecting target RNA using a novel CRISPR / Cas13 system, comprising: (a) A process of mixing the novel CRISPR / Cas13 composition, sample, and detection reagent of Article 3; Here, the guide domain of the programmable guide RNA of the novel CRISPR / Cas13 composition can complementarily bind to the target RNA, The above detection reagent comprises a reporter, and the above reporter comprises a probe nucleic acid, When the above probe nucleic acid is cleaved, the above reporter produces a detectable signal. Here, when the above sample contains the above target RNA, the protein-RNA complex of the above novel CRISPR / Cas13 composition complementarily binds to the above target RNA, the auxiliary cleavage function of the above protein-RNA complex is activated, and the above probe nucleic acid is cleaved accordingly; and (b) a process of measuring a detectable signal from the above mixture; By measuring the detectable signal above, information about the target RNA can be obtained.

10. In the method for detecting the target RNA of Article 9, The above (b) process is a process of measuring whether the detectable signal occurs; the intensity of the signal that occurs; the change in signal intensity over time; or any combination of the above.

11. In a method for detecting a target RNA of any one of claims 9 to 10, Information about the above target RNA is whether the above target RNA is present, how much of the above target RNA is contained in the sample, or both.

12. In a method for detecting a target RNA of any one of claims 9 to 11, The above detectable signal is a fluorescence signal.

13. Methods for providing COVID-19 diagnostic information, including: (a) a process of mixing a novel CRISPR / Cas13 complex, a sample, and a detection reagent; Here, the above specimen is a specimen collected from a patient suspected of having COVID-19 infection. Here, the novel CRISPR / Cas13 complex comprises: Cas13 protein of sequence number 1; and guide RNA, Here, the above guide RNA has a structure in which the direct repeat portion of sequence number 2 and the guide domain are sequentially connected from the 5' end to the 3' end. The above guide domain comprises an RNA sequence selected from among SEQ ID NO: 37 to SEQ ID NO: 51 and SEQ ID NO: 52 to SEQ ID NO:

56. Here, the above detection reagent comprises a reporter, and the above reporter comprises a probe nucleic acid, When the above probe nucleic acid is cleaved, the above reporter generates a fluorescent signal; (b) a process of measuring the fluorescence signal in the above mixture; and, (c) A process of analyzing the above fluorescent signal to obtain information on whether the patient who provided the sample is infected with COVID-19.

14. In the method of providing COVID-19 diagnostic information in Article 13, The above (b) process is a process of measuring whether a fluorescence signal is generated; the intensity of the generated fluorescence signal; the change in the fluorescence signal over time; or any combination thereof.

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