WO2024167317A1

WO2024167317A1 - Novel crispr/cas12a composition and use thereof for detecting target nucleic acid

Info

Publication number: WO2024167317A1
Application number: PCT/KR2024/001849
Authority: WO
Inventors: 가시우나스게드리우스; 스티티리트미글; 이봉희; 이재원; 이재석; 바야르사이칸델거; 바야르사이칸고비저럴; 구옥재; 강현아
Original assignee: 주식회사 엔세이지
Priority date: 2023-02-10
Filing date: 2024-02-07
Publication date: 2024-08-15

Abstract

The present specification addresses the technical problem of providing a novel CRISPR/Cas12a system that can be used for detecting a target nucleic acid, wherein the previously unused CRISPR/Cas12a system having the Cas12a protein and guide RNA was discovered from a known DB through metagenome analysis and manufactured, and the auxiliary cleavage activity of the CRISPR/Cas12a system was verified. In order to solve the technical problem, the components of the CRISPR/Cas12a system, which was confirmed to be capable of recognizing a target nucleic acid and have auxiliary cleavage activity, are disclosed in detail in the present specification.

Description

Novel CRISPR/Cas12a composition and its use for detecting target nucleic acid

The technology disclosed in this specification is a technology for a CRISPR/Cas system, particularly a CRISPR/Cas12a system having a collateral cleavage function, and a method for detecting a target nucleic acid using the same.

The CRISPR/Cas12a system, also called the CRISPR/Cpf1 system, is a CRISPR/Cas system belonging to the A subtype of Type V of Class 2. The CRISPR/Cas12a system was first reported by Feng Zhang's research team at the Broad Institute. The CRISPR/Cas12a system has a simpler guide RNA structure than the CRISPR/Cas9 system, has a lower off-target cleavage rate, and thus enables more precise gene editing, and many researchers are actively studying its utilization methods. The inventors of the present specification discovered a CRISPR/Cas12a system having a Cas12a protein and guide RNA that has not been used previously through metagenome analysis from a known DB, and completed a novel CRISPR/Cas12a system that can be used for target nucleic acid detection based on this.

The technical task of this specification is to discover a CRISPR/Cas12a system having a Cas12a protein and guide RNA that has not been used previously through metagenome analysis from a known DB, to manufacture the same, and to verify the secondary cleavage activity function of the CRISPR/Cas12a system, thereby providing a novel CRISPR/Cas12a system that can be used for target nucleic acid detection.

To solve the above technical problems, the present specification discloses in detail components of a CRISPR/Cas12a system that can actually recognize a target nucleic acid and has a secondary cleavage activity. Specifically, the composition of a Cas12a protein having an amino acid sequence of No entry found and a program guide RNA capable of operating together with the Cas12a protein is disclosed.

The novel CRISPR/Cas12a system described herein is characterized by the activation of a collateral-editing function when a target nucleic acid is present, and this effect can be utilized to detect the presence of the target nucleic acid and further to measure how much of the target nucleic acid is contained.

Figure 1 shows the results of verifying the Cas12a protein manufactured according to Experimental Example 1.3 through ethidium bromide staining.

Figure 2 shows the results of analyzing the expression of Cas12a protein manufactured according to Experimental Example 1.4 using SDS-PAGE.

Figure 3 shows the results of confirming the expression of guide RNA through ethidium bromide staining.

Figure 4 shows the results of an in vitro target-specific nucleic acid cleavage activity experiment of the novel CRISPR/Cas12a system shown in [Table 2] of Experimental Example 2, with the reaction products visualized by Ethidium Bromide staining. The culture conditions are as follows: pH 7.9, 25℃.

Figure 5 shows the results of an in vitro target-specific nucleic acid cleavage activity experiment of the novel CRISPR/Cas12a system shown in [Table 2] of Experimental Example 2, with the reaction products visualized by Ethidium Bromide staining. The culture conditions are as follows: pH 7.9, 37℃.

Figure 6 shows the results of an in vitro target-specific nucleic acid cleavage activity experiment of the novel CRISPR/Cas12a system shown in Table 2 of Experimental Example 2, visualizing the reaction products with Ethidium Bromide staining. The culture conditions are as follows: pH 7.9, 60℃.

Figure 7 shows the results of an in vitro target-specific nucleic acid cleavage activity experiment of the novel CRISPR/Cas12a system shown in Table 2 of Experimental Example 2, visualizing the reaction products with Ethidium Bromide staining. The culture conditions are as follows: pH 6.5, 25℃.

Figure 8 shows the results of an in vitro target-specific nucleic acid cleavage activity experiment of the novel CRISPR/Cas12a system shown in [Table 2] of Experimental Example 2, with the reaction products visualized by Ethidium Bromide staining. The culture conditions are as follows: pH 6.5, 37℃.

Figure 9 shows the results of an in vitro target-specific nucleic acid cleavage activity experiment of the novel CRISPR/Cas12a system shown in [Table 2] of Experimental Example 2, with the reaction products visualized by Ethidium Bromide staining. The culture conditions are as follows: pH 6.5, 60℃.

Figure 10 shows the results of an in vitro target-specific nucleic acid cleavage activity experiment of the novel CRISPR/Cas12a system shown in Table 2 of Experimental Example 2, visualizing the reaction products with Ethidium Bromide staining. The culture conditions are as follows: pH 4.5, 25℃.

Figure 11 shows the results of an in vitro target-specific nucleic acid cleavage activity experiment of the novel CRISPR/Cas12a system shown in [Table 2] of Experimental Example 2, and the reaction products were visualized by Ethidium Bromide staining. The culture conditions were as follows: pH 4.5, 37℃.

Figure 12 shows the results of an in vitro target-specific nucleic acid cleavage activity experiment of the novel CRISPR/Cas12a system shown in [Table 2] of Experimental Example 2, with the reaction products visualized by Ethidium Bromide staining. The culture conditions are as follows: pH 4.5, 60℃.

Figure 13 shows the results of confirming the collateral cleavage function of the CRISPR/Cas12a system of NGS-01 of [Table 2] according to Experimental Example 3. Here, the horizontal axis represents the time point at which fluorescence was measured, and the vertical axis represents the normalized fluorescence signal intensity.

Figure 14 shows the results of confirming the collateral cleavage function of the CRISPR/Cas12a system of NGS-02 of [Table 2] according to Experimental Example 3. Here, the horizontal axis represents the time point at which fluorescence was measured, and the vertical axis represents the normalized fluorescence signal intensity.

Figure 15 shows the results of confirming the collateral cleavage function of the CRISPR/Cas12a system of NGS-03 of [Table 2] according to Experimental Example 3. Here, the horizontal axis represents the time point at which fluorescence was measured, and the vertical axis represents the normalized fluorescence signal intensity.

Figure 16 shows the results of confirming the collateral cleavage function of the CRISPR/Cas12a system of NGS-04 of [Table 2] according to Experimental Example 3. Here, the horizontal axis represents the time point at which fluorescence was measured, and the vertical axis represents the normalized fluorescence signal intensity.

Figure 17 shows the results of confirming the collateral cleavage function of the CRISPR/Cas12a system of NGS-05 of [Table 2] according to Experimental Example 3. Here, the horizontal axis represents the time point at which fluorescence was measured, and the vertical axis represents the normalized fluorescence signal intensity.

Figure 18 shows the results of confirming the collateral cleavage function of the CRISPR/Cas12a system of NGS-06 of [Table 2] according to Experimental Example 3. Here, the horizontal axis represents the time point at which fluorescence was measured, and the vertical axis represents the normalized fluorescence signal intensity.

Figure 19 shows the results of confirming the collateral cleavage function of the CRISPR/Cas12a system of NGS-07 in [Table 2] according to Experimental Example 3. Here, the horizontal axis represents the time point at which fluorescence was measured, and the vertical axis represents the normalized fluorescence signal intensity.

Figure 20 shows the results of confirming the collateral cleavage function of the CRISPR/Cas12a system of NGS-08 of [Table 2] according to Experimental Example 3. Here, the horizontal axis represents the time point at which fluorescence was measured, and the vertical axis represents the normalized fluorescence signal intensity.

Figure 21 shows the results of confirming the collateral cleavage function of the CRISPR/Cas12a system of NGS-09 in [Table 2] according to Experimental Example 3. Here, the horizontal axis represents the time point at which fluorescence was measured, and the vertical axis represents the normalized fluorescence signal intensity.

Figure 22 shows the results of confirming the collateral cleavage function of the CRISPR/Cas12a system of NGS-10 of [Table 2] according to Experimental Example 3. Here, the horizontal axis represents the time point at which fluorescence was measured, and the vertical axis represents the normalized fluorescence signal intensity.

Figure 23 shows the results of confirming the collateral cleavage function of the CRISPR/LbaCas12a system as a control group according to Experimental Example 3. Here, the horizontal axis represents the time point at which fluorescence was measured, and the vertical axis represents the normalized fluorescence signal intensity.

Figure 24 shows the results of confirming the expression results of the programmable guide RNA manufactured according to Experimental Example 2.

Hereinafter, the best mode for carrying out the invention is disclosed by way of example. This includes some embodiments of the invention disclosed in this specification, but not all embodiments. The embodiments described in this paragraph are merely examples, and the embodiments described in this paragraph should not be construed as the "best mode of the invention." Those skilled in the art will be able to think of many modifications and more desirable embodiments of the embodiments described in this paragraph, and such contents should also be considered to be included in the best mode for carrying out the invention.

The present disclosure provides a non-target nucleic acid cleavage method comprising:

Process of mixing Cas12a protein, guide RNA, and sample;

wherein the sample comprises a target nucleic acid and a non-target nucleic acid,

The above Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10; and SEQ ID NO: 109;

The above guide RNA comprises a guide domain and a scaffold,

The above guide RNA has scaffold and guide domains sequentially connected from the 5' end to the 3' end.

The above scaffold can allow the guide RNA to interact with the Cas12a protein to form a protein-RNA complex,

The above guide domain can hybridize with the target nucleic acid,

The above guide domain cannot hybridize with the non-target nucleic acid;

As a result of performing the above process, the Cas12a protein and the guide RNA combine to form a protein-RNA complex,

The above protein-RNA complex binds to the target nucleic acid through the guide domain, thereby activating the secondary cleavage function.

The non-target nucleic acid is cleaved by the protein-RNA complex.

Ribonucleoprotein, and the process of mixing samples;

The above ribonucleoprotein comprises Cas12a protein and guide RNA,

The above Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10,

The above guide RNA comprises a guide domain and a scaffold,

The above scaffold can enable the guide RNA to interact with the Cas12a protein to form a ribonucleoprotein,

The above guide domain can hybridize with the target nucleic acid,

The above guide domain cannot hybridize with the non-target nucleic acid;

The ribonucleoprotein binds to the target nucleic acid through the guide domain, thereby activating the secondary cleavage function.

The non-target nucleic acid is cleaved by the ribonucleoprotein.

In one embodiment, the Cas12a protein may be composed of an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10.

In one embodiment, the scaffold of the guide RNA may comprise an RNA sequence selected from SEQ ID NO: 11 to SEQ ID NO: 20.

In one embodiment, the scaffold of the Cas12a protein and the guide RNA can be a selected combination of:

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 1, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 11;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 2, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 12;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 3, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 13;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 4, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 14;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 5, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 15;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 6, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 16;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 7, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 17;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 8, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 18;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 9, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 19; or

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 10, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 20.

As an example,

The above guide RNA further comprises a stem-loop,

The above stem-loop is connected to the 5' end of the above scaffold,

The above stem-loop may comprise an RNA sequence of SEQ ID NO: 21.

As an example,

The target nucleic acid may be double-stranded DNA or single-stranded DNA, and the non-target nucleic acid may be double-stranded DNA or single-stranded DNA.

The present specification provides a method for detecting a target nucleic acid in a sample, comprising:

Process of mixing Cas12a protein, guide RNA, detection composition and sample;

Here, the detection composition comprises a probe nucleic acid,

The above probe nucleic acid produces a detectable signal upon cleavage,

The above guide RNA comprises a guide domain and a scaffold,

The above guide domain can hybridize with the target nucleic acid,

When the target nucleic acid is contained in the sample, the protein-RNA complex binds to the target nucleic acid through the guide domain, thereby activating the secondary cleavage function.

When the probe nucleic acid is cleaved by the protein-RNA complex, the detectable signal is measured.

By measuring the detectable signal, it is possible to determine whether the target nucleic acid is present in the sample and/or the amount of the target nucleic acid contained in the sample.

A process of mixing ribonucleoprotein, a composition for detection and a sample;

Here, the detection composition comprises a probe nucleic acid,

The above probe nucleic acid produces a detectable signal upon cleavage,

The above ribonucleoprotein comprises Cas12a protein and guide RNA,

The above guide RNA comprises a guide domain and a scaffold,

The above guide domain can hybridize with the target nucleic acid,

If the target nucleic acid is contained in the sample, the ribonucleoprotein binds to the target nucleic acid through the guide domain, thereby activating the secondary cleavage function.

When the probe nucleic acid is cleaved by the ribonucleoprotein, the detectable signal is measured,

Process of mixing the composition for detection and the sample;

Here, the detection composition comprises ribonucleoprotein and probe nucleic acid,

The above probe nucleic acid produces a detectable signal upon cleavage,

The above ribonucleoprotein comprises Cas12a protein and guide RNA,

The above guide RNA comprises a guide domain and a scaffold,

The above guide domain can hybridize with the target nucleic acid,

In one embodiment, the guide RNA further comprises a stem-loop, wherein the stem-loop is connected to the 5' end of the scaffold, and wherein the stem-loop comprises an RNA sequence of SEQ ID NO: 21.

In one embodiment, the target nucleic acid may be double-stranded DNA or single-stranded DNA, and the non-target nucleic acid may be double-stranded DNA or single-stranded DNA.

In one embodiment, the detectable signal generated when the probe nucleic acid is cleaved may be a fluorescent signal.

The present specification provides a composition for detecting a target nucleic acid, comprising:

Cas12a protein; programmable guide RNA; and probe nucleic acid;

Here, the Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10,

The above programmable guide RNA has a 5'-[direct repeat]-[guide domain]-3' structure,

The above direct repeat portion enables the guide RNA to interact with the Cas12a protein to form a ribonucleoprotein (RNP),

The above guide domain is artificially designed to target the target nucleic acid,

The above probe nucleic acid is one which, when cleaved, produces a detectable signal.

In one embodiment, the Cas12a protein and the programmable guide RNA may be combined to form an RNP.

In one embodiment, the direct repeat portion of the guide RNA may comprise an RNA sequence selected from SEQ ID NO: 11 to SEQ ID NO: 20.

As an example,

When the above Cas12a protein comprises an amino acid sequence of sequence number 1, the direct repeat portion of the guide RNA comprises an RNA sequence of sequence number 11,

When the above Cas12a protein comprises an amino acid sequence of sequence number 2, the direct repeat portion of the guide RNA comprises an RNA sequence of sequence number 12,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 3, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 13,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 4, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 14,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 5, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 15,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 6, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 16,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 7, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 17,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 8, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 18,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 9, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 19,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 10, the direct repeat portion of the guide RNA may comprise an RNA sequence of SEQ ID NO: 20.

In one embodiment, a composition for detecting a target nucleic acid, wherein the detectable signal generated when the probe nucleic acid is cleaved is a fluorescent signal.

The present specification provides a target nucleic acid detection kit comprising:

Cas12a protein; programmable guide RNA; and probe nucleic acid;

In one embodiment, the target nucleic acid detection kit comprises two or more containers,

The above Cas12a protein and the probe nucleic acid may be contained in different containers.

As an example,

The present disclosure provides a Cas12a composition comprising:

Cas12a protein or a nucleic acid encoding said Cas12a protein; and

A programmable guide RNA or a nucleic acid encoding said programmable guide RNA;

Here, the programmable guide RNA has a 5'-[direct repeat]-[guide domain]-3' structure,

The above direct repeat portion and the Cas12a protein are a selected combination of:

In one embodiment, the Cas12a composition may comprise the Cas12a protein and the programmable guide RNA.

In one example, the Cas12a protein and the programmable guide RNA may be combined to form a ribonucleoprotein.

The present specification provides a use of the Cas12a composition in the non-target nucleic acid cleavage method.

The present specification provides a use of the Cas12a composition in the method for detecting a target nucleic acid.

Hereinafter, with reference to the attached drawings, the content of the invention will be described in more detail through specific implementation examples and examples. It should be noted that the attached drawings include some implementation examples of the invention, but not all implementation examples. The content of the invention disclosed by this specification can be implemented in various ways and is not limited to the specific implementation examples described herein. It should be understood that these implementation examples are 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 implementation examples of the content of the invention disclosed herein. Therefore, the content of the invention disclosed herein is not limited to the specific implementation examples described herein, and modifications and other implementations thereof are also intended to be included within the scope of the claims.

용어의 정의Definition of terms

approximately

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.

NLS (Nuclear Localization Sequence)

The term "NLS" herein refers to a peptide of a certain length, or its sequence, which acts as a kind of "tag" by attaching to a protein that is the target of transport when transporting a material outside the nucleus into the nucleus by nuclear transport. Specifically, the NLS is an NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 88); an NLS from nucleoplasmin (e.g., a nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 89)); a c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 90) or RQRRNELKRSP (SEQ ID NO: 91); an hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 92); The sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 93) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 94) and PPKKARED (SEQ ID NO: 95) of myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 96) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 97) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 98) and PKQKKRK (SEQ ID NO: 99) of influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 100) of hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 101) of mouse Mx1 protein; The NLS sequence may be derived from, but is not limited to, the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 102) of human poly(ADP-ribose) polymerase; or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 103) of the steroid hormone receptor (human) glucocorticoid. The term "NLS" as used herein has all meanings that can be recognized by a person skilled in the art and may be appropriately interpreted depending on the context.

Amino acid sequence notation

Unless otherwise stated, when describing an amino acid sequence in this specification, the amino acid single-letter notation or three-letter notation is used, and is described in the direction from the N-terminal to the C-terminal. For example, in the case of notation as RNVP, it means a peptide in which arginine, asparagine, valine, and proline are sequentially connected in the direction from the N-terminal to the C-terminal. As another example, in the case of notation as Thr-Leu-Lys, it means a peptide in which threonine, leucine, and lysine are sequentially connected in the direction 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.

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).

Nucleic acid sequence notation

The symbols A, T, C, G, and U used herein are to be interpreted as having the meanings understood by a person skilled in the art. They may be appropriately interpreted as bases, nucleosides, or nucleotides on 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, and 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 including each of the above nucleosides.

target gene or target nucleic acid

The "target gene" or "target nucleic acid" used in this specification basically means a gene or nucleic acid within a cell that is the target of gene editing. The target gene or target nucleic acid may be used interchangeably and may refer to the same subject. Unless otherwise specified, the target gene or target nucleic acid may refer to both a unique gene or nucleic acid possessed by the target cell, or an externally derived gene or nucleic acid, and is not particularly limited as long as it can be the target of gene editing. The target gene or target nucleic acid may be single-stranded DNA, double-stranded DNA, and/or RNA. In addition, the above terms include all meanings that can be recognized by a person skilled in the art, and may be appropriately interpreted according to the context.

Target sequence

As used herein, the term "target sequence" refers to a specific sequence recognized by the CRISPR/Cas complex to cleave a target gene or target nucleic acid. The target sequence may be appropriately selected depending on the purpose. Specifically, the term "target sequence" refers to a sequence included in a target gene or target nucleic acid sequence, and a sequence having complementarity with a spacer sequence included in a guide RNA provided herein, or an engineered guide RNA. In general, the spacer sequence is determined in consideration of the sequence of the target gene or target nucleic acid and the PAM sequence recognized by the effector protein of the CRISPR/Cas system. The target sequence may refer only to a specific strand that complementarily binds to the guide RNA of the CRISPR/Cas complex, or may refer to the entire target double strand including the specific strand portion, which is appropriately interpreted depending on the context. In addition, the term includes all meanings that can be recognized by a person skilled in the art, and may be appropriately interpreted depending on the context.

vector

As used herein, "vector" refers to any material capable of transporting genetic material into a cell, unless otherwise specified. For example, a vector may be, but is not limited to, a DNA molecule containing the genetic material of interest, for example, a nucleic acid encoding an effector protein of a CRISPR/Cas system, and/or a nucleic acid encoding a guide RNA. The above terms include all meanings that can be recognized by a person skilled in the art, and may be appropriately interpreted according to the context.

배경기술 - CRISPR/Cas12a 시스템Background - CRISPR/Cas12a system

Overview of the CRISPR/Cas12a System

The CRISPR/Cas12a system is a CRISPR/Cas system that has been studied extensively along with the CRISPR/Cas9 system. The CRISPR/Cas12a system, like the CRISPR/Cas9 system, is known to have target-specific cleavage activity for double-stranded nucleic acids, and therefore has high utility for gene editing in eukaryotic cells. Compared to the CRISPR/Cas9 system, the CRISPR/Cas12a system has the following characteristics: 1) the Cas12a protein has only one type of nucleic acid cleavage domain (RuvC domain), 2) the guide RNA consists only of crRNA, and 3) it cleaves nucleic acids in a sticky end form that generates 4-5 nucleotide overhangs during gene cleavage. The CRISPR/Cas12a system and its components will be described in more detail below.

CRISPR/Cas12a classification

The CRISPR/Cas12a system belongs to the A subtype of the Class 2 type V CRISPR/Cas system, and is also called the CRISPR/Cpf1 system.

CRISPR/Cas12a system configuration and function

The CRISPR/Cas12a system comprises a Cas12a protein and a guide RNA therefor. The Cas12a protein and the guide RNA act as a complex and exhibit target-specific nucleic acid cleavage activity and secondary cleavage activity. The Cas12a protein recognizes a PAM sequence and functions to cleave a nucleic acid of a target sequence targeted by the guide RNA. The guide RNA functions to target a nucleic acid of a target sequence.

Structure of the Cas12a protein

The Cas12a protein can be broadly divided into REC (RECognition) lobe and NUC (NUClease) lob, and the NUC lob is further divided into the RuvC domain, NUC domain, WED domain, PI (PAM Interacting) domain, and BH (Bridge Helix) domain. The specific structure of the Cas12a protein has already been reported by several researchers and is described in detail in Paul et.al (CRISPR-Cas12a: Functional overview and applications. Biomedical Journal, Vol.43, Issue 1, pp.8-17, 2020). Among these, the WED II, WED III, REC1, and PI domains play an important role in allowing the Cas12a protein to recognize the PAM sequence. Meanwhile, the domains that play an important role in the Cas12a protein cleaving the nucleic acid of the target sequence are the RuvC domain and the NUC domain.

Structure of guide RNA

Unlike the guide RNA of the CRISPR/Cas9 system, the guide RNA of the CRISPR/Cas12a system is composed of a single RNA molecule, called crRNA. The guide RNA includes a direct repeat and a guide domain. Specifically, the guide RNA has the direct repeat and the guide domain sequentially connected in the direction from the 3' end to the 5' end. The direct repeat is a part involved in the guide RNA interacting with the Cas12a protein to form a complex, and the guide domain is a part that binds to the nucleic acid of the target sequence to enable the CRISPR/Cas12a system to exhibit target-specific cleavage activity and secondary cleavage activity.

Target-specific nucleic acid cleavage activity and PAM (Protospacer Adjacent Motif) of the CRISPR/Cas12a system

The CRISPR/Cas12a system has target-specific nucleic acid cleavage activity, but two conditions are required to exhibit this target-specific nucleic acid cleavage activity. First, there must be a base sequence of a certain length that the Cas12a protein can recognize in the nucleic acid. Second, there must be a sequence that can complementarily bind to the guide domain included in the guide RNA around the base sequence of the certain length. When the above two conditions are satisfied and 1) the Cas12a protein recognizes the base sequence of the certain length, and 2) the guide domain complementarily binds to a portion of the sequence around the base sequence of the certain length, nucleic acid cleavage activity is exhibited. At this time, the base sequence of a certain length recognized by the Cas12a protein is called a Protospacer Adjacent Motif (PAM) sequence.

The above PAM sequence is a unique sequence determined by the Cas12a protein. If the PAM sequence of the Cas12a protein is known, it can be used to design a CRISPR/Cas12a system that targets a nucleic acid of a predetermined target sequence around the PAM sequence.

Collateral cleavage activity of the CRISPR/Cas12a system

Some CRISPR/Cas12a systems have a collateral cleavage function in addition to the target-specific nucleic acid cleavage activity. This refers to the effect of randomly cleaving single-stranded nucleic acids in the vicinity when the CRISPR/Cas12a system recognizes the target nucleic acid and is activated. Specifically, researchers have reported that the CRISPR/Cas12a system has the activity of randomly cleaving single-stranded DNA in the vicinity when binding to the target DNA when targeting DNA (J. S. Chen, et al., CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360(2018) 436). By utilizing this collateral cleavage activity, it is possible to detect whether a target nucleic acid exists in a sample using the CRISPR/Cas12a system.

공지 데이터베이스로부터 신규 CRISPR/Cas12a 시스템 발굴Discovering a novel CRISPR/Cas12a system from the public database

In this specification, a novel CRISPR/Cas12a system discovered from a known database is disclosed. The inventors of this specification synthesized a Cas12a protein and a guide RNA therefor specified from microbial genome information of a known database, and prepared a CRISPR/Cas12a complex, and confirmed that it has a target-specific nucleic acid cleavage function and ancillary cleavage function, thereby completing the present invention.

Specifically, the inventors utilized data known to the National Center for Biotechnology Information (NCBI). First, based on the camel stomach metagenome sequencing data (Accession: PRJNA746430) known to the NCBI, they assembled it to obtain genome information on each microorganism contained in the camel stomach. Then, based on the homology information with the known Cas12a protein, they found genes encoding the CRISPR/Cas12a system among the genes of the microorganisms contained in the camel stomach, and specified the amino acid sequence of each component thereof (i.e., Cas12a protein, guide RNA, etc.). The inventors selected a candidate having an excellent collateral cleavage function among the CRISPR/Cas12a system candidates thus obtained, and thus reached the present invention.

신규 CRISPR/Cas12a 시스템A novel CRISPR/Cas12a system

Overview of the Novel CRISPR/Cas12a System

In this specification, 11 novel CRISPR/Cas12a systems, among the CRISPR/Cas12a systems discovered from the above-mentioned public database, are disclosed, which have been confirmed to have collateral cleavage activity. The inventors of the present specification directly synthesized the Cas12a protein and guide RNA of the CRISPR/Cas12a system discovered from the above-mentioned public database. The inventors used the synthesized Cas12a protein and guide RNA to identify the PAM sequence recognized by the Cas12a protein, and confirmed that the corresponding CRISPR/Cas12a system has collateral cleavage function, thereby completing the present invention. In addition, the guide RNA was optimized by adding an additional component (stem-loop).

The novel CRISPR/Cas12a system disclosed herein comprises a Cas12a protein and a programmable guide RNA as its components. The novel CRISPR/Cas12a system has a collateral cleavage function that randomly cleaves single-stranded DNA present in the vicinity when the programmable guide RNA binds to a target nucleic acid. Since the novel CRISPR/Cas12a system has a collateral cleavage function, it can be used for the purpose of detecting a target nucleic acid.

The CRISPR/Cas12a system disclosed herein is described in more detail below.

Cas12a protein - amino acid sequence

The novel CRISPR/Cas12a system disclosed herein comprises a novel Cas12a protein. The novel Cas12a protein is an ortholog of the Cas12a protein, which has been identified from a known database, but its function or use has not been elucidated in the existing literature. The novel Cas12a protein is a Cas12a protein represented by an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 10, respectively.

Cas12a protein - PAM sequence

The inventors of the present invention have experimentally discovered a PAM sequence that the novel Cas12a protein can recognize. The PAM sequence is described in the section “Background Art - CRISPR/Cas12a System.”

In one embodiment, the Cas12a protein represented by the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 10 can recognize the PAM sequence of 5'-TTTV-3'.

Programmable Guide RNA - Guide RNA Structure

Disclosed herein is a programmable guide RNA for a novel Cas12a protein. The guide RNA can form a complex with the novel Cas12a protein and target a nucleic acid having a target sequence. As described in the section “Background Art - CRISPR/Cas12a System”, unlike the CRISPR/Cas9 system, the guide RNA of the CRISPR/Cas12a system does not have a tracrRNA, so the guide RNA and crRNA can be used interchangeably in the present specification. The guide RNA includes a direct repeat and a guide domain, and may additionally include a stem-loop. The direct repeat is a structure that interacts with the Cas12a protein to allow the Cas12a protein and the guide RNA to form a complex. The guide domain is a structure that enables the CRISPR/Cas12a system to exhibit target-specific nucleic acid cleavage activity and/or ancillary cleavage activity. The above guide domain is designed to target a nucleic acid having a target sequence. The above stem-loop is a configuration that can increase the stability of the guide RNA.

Programmable Guide RNA - Direct Repeats

As described above, the direct repeat portion of the programmable guide RNA is a structure that interacts with the Cas12a protein and participates in forming a complex between the guide RNA and the Cas12a protein. The nucleic acid sequence of the direct repeat portion may vary depending on the type of the Cas12a protein. The direct repeat portion may also be referred to as a “scaffold.”

In one embodiment, in one embodiment, when the Cas12a protein included in the novel CRISPR/Cas12a system of the present specification is represented by the amino acid sequence of SEQ ID NO: 1, the direct repeat portion is represented by SEQ ID NO: 11.

In one embodiment, in one embodiment, when the Cas12a protein included in the novel CRISPR/Cas12a system of the present specification is represented by the amino acid sequence of SEQ ID NO: 2, the direct repeat portion is represented by SEQ ID NO: 12.

In one embodiment, in one embodiment, when the Cas12a protein included in the novel CRISPR/Cas12a system of the present specification is represented by the amino acid sequence of SEQ ID NO: 3, the direct repeat portion is represented by SEQ ID NO: 13.

In one embodiment, in one embodiment, when the Cas12a protein included in the novel CRISPR/Cas12a system of the present specification is represented by the amino acid sequence of SEQ ID NO: 4, the direct repeat portion is represented by SEQ ID NO: 14.

In one embodiment, in one embodiment, when the Cas12a protein included in the novel CRISPR/Cas12a system of the present specification is represented by the amino acid sequence of SEQ ID NO: 5, the direct repeat portion is represented by SEQ ID NO: 15.

In one embodiment, in one embodiment, when the Cas12a protein included in the novel CRISPR/Cas12a system of the present specification is represented by the amino acid sequence of SEQ ID NO: 6, the direct repeat portion is represented by SEQ ID NO: 16.

In one embodiment, in one embodiment, when the Cas12a protein included in the novel CRISPR/Cas12a system of the present specification is represented by the amino acid sequence of SEQ ID NO: 7, the direct repeat portion is represented by SEQ ID NO: 17.

In one embodiment, in one embodiment, when the Cas12a protein included in the novel CRISPR/Cas12a system of the present specification is represented by the amino acid sequence of SEQ ID NO: 8, the direct repeat portion is represented by SEQ ID NO: 18.

In one embodiment, in one embodiment, when the Cas12a protein included in the novel CRISPR/Cas12a system of the present specification is represented by the amino acid sequence of SEQ ID NO: 9, the direct repeat portion is represented by SEQ ID NO: 19.

In one embodiment, in one embodiment, when the Cas12a protein included in the novel CRISPR/Cas12a system of the present specification is represented by the amino acid sequence of SEQ ID NO: 10, the direct repeat portion is represented by SEQ ID NO: 20.

Programmable guide RNA - guide domain

The above programmable guide RNA comprises a guide domain. The guide domain is a structure capable of targeting a nucleic acid of a target sequence and is involved in activating a target-specific nucleic acid cleavage effect and/or a collateral cleavage effect of the CRISPR/Cas12a system. Specifically, the guide domain can complementarily bind to the target nucleic acid. In order to use the novel CRISPR/Cas12a system disclosed herein for the purpose of gene editing and/or nucleic acid detection of the target sequence, the guide domain is artificially designed to target a gene to be edited or a nucleic acid of the target sequence.

Programmable guide RNA - relationship between guide domain and target sequence

In order for the novel CRISPR/Cas12a system to cleave a specific nucleic acid, first of all, the guide domain must be able to bind to the nucleic acid of the target sequence. Therefore, the guide domain may have a sequence complementary to the target sequence, or in some cases, may have a sequence equivalent to the target sequence. The relationship between the above-mentioned guide domain and the target sequence varies depending on the type of nucleic acid having the target sequence and/or the location of the target sequence within the nucleic acid.

In one embodiment, when the nucleic acid of the target sequence is a single-stranded nucleic acid, the guide domain may have a sequence complementary to the target sequence. In another embodiment, when the nucleic acid of the target sequence is a double-stranded nucleic acid, and the target sequence is located on the same strand as the strand on which the PAM sequence of the CRISPR/Cas12a system is located, the guide domain may be a sequence equivalent to the target sequence. In another embodiment, when the nucleic acid of the target sequence is a double-stranded nucleic acid, and the target sequence is located on a different strand from the strand on which the PAM sequence of the CRISPR/Cas12a system is located, the guide domain may be a sequence complementary to the target sequence.

Programmable Guide RNA - Stem-Loop

The above programmable guide RNA may optionally further include a stem-loop. The stem-loop refers to an RNA fragment capable of forming a stem-loop. The stem-loop may be linked to the 5' end of the direct repeat portion of the guide RNA. The stem-loop may prevent the guide RNA from being easily degraded and may allow it to function more stably. When the above programmable guide RNA includes the stem-loop, the direct repeat portion and the stem-loop portion may be collectively referred to as a "scaffold."

CRISPR/Cas12a complex

Disclosed herein is a novel CRISPR/Cas12a complex. The novel CRISPR/Cas12a complex comprises a novel Cas12a protein and a programmable guide RNA in a complex, and has direct target-specific nucleic acid cleavage function and/or accessory cleavage function.

Here, the novel Cas12a protein is as described in the “Cas12a protein” section. In addition, the programmable guide RNA is as described in the “Programmable guide RNA” section.

Features of the new CRISPR/Cas12a system - collateral cutting function

When the novel CRISPR/Cas12a system binds to an external specific nucleic acid (i.e., a target nucleic acid), the aforementioned ancillary cleavage function is activated to randomly cleave nucleic acids present in the vicinity. Here, the “external specific nucleic acid” refers to a nucleic acid to which the guide domain of the programmable guide RNA can complementarily bind. Therefore, the novel CRISPR/Cas12a system can utilize the ancillary cleavage function by designing the guide domain of the programmable guide RNA according to the purpose.

Function of the novel CRISPR/Cas12a system - Target-specific nucleic acid cleavage function

The novel CRISPR/Cas12a system can bind to a specific external nucleic acid (i.e., a target nucleic acid) and cleave the nucleic acid. The target-specific nucleic acid cleavage function has been described above.

Here, the "external specific nucleic acid" refers to a nucleic acid to which the guide domain of the programmable guide RNA can complementarily bind. Therefore, the novel CRISPR/Cas12a system can utilize its target-specific cleavage function by designing the guide domain of the programmable guide RNA according to the purpose.

Uses of the Novel CRISPR/Cas12a System #1 - Non-targeted Nucleic Acid Cleavage

The novel CRISPR/Cas12a system described above is characterized by having a secondary cleavage function, and thus can be used for non-target nucleic acid cleavage purposes. Here, the term "non-target nucleic acid" means a nucleic acid having any sequence that cannot complementarily bind to the guide domain of the guide RNA of the novel CRISPR/Cas12a system.

For example, in the case where a non-target nucleic acid is to be cut by the novel CRISPR/Cas12a system, after the novel CRISPR/Cas12a system is added to the non-target nucleic acid, a nucleic acid having a specific sequence that can bind to the guide RNA of the novel CRISPR/Cas12a system (i.e., a target nucleic acid) is added to induce binding to the novel CRISPR/Cas12a system. When the target nucleic acid binds to the novel CRISPR/Cas12a system, its secondary cleavage function is activated, and as a result, the non-target nucleic acid can be cleaved.

Uses of the Novel CRISPR/Cas12a System #2 - Target Nucleic Acid Detection

By utilizing the ancillary cleavage function of the novel CRISPR/Cas12a system, the novel CRISPR/Cas12a system can be used for target nucleic acid detection purposes. Hereinafter, a composition used for target nucleic acid detection purposes and a method for target nucleic acid detection are described.

신규 CRISPR/Cas12a 시스템을 포함하는 표적 핵산 검출용 조성물Composition for detecting target nucleic acid comprising novel CRISPR/Cas12a system

Overview of Composition for Detection of Target Nucleic Acid

The present specification discloses a composition for detecting a target nucleic acid. The composition for detecting a target nucleic acid comprises a detecting agent comprising a novel CRISPR/Cas12a complex and a probe nucleic acid, and optionally may further comprise a composition for signal amplification. The composition for detecting a target nucleic acid is designed to detect a nucleic acid having a target sequence by utilizing the secondary cleavage activity of the CRISPR/Cas12a system.

Composition for target nucleic acid detection #1 - Novel CRISPR/Cas12a complex

The composition for detecting the target nucleic acid comprises a novel CRISPR/Cas12a complex. The novel CRISPR/Cas12a composition is as described in the section “Novel CRISPR/Cas12a System”.

Composition for detecting target nucleic acid #2 - Detecting agent comprising probe nucleic acid

The composition for detecting the target nucleic acid comprises a detecting agent comprising a probe nucleic acid. Here, the probe nucleic acid may be DNA and/or RNA. The probe nucleic acid may be a DNA and/or RNA with an additional component bound thereto. The detecting agent is characterized in that when the probe nucleic acid is cleaved, a detectable signal is generated. The detectable signal that the probe nucleic acid can generate is not particularly limited as long as it can confirm that the probe nucleic acid has been cleaved. Therefore, a person skilled in the art can appropriately select other components of the probe nucleic acid and the detecting agent with reference to known techniques.

Composition for target nucleic acid detection #3 - Probe nucleic acid sequence example

In one embodiment, the probe nucleic acid sequence may be one or more selected from the following:

5'-TTATT-3'; 5'-TTATTATT-3'; 5'-TTTTTTTT-3'; 5'-AAAAAAAA-3'; 5'-GGGGGGGG-3'; 5'-CCCCCCCC-3'; TAGCATGTCA (SEQ ID NO: 85); CCAAGGTTCCAAGGTTCCAAGGTTC (SEQ ID NO: 86); and CAGTCAGTCAGTCAGTCAGTCAGTCAGTC (SEQ ID NO: 87).

Composition for target nucleic acid detection #4 - Additional composition for signal amplification

The composition for detecting the target nucleic acid may include an additional component for signal amplification. This is not limited to anything else as long as it can directly/indirectly utilize the secondary cleavage function of the CRISPR/Cas12a complex and amplify a detectable signal generated from the detection agent. Accordingly, a person skilled in the art can appropriately select an additional component for signal amplification by referring to known techniques.

Examples of detectable signals

The detectable signal that the above probe nucleic acid can generate is not particularly limited as long as it can confirm that the probe nucleic acid has been cleaved.

In one embodiment, the detectable signal may be a fluorescent signal. In one embodiment, the fluorescent signal may not be detected before cleavage of the probe nucleic acid, but may be detected after cleavage of the probe nucleic acid. In another embodiment, the fluorescent signal may be stronger after cleavage of the probe nucleic acid than before cleavage of the probe nucleic acid. In another embodiment, the fluorescent signal may be weaker after cleavage of the probe nucleic acid than before cleavage of the probe nucleic acid. In another embodiment, the fluorescent signal may be detected before cleavage of the probe nucleic acid, but may not be detected after cleavage of the probe nucleic acid. Here, the configuration for generating the fluorescent signal may be a configuration appropriately selected by a person skilled in the art known in the art.

표적 핵산 검출용 키트Kit for detection of target nucleic acid

The present specification discloses a kit for detecting a target nucleic acid. The kit for detecting a target nucleic acid comprises a novel CRISPR/Cas12a protein, a programmable guide RNA, a detecting agent comprising a probe nucleic acid, and optionally, a signal amplification composition. The kit for detecting a target nucleic acid may comprise one or more vessels, which may be, in some cases, a well; a plate; a reactor; a vial; a test-tube; a cassette; and/or media. The novel CRISPR/Cas12a protein, the programmable guide RNA, the detecting agent comprising a probe nucleic acid, and optionally, a signal amplification composition of the kit for detecting a target nucleic acid may be contained in separate vessels. Two or more of the aforementioned components of the kit for detecting a target nucleic acid may be contained in the same vessel. Two or more of the aforementioned components of the kit for detecting a target nucleic acid may be contained in different vessels. For example, among the above-mentioned components of the kit for detecting the target nucleic acid, the novel CRISPR/Cas12a protein may be contained in a different container from the detection agent containing the probe nucleic acid.

표적 핵산 검출 방법Target nucleic acid detection method

Overview of Target Nucleic Acid Detection Methods

The present specification discloses a target nucleic acid detection method using a novel CRISPR/Cas12a system. The target nucleic acid detection method is a method for finding out information such as the presence or absence and the amount of a target nucleic acid having a specific nucleic acid sequence in a sample. The target nucleic acid detection method utilizes the secondary cleavage function of the novel CRISPR/Cas12a system. Since the secondary cleavage function is activated only when the novel CRISPR/Cas12a system binds to a target nucleic acid, by utilizing this property, it is possible to determine whether a target nucleic acid exists and how much of it exists.

In one embodiment, the method for detecting a target nucleic acid comprises a process of mixing a sample, a novel CRISPR/Cas12a complex, and a detection agent comprising a probe nucleic acid. Each component appearing in the method is described in more detail below.

Sample

The above sample means a specimen that is expected to contain the target nucleic acid or is not expected to contain the target nucleic acid. The above sample is taken from the subject's body and is not otherwise limited as long as it is likely to contain the target nucleic acid.

In one embodiment, the sample can be in a liquid, solid, and/or gaseous form. In another embodiment, the sample can be a mixture of two or more substances. In another embodiment, the sample can be a body tissue and/or a bodily fluid of the subject. Here, the subject can be a human, a non-human animal, and/or a plant. The bodily fluid can be human blood, sweat, urine, and/or other secretions.

Novel CRISPR/Cas12a complex

The novel CRISPR/Cas12a complex is as described in the above paragraph “Novel CRISPR/Cas12a System”. The present target nucleic acid detection method encompasses not only the case where the novel CRISPR/Cas12a complex is formed at the time when the target nucleic acid detection method starts to be performed, but also the case where the novel CRISPR/Cas12a protein and the programmable guide RNA do not form a complex but exist separately at the time when the target nucleic acid detection method starts to be performed, and then the novel CRISPR/Cas12a complex is formed at a specific time. Here, the programmable guide RNA of the novel CRISPR/Cas12a complex targets the target nucleic acid of the target nucleic acid detection method. At this time, the programmable guide RNA can complementarily bind to the target nucleic acid. When the novel CRISPR/Cas12a complex binds to the target nucleic acid, the collateral cleavage activity of the novel CRISPR/Cas12a complex is activated.

A detection agent comprising a probe nucleic acid

The detection agent comprising the above probe nucleic acid is as described in the above paragraph “Composition for detecting target nucleic acid”. Here, when the probe nucleic acid is cleaved, the detection agent comprising the probe nucleic acid generates a detectable signal.

Mix each component

The target nucleic acid detection method provided herein includes a process of mixing a sample, a novel CRISPR/Cas12a complex, and a detection agent including a probe nucleic acid. The target nucleic acid detection method utilizes a phenomenon in which the novel CRISPR/Cas12a complex binds to the target nucleic acid and activates a secondary cleavage function. Therefore, the mixing process can be implemented in various forms, as long as 1) when a target nucleic acid exists in the sample, the target nucleic acid can bind to the novel CRISPR/Cas12a complex, and 2) the CRISPR/Cas12a complex with the secondary cleavage function activated can interact with the detection agent including the probe nucleic acid. For example, the mixing process can be a process in which the three components are mixed simultaneously, or can be a process in which the three components are mixed sequentially with a time difference. For another example, the mixing process can be performed in a state in which two of the three components are already mixed.

Kit available

The above target nucleic acid detection method can be performed in one or more kits. In this case, the mixing of each component occurs within the kit, and the mixing process includes a process of providing each component to the kit. The kit is not particularly limited as long as each component can be appropriately mixed, and a person skilled in the art can appropriately select it by referring to known techniques. For example, the kit can be at least one selected from a vessel; a well; a plate; a reactor; a vial; a test-tube; a cassette; and media, but is not limited thereto.

Signal detection and information acquisition about target nucleic acids

As a result of performing the above method, a detectable signal is generated from the detector, and the detectable signal reflects information about the target nucleic acid. Therefore, the method may additionally include a process of measuring the detectable signal to derive information about the target nucleic acid.

Information about the target nucleic acid may include, but is not limited to, for example, the presence or absence of a predetermined target sequence in the sample, the amount of the predetermined target sequence in the sample, the concentration of the predetermined target sequence in the sample, the count of the predetermined target sequence in the sample, and the expression level of the predetermined target sequence in the sample.

Possible embodiments of the invention

Hereinafter, possible embodiments of the invention provided in this specification are listed. The following embodiments provided in this paragraph are merely examples of the invention. Therefore, the invention provided in this specification should not be construed as being limited to the following embodiments. The brief descriptions described with the embodiment numbers are also only for the convenience of distinguishing between each embodiment and should not be construed as limitations to the invention disclosed in this specification.

신규 Cas12a 단백질Novel Cas12a protein

Example 1, novel Cas12a protein

A novel Cas12a protein represented by an amino acid sequence selected from the following:

HQLLEDVISVGNENLTPSYIVLEKLSREMKRGRQKIEKQVYQKFELALASKLGFYVNKDIEEEKPGSIQMPLQFVPPVNTFDQIDKKDSFGIMLYTRANYTSVTDPLTGWRKTIYITNGNNEDILKELTEAFDDIFFDGVDYVFSYVEKNSGQRWNIYSGNHGKSLDRFEYNKDTKCYESYNIVEVLDVLFADFNKSSSLLE QMRRGVKLKKAEENRTAYDSLRKAINMIQQIRNTGNEIKDNNFLQSPIRKDGKHFDTRNANDFRDLNLHFIKDADANGAYNIARKGLIMDAHYKYWMEQGKPNVKKKNKKEKEVSALSLYVSDREWDMWLLNREMWEKHLSEFSLRKQD (SEQ ID NO: 1); QVYQNFEKALITKLNYLVFKEIQDYNTAGSVLIGYQLTDKFVSFEKMGKQTGALFYVPAAYTSKIDPTTGFANLFNTRKCTNVESRKVFLMTFDAIKYNAVRKSFAFSFDYAKFQTSAKSFQNKWTVYTSQKRLVFNPLTRQDTAVNPTQMIVDALNKRGVSLSDEFDLKAYLQNTDAIKANAEFFKTIFIAL EYTLQMRNSNSSSGEDYIESPVLNKSGEFFRSSEDNPALPIDADANGAYHIAMKGLFLLLNVFNQGKKDLKIEHQAWFEFMQKRNM (SEQ ID NO: 2); GFINQIQFSTKSTLSAKRDLLCNIDSIRYDPSEECYIFRIDYTKVGDRTKEFKNVWDIYTRGDRIVYSTKERCDKHLHPTEIMTKALESAGIGKEGELKEKICSADSKVIEAVVYALDLTLRLRNEDRETDYILSPVRNADGKFFCTLDGDLSLPLDSDANGAYNIALKGLLMLKMTEESNGPDADKYDISLVTNSDWLTFCQTGHRTWKS (SEQ ID NO: 3); FVNLFGSRQLTYTSVSAAKAFFGTMREIGYDAQRDCYRFAFRYSDYDLYQQDHRDSWTVFTAGKGRIAHTVKNGRHTFEEIDVTERMKELFQRFGLDPYAADLRGAIDAADKKDFFSELLWLFKVTVQLRYEDSEQDFILSPIEKDGRFFDSRQAKKDEPQDGDANGAYHIALQGLRLARERIKDGRVQKDPKGKQTLNWLRFA QRKDYLE (SEQ ID NO: 4); LTNAFESFEKMGKQNGFIFYVPAYLTSKIDPTTGFADLLHPSSKQSKESMRDFVGRFDSITFNKTENYFEFELDYNKFPRCNTDYRKKWTVCTYGSRIKTFRNPEKNSEWDNKTVELTPAFMALFEKYSIDVNGDIKAQIMSVDKKDFFVELIGLLRLTLQMRNSETGKVDRDYLISPVKNSEGVFYNSDDYKGIENASLPKDADANGAYNI ARKGLWIIEQIKACENDAELNKIRLAISNAEWLEYAQKK (SEQ ID NO: 5); KYESVEKAKSVIESFDFIRYNKGSDMFEIGLDYSKTERGITSYKKKWTICTNGNRIRIFRNKAKNNEWDDETVYLTEAFKKLFNEYGIDISADIKEQLLAKTDKDFFNGFINLLSLTLQMRNSIPNSDTDYLISPVRGKDGKFYCSDDYQGKNALLPENADANGAYNIARKALWCIEQIKAADDDKLNEVKLAIKNKEWLEYAQKG (SEQ ID NO: 6); LEDLNSEMKRGRQKIEKQVYQNLELALAKKFNFVVDKEAKQGELGSVSKALQLTPPVNNYQDIEGKKQFGVMLYTRANYTSITDPTTGWRKTIYIKNGKDEEIRKQILEKFTDFGFAGKDYYFEYTEENAGHTWRMYSGKNGEPLPRFQNKKQMEQDKNIWVPEQINVKEILDKLFANFDKGRSFKEQVNE EVKLEKIEGRSETAWQSLRYALNMIQQIRNSGVEKTDDNFLYSPVRNENGEHFDTRNHENNGDLSPIVDADANGAFNIARKGLIMDAHIKHWINSGRPKIKKDGKETPDLDLFISDREWDLWLLDREEWKKYLPVFASKSKKDADKTTSTKSPKRKKK (SEQ ID NO: 7); ENNAIVVLEDLNFGFKRGRFCVERQVYQKFEKMLIDKLNYLVFKNKPEGDVGGVLKGYQLAEKFDSFQKLGKQSGFLFYIPAAYTSKIDPTTGFANLFNMTELTSAEKKKEFLSHFEDITYDGKNDRFLFSFDYKNFKCFQTDYIKKWTVYSQGKRIVYDKESKSAKEISPVEIIKAALAKQNIALTDQLDVLSAINSAEASPK SASFFGDICYAFEKTLQMRNSIPNTDEDYLVSPVLNKKGEFYDSRSCGDTLPKNADANGAYHIALKGLYLIKNVFDAGGKDLKISHEDWFKFAQSRNS (SEQ ID NO: 8); LLHPKYTSVEESHNLFGNFDDIRYNAVTDMFEFDLDYSKFPKSKADFKKKWTVCTNSDRIMTFRNPAKNSEWDNKRVILTDEFKKLFEEFGVDYKSNLREKILGISNPDFNKRLVKFLALTLQMRNSVTGSTNPEDDYLISPVRDKNGVFYDSRNFIGNDAVLPADADANGAYNIARKGLWAIDVIKSTSSDMLDKADLSISNAEWLEYVQK( SEQ ID NO: 9); and

GMLHALQLANKFKSFSKMGKQCGCLFYIPAWNTSKIDPVTGFVNLLDTRYESVKASKEFFSHFDCITFNATMDWFEFSFDYDNFHKKAEGTQTQWTVFSRGSRIHTFRNPEKNNQWDDEEINLTDKFKELFAKYSINYRGNLKDAMQDSNEATFFKELMGLMKLLMQMRNSKANSEIDYMLSPVSDKNGNFYDSRVCGGSLP ENADANGAYNIARKGLMLIRNIQAAPDDKKPSLTITNKDWLCFAQTKPYLED (SEQ ID NO: 10).

Example 2, including similar sequences

An amino acid sequence selected from among SEQ ID NOs: 1 to 10 is 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, or about 99% or more identical, same, or matched, and/or a Cas12a protein having an equivalent sequence;

Here, the meaning of the term "medicine" is as defined in the "medicine" section of the "Definitions of Terms".

Example 3, PAM sequence

In any one of Examples 1 to 2, the Cas12a protein can recognize a PAM (Protospacer Adjacent Motif) sequence of 5'-TTTV-3'.

Example 4, including NLS

In any one of Examples 1 to 3, the Cas12a protein further comprises at least one NLS (Nuclear Localization Signal) at the N-terminus and/or C-terminus of the selected amino acid sequence.

Example 5, NLS limited

In Example 4, the NLS is each independently PKKKRKV (SEQ ID NO: 88), KRPAATKKAGQAKKKK (SEQ ID NO: 89), PAAKRVKLD (SEQ ID NO: 90), RQRRNELKRSP (SEQ ID NO: 91), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 92), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 93), VSRKRPRP (SEQ ID NO: 94), PPKKARED (SEQ ID NO: 95), PQPKKKPL (SEQ ID NO: 96), SALIKKKKKMAP (SEQ ID NO: 97), DRLRR (SEQ ID NO: 98), PKQKKRK (SEQ ID NO: 99), RKLKKKIKKL (SEQ ID NO: 100), At least one selected from REKKKFLKRR (SEQ ID NO: 101), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 102), and RKCLQAGMNLEARKTKK (SEQ ID NO: 103).

프로그램 가능한 가이드 RNAProgrammable Guide RNA

Example 6, Programmable Guide RNA

Programmable guide RNA containing:

A direct repeat (DR) represented by a nucleic acid sequence selected from among UAAUUUCUACUAUUCGUAGAU (SEQ ID NO: 11), UUAAUUUCUACUUUCGUAGAU (SEQ ID NO: 12), CAAUUUCUACUUUCAGUAGAU (SEQ ID NO: 13), UUAAUUUCUACUUGUGUAGAU (SEQ ID NO: 14), UAAAUUUCUACUAUUGUAGAU (SEQ ID NO: 15), AUAAUUUCUACUAUUGUAGAU (SEQ ID NO: 16), AAAAUUUCUACUAUUGUAGAU (SEQ ID NO: 17), AAAAUUUCUACUCUUGUAGAU (SEQ ID NO: 18), AUAAUUUCUACUGUUGUAGAU (SEQ ID NO: 19), and AUAAUUUCUACUGUUGUAGAU (SEQ ID NO: 20); and

Guide domain,

Here, the direct repeat portion can interact with the Cas12a protein to allow the guide RNA to form a complex with the Cas12a protein,

The above guide domain is artificially designed to target a target nucleic acid of a predetermined target sequence and/or to be able to hybridize with a target nucleic acid of the predetermined target sequence.

The above direct repeat portion and the above guide domain are sequentially connected in the 5' end to the 3' end direction.

Example 7, with stem loop

In Example 6,

The above programmable guide RNA further comprises a stem-loop,

The above stem-loop is connected to the 5' end of the above direct repeat.

Example 8, Stem-loop sequence limitation

In Example 7, the stem-loop has a nucleic acid sequence of GUUUCAAAGAUUAAAU (SEQ ID NO: 21).

Example 9, limiting the length of the guide domain

A programmable guide RNA according to any one of Examples 6 to 8, wherein the guide domain is 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt in length.

Example 10, Relationship between guide domain and target sequence

A programmable guide RNA according to any one of embodiments 6 to 9, wherein the guide domain has a sequence equivalent to a nucleic acid of the predetermined target sequence or a sequence complementary to a nucleic acid of the predetermined target sequence.

Example 11, Relationship between guide domain and target sequence

In any one of Examples 6 to 10,

The nucleic acid of the above predetermined target sequence is a double-stranded nucleic acid,

The double-stranded nucleic acid comprises a first strand comprising a nucleic acid of a target sequence and a second strand having a nucleic acid sequence complementary to the first strand,

The above guide domain can complementarily bind to the first strand or complementarily bind to the second strand.

Example 12, including PAM sequence

In Example 11,

When the above guide domain complementarily binds to the first strand, the second strand comprises a 5'-TTTV-3' sequence,

When the above guide domain complementarily binds to the second strand, the first strand comprises a 5'-TTTV-3' sequence.

Example 13, including mismatch

In Example 11, the guide domain has 0, 1, 2, 3, 4, or 5 mismatches compared to the sequence of the complementarily binding strand.

Example 14, Target Nucleic Acid Specification

In any one of Examples 6 to 13, the target nucleic acid is selected from double-stranded DNA, single-stranded DNA, and RNA.

프로브 핵산을 포함하는 검출제A detection agent comprising a probe nucleic acid

Example 15, Detector Comprising Probe Nucleic Acid

A detecting agent comprising a probe nucleic acid, which produces a detectable signal when the probe nucleic acid is cleaved.

Example 16, Probe Nucleic Acid Composition

In Example 15, the probe nucleic acid comprises a nucleic acid and one or more molecules of a signal generator.

Example 17, Probe Nucleic Acid Sequence Definition

In any one of Examples 15 to 16, the nucleic acid sequence of the probe nucleic acid is at least one selected from the following:

Example 18, addition of signal amplifying agent

In any one of Examples 15 to 17, the detector further comprises a signal amplifying substance.

Example 19, Limiting the detectable signal

In any one of Examples 15 to 18, the detectable signal is a fluorescence signal.

Example 20, Nucleic Acid Specification

In any one of Examples 15 to 19, the nucleic acid contained in the probe nucleic acid is at least one selected from single-stranded DNA, double-stranded DNA, and RNA.

신규 CRISPR/Cas12a 복합체Novel CRISPR/Cas12a complex

Example 21, Novel CRISPR/Cas12a Complex

A novel CRISPR/Cas12a complex containing:

The Cas12a protein of any one of Examples 1 to 5; and

A programmable guide RNA of any one of Examples 6 to 14,

Here, the Cas12a protein and the programmable guide RNA are combined to form a ribonucleoprotein,

The above CRISPR/Cas12a complex has a collateral cleavage function,

When the CRISPR/Cas12a complex binds to a target nucleic acid targeted by the guide domain of the programmable guide RNA, the secondary cleavage function is activated.

Example 22, Cas12a protein and direct repeat matching

In Example 21, the direct repeat portion of the Cas12a protein and the programmable guide RNA is a combination selected from the following:

A Cas12a protein having an amino acid sequence of SEQ ID NO: 1 and a direct repeat portion having a nucleic acid sequence of SEQ ID NO: 11;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 2 and a direct repeat portion having a nucleic acid sequence of SEQ ID NO: 12;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 3 and a direct repeat portion having a nucleic acid sequence of SEQ ID NO: 13;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 4 and a direct repeat portion having a nucleic acid sequence of SEQ ID NO: 14;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 5 and a direct repeat portion having a nucleic acid sequence of SEQ ID NO: 15;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 6 and a direct repeat portion having a nucleic acid sequence of SEQ ID NO: 16;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 7 and a direct repeat portion having a nucleic acid sequence of SEQ ID NO: 17;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 8 and a direct repeat portion having a nucleic acid sequence of SEQ ID NO: 18;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 9 and a direct repeat portion having a nucleic acid sequence of SEQ ID NO: 19; and

A Cas12a protein having an amino acid sequence of SEQ ID NO: 10 and a direct repeat portion having a nucleic acid sequence of SEQ ID NO: 20.

Example 23, sequence similar to direct repeat

A Cas12a protein having an amino acid sequence of SEQ ID NO: 1 and a direct repeat portion having a sequence similar to the nucleic acid sequence of SEQ ID NO: 11;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 2 and a direct repeat portion having a sequence similar to the nucleic acid sequence of SEQ ID NO: 12;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 3 and a direct repeat portion having a sequence similar to a nucleic acid sequence of SEQ ID NO: 13;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 4 and a direct repeat portion having a sequence similar to the nucleic acid sequence of SEQ ID NO: 14;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 5 and a direct repeat portion having a sequence similar to the nucleic acid sequence of SEQ ID NO: 15;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 6 and a direct repeat portion having a sequence similar to the nucleic acid sequence of SEQ ID NO: 16;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 7 and a direct repeat portion having a sequence similar to the nucleic acid sequence of SEQ ID NO: 17;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 8 and a direct repeat portion having a sequence similar to the nucleic acid sequence of SEQ ID NO: 18;

A Cas12a protein having an amino acid sequence of SEQ ID NO: 9 and a direct repeat portion having a sequence similar to a nucleic acid sequence of SEQ ID NO: 19; and

A Cas12a protein having an amino acid sequence of SEQ ID NO: 10 and a direct repeat portion having a sequence similar to a nucleic acid sequence of SEQ ID NO: 20;

Here, a sequence similar to the nucleic acid sequence of the above SEQ ID NOs: 11 to 20 means a sequence that is 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 identical to the above nucleic acid sequence, Same, matched, and/or equivalent nucleic acid sequences, wherein the term "about" has the same meaning as defined in the "About" section of the Definitions section.

신규 CRISPR/Cas12a 벡터Novel CRISPR/Cas12a vector

Example 24, Expression Vector

Expression vectors of novel CRISPR/Cas12a system components comprising one or more of the following:

A nucleic acid encoding the Cas12a protein of any one of Examples 1 to 5; and

A nucleic acid encoding a programmable guide RNA of any one of Examples 6 to 14.

Here, the expression vector can enable the Cas12a protein and the guide RNA to be expressed intracellularly and/or extracellularly,

As a result of the above expression, a CRISPR/Cas12a complex of any one of Examples 21 to 23 can be formed,

The above CRISPR/Cas12a complex has a collateral cleavage function,

Example 25, Cas12a protein and direct repeat matching

In Example 24, the direct repeat portion of the Cas12a protein and the programmable guide RNA is a combination selected from the following:

Example 26, sequence similar to direct repeat

A Cas12a protein having an amino acid sequence of SEQ ID NO: 9 and a direct repeat portion having a sequence similar to the nucleic acid sequence of SEQ ID NO: 19;

Here, the sequence similar to the nucleic acid sequence of the above SEQ ID NOs: 11 to 20 and SEQ ID NO: 110 means a sequence similar to the nucleic acid sequence of 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 It means a nucleic acid sequence that is more than 99% identical, same, matched, and/or equivalent, wherein the term "about" has the same meaning as defined in the "About" section of the Definition of Terms.

Example 27, Cas12a encoding nucleic acid limitation

In any one of Examples 24 to 26, the nucleic acid encoding the Cas12a protein has a nucleic acid sequence selected from SEQ ID NO: 32 to SEQ ID NO: 41.

Example 28, Additional Configuration

An expression vector according to any one of Examples 24 to 27, characterized in that the expression vector further comprises at least one selected from the following:

Promoter; enhancer; intron; polyadenylation signal; Kozak consensus sequence; internal ribosome entry site (IRES); splice acceptor; 2A sequence and/or replication origin.

Example 29, Promoter Only

An expression vector according to any one of embodiments 24 to 28, wherein the expression vector comprises a promoter, wherein the promoter is at least one selected from the group consisting of the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), the rous sarcoma virus (RSV) promoter, the human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1;31(17)), the human H1 promoter (H1), and 7SK.

Example 30, replication origin limitation

An expression vector according to any one of Examples 24 to 29, wherein the expression vector comprises an origin of replication, and the origin of replication is at least one selected from an f1 origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, and a BBV origin of replication.

Example 31, non/viral vector

An expression vector according to any one of Examples 24 to 30, characterized in that the expression vector is a non-viral vector or a viral vector.

Example 32, virus type limitation

In Example 31, an expression vector selected from retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus and herpes simplex virus.

Example 33, Nonviral vector type limitation

In Example 31, an expression vector selected from a plasmid, a phage, naked DNA, a DNA complex, mRNA, and a PCR amplicon.

Example 34, Use of Vectors

In any one of Examples 24 to 33, the vector is for expressing at least one selected from the Cas12a protein and the programmable guide RNA.

신규 CRISPR/Cas12a 조성물Novel CRISPR/Cas12a compositions

Example 35, novel CRISPR/Cas12a composition

Novel CRISPR/Cas12a compositions comprising:

The Cas12a protein of any one of Examples 1 to 5, or a nucleic acid encoding said Cas12a protein; and

A programmable guide RNA of any one of Examples 6 to 14, or a nucleic acid encoding said guide RNA;

Here, the Cas12a protein and the programmable guide RNA can interact to form a CRISPR/Cas12a complex,

The above CRISPR/Cas12a complex has a collateral cleavage function,

Example 36, Cas12a protein and direct repeat matching

In Example 35, the direct repeat portion of the Cas12a protein and the programmable guide RNA is a combination selected from the following:

Example 37, sequence similar to direct repeat

Example 38, including complex

In any one of Examples 35 to 37, the CRISPR/Cas12a composition comprises the Cas12a protein and the programmable guide RNA in the form of a CRISPR/Cas12a complex of any one of Examples 21 to 23.

Example 39, including vectors

In any one of Examples 35 to 38, the CRISPR/Cas12a composition comprises a nucleic acid encoding the Cas12a protein and a nucleic acid encoding the programmable guide RNA in the form of an expression vector of any one of Examples 24 to 34.

비표적 핵산 절단 방법Non-target nucleic acid cleavage method

Example 40, Non-target Nucleic Acid Cleavage Method

Non-target nucleic acid cleavage methods comprising:

A process of mixing, reacting, blending or admixing the CRISPR/Cas12a composition and the sample of any one of Examples 35 to 39, adding the CRISPR/Cas12a composition to the sample, and/or adding the sample to the CRISPR/Cas12a composition;

The guide domain of the programmable guide RNA of the CRISPR/Cas12a composition can hybridize and/or complementarily bind to the target nucleic acid,

The guide domain of the programmable guide RNA of the CRISPR/Cas12a composition is such that the guide domain cannot hybridize with and/or cannot complementarily bind to the non-target nucleic acid.

표적 핵산 검출용 조성물Composition for detecting target nucleic acid

Example 41, Composition for detecting target nucleic acid

Composition for detecting target nucleic acid comprising:

A CRISPR/Cas12a composition of any one of Examples 35 to 39; and

A detection agent comprising a probe nucleic acid according to any one of Examples 15 to 20.

표적 핵산 검출용 키트Kit for detection of target nucleic acid

Example 42, Kit for detection of target nucleic acid

Kit for detection of target nucleic acids comprising:

The Cas12a protein of any one of Examples 1 to 5;

A programmable guide RNA of any one of Examples 6 to 14; and

A detector according to any one of Examples 15 to 20.

Example 43, comprising one or more containers

In Example 42, the kit for detecting the target nucleic acid comprises one or more vessels; wells; plates; reactors; vials; test tubes; cassettes; and/or media.

Example 44, Limited inclusion relationship within the container

In Example 43, the kit for detecting target nucleic acid has the following features:

The above target nucleic acid detection kit comprises a first container, a second container, and a third container,

The above Cas12a protein is contained in the first container,

The above programmable guide RNA is contained in the second container,

The above detection agent is contained in the third container;

The above target nucleic acid detection kit comprises a fourth container and a fifth container,

The above Cas12a protein and the above programmable guide RNA are contained in the fourth container,

The above detection agent is contained in the fifth container;

The above target nucleic acid detection kit is contained in the 6th container and the 7th container,

The above Cas12a protein is contained in the sixth container,

The above programmable guide RNA and the above detection agent are contained in the seventh container;

The above target nucleic acid detection kit is contained in the 8th container and the 9th container,

The above Cas12a protein and the above detection agent are contained in the 8th container,

wherein said programmable guide RNA is contained in said ninth container; or

The above target nucleic acid detection kit comprises a 10th container,

The Cas12a protein, the guide RNA, and the detection agent are contained in the 10th container,

Here, each of the above containers can be replaced with a selected one from among a vessel; a well; a plate; a reactor; a vial; a test-tube; a cassette; and media.

Example 45, where Cas12a and guide RNA are contained in the same container

In Example 44, when the Cas12a and the programmable guide RNA are contained in the same container, the Cas12a and the programmable guide RNA may be combined to form a protein-RNA complex.

Here, the protein-RNA complex may also be referred to as a novel CRISPR/Cas12a complex, or ribonucleoprotein (RNP).

Example 46, Cas12a protein and detection agent are present in different containers

In any one of Examples 44 to 45, the Cas12a protein and the detection agent are contained in different containers.

표적 핵산 검출 방법Target nucleic acid detection method

Example 47, Method for detecting target nucleic acid

A method for detecting whether a target nucleic acid having a target sequence is present in a sample, comprising:

A process of mixing the above sample, the CRISPR/Cas12a composition of any one of Examples 35 to 39, and the detection agent of any one of Examples 15 to 20,

Here, the Cas12a protein of the CRISPR/Cas12a composition and the programmable guide RNA can form a CRISPR/Cas12a complex,

The guide domain of the programmable guide RNA of the above CRISPR/Cas12a composition targets the target nucleic acid,

Accordingly, when the target nucleic acid of the target sequence is present in the sample, the collateral cleavage activity of the CRISPR/Cas12a complex is activated,

The above probe nucleic acid, when cleaved, produces a detectable signal.

Example 48, mixed aspect

In Example 47, the mixing process is selected from the following:

Simultaneously mixing the above sample, the CRISPR/Cas12a composition, and the detection agent;

Mixing the above sample and the CRISPR/Cas12a composition, and then mixing the above detection agent;

Mixing the above sample and the above detector, and then mixing the CRISPR/Cas12a composition;

Mixing the CRISPR/Cas12a composition and the detection agent, and then mixing the sample;

Mixing the above sample and the CRISPR/Cas12a composition, and the detection agent;

Mixing the mixture of the above sample and the above detector, and the CRISPR/Cas12a composition; and

Mixing the mixture of the CRISPR/Cas12a composition and the detection agent, and the sample.

Example 49, composition for detection

In Example 48, the mixture of the CRISPR/Cas12a composition and the detection agent is the composition for detecting target nucleic acid of Example 41.

Example 50, Detectable Signal Analysis

In any one of Examples 47 to 49, the method further comprises the following steps:

A process of obtaining, measuring, and/or counting the above detectable signal,

Here, as a result of performing the above process, at least one indicator selected from the following can be determined, evaluated, calculated, predicted/forecast, and/or derived:

The presence or absence of a predetermined target sequence in the sample; the amount of the predetermined target sequence in the sample; the concentration of the predetermined target sequence in the sample; the count of the predetermined target sequence in the sample; and the expression level of the predetermined target sequence in the sample.

Here, the above indicators include both absolute and relative values.

Example 51, Use of the Kit

In any one of Examples 47 to 50, the mixing process is performed in one or more kits.

Example 52, Kit Limited

In Example 51, the kit comprises at least one selected from the following:

A vessel; a well; a plate; a reactor; a vial; a test-tube; a cassette; media; and any suitable combination of the above examples.

CRISPR/Cas12a 시스템의 용도Uses of the CRISPR/Cas12a System

Example 53, for use in detecting target nucleic acid

Use of the Cas12a protein of any one of Examples 1 to 5, the programmable guide RNA of any one of Examples 6 to 14, and/or the CRISPR/Cas12a composition of any one of Examples 35 to 39 for detecting a target nucleic acid of any one of Examples 47 to 52.

Example 54, Non-target nucleic acid cleavage use

Use of the Cas12a protein of any one of Examples 1 to 5, the programmable guide RNA of any one of Examples 6 to 14, and/or the CRISPR/Cas12a composition of any one of Examples 35 to 39 for non-target nucleic acid cleavage of Example 40.

Experimental example

Hereinafter, the invention provided by this specification will be described in more detail through experimental examples and examples. It will be apparent to those skilled in the art that these examples are only intended to illustrate the content disclosed by this specification, and the scope of the content disclosed by this specification is not to be construed as being limited by these examples.

실험예 1. 실험 방법 및 재료Experimental Example 1. Experimental Method and Materials

Experimental Example 1.1. Discovery of CRISPR/Cas12a System from Known Databases

We used metagenome data from the rumen of herbivores (camels, cattle, and sheep) published in the NCBI (National Center for Biotechnology Information) database.

Using the above NGS data, the CRISPR/Cas12a system was specified in the following way:

(1) The sequence data were assembled with MEGAHIT v.1.2.9 (NGS assembly software) to derive the genome information of microorganisms. (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).

(2) With reference to the known Cas12a sequence information (e.g., Cas12a derived from Lachnospiraceae bacterium), a homology analysis using the (BLAST) tool was performed to identify a group of Cas12a protein candidates included in the genome information of the microorganism.

(3) In the same manner as (2), guide RNA candidates for each Cas12a protein candidate were specified.

Below, an experiment was conducted using the sequence information of the CRISPR/Cas12a system derived by the above method.

Experimental Example 1.2. Production of a novel Cas12a protein expression vector

In Experimental Example 1.1, an E. coli codon-optimized nucleic acid sequence was specified for the amino acid sequence of the novel Cas12a protein discovered, and this was cloned into a pET28 vector to produce a novel Cas12a protein expression vector. Here, the vector can express a fusion protein including a 6-His-tag and an SV40-derived NLS (nuclear localization signal) at the C-terminus of the novel Cas12a protein, and an SV40-derived NLS at the N-terminus.

Experimental Example 1.3. Verification of a Novel Cas12a Protein

The expression vector prepared according to Experimental Example 1.2 was transformed into different E. coli strains (NiCo21 (DE3), T7 Express lysY / Iq) to test its expression. The E. coli colonies transformed with the expression vector were cultured overnight in Luria-Bertani broth containing 50 mg / ml of ampicillin at 37 ° C. The E. coli colonies were diluted 1:200 in 200 ml of the same medium as above and cultured at 37 ° C until the OD600 reached 0.70 to 0.75. The cultures were incubated at room temperature for 1 hour and then cultured with 0.5 mM isopropyl-b-D-1-thiogalactopyranoside for 16 to 18 hours at 23 ° C with shaking at 220 rpm to induce the expression of the Cas12a protein.

The expressed Cas12a protein was verified by Restriction Digest Analysis. Briefly, 1 ug of plasmid sample (NiCo21(DE3), T7 Express lysY/Iq Competent E. coli) was mixed with 1 uL of restriction enzyme, 2 uL of digestion buffer, and nuclease-free water in a final volume of 20 μl and incubated. The expressed Cas12a protein was verified by visualizing the reaction products separated on a 1% agarose gel using 1X TAE (Tris acetate EDTA) buffer at 37 °C for 30 min by ethidium bromide staining (Fig. 1).

Experimental Example 1.4. Expression and Purification of Novel Cas12a Protein

Expression of the novel Cas12a ortholog prepared by Example 1.2 and verified by Example 1.3 was tested in different E. coli strains (NiCo21(DE3), T7 Express lysY/Iq Competent E. coli). Specifically, the E. coli cells were transformed with the plasmid vector prepared in 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 37C. The cells were diluted 1:200 in 250 ml of the same medium and grown at 37C until the OD600 reached 0.70 to 0.75. Cultures were incubated at room temperature for 1 h and protein expression was induced with 0.5 mM isopropyl-b-D-1-thiogalactopyranoside by incubation for 16–18 h at 23 °C with shaking (220 rpm). Cell pellets were analyzed for protein expression using 10% SDS-PAGE (Fig. 2). Protein purification was performed with the Ni-NTA Fast start kit (Qiagen; 30600) followed by subsequent purification on a HiTrap HP SP (GE Healthcare; 17115101). The purified Cas12a protein was stored in 20 mM sodium acetate (pH 6.0), 500 mM NaCl, 0.1 mM EDTA, 0.1 mM TCEP and 50% (v/v) glycerol at -80 °C.

Experimental Example 1.5. Production of programmable guide RNA

sgRNA synthesis was performed by in vitro transcription using the MEGAscript® T7 transcription kit (ThermoFisher Scientific). The template for sgRNA transcription was generated by PCR amplification of the synthesized fragment (CloneAmp HiFi PCR Premix, Takara). The DNA template was then removed with DNaseI (ThermoFisher Scientific), and the transcribed RNA product was purified with a PCR purification kit (MEGAclear® Transcription Clean-Up Kit) and eluted with nuclease-free water.

The primers used in the above method are shown in [Table 1].

LABELLABEL	PRIMER SEQUENCE 5' to 3'PRIMER SEQUENCE 5' to 3'	SEQ ID NOSEQ ID NO
NSG-01-FWDNSG-01-FWD	GAAATTAATACGACTCACTATAGGgtttcaaagattaaatTAATTTCTACTATTCGTAGATGAAATTAATACGACTCACTATAGGgtttcaaagattaaatTAATTTCTACTATTCGTAGAT	5252
NSG-01-RG1-REVNSG-01-RG1-REV	gagaatctcttgcggctattATCTACGAATAGTAGAAATTAgagaatctcttgcggctattATCTACGAATAGTAGAAATTA	6262
NSG-01-RG2-REVNSG-01-RG2-REV	tgcaggatgacaagatggagATCTACGAATAGTAGAAATTAtgcaggatgacaagatggagATCTACGAATAGTAGAAATTA	6363
NSG-02-FWDNSG-02-FWD	GAAATTAATACGACTCACTATAGGgtttcaaagattaaatTTAATTTCTACTTTCGTAGATGAAATTAATACGACTCACTATAGGgtttcaaagattaaatTTAATTTCTACTTTCGTAGAT	5353
NSG-02-RG1-REVNSG-02-RG1-REV	gagaatctcttgcggctattATCTACGAAAGTAGAAATTAAgagaatctcttgcggctattATCTACGAAAAGTAGAAATTAA	6464
NSG-02-RG2-REVNSG-02-RG2-REV	tgcaggatgacaagatggagATCTACGAAAGTAGAAATTAAtgcaggatgacaagatggagATCTACGAAAAGTAGAAATTAA	6565
NSG-03-FWDNSG-03-FWD	GAAATTAATACGACTCACTATAGGgtttcaaagattaaatCAATTTCTACTTTCAGTAGATGAAATTAATACGACTCACTATAGGgtttcaaagattaaatCAATTTCTACTTTCAGTAGAT	5454
NSG-03-RG1-REVNSG-03-RG1-REV	gagaatctcttgcggctattATCTACTGAAAGTAGAAA gagaatctcttgcggctattATCTACTGAAAGTAGAAA	6666
NSG-03-RG2-REVNSG-03-RG2-REV	tgcaggatgacaagatggagATCTACTGAAAGTAGAAA tgcaggatgacaagatggagATCTACTGAAAGTAGAAA	6767
NSG-04-FWDNSG-04-FWD	GAAATTAATACGACTCACTATAGGgtttcaaagattaaatTTAATTTCTACTTGTGTAGATGAAATTAATACGACTCACTATAGGgtttcaaagattaaatTTAATTTCTACTTGTGTAGAT	5555
NSG-04-RG1-REVNSG-04-RG1-REV	gagaatctcttgcggctattATCTACACAAGTAGAAAT gagaatctcttgcggctattATCTACACAAGTAGAAAT	6868
NSG-04-RG2-REVNSG-04-RG2-REV	tgcaggatgacaagatggagATCTACACAAGTAGAAAT tgcaggatgacaagatggagATCTACACAAGTAGAAAT	6969
NSG-05-FWDNSG-05-FWD	GAAATTAATACGACTCACTATAGGgtttcaaagattaaatTAAATTTCTACTATTGTAGAT GAAATTAATACGACTCACTATAGGgtttcaaagattaaatTAAATTTCTACTATTGTAGAT	5656
NSG-05-RG1-REVNSG-05-RG1-REV	gagaatctcttgcggctattATCTACAATAGTAGAAATTTAgagaatctcttgcggctattATCTACAATAGTAGAAATTTA	7070
NSG-05-RG2-REVNSG-05-RG2-REV	tgcaggatgacaagatggagATCTACAATAGTAGAAATTTAtgcaggatgacaagatggagATCTACAATAGTAGAAATTTA	7171
NSG-06-FWDNSG-06-FWD	GAAATTAATACGACTCACTATAGGgtttcaaagattaaatATAATTTCTACTATTGTAGAT GAAATTAATACGACTCACTATAGGgtttcaaagattaaatATAATTTCTACTATTGTAGAT	5757
NSG-06-RG1-REVNSG-06-RG1-REV	gagaatctcttgcggctattATCTACAATAGTAGAAATTATgagaatctcttgcggctattATCTACAATAGTAGAAAATTAT	7272
NSG-06-RG2-REVNSG-06-RG2-REV	tgcaggatgacaagatggagATCTACAATAGTAGAAATTATtgcaggatgacaagatggagATCTACAATAGTAGAAAATTAT	7373
NSG-07-FWDNSG-07-FWD	GAAATTAATACGACTCACTATAGGgtttcaaagattaaatAAAATTTCTACTATTGTAGATGAAATTAATACGACTCACTATAGGgtttcaaagattaaatAAAATTTCTACTATTGTAGAT	5858
NSG-07-RG1-REVNSG-07-RG1-REV	gagaatctcttgcggctattATCTACAATAGTAGAAATTTTgagaatctcttgcggctattATCTACAATAGTAGAAATTTT	7474
NSG-07-RG2-REVNSG-07-RG2-REV	tgcaggatgacaagatggagATCTACAATAGTAGAAATTTTtgcaggatgacaagatggagATCTACAATAGTAGAAATTTT	7575
NSG-08-FWDNSG-08-FWD	GAAATTAATACGACTCACTATAGGgtttcaaagattaaatAAAATTTCTACTCTTGTAGAT GAAATTAATACGACTCACTATAGGgtttcaaagattaaatAAAATTTCTACTCTTGTAGAT	5959
NSG-08-RG1-REVNSG-08-RG1-REV	gagaatctcttgcggctattATCTACAAGAGTAGAAAT gagaatctcttgcggctattATCTACAAGAGTAGAAAT	7676
NSG-08-RG2-REVNSG-08-RG2-REV	tgcaggatgacaagatggagATCTACAAGAGTAGAAAT tgcaggatgacaagatggagATCTACAAGAGTAGAAAT	7777
NSG-09-FWDNSG-10-FWDNSG-09-FWDNSG-10-FWD	GAAATTAATACGACTCACTATAGGgtttcaaagattaaatATAATTTCTACTGTTGTAGAT GAAATTAATACGACTCACTATAGGgtttcaaagattaaatATAATTTCTACTGTTGTAGAT	6060
NSG-09-RG1-REVNSG-10-RG1-REVNSG-09-RG1-REVNSG-10-RG1-REV	gagaatctcttgcggctattATCTACAACAGTAGAAATTATgagaatctcttgcggctattATCTACAACAGTAGAAAATTAT	7878
NSG-09-RG2-REVNSG-10-RG2-REVNSG-09-RG2-REVNSG-10-RG2-REV	tgcaggatgacaagatggagATCTACAACAGTAGAAATTATtgcaggatgacaagatggagATCTACAACAGTAGAAAATTAT	7979
Lba_FWDLba_FWD	GAAATTAATACGACTCACTATAGGgtttcaaagattaaatTAATTTCTACTAAGTGTAGAT GAAATTAATACGACTCACTATAGGgtttcaaagattaaatTAATTTCTACTAAGTGTAGAT	104104
Lba_RG1_REVLba_RG1_REV	gagaatctcttgcggctattATCTACACTTAGTAGAAATTAgagaatctcttgcggctattATCTACACTTAGTAGAAATTA	105105
Lba_RG2_REVLba_RG2_REV	tgcaggatgacaagatggagATCTACACTTAGTAGAAATTAtgcaggatgacaagatggagATCTACACTTAGTAGAAATTA	106106

The concentration and purity of the above guide RNA were measured by NanoDrop, and RNA integrity was visualized by ethidium bromide staining of the reaction products separated on a 2% agarose gel with 1X TAE (Tris acetate EDTA) buffer (Fig. 3).

Experimental Example 1.6. Preparation of CRISPR/Cas12a complex

Each novel CRISPR/Cas12a complex was individually prepared by incubating 0.5uM Cas12a protein and 1uM crRNA in NEB r2.1 buffer containing 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 100ug/mL BSA (pH=7.9) at room temperature for 15 minutes.

Experimental Example 1.7. PAM sequence of CRISPR/Cas12a complex

Most Cas12a proteins are known to recognize the PAM of 5'-TTTV-3', and the CRISPR/Cas12a system manufactured according to Experimental Examples 1.1 to 1.6 was also found to recognize the PAM of 5'-TTTV-3' as a result of the experiment.

The following experiments can be conducted to determine whether the novel Cas12a proteins can recognize PAM sequences other than 5'-TTTV-3'.

To elucidate the PAM sequence of the CRISPR/Cas12a complex manufactured by Experimental Examples 1.1 to 1.6, the following experiments are conducted.

To generate sufficient DNA for sequencing, DNA fragments containing the PAM sequence recognized upon cleavage are amplified by PCR using the adapter primer and another primer adjacent to the PAM sequence. The resulting PCR-amplified Cas12a PAM library is PCR-amplified by adding an index sequence and a single-read deep sequence to the adapter side of the amplicon. To ensure adequate coverage, the PAM library is sequenced to a depth greater than the diversity of the initial random PAM library (e.g., 5x or more) (>80,000 reads). Only when the 12 nt long sequences on both sides of the 7 nt PAM sequence are perfectly matched, they are used as PAM sequence identification data, and the PAM sequence is analyzed only when the Cas12a-guide RNA perfectly recognizes and cleaves the target site. The PAM sequence analysis range is limited to the most frequent upper specific fraction (e.g., 10%) of PAMs to reduce the influence of non-specific noise due to other components of the reaction mixture.

실험예 2. 신규 CRISPR/Cas12a 시스템의 표적-특이적 핵산 절단 기능 확인Experimental Example 2. Confirmation of the target-specific nucleic acid cleavage function of the novel CRISPR/Cas12a system

The target-specific nucleic acid cleavage function of each novel CRISPR/Cas12a complex obtained according to Experimental Examples 1.1 to 1.6 was confirmed. The sequence information of the CRISPR/Cas12a complex used in the Experimental Examples is shown in the following [Table 2]:

LABELLABEL	Cas12a protein (SEQ ID NO)Cas12a protein (SEQ ID NO)	Stem-loopStem-loop	guide RNA Direct repeatguide RNA Direct repeat	Guide domain of guide RNAGuide domain of guide RNA
NSG-01NSG-01	11	GUUUCAAAGAUUAAAU (SEQ ID NO: 21)GUUUCAAAGAUUAAAU (SEQ ID NO: 21)	UAAUUUCUACUAUUCGUAGAU (SEQ ID NO: 11)UAAUUUCUACUAUUCGUAGAU (SEQ ID NO: 11)	AATAGCCGCAAGAGATTCTC (SEQ ID NO: 107) for RG1 or CTCCATCTTGTCATCCTGCA (SEQ ID NO: 108) for RG2AATAGCCGCAAGAGATTCTC (SEQ ID NO: 107) for RG1 or CTCCATCTTGTCATCCTGCA (SEQ ID NO: 108) for RG2
NSG-02NSG-02	22	SEQ ID NO: 21SEQ ID NO: 21	UUAAUUUCUACUUUCGUAGAU(SEQ ID NO: 12)UUAAUUUCUACUUUCGUAGAU(SEQ ID NO: 12)	SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2
NSG-03NSG-03	33	SEQ ID NO: 21SEQ ID NO: 21	CAAUUUCUACUUUCAGUAGAU(SEQ ID NO: 13)CAAUUUCUACUUUCAGUAGAU(SEQ ID NO: 13)	SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2
NSG-04NSG-04	44	SEQ ID NO: 21SEQ ID NO: 21	UUAAUUUCUACUUGUGUAGAU(SEQ ID NO: 14)UUAAUUUCUACUUGUGUAGAU(SEQ ID NO: 14)	SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2
NSG-05NSG-05	55	SEQ ID NO: 21SEQ ID NO: 21	UAAAUUUCUACUAUUGUAGAU(SEQ ID NO: 15)UAAAUUUCUACUAUUGUAGAU(SEQ ID NO: 15)	SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2
NSG-06NSG-06	66	SEQ ID NO: 21SEQ ID NO: 21	AUAAUUUCUACUAUUGUAGAU(SEQ ID NO: 16)AUAAUUUCUACUAUUGUAGAU(SEQ ID NO: 16)	SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2
NSG-07NSG-07	77	SEQ ID NO: 21SEQ ID NO: 21	AAAAUUUCUACUAUUGUAGAU(SEQ ID NO: 17)AAAAUUUCUACUAUUGUAGAU(SEQ ID NO: 17)	SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2
NSG-08NSG-08	88	SEQ ID NO: 21SEQ ID NO: 21	AAAAUUUCUACUCUUGUAGAU(SEQ ID NO: 18)AAAAUUUCUACUCUUGUAGAU(SEQ ID NO: 18)	SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2
NSG-09NSG-09	99	SEQ ID NO: 21SEQ ID NO: 21	AUAAUUUCUACUGUUGUAGAU(SEQ ID NO: 19)AUAAUUUCUACUGUUGUAGAU(SEQ ID NO: 19)	SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2
NSG-10NSG-10	1010	SEQ ID NO: 21SEQ ID NO: 21	AUAAUUUCUACUGUUGUAGAU(SEQ ID NO: 20)AUAAUUUCUACUGUUGUAGAU(SEQ ID NO: 20)	SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2SEQ ID NO: 107 for RG1 Or SEQ ID NO: 108 for RG2

* Here, the guide RNA is in the form of a Stem-loop, Direct repeat, and Guide domain sequentially connected in the direction from the 5' end to the 3' end.

** Here, the Guide sequence is specific based on the PAM sequence of 5'-TTTV-3'.

Specifically, the DNA template of the SCN1A gene with sequence number 82 was added to the novel CRISPR/Cas12a complex obtained according to Experimental Example 1.6, and the reaction was performed for 1 hour under each culture condition. Each culture condition and experimental results are shown in [Table 3] below:

LABELLABEL	pH = 7.9pH = 7.9			pH = 6.5pH = 6.5			pH = 4.5pH = 4.5
LABELLABEL	25 ℃25 ℃	37 ℃37℃	60 ℃60℃	25 ℃25 ℃	37 ℃37℃	60 ℃60℃	25 ℃25 ℃	37 ℃37℃	60 ℃60℃
NSG-01NSG-01	++++++	++++++	++++++	++++++	++++++	++++++	++	++++++	++
NSG-02NSG-02	++++++	++++++	++++	++++++	++++++	++++++	++++	++++++	++++
NSG-03NSG-03	++++++	++++++	++++++	++++++	++++++	++++++	++	++++	++
NSG-04NSG-04	++++++	++++++	++++++	++++++	++++++	++++++	++++	++++++	++++
NSG-05NSG-05	++++++	++++++	++++++	++++++	++++++	++++	++	++++	++
NSG-06NSG-06	++++++	++++++	++	++++++	++++++	++++++	++	++++	++
NSG-07NSG-07	++++++	++++++	++++++	++++++	++++++	++++++	++	++++++	++++
NSG-08NSG-08	++++++	++++++	++++++	++++++	++++++	++++++	++	++++++	--
NSG-09NSG-09	++++++	++++++	++++++	++++++	++++++	++++++	++++	++++	++
NSG-10NSG-10	++++++	++++++	++++++	++++++	++++++	++++++	++++	++++++	++

* The meaning of each symbol is as follows: (+++) Excellent cleavage activity; (++) Medium cleavage activity; (+ ) Poor cleavage activity; ( - ) No cleavage activity

** Each pH culture condition is as follows: pH = 7.9 (NEB buffer 2.1), pH = 6.5 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 ug/mL BSA), and pH = 4.5 (50 mM NaCl, 10 mM sodium acetate, 10 mM MgCl2, 0.1 mM DTT)

The target-specific nucleic acid cleavage function of each CRISPR/Cas12a complex was visualized by ethidium bromide staining of reaction products separated on 1% agarose gels using 1X Tris acetate EDTA (TAE) buffer (Figs. 4 to 12).

Experimental results showed that the novel CRISPR/Cas12a complex in [Table 2] has a target-specific nucleic acid cleavage function for DNA.

실험예 3. 신규 CRISPR/Cas12a 시스템의 부수적 절단 기능 확인Experimental Example 3. Confirmation of the secondary cutting function of the novel CRISPR/Cas12a system

Experimental Example 3.1. Preparation of dsDNA template of SCN1A gene

Linear dsDNA of the SCN1A gene was synthesized by PCR amplification using gDNA of human HEK cells, the forward primer of AGGTTCTTTCCAGCTTCCAA (SEQ ID NO: 83), and the reverse primer of TGCCTTTATACGCACAGTCT (SEQ ID NO: 84) in nuclease-free water in a total volume of 20 uL. The generated template DNA was purified with LaboPass(TM) Gel and PCR Clean-up Kit (LaboPass, CMA0112) for ancillary activity test analysis.

Experimental Example 3.2. Confirmation of the secondary cutting function of the novel CRISPR/Cas12a system

It was experimentally confirmed whether the CRISPR/Cas12a complex of the above [Table 2] has a secondary cleavage function. Specifically, first, each CRISPR/Cas12a complex of [Table 2] was prepared in a total of 20 uL with reference to Experimental Examples 1.1 to 1.6. Thereafter, the prepared complex was mixed with 30 uL of nuclease free water, 50 nM DNase Alert substrate (final concentration; IDT, 11-04-03-04) as a detector, and various contents of target DNA (0.2 ng/uL; 0.5 ng/uL; 1.0 ng/uL, and 2.0 ng/uL, respectively). The above mixture was loaded into 384-well (SPL) and incubated at 37°C for up to 90 min in a fluorescence plate reader (TECAN, Infinite 200 Pro M Plex), and fluorescence measurements were performed every 3 min (λex: 535 nm; λem: 570 nM, gain: 114).

The experimental results are shown in Figs. 13 to 23.

Experimental results showed that all CRISPR/Cas12a complexes in [Table 2] activated the secondary cleavage function when bound to the target nucleic acid.

The novel CRISPR/Cas12a system disclosed herein is characterized by the activation of a collateral-editing function when a target nucleic acid is present, and this effect can be used to measure the presence and quantitative properties of the target nucleic acid. In other words, the novel CRISPR/Cas12a system disclosed herein can be used to diagnose a disease having a specific nucleic acid as a biomarker.

Claims

Non-target nucleic acid cleavage methods comprising:

Process of mixing Cas12a protein, guide RNA, and sample;

wherein the sample comprises a target nucleic acid and a non-target nucleic acid,

The above Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10,

The above guide RNA comprises a guide domain and a scaffold,

The above guide RNA has scaffold and guide domains sequentially connected from the 5' end to the 3' end.

The above scaffold can allow the guide RNA to interact with the Cas12a protein to form a protein-RNA complex,

The above guide domain can hybridize with the target nucleic acid,

The above guide domain cannot hybridize with the non-target nucleic acid;

As a result of performing the above process, the Cas12a protein and the guide RNA combine to form a protein-RNA complex,

The above protein-RNA complex binds to the target nucleic acid through the guide domain, thereby activating the secondary cleavage function.

The non-target nucleic acid is cleaved by the protein-RNA complex.
Non-target nucleic acid cleavage methods comprising:

Ribonucleoprotein, and the process of mixing samples;

wherein the sample comprises a target nucleic acid and a non-target nucleic acid,

The above ribonucleoprotein comprises Cas12a protein and guide RNA,

The above Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10,

The above guide RNA comprises a guide domain and a scaffold,

The above guide RNA has scaffold and guide domains sequentially connected from the 5' end to the 3' end.

The above scaffold can enable the guide RNA to interact with the Cas12a protein to form a ribonucleoprotein,

The above guide domain can hybridize with the target nucleic acid,

The above guide domain cannot hybridize with the non-target nucleic acid;

The ribonucleoprotein binds to the target nucleic acid through the guide domain, thereby activating the secondary cleavage function.

The non-target nucleic acid is cleaved by the ribonucleoprotein.
A method for non-target nucleic acid cleavage in any one of claims 1 to 2, wherein the Cas12a protein consists of an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10.
A method for non-target nucleic acid cleavage in any one of claims 1 to 3, wherein the scaffold of the guide RNA comprises an RNA sequence selected from SEQ ID NO: 11 to SEQ ID NO: 20.
In the fourth paragraph, the Cas12a protein and the scaffold of the guide RNA are a combination selected from the following, a non-target nucleic acid cleavage method:

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 1, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 11;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 2, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 12;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 3, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 13;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 4, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 14;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 5, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 15;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 6, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 16;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 7, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 17;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 8, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 18;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 9, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 19; or

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 10, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 20.
In any one of paragraphs 4 and 5,

The above guide RNA further comprises a stem-loop,

The above stem-loop is connected to the 5' end of the above scaffold,

The above stem-loop comprises an RNA sequence of sequence number 21.

A method for non-target nucleic acid cleavage.
In any one of paragraphs 1 to 6,

The target nucleic acid is double-stranded DNA or single-stranded DNA, and the non-target nucleic acid is double-stranded DNA or single-stranded DNA.

A method for non-target nucleic acid cleavage.
A method for detecting a target nucleic acid in a sample, comprising:

Process of mixing Cas12a protein, guide RNA, detection composition and sample;

Here, the detection composition comprises a probe nucleic acid,

The above probe nucleic acid produces a detectable signal upon cleavage,

The above Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10,

The above guide RNA comprises a guide domain and a scaffold,

The above guide RNA has scaffold and guide domains sequentially connected from the 5' end to the 3' end.

The above scaffold can allow the guide RNA to interact with the Cas12a protein to form a protein-RNA complex,

The above guide domain can hybridize with the target nucleic acid,

As a result of performing the above process, the Cas12a protein and the guide RNA combine to form a protein-RNA complex,

When the target nucleic acid is contained in the sample, the protein-RNA complex binds to the target nucleic acid through the guide domain, thereby activating the secondary cleavage function.

When the probe nucleic acid is cleaved by the protein-RNA complex, the detectable signal is measured.

By measuring said detectable signal, it is possible to determine whether said target nucleic acid is present in said sample, and/or the amount of said target nucleic acid contained in said sample.
A method for detecting a target nucleic acid in a sample, comprising:

A process of mixing ribonucleoprotein, a composition for detection and a sample;

Here, the detection composition comprises a probe nucleic acid,

The above probe nucleic acid produces a detectable signal upon cleavage,

The above ribonucleoprotein comprises Cas12a protein and guide RNA,

The above Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10,

The above guide RNA comprises a guide domain and a scaffold,

The above guide RNA has scaffold and guide domains sequentially connected from the 5' end to the 3' end.

The above scaffold can enable the guide RNA to interact with the Cas12a protein to form a ribonucleoprotein,

The above guide domain can hybridize with the target nucleic acid,

If the target nucleic acid is contained in the sample, the ribonucleoprotein binds to the target nucleic acid through the guide domain, thereby activating the secondary cleavage function.

When the probe nucleic acid is cleaved by the ribonucleoprotein, the detectable signal is measured,

By measuring said detectable signal, it is possible to determine whether said target nucleic acid is present in said sample, and/or the amount of said target nucleic acid contained in said sample.
A method for detecting a target nucleic acid in a sample, comprising:

Process of mixing the composition for detection and the sample;

Here, the detection composition comprises ribonucleoprotein and probe nucleic acid,

The above probe nucleic acid produces a detectable signal upon cleavage,

The above ribonucleoprotein comprises Cas12a protein and guide RNA,

The above Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10,

The above guide RNA comprises a guide domain and a scaffold,

The above guide RNA has scaffold and guide domains sequentially connected from the 5' end to the 3' end.

The above scaffold can enable the guide RNA to interact with the Cas12a protein to form a ribonucleoprotein,

The above guide domain can hybridize with the target nucleic acid,

If the target nucleic acid is contained in the sample, the ribonucleoprotein binds to the target nucleic acid through the guide domain, thereby activating the secondary cleavage function.

When the probe nucleic acid is cleaved by the ribonucleoprotein, the detectable signal is measured,

By measuring said detectable signal, it is possible to determine whether said target nucleic acid is present in said sample, and/or the amount of said target nucleic acid contained in said sample.
A method for detecting a target nucleic acid, wherein in any one of claims 8 to 10, the Cas12a protein is composed of an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10.
A method for detecting a target nucleic acid, wherein the scaffold of the guide RNA comprises an RNA sequence selected from SEQ ID NO: 11 to SEQ ID NO: 20, in any one of claims 8 to 11.
In the 12th paragraph, the Cas12a protein and the scaffold of the guide RNA are a selected combination of the following, a method for detecting a target nucleic acid:

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 1, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 11;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 2, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 12;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 3, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 13;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 4, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 14;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 5, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 15;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 6, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 16;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 7, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 17;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 8, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 18;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 9, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 19; or

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 10, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 20.
In any one of Articles 12 to 13,

The above guide RNA further comprises a stem-loop,

The above stem-loop is connected to the 5' end of the above scaffold,

The above stem-loop comprises an RNA sequence of sequence number 21.

Method for detecting target nucleic acid.
In any one of Articles 8 to 14,

The target nucleic acid is double-stranded DNA or single-stranded DNA, and the non-target nucleic acid is double-stranded DNA or single-stranded DNA.

Method for detecting target nucleic acid.
A method for detecting a target nucleic acid according to any one of claims 8 to 15, wherein the detectable signal generated when the probe nucleic acid is cleaved is a fluorescent signal.
Composition for detecting target nucleic acid comprising:

Cas12a protein; programmable guide RNA; and probe nucleic acid;

Here, the Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10,

The above programmable guide RNA has a 5'-[direct repeat]-[guide domain]-3' structure,

The above direct repeat portion enables the guide RNA to interact with the Cas12a protein to form a ribonucleoprotein (RNP),

The above guide domain is artificially designed to target the target nucleic acid,

The above probe nucleic acid is one which, when cleaved, produces a detectable signal.
A composition for detecting a target nucleic acid, wherein in claim 17, the Cas12a protein and the programmable guide RNA are combined to form an RNP.
A composition for detecting a target nucleic acid, wherein the direct repeat portion of the guide RNA in any one of claims 17 to 18 comprises an RNA sequence selected from SEQ ID NO: 11 to SEQ ID NO: 20.
In Article 19,

When the above Cas12a protein comprises an amino acid sequence of sequence number 1, the direct repeat portion of the guide RNA comprises an RNA sequence of sequence number 11,

When the above Cas12a protein comprises an amino acid sequence of sequence number 2, the direct repeat portion of the guide RNA comprises an RNA sequence of sequence number 12,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 3, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 13,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 4, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 14,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 5, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 15,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 6, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 16,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 7, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 17,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 8, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 18,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 9, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 19,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 10, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 20.

A composition for detecting a target nucleic acid.
A composition for detecting a target nucleic acid, wherein the detectable signal generated when the probe nucleic acid is cleaved in any one of claims 17 to 20 is a fluorescent signal.
Target nucleic acid detection kit containing:

Cas12a protein; programmable guide RNA; and probe nucleic acid;

Here, the Cas12a protein comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 10,

The above programmable guide RNA has a 5'-[direct repeat]-[guide domain]-3' structure,

The above direct repeat portion enables the guide RNA to interact with the Cas12a protein to form a ribonucleoprotein (RNP),

The above guide domain is artificially designed to target the target nucleic acid,

The above probe nucleic acid is one which, when cleaved, produces a detectable signal.
In claim 22, the target nucleic acid detection kit comprises two or more containers,

A target nucleic acid detection kit, characterized in that the Cas12a protein and the probe nucleic acid are contained in different containers.
A kit for detecting a target nucleic acid, wherein the direct repeat portion of the guide RNA in any one of claims 22 to 23 comprises an RNA sequence selected from SEQ ID NO: 11 to SEQ ID NO: 20.
In Article 24,

When the above Cas12a protein comprises an amino acid sequence of sequence number 1, the direct repeat portion of the guide RNA comprises an RNA sequence of sequence number 11,

When the above Cas12a protein comprises an amino acid sequence of sequence number 2, the direct repeat portion of the guide RNA comprises an RNA sequence of sequence number 12,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 3, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 13,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 4, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 14,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 5, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 15,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 6, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 16,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 7, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 17,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 8, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 18,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 9, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 19,

When the above Cas12a protein comprises an amino acid sequence of SEQ ID NO: 10, the direct repeat portion of the guide RNA comprises an RNA sequence of SEQ ID NO: 20.

Target nucleic acid detection kit.
A composition for detecting a target nucleic acid, wherein the detectable signal generated when the probe nucleic acid is cleaved in any one of claims 22 to 25 is a fluorescent signal.
A target nucleic acid detection kit according to any one of claims 22 to 26, wherein the Cas12a protein and the programmable guide RNA are combined to form an RNP.
A Cas12a composition comprising:

Cas12a protein or a nucleic acid encoding said Cas12a protein; and

A programmable guide RNA or a nucleic acid encoding said programmable guide RNA;

Here, the programmable guide RNA has a 5'-[direct repeat]-[guide domain]-3' structure,

The above direct repeat portion enables the guide RNA to interact with the Cas12a protein to form a ribonucleoprotein (RNP),

The above guide domain is artificially designed to target the target nucleic acid,

The above direct repeat portion and the Cas12a protein are a selected combination of:

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 1, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 11;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 2, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 12;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 3, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 13;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 4, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 14;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 5, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 15;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 6, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 16;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 7, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 17;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 8, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 18;

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 9, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 19; or

The Cas12a protein comprises an amino acid sequence of SEQ ID NO: 10, and the guide RNA scaffold comprises an RNA sequence of SEQ ID NO: 20.
In claim 28, the Cas12a composition comprises the Cas12a protein and the programmable guide RNA.
A Cas12a composition in claim 29, wherein the Cas12a protein and the programmable guide RNA are combined to form a ribonucleoprotein.
Use of a Cas12a composition according to any one of claims 28 to 30 in a non-target nucleic acid cleavage method according to any one of claims 1 to 7.
Use of a Cas12a composition according to any one of claims 28 to 30 in a method for detecting a target nucleic acid according to any one of claims 8 to 16.