KR20230132341A - Cpf1 mutant protein for rapid DNA diagnosis - Google Patents

Abstract

The present invention relates to Cpf1 mutant protein for rapid DNA diagnosis, and more specifically, to the 1025th, 1026, 1032, 1033, 1050, 1061, 1062, 1079, 1080, 1096, 1097, 1101, 1120 and 1121st of LbCpf1 protein. A Cpf1 mutant protein in which at least one of the amino acids is mutated can exhibit excellent trans-cleavage activity while maintaining cis-cleavage activity.

Description

Cpf1 mutant protein for rapid DNA diagnosis {Cpf1 mutant protein for rapid DNA diagnosis}

The present invention relates to Cpf1 mutant protein.

Rapid detection of nucleic acid molecules is a key technology in fields such as public health, environmental detection, and criminal investigation. Nucleic acid molecule detection can not only detect whether humans, animals, plants, etc. are infected with pathogens, but also detect genetic diseases or cancer risk, provide an auxiliary reference for personal drug use, whether microbial contamination exists in water bodies, etc. It can also be used for detection. Currently, many nucleic acid detection methods have been developed, such as real-time PCR and FISH hybridization (Fluorescence in situ hybridization), but there is still a need to develop nucleic acid detection technology that is fast, inexpensive, and has excellent sensitivity.

Recently, CRISPR technology has shown great application value in the field of genome editing. This task is mainly performed by DNA (or RNA) targeting endonucleases such as Cas9, allowing people to guide proteins such as Cas9 by simply designing a simple RNA containing a PAM (for example, the PAM sequence of Cas9 is NGG). It can cause breakage of double-stranded DNA (or cleavage of RNA) by targeting any nucleic acid target sequence. In addition to genome editing, CRISPR can also be used for gene regulation, epigenetic editing, functional gene screening, and genome imaging. Recently, CRISPR, a rich toolbox, has begun to appear in the field of nucleic acid detection and is attracting attention.

Among CRISPR proteins, LbCpfl (CRISPR from Prevotella and Francisella 1 (Cpfl) proteins from Lachnospiraceae bacterium ND2006), which is used for diagnosis, has cis-cleavage activity to accurately cleave the DNA double helix portion complementary to the sequence of crRNA, and at the same time has collateral DNase activity. It has a trans-cleavage activity that cuts a single strand of DNA and has recently been used in diagnosis. However, the trans-cleavage activity of LbCpf1 is slow at 5.0x10 ⁶ s ^-1 M ^-1 (Kcat/Km), ~3 turnovers/sec, so there is a problem that it takes more than 30 minutes for accurate diagnosis.

Korean Patent Publication No. 10-2020-0132924

The purpose of the present invention is to provide a Cpf1 mutant protein.

The purpose of the present invention is to provide a polynucleotide encoding a Cpf1 mutant protein.

The purpose of the present invention is to provide a vector containing a polynucleotide encoding a Cpf1 mutant protein.

The purpose of the present invention is to provide a complex containing a Cpf1 mutant protein and a guide RNA.

The purpose of the present invention is to provide a kit for detecting target nucleic acid molecules containing Cpf1 mutant proteins.

The purpose of the present invention is to provide a method for detecting a target nucleic acid including the step of treating a Cpf1 mutant protein in a sample.

1. Cpf1 mutation in which at least one of amino acids 1025, 1026, 1032, 1033, 1050, 1061, 1062, 1079, 1080, 1096, 1097, 1101, 1120, and 1121 of the LbCpf1 ( Lachnospiraceae bacterium Cpf1) protein is mutated Protein.

2. In 1 above, the Cpf1 mutant protein has at least one of amino acids 1025, 1026, 1032, 1033, 1050, 1061, 1062, 1079, 1080, 1096, 1097, 1101, 1120, and 1121 of the LbCpf1 protein, respectively. A Cpf1 variant protein substituted with a hydrophobic amino acid independently selected from the group consisting of alanine, isoleucine, phenylalanine, valine and leucine.

3. In 1 above, the Cpf1 mutant protein is one in which i) the amino acids K1025 and K1026 of the LbCpf1 protein are substituted with alanine; ii) the amino acids at D1032 and R1033 are replaced with alanine; iii) the amino acid at K1050 is replaced with alanine; iv) the amino acids at K1061 and K1062 are replaced with alanine; v) the amino acids at K1079 and K1080 are replaced with alanine; vi) the amino acids of K1096 and E1097 are substituted with a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, phenylalanine, valine and leucine; vii) the amino acid of K1101 is replaced with alanine; viii) the amino acids D1120 and K1121 are substituted with alanine; ix) the amino acids K1025, K1026, K1079 and K1080 are substituted with alanine; x) the amino acids K1025, K1026, K1096 and E1097 are replaced with alanine; xi) the amino acids K1079, K1080, K1096 and E1097 are substituted with alanine; or xii) a Cpf1 mutant protein in which the amino acids K1025, K1026, K1079, K1080, K1096 and E1097 are substituted with alanine.

4. The Cpf1 mutant protein of 1 above, wherein the LbCpf1 protein is the sequence of SEQ ID NO: 1.

5. Polynucleotide encoding the Cpf1 mutant protein of 1 above.

6. A vector containing a polynucleotide encoding the Cpf1 mutant protein of 1 above.

7. A complex containing the Cpf1 mutant protein of 1 above and a guide RNA that can bind complementary to a target nucleic acid.

8. A kit for detecting a target nucleic acid containing at least one of the Cpf1 mutant protein of any one of 1 to 4 above, the polynucleotide of 5 above, the vector of 6 above, and the complex of 7 above.

9. The kit for detecting target nucleic acid according to item 8 above, further comprising at least one of guide RNA or labeled ssDNA capable of complementary binding to the target nucleic acid.

10. A target nucleic acid detection method comprising treating a sample with at least one of the Cpf1 mutant protein of any one of 1 to 4 above, the polynucleotide of 5 above, the vector of 6 above, and the complex of 7 above.

11. The method of detecting a target nucleic acid according to item 10 above, further comprising treating the sample with at least one of guide RNA or labeled ssDNA capable of binding complementary to the target nucleic acid.

The Cpf1 mutant protein of the present invention can exhibit excellent trans-cleavage activity while maintaining cis-cleavage activity.

The Cpf1 mutant protein of the present invention can detect target nucleic acids quickly and with high intensity.

The Cpf1 mutant protein of the present invention can detect target nucleic acids quickly and with high intensity, and can be used for point-of-care (POC) essential for various infectious diseases, including virus-borne infectious diseases.

Figure 1 shows a schematic diagram of the domains of the wild-type LbCpf1 protein and the 3D structure of the wild-type LbCpf1 protein.
Figures 2 to 9 show the 3D structure of each LbCpf1 mutant protein according to an example and the amino acid position where the mutation occurred. The meaning of the colors shown in the 3D structures of Figures 2 to 9 is indicated at the bottom left of Figure 2.
Figures 10 and 11 show the results of SDS-PAGE gel analysis confirming the purified LbCpf1 mutant protein. In Figures 10 and 11, D1181A is a negative control and WT means wild type LbCpf1 protein.
Figures 12 and 13 show the results of cis-cleavage analysis of the LbCpf1 mutant protein. 12 and 13, WT refers to wild-type LbCpf1 protein.
Figures 14 and 15 show the results of trans-cleavage analysis of the LbCpf1 mutant protein. 14 and 15, WT refers to wild-type LbCpf1 protein.
Figure 16 shows the results of lateral flow assay using the LbCpf1 mutant protein.
Figure 17 shows the 3D structure of AsCpf1 protein. The mutation positions of each AsCpf1 mutant protein in Table 4 are displayed at once in the 3D structure of Figure 17.
Figure 18 shows the results of analysis of trans-cleavage activity of AsCpf1 mutant protein. In Figure 18, WT refers to wild-type AsCpf1 protein.
Figure 19 schematically illustrates the range of applications in which the Cpf1 mutant protein of the present invention can be utilized. Using the Cpf1 mutant protein of the present invention, a POC test can be effectively performed through CRISPR reaction.

Hereinafter, the present invention will be described in detail.

The present invention provides a Cpf1 (Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1) mutant protein.

The present specification can provide a technology that can further enhance the trans-cleavage activity of Cpf1 while maintaining its cis-cleavage activity. More specifically, mutant proteins of Cpf1 can exhibit superior collateral DNase activity compared to wild-type Cpf1 in the presence of target nucleic acids. Therefore, the Cpf1 mutant protein of the present invention can be used in fields that require more rapid detection of target nucleic acids (for example, point-of-care diagnosis, etc.).

Cpf1 is an RNA-guided endonuclease and can also be expressed as Cas12 or Cas12a.

“Mutant protein” refers to a protein that has undergone mutation. For example, a mutant protein may be a mutation in one amino acid from a wild-type protein, or it may be a mutation in a wide range of amino acids due to cleavage. Mutations may be artificially introduced, for example, amino acid mutations may be introduced by changing some nucleotides included in a polynucleotide encoding a wild-type protein.

A “variation” may include the insertion, substitution, or deletion of one or more consecutive or non-consecutive amino acids.

“Cpf1 mutant protein” refers to a protein that has mutated from wild-type Cpf1. For example, the Cpf1 mutant protein has at least one amino acid substituted and/or deleted in the amino acid sequence of the wild-type Cpf1 protein, a foreign amino acid is inserted into the amino acid sequence of the wild-type Cpf1 protein, or at least one amino acid is substituted in the amino acid sequence of the wild-type Cpf1 protein. And/or it may be deleted and a foreign amino acid may be inserted.

The Cpf1 mutant protein may be mutated at a position expected to affect trans-cleavage activity in the three-dimensional structure of the wild-type Cpf1 protein.

The Cpf1 mutant protein may include a mutation in at least one amino acid among amino acids 1025 to 1121 of the LbCpf1 protein.

The Cpf1 mutant protein may contain a mutation in at least one of amino acids 1025, 1026, 1032, 1033, 1050, 1061, 1062, 1079, 1080, 1096, 1097, 1101, 1120, and 1121 of the LbCpf1 protein.

The Cpf1 mutant protein may be one in which at least one of amino acids 1025, 1026, 1032, 1033, 1050, 1061, 1062, 1079, 1080, 1096, 1097, 1101, 1120, and 1121 of the LbCpf1 protein is mutated.

The LbCpf1 protein refers to the wild type LbCpf1 protein. The wild-type LbCpf1 protein may include the sequence of SEQ ID NO: 1. The wild-type LbCpf1 protein may be a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity to the sequence of SEQ ID NO: 1. The wild-type LbCpf1 protein may have the sequence of SEQ ID NO: 1.

LbCpf1 protein refers to Cpf1 from Lachnospiraceae_bacterium_ND2006 .

SEQ ID NO: 1:

The Cpf1 mutant protein may be one in which at least one of the non-hydrophobic amino acids of the wild-type Cpf1 protein is replaced with a hydrophobic amino acid. A non-hydrophobic amino acid may be an amino acid that is not hydrophobic, for example, a hydrophilic amino acid. The hydrophobic amino acid introduced by substitution into wild-type Cpf1 is not bulky, has a simple structure, and may not cause side reactions other than trans-cleavage.

The hydrophobic amino acid may be at least one selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, proline, and methionine. Specifically, the hydrophobic amino acid may be selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Alanine (Ala, A) is a simple amino acid that is hydrophobic and does not have a large residue size. Isoleucine (Ile, I), phenylalanine (Phe, F), valine (Val, V), and leucine (Leu, L) have relatively high hydrophobic properties compared to other hydrophobic amino acids.

The Cpf1 mutant protein may include a mutation in which at least one of amino acids 1025 to 1121 of the LbCpf1 protein is substituted with a hydrophobic amino acid.

CPF1 variant protein is at least one of the 1025, 1026, 1032, 1033, 1050, 1061, 1062, 1079, 1080, 1096, 1097, 1101, 1120 and 1121th amino acids, respectively and a mutation substituted with a hydrophobic amino acid selected from the group consisting of leucine. CPF1 variant protein is at least one of the 1025, 1026, 1032, 1033, 1050, 1061, 1062, 1079, 1080, 1096, 1097, 1101, 1120 and 1121th amino acids, respectively and may be substituted with a hydrophobic amino acid selected from the group consisting of leucine.

According to one embodiment, the Cpf1 mutant protein may be one in which amino acids 1025 to 1026 of LbCpf1 (SEQ ID NO: 1) are mutated (mutation of amino acids K1025 to K1026 in LbCpf1). Specifically, the Cpf1 mutant protein may be one in which amino acids 1025 to 1026 of LbCpf1 (SEQ ID NO: 1) are each independently substituted with a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Specifically, the Cpf1 mutant protein may be one in which amino acids 1025 to 1026 of LbCpf1 (SEQ ID NO: 1) are substituted with alanine.

According to one embodiment, the Cpf1 mutant protein may be one in which amino acids 1079 to 1080 of LbCpf1 (SEQ ID NO: 1) are mutated (mutation of amino acids K1079 to K1080 in LbCpf1). Specifically, the Cpf1 mutant protein may be one in which amino acids 1079 to 1080 of LbCpf1 (SEQ ID NO: 1) are each independently substituted with a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Specifically, the Cpf1 mutant protein may be one in which amino acids 1079 and 1080 of LbCpf1 (SEQ ID NO: 1) are substituted with alanine.

According to one embodiment, the Cpf1 mutant protein may be one in which amino acids 1096 to 1097 of LbCpf1 (SEQ ID NO: 1) are mutated (mutation of amino acids K1096 to E1097 in LbCpf1). Specifically, the Cpf1 mutant protein may be one in which amino acids 1096 and 1097 of LbCpf1 (SEQ ID NO: 1) are each independently substituted with hydrophobic amino acids selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Specifically, the Cpf1 mutant protein is LbCpf1 (SEQ ID NO: 1) in which both the 1079th and 1080th amino acids are substituted with alanine, both the 1079th and 1080th amino acids are substituted with isoleucine, both the 1079th and 1080th amino acids are substituted with phenylalanine, or Both the 1079th and 1080th amino acids may be substituted with valine, or both the 1079th and 1080th amino acids may be substituted with leucine.

According to one embodiment, the Cpf1 mutant protein may be one in which amino acids 1032 to 1033 of LbCpf1 (SEQ ID NO: 1) are mutated (mutation of amino acids D1032 to R1033 in LbCpf1). Specifically, the Cpf1 mutant protein may be one in which amino acids 1032 to 1033 of LbCpf1 (SEQ ID NO: 1) are each independently substituted with a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Specifically, the Cpf1 mutant protein may be one in which amino acids 1032 to 1033 in LbCpf1 (SEQ ID NO: 1) are substituted with alanine.

According to one embodiment, the Cpf1 mutant protein may be one in which the 1050th amino acid in LbCpf1 (SEQ ID NO: 1) is mutated (mutation of the K1050 amino acid in LbCpf1). Specifically, the Cpf1 mutant protein may be one in which the 1050th amino acid in LbCpf1 (SEQ ID NO: 1) is substituted with a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Specifically, the Cpf1 mutant protein may be one in which the 1050th amino acid in LbCpf1 (SEQ ID NO: 1) is substituted with alanine.

According to one embodiment, the Cpf1 mutant protein may be one in which amino acids 1061 to 1062 of LbCpf1 (SEQ ID NO: 1) are mutated (mutation of amino acids K1061 to K1062 in LbCpf1). Specifically, the Cpf1 mutant protein may be one in which amino acids 1061 to 1062 of LbCpf1 (SEQ ID NO: 1) are each independently substituted with a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Specifically, the Cpf1 mutant protein may be one in which amino acids 1061 to 1062 of LbCpf1 (SEQ ID NO: 1) are substituted with alanine.

According to one embodiment, the Cpf1 mutant protein may be one in which the 1101st amino acid in LbCpf1 (SEQ ID NO: 1) is mutated (mutation of the K1101 amino acid in LbCpf1). Specifically, the Cpf1 mutant protein may be one in which the 1101st amino acid in LbCpf1 (SEQ ID NO: 1) is substituted with a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Specifically, the Cpf1 mutant protein may be one in which the 1101st amino acid in LbCpf1 (SEQ ID NO: 1) is substituted with alanine.

According to one embodiment, the Cpf1 mutant protein may be one in which the 1120th and 1121st amino acids in LbCpf1 (SEQ ID NO: 1) are mutated (mutation of the D1120 and K1121 amino acids in LbCpf1). Specifically, the Cpf1 mutant protein may be one in which amino acids 1120 and 1121 in LbCpf1 (SEQ ID NO: 1) are substituted with hydrophobic amino acids selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Specifically, the Cpf1 mutant protein may be one in which the 1120th and 1121st amino acids in LbCpf1 (SEQ ID NO: 1) are substituted with alanine.

According to one embodiment, the Cpf1 mutant protein may be one in which amino acids 1025, 1026, 1079, and 1080 of LbCpf1 (SEQ ID NO: 1) are mutated. Specifically, the Cpf1 mutant protein may be one in which amino acids 1025, 1026, 1079, and 1080 of LbCpf1 (SEQ ID NO: 1) are each independently substituted with hydrophobic amino acids selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Specifically, the Cpf1 mutant protein may be one in which amino acids 1025, 1026, 1079, and 1080 of LbCpf1 (SEQ ID NO: 1) are substituted with alanine.

According to one embodiment, the Cpf1 mutant protein may be one in which amino acids 1025, 1026, 1096, and 1097 of LbCpf1 (SEQ ID NO: 1) are mutated. Specifically, the Cpf1 mutant protein may be one in which amino acids 1025, 1026, 1096, and 1097 of LbCpf1 (SEQ ID NO: 1) are each independently substituted with hydrophobic amino acids selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Specifically, the Cpf1 mutant protein may be one in which amino acids 1025, 1026, 1096, and 1097 of LbCpf1 (SEQ ID NO: 1) are substituted with alanine.

According to one embodiment, the Cpf1 mutant protein may be one in which amino acids 1079, 1080, 1096, and 1097 of LbCpf1 (SEQ ID NO: 1) are mutated. Specifically, the Cpf1 mutant protein may be one in which amino acids 1079, 1080, 1096, and 1097 of LbCpf1 (SEQ ID NO: 1) are each independently substituted with hydrophobic amino acids selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. Specifically, the Cpf1 mutant protein may be one in which amino acids 1079, 1080, 1096, and 1097 of LbCpf1 (SEQ ID NO: 1) are substituted with alanine.

According to one embodiment, the Cpf1 mutant protein may be one in which amino acids 1025, 1026, 1079, 1080, 1096, and 1097 of LbCpf1 (SEQ ID NO: 1) are mutated. Specifically, the Cpf1 mutant protein is one in which amino acids 1025, 1026, 1079, 1080, 1096, and 1097 in LbCpf1 (SEQ ID NO: 1) are each independently substituted with hydrophobic amino acids selected from the group consisting of alanine, isoleucine, phenylalanine, valine, and leucine. You can. Specifically, the Cpf1 mutant protein may be one in which amino acids 1025, 1026, 1079, 1080, 1096, and 1097 of LbCpf1 (SEQ ID NO: 1) are substituted with alanine.

The Cpf1 mutant protein may be an LbCpf1 mutant protein. According to one embodiment, the LbCpf1 mutant protein has a superior trans-cleavage activity enhancement effect compared to the AsCpf1 mutant protein containing a mutation at the corresponding position.

Additionally, the present invention can provide a polynucleotide encoding the above-described Cpf1 mutant protein.

For example, the polynucleotide encoding the Cpf1 mutant protein may be one in which certain nucleotides are mutated in the polynucleotide sequence of SEQ ID NO: 3. SEQ ID NO: 3 may be a sequence encoding wild-type LbCpf1.

The Cpf1 mutant protein may be produced by inducing site-directed mutation at positions 3073 to 3078 in the polynucleotide sequence of SEQ ID NO: 3 (mutation at positions 3073 to 3078: aag aag -> gcg gcg ). The Cpf1 mutant protein may be produced by inducing a specific site mutation at positions 3094 to 3099 in the polynucleotide sequence of SEQ ID NO: 3 (mutation at positions 3094 to 3099: gac agg -> gcc gcg ).

The Cpf1 mutant protein may be produced by inducing a specific site mutation at positions 3148 to 3150 in the polynucleotide sequence of SEQ ID NO: 3 (mutation at positions 3148 to 3150: aag -> gcg ).

The Cpf1 mutant protein may be produced by inducing a mutation at a specific site at positions 3181 to 3186 in the polynucleotide sequence of SEQ ID NO: 3 (mutation at positions 3181 to 3186: aag aag -> gcg gcg ).

The Cpf1 mutant protein may be produced by inducing a specific site mutation at positions 3235 to 3240 in the polynucleotide sequence of SEQ ID NO: 3 (mutation at positions 3235 to 3240: aag aag -> gcg gcg ).

The Cpf1 mutant protein may be produced by inducing a mutation at a specific site at positions 3286 to 3291 in the polynucleotide sequence of SEQ ID NO: 3 (mutation at positions 3286 to 3291: aag gag -> gcg gcg /aag gag -> ata ata /aag gag -> ctg ctg /aag gag -> gtg gtg /aag gag -> ttt ttt ).

The Cpf1 mutant protein may be produced by inducing a specific site mutation at positions 3301 to 3303 in the polynucleotide sequence of SEQ ID NO: 3 (mutation at positions 3301 to 3303: aag -> gcg ).

The Cpf1 mutant protein may be produced by inducing a specific site mutation at positions 3358 to 3363 in the polynucleotide sequence of SEQ ID NO: 3 (mutation at positions 3358 to 3363: gac aag -> gcc gcg ).

The Cpf1 mutant protein may be produced by inducing specific site mutations at positions 3073 to 3078 and 3235 to 3240 in the polynucleotide sequence of SEQ ID NO: 3 (mutation at positions 3073 to 3078: aag aag -> gcg gcg , and the mutation at positions 3235 to 3240: aag aag -> gcg gcg ).

The Cpf1 mutant protein may be prepared by inducing specific site mutations at positions 3073 to 3078 and 3286 to 3291 of the polynucleotide of SEQ ID NO: 3 (mutation at positions 3073 to 3078: aag aag -> gcg gcg, and mutations at positions 3286 to 3291: aag gag -> gcg gcg ).

The Cpf1 mutant protein may be produced by inducing specific site mutations at positions 3235 to 3240 and 3286 to 3291 of the polynucleotide of SEQ ID NO: 3 (mutation at positions 3235 to 3240: aag aag -> gcg gcg , and mutations at positions 3286 to 3291: aag gag -> gcg gcg ).

The Cpf1 mutant protein may be prepared by inducing specific site mutations at positions 3073 to 3078, 3235 to 3240, and 3286 to 3291 of the polynucleotide of SEQ ID NO: 3 (mutation at positions 3073 to 3078: aag aag -> gcg gcg , mutation at positions 3235 to 3240: aag aag -> gcg gcg and mutations at positions 3286 to 3291: aag gag -> gcg gcg ).

The polynucleotide of the present invention is sufficient as long as it encodes a Cpf1 mutant protein, and the specific sequence is not limited.

Since the Cpf1 mutant protein has been described above, detailed description will be omitted.

Additionally, the present invention can provide a vector containing a polynucleotide encoding the above-described Cpf1 mutant protein.

The vector may be, but is not limited to, a plasmid vector or a viral vector.

The vector may further include a nucleic acid sequence encoding a guide RNA that can bind complementary to the target nucleic acid to be detected. The target nucleic acid may be target DNA or target RNA, and may be double-stranded or single-stranded.

Guide RNA refers to an RNA molecule required for recognition of the target sequence within the target strand of the target nucleic acid to be detected by Cpf1. The term target strand refers to the nucleic acid strand that interacts with the guide RNA to form a guide RNA-DNA hybrid. The non-target strand is complementary to the target strand. When the Cpf1 protein is activated and cleaves the nucleic acid strand, the specific nucleic acid target site may be displaced into the RuvC/NuC pocket of the endonuclease. Guide RNA can be designed to target some nucleotide sequences (target sequences) included in the target nucleic acid to be detected. The guide RNA may be crRNA.

Since the Cpf1 mutant protein has been described above, detailed description will be omitted.

Additionally, the present invention can provide a complex containing a Cpf1 mutant protein and a guide RNA capable of binding complementary to a target nucleic acid to be detected. The target nucleic acid may be target DNA. The guide RNA may be crRNA. Guide RNA refers to the RNA molecule required for recognition of the target sequence within the target strand by Cpf1.

Cpf1 can form a crRNA-Cpf1 complex when associated with an appropriate crRNA.

Since the Cpf1 mutant protein has been described above, detailed description will be omitted.

Additionally, the present invention can provide a kit for detecting a target nucleic acid containing at least one selected from the group consisting of the Cpf1 mutant protein, the polynucleotide, the vector, and the complex.

The kit of the present invention may further include a guide RNA capable of binding to the target nucleic acid to be detected. The guide RNA may be crRNA. The bond may be a complementary bond. crRNA can recognize target sequences with high specificity.

The kit of the present invention may further include ssDNA (single-stranded DNA).

The ssDNA may be labeled with a set of at least one interacting label (e.g., comprising at least one fluorophore and at least one quencher), wherein the interacting label may provide a signal upon cleavage of the ssDNA. there is.

The ssDNA may be labeled ssDNA. Labeled ssDNA may be one in which at least one of a fluorescent source, a quencher, and a reporter is bound to ssDNA.

When the labeled ssDNA is cleaved by the Cpf1 mutant protein, the luminescent group can emit light. The Cpf1 mutant protein of the present invention can degrade labeled ssDNA and generate a signal.

As long as a detectable difference occurs after the labeled ssDNA is cut, any other labeling method that can theoretically be detected can be applied. Labeled ssDNA can be designed to emit fluorescence after binding to a compound to detect whether the labeled ssDNA has been cleaved.

The kit may further include DNA polymerase and predetermined primers for amplifying target DNA. DNA amplification may be to amplify a target sequence within a target nucleic acid, or may be performed to increase the accuracy of diagnosis by further enhancing the intensity of the signal generated by decomposition of labeled ssDNA. DNA amplification may be performed before the step of treating the Cpf1 mutant protein in the sample to be detected.

Meanwhile, the Cpf1 mutant protein has high trans-cleavage activity, so it can effectively detect target nucleic acids without performing a DNA amplification step. Even if a DNA amplification step is performed, the target nucleic acid can be detected even with a short amplification time. . Accordingly, it can provide advantages in terms of detection time and cost.

As described above, the Cpf1 mutant protein can effectively detect target nucleic acids, and therefore, the Cpf1 mutant protein of the present invention can be used for SNP detection, cancer diagnosis, bacterial infection diagnosis, and viral infection diagnosis.

Since the Cpf1 mutant protein, the polynucleotide encoding the Cpf1 mutant protein, the vector, and the complex have been described above, detailed descriptions will be omitted.

Additionally, the present invention can provide a method for detecting a target nucleic acid using a Cpf1 mutant protein.

The target nucleic acid detection method may include treating a sample with at least one selected from the group consisting of a Cpf1 mutant protein, the polynucleotide, the vector, and the complex. Cpf1 protein can be processed in the form of complexes with proteins, polynucleotides, vectors, and guide RNAs.

The sample is a sample to be detected to determine whether the target nucleic acid is present. For example, the sample to be detected may be a sample isolated from an animal, including a human, or a plant, but is not limited thereto. The plant may be a plant for which genetic information is known. For example, it may be blood, serum, urine, saliva, skin, plant tissue, etc. isolated from animals, including humans.

The target nucleic acid detection method may further include treating the sample with a guide RNA capable of binding complementary to the target nucleic acid. The guide RNA may be processed simultaneously or simultaneously with at least one selected from the group consisting of the Cpf1 mutant protein, the polynucleotide, the vector, and the complex. The guide RNA may be composed of a sequence that can recognize the target sequence to be detected. The guide RNA may be crRNA. Guide RNA may be processed in the form of an RNA sequence or a vector encoding it.

The target nucleic acid detection method may further include processing ssDNA labeled in the sample. The labeled ssDNA may be processed simultaneously or simultaneously with at least one selected from the group consisting of the Cpf1 mutant protein, the polynucleotide, the vector, and the complex.

The target nucleic acid detection method may further include determining the presence, level, and/or concentration of the target nucleic acid. The step of determining the presence, level and/or concentration of the target nucleic acid may be determined based on the signal intensity resulting from cleavage of the labeled ssDNA by the trans-cleavage activity of the Cpf1 variant protein.

The Cpf1 mutant protein, the polynucleotide encoding the Cpf1 mutant protein, the vector, the complex, guide RNA, and labeled ssDNA have been described above, and detailed descriptions thereof will be omitted.

The target nucleic acid to be detected may be a nucleic acid fragment that causes a certain disease. For example, the target nucleic acid to be detected may be a genomic nucleic acid fragment of an infectious agent that causes a disease.

Additionally, the present invention can provide a method for diagnosing diseases using the Cpf1 mutant protein.

The disease may be any disease associated with increased/reduced gene expression and/or the presence of exogenous genetic material. The disease may be an infectious disease.

A method for diagnosing a disease may include treating a sample isolated from an individual to be diagnosed with at least one selected from the group consisting of a Cpf1 mutant protein, the polynucleotide, the vector, and the complex. Cpf1 variant proteins can be processed in the form of proteins, polynucleotides, vectors, and/or complexes.

The entity to be diagnosed may be an entity that contains or is suspected of containing the target nucleic acid. The target nucleic acid to be detected may be a nucleic acid fragment that causes the disease. For example, the target nucleic acid to be detected may be a genomic nucleic acid fragment of an infectious agent that causes a disease.

The entity may be an animal, including a human, or a plant, for example, the animal may be a human, dog, cat, rabbit, cow, horse, or goat.

Samples may be blood, serum, urine, saliva, skin, tissue, etc., but are not limited thereto.

The method for diagnosing a disease may further include treating the sample with a guide RNA that can bind complementary to the target nucleic acid. The guide RNA may be processed simultaneously or simultaneously with at least one selected from the group consisting of the Cpf1 mutant protein, the polynucleotide, the vector, and the complex.

The guide RNA may be composed of a sequence that can recognize the target sequence to be detected. The guide RNA may be crRNA. Guide RNA may be processed in the form of an RNA sequence or a vector encoding it.

The method for diagnosing a disease may further include processing ssDNA labeled in the sample. The labeled ssDNA may be processed simultaneously or simultaneously with at least one selected from the group consisting of the Cpf1 mutant protein, the polynucleotide, the vector, and the complex.

The method for diagnosing a disease may further include determining the presence, level, and/or concentration of a target nucleic acid. The step of determining the presence, level and/or concentration of the target nucleic acid may be determined based on the signal intensity resulting from cleavage of the labeled ssDNA by the trans-cleavage activity of the Cpf1 variant protein.

Methods for detecting target nucleic acids and diagnosing diseases can be performed by humans or devices.

The Cpf1 mutant protein, the polynucleotide encoding the Cpf1 mutant protein, the vector, the complex, guide RNA, and labeled ssDNA have been described above, and detailed descriptions thereof will be omitted.

The Cpf1 mutant protein of the present invention has a high trans-cutting activity in addition to cis-cutting activity that recognizes and cuts a specific target sequence, so it can cut ssDNA more quickly and generate a strong signal. Accordingly, the Cpf1 mutant protein of the present invention can be used to detect target DNA.

Hereinafter, the present invention will be described in detail with reference to examples.

Example

The present inventors analyzed the domain that acts as an enzyme that cleaves DNA of the wild-type LbCpf1 protein, searched for parts that may affect trans-cleavage activity, and changed amino acids through point mutation (In site directed mutagenesis). Trans-cleavage activity was confirmed.

1. Preparation of Cpf1 mutant protein

(1) Cpf1 mutant protein design

First, the structure of the wild-type LbCpf1 protein was analyzed using the RCSB PBD database, which can predict protein tertiary structure (see Figure 1). Through structural analysis of the wild-type LbCpf1 protein, the TSL domain near RuvC, where cis-cleavage occurs to cut the target DNA double helix, was found, and the location at which mutations would occur in this region was selected. Specifically, in order to destabilize the binding of the target DNA at the corresponding site, it was designed to replace non-hydrophobic amino acids with hydrophobic amino acids (DOI: 10.2210/pdb5XUT/pdb NDB: 5XUT). Specifically, a mutant was designed in which the amino acid at a position expected to affect trans-cleavage activity in the wild-type LbCpf1 protein was replaced with alanine (Ala, A), which is hydrophobic, has a small residue size, and has a simple structure. In addition, in the wild-type LbCpf1 protein, the amino acids at positions expected to affect trans-cleavage activity are other hydrophobic amino acids besides alanine (top 4 in order of increasing hydrophobicity: isoleucine (Ile, I), phenylalanine (Phe, F), Mutants substituted with valine (Val, V), or leucine (Leu, L)) were designed.

Information on the various LbCpf1 mutant proteins designed as above is listed in Table 2 below.

LbCpf1 variant protein name Mutated portion compared to wild type LbCpf1 (SEQ ID NO: 1) KK1025AA K1025A, K1026A DR1032AA D1032A, R1033A K1050A K1050A KK1061AA K1061A, K1062A KK1079AA K1079A, K1080A KE1096AA K1096A, E1097A KE1096II K1096I, E1097I KE1096LL K1096L, E1097L KE1096VV K1096V, E1097V KE1096FF K1096F, E1097F K1101A K1101A DK1120AA D1120A, K1121A KK1025AA/KK1079AA K1025A, K1026A, K1079A, K1080A KK1025AA/KE1096AA K1025A, K1026A, K1096A, E1097A KK1079AA/KE1096AA K1079A, K1080A, K1096A, E1097A KK1025AA/KK1079AA/KE1096AA K1025A, K1026A, K1079A, K1080A, K1096A, E1097A KTK1015ATA K1015A, K1017A RIR1071AIA R1071A, R1073A

The second column in Table 2 indicates the mutation position and amino acid change based on the wild-type LbCpf1 sequence of SEQ ID NO: 1. For example, K1025A refers to a mutation in which the amino acid lysine (K) at position 1025 of the LbCpf1 (SEQ ID NO: 1) sequence is replaced with alanine (A), and E1097A refers to a mutation at position 1097 of the LbCpf1 (SEQ ID NO: 1) sequence. It refers to a mutation in which the amino acid glutamic acid (E) is replaced with alanine (A). For example, in Table 2, in the LbCpf1 mutant protein KK1025AA, the amino acid lysine (K) at the 1025th position of the LbCpf1 (SEQ ID NO: 1) sequence is replaced with alanine (A), and the amino acid lysine (K) at the 1026th position is replaced with alanine (A). It is a mutant protein substituted with . In addition, the LbCpf1 mutant protein KK1025AA/KE1096AA has lysine (K) replaced with alanine (A) at amino acids 1025, 1026, and 1096 of the LbCpf1 (SEQ ID NO: 1) sequence, and amino acid 1097 substituted from glutamic acid (E) to alanine (A). It is a mutant protein substituted with . In the LbCpf1 mutant protein KK1025AA/KK1079AA/KE1096AA, lysine (K) is replaced with alanine (A) at amino acids 1025, 1026, 1079, 1080, and 1096 of the LbCpf1 (SEQ ID NO: 1) sequence, and amino acid 1097 is glutamic acid (E). It is a mutant protein substituted with alanine (A).

Figures 2 to 9 show the 3D structure and amino acid mutated positions of each LbCpf1 mutant protein.

(2) Construction of plasmid for producing Cpf1 mutant protein

DNA sequences encoding non-hydrophobic amino acids can be compared to DNA sequences encoding hydrophobic amino acids (alanine: GCG, GCA, GCC, GCT/isoleucine: ATA, ATC, ATT/phenylalanine:TTT, TTC/valine:GTG, GTA, GTC, GTT / leucine: TTG, TTA, CTT, CTC, CTA, CTG) primers for mutagenesis that can be substituted were designed and in site directed mutagenesis was performed (see Table 3 below).

division order Primer for alanine substitution KTK 1015 ATA Forward 5'-acc ggc　ttt　gtg　aac　ctg　ctg　GCa　acc　GCg　tat　acc　agc　atc　gcc　gat　tcc　-3' (SEQ ID NO: 4) KK1025AA Forward 5'-acc　agc　atc　gcc　gat　tcc　gcg　gcg　ttc　atc　agc　tcc　ttt　gac　-3' (SEQ ID NO: 5) DR1032AA Forward 5'-aag　aag　ttc　atc　agc tcc ttt gCc GCg atc atg tac gtg ccc gag gag-3' (SEQ ID NO: 6) K1050A Forward 5'-ttc gag ttt gcc ctg gac tat GCg aac ttc tct cgc aca gac gcc-3' (SEQ ID NO: 7) KK1061AA Forward 5'-cgc　aca　gac　gcc　gat　tac　atc　GCg　GCg　tgg　aag　ctg　tac　tcc　tac　ggc-3' (SEQ ID NO: 8) RIR1071AIA Forward 5'-aag　ctg　tac　tcc　tac　ggc aac GCg atc GCa atc ttc cgg aat cct aag aag-3' (SEQ ID NO: 9) KK1079AA Forward 5'-aga　atc　ttc　cgg　aat　cct　gcg　gcg　aac　aac　gtg　ttc　gac　tgg　-3' (SEQ ID NO: 10) KE1096AA Forward 5'-tgc　ctg　acc　agc　gcc　tat　gcg　gcg　ctg　ttc　aac　aag　tac　ggc　-3' (SEQ ID NO: 11) K1101A Forward 5'-gcc　tat　aag　gag　ctg　ttc　aac　GCg　tac　ggc　atc　aat　tat　cag　cag　-3' (SEQ ID NO: 12) DK1120AA Forward 5'-gcc　ctg　ctg　tgc　gag　cag　tcc　gCc　GCg　gcc　ttc　tac　tct　agc　ttt　atg　-3' (SEQ ID NO: 13) Primer for isoleucine substitution KE1096II Forward 5'-gtg tgc ctg acc agc gcc tat aTA ATA ctg ttc aac aag tac ggc atc-3' (SEQ ID NO: 14) Primer for phenylalanine substitution KE1096FF Forward 5'-gtg tgc ctg acc agc gcc tat TTT TTT ctg ttc aac aag tac ggc atc-3' (SEQ ID NO: 15) Primer for valine substitution KE1096VV Forward 5'-gtg tgc ctg acc agc gcc tat GTg gTg ctg ttc aac aag tac ggc atc-3' (SEQ ID NO: 16) Primer for leucine substitution KE1096LL Forward 5'-gtg tgc ctg acc agc gcc tat CTg CTg ctg ttc aac aag tac ggc atc-3' (SEQ ID NO: 17)

Specifically, PCR was performed using pET28a-LbCpf1 plasmid DNA, which encodes the DNA of LbCpf1 wild type, as a template, using the primers for mutagenesis designed above. 5X phusion GC 6ul, 10uM Forward primer 1ul, 100ng/ul plasmid 1ul, 10mM dNTPP 0.5ul, DMSO 1.5ul, D.W. Pre-extension reaction for 5 minutes at 98℃ with a total of 30ul, then 98℃ for 30 seconds, 55℃ After chain reaction for a total of 25 cycles of 1 minute at 72°C and 4 minutes and 30 seconds at 72°C, the PCR reaction was terminated by reacting at 72°C for 8 minutes. After purifying the PCR product using a DNA purification kit, the original plasmid that did not contain the mutation was removed by treating it with 0.5ul of Dpn1 enzyme per 10ul at 37°C for 1 hour. Then, 5ul of sample was added to E.coli DH5alpha competent cells, incubated on ice for 30 minutes, heat shocked at 42 degrees for 1 minute, applied to an agar plate containing 100ug/ml kanamycin, and incubated for one day at 37 degrees to generate individual colonies. I ordered it. Purify each plasmid DNA and sequence primer T7 promoter_Forward 5'- taatacgactcactataggg-3' (SEQ ID NO: 18), LbCpf1 seq primer2 5'- GAGGATTTCTTTGAGGGCGAG-3' (SEQ ID NO: 19), LbCpf1 seq primer3 5'- GGCAAGGAGACAAACAGGGA-3' (SEQ ID NO: 20), LbCpf1 seq primer 4 5'- CCAGATAATCCCAAGAAAACC-3' (SEQ ID NO: 21), LbCpf1 seq primer 5 5'- ATTCCAAGAAGTTCATCAGC-3' (SEQ ID NO: 22), and each LbCpf1 mutant protein candidate was located at the desired position. It was confirmed that the amino acid was mutated and that no mutation occurred in other areas. The LbCpf1 mutant protein in which the amino acid was mutated at the desired position and no mutation occurred at other sites was finally selected.

(3) Purification of Cpf1 mutant protein

The plasmids prepared in step 1.(2) above were each transformed into E.coli DE3. Each colony was inoculated into 5ml of Lb broth containing kanamycin at a concentration of 50ug/ml and incubated for primary culture by shaking incubation at 37°C and 220rpm for 16 hours. Second, the primary culture was inoculated into 200ml Lb broth at a ratio of 1:10000. The cells were inoculated and cultured at 37°C and 220rpm for about 5 hours until the OD600 value reached 0.6. When the OD600 reached 0.6, protein expression was induced by adding 0.8mM of IPTG (Isopropyl β) and the protein was expressed for more than 16 hours at 16°C and 220rpm. E.coli with the expressed protein was cultured in Lb broth using a centrifuge. Remove, collect in a tube, and resuspend using lysis buffer containing 50mM NaH ₂ PO ₄ , 300mM NaCl, 10mM imidazole, 2mg/ml lysozyme, and 1mM PMSF pH 7.4. Afterwards, 15% on/off cycle 3 times for 15 seconds each. Sonicate at amplification intensity to break the cell membrane, settle the E. coli residues using a centrifuge, collect the supernatant, mix with Ni-NTA Agarose beads, and react at 4°C for 1 hour to form an agarose bead-protein complex. Afterwards, the lysate containing the above complex was added to the gravity column to remove flow through, and then 10ml of lysis buffer was flowed once more to remove substances that could bind non-specifically. NaH ₂ was added to separate only the proteins from the beads. 3ml of elution buffer with 50mM PO ₄ , 300mM NaCl, 250mM imidazole, pH 7.4 was added to the column, and the solution extracted from the column was centrifuged on centricon to obtain protein: 100mM HEPES, 150mM NaCl, 0.1mM EDTA, 1mM DTT. , the buffer was replaced with a storage solution containing 2% sucrose and 20% glycerol, and the concentration was measured. The results of confirming the expression of each Cpf1 mutant protein are shown in Figures 10 and 11.

2. Analysis of cis-cleavage activity of Cpf1 mutant protein

The present inventors performed a cis-cleavage analysis as follows to confirm whether the LbCpf1 mutant protein prepared in 1 above maintains the ability to cleave target DNA (see Figures 12 and 13).

Specifically, to form _an RNP (mutated protein: crRNA) complex, crRNA targeting the DNMT1 gene (SEQ ID NO: 23: 5'- 200 nM of AAUUUCUACUAAGUGUAGAUCUGAUGGUCCAUGUCUGUUACUC-3') and 200 nM of the above-mentioned mutant protein were first reacted at 37°C for 5 minutes. As a substrate, a 51bp DNA fragment of the DNMT1 gene (target strand (SEQ ID NO: 24): 5'-CACTTGACAGGCGAGTAACAGACATGGACCATCAGGAAACATTAACGTACT-3', non-target strand (SEQ ID NO: 25): 5'-GTGAACTGTCCGCTCATTGTCTGTACCTGGTAGTCCTTTGTAATTGCATGA-3') was labeled with Cy5 fluorescent protein. .

The annealed substrate was added to the complex (mutant protein:crRNA=200nM:200nM) to a final reaction concentration of 20nM and reacted at 37°C for 10 minutes. After the reaction, the obtained material was loaded onto a 10% TBE-UREA PAGE gel and the size was confirmed. The target strand had a size of 13 nt, and the non-target strand had a size of 18 nt, which means that the substrate was cut by the complex. Through the results of this experiment, it was confirmed that all mutant proteins of the present invention exhibited cis activity.

3. Trans-cleavage activity analysis of Cpf1 mutant protein

The present inventors performed a trans-cleavage analysis as follows to determine how much the LbCpf1 mutant protein prepared in step 1 above has improved trans-cleavage activity compared to the wild-type LbCpf1 protein (see Figures 14 and 15).

Specifically, cis-cleavage was performed on 1uM crRNA, 1uM mutant protein, and 100nM DNMT1 DNA at 37°C for 10 minutes under conditions of 100mM NaCl, 10mM MgCl ₂ , 1mM DTT, 10mM Tris-Cl pH 7.4, and then the reaction was placed in a 96 well. 10ul each was dispensed onto a light-shielding plate. The reporter molecule (5'-BHQ-TTTTTTTT (SEQ ID NO: 26)-FAM-3') was prepared at a concentration of 125 nM under the same buffer conditions as the cis cleavage reaction. 40ul of the prepared reporter molecule was added to each well, and the final concentrations of RNP, DNMT1 DNA substrate, and reporter molecule were as follows; Mutant protein: crRNA: DNMT1 DNA substrate: Reporter molecule=200nM:200nM:20nM:100nM. Afterwards, after 494nm excitation in the spectrometer equipment, the 515nm emission value was measured once every 30 seconds for 30 minutes, and the measured values were displayed in a graph using the graph pad program. As shown in Figures 14 and 15, it was confirmed that the trans-cleavage function of the mutant proteins except KTK1015ATA and RIR1071AIA was enhanced compared to the wild type.

4. Lateral flow analysis using Cpf1 mutant protein

The present inventors performed a lateral flow assay (LFA) using a mutant protein with excellent trans-cleavage activity among the LbCpf1 mutant proteins prepared in step 1 above (see FIG. 16).

Specifically, cis cleavage was performed at 37°C for 10 minutes at 37°C in a 10ul volume with 1uM crRNA, 1uM mutant protein, and 100nM DNMT1 DNA under 100mM NaCl, 10mM MgCl2, 1mM DTT, 10mM Tris-Cl pH 7.4 conditions, and then under the same buffer conditions. 10ul of 10uM reporter molecule (5'-Biotin-TTTTTTTT (SEQ ID NO: 26)-FAM-3') at a concentration of 0.5uM was added to the cis-cleaved sample, and trans-cleavage was performed at the same temperature for 30 minutes. Add 80 ul of 1 did. As shown in Figure 16, compared to the sample using the wild-type protein, the sample using the mutant protein of the present invention showed a distinct signal in the lfa kit. In particular, unlike samples using the wild-type protein, samples using the mutant protein of the present invention showed a strong signal within 5 minutes of application to the lfa kit, confirming that the mutant protein of the present invention can be used for rapid diagnosis.

5. Trans-cleavage activity analysis of AsCpf1 mutant protein

(1) Preparation of AsCpf1 mutant protein

An AsCpf1 mutant protein in which several amino acid sequences were mutated from the sequence of wild-type AsCpf1 (SEQ ID NO: 2) was designed and produced in a similar manner to the method in 1 above. Specifically, information on the AsCpf1 mutant protein used in the experiments below is listed in Table 4 below. The location of mutations in each mutant protein is shown in Figure 17.

AsCpf1 mutant protein name Mutated portion compared to wild type AsCpf1 AsCpf1 mutant protein description RKH1094AAA R1094A, K1095A, H1096A In SEQ ID NO: 2, the 1094th amino acid R is substituted with A, the 1095th amino acid K is substituted with A, and the 1096th amino acid H is substituted with A. Δ1172-5 1172,1173,1174 and 1175 amino acid deletions In SEQ ID NO: 2, the 1172nd amino acid R, 1173rd amino acid Y, 1174th amino acid R, and 1175th amino acid D are deleted. EEK1187AAA E1187A, E1188A, K1189A In SEQ ID NO: 2, the 1187th amino acid E is substituted with A, the 1188th amino acid E is substituted with A, and the 1189th amino acid K is substituted with A. RD1194AA R1194A, D1195A In SEQ ID NO: 2, the 1194th amino acid R is substituted with A, and the 1195th amino acid D is substituted with A.

SEQ ID NO: 2:

(2) Trans-cleavage activity analysis of AsCpf1 mutant protein

Similar to the method in 3. above, the trans-cleavage activity of the AsCpf1 mutant protein prepared in 5.(1) above was confirmed (see FIG. 18). As a result, unlike the LbCpf1 mutant protein, the AsCpf1 mutant protein showed similar or lower trans-cleavage activity than the wild-type AsCpf1 protein.

Claims (11)

A Cpf1 mutant protein in which at least one of amino acids 1025, 1026, 1032, 1033, 1050, 1061, 1062, 1079, 1080, 1096, 1097, 1101, 1120, and 1121 of the LbCpf1 ( Lachnospiraceae bacterium Cpf1) protein is mutated.
The method of claim 1, wherein in the Cpf1 mutant protein, at least one of amino acids 1025, 1026, 1032, 1033, 1050, 1061, 1062, 1079, 1080, 1096, 1097, 1101, 1120, and 1121 of the LbCpf1 protein is each independently A Cpf1 mutant protein substituted with a hydrophobic amino acid selected from the group consisting of alanine, isoleucine, phenylalanine, valine and leucine.
The method according to claim 1, wherein the Cpf1 mutant protein is one in which i) the amino acids K1025 and K1026 of the LbCpf1 protein are substituted with alanine; ii) the amino acids at D1032 and R1033 are replaced with alanine; iii) the amino acid at K1050 is replaced with alanine; iv) the amino acids at K1061 and K1062 are replaced with alanine; v) the amino acids at K1079 and K1080 are replaced with alanine; vi) the amino acids of K1096 and E1097 are substituted with hydrophobic amino acids selected from the group consisting of alanine, isoleucine, phenylalanine, valine and leucine; vii) the amino acid of K1101 is replaced with alanine; viii) the amino acids D1120 and K1121 are substituted with alanine; ix) the amino acids K1025, K1026, K1079 and K1080 are substituted with alanine; x) the amino acids K1025, K1026, K1096 and E1097 are replaced with alanine; xi) the amino acids K1079, K1080, K1096 and E1097 are substituted with alanine; or xii) a Cpf1 mutant protein in which the amino acids K1025, K1026, K1079, K1080, K1096 and E1097 are substituted with alanine.
The method according to claim 1, wherein the LbCpf1 protein is a Cpf1 mutant protein having the sequence of SEQ ID NO: 1.
A polynucleotide encoding the Cpf1 mutant protein of claim 1.
A vector containing a polynucleotide encoding the Cpf1 mutant protein of claim 1.
A complex comprising the Cpf1 mutant protein of claim 1 and a guide RNA capable of complementary binding to a target nucleic acid.
A kit for detecting a target nucleic acid comprising at least one of the Cpf1 mutant protein of any one of claims 1 to 4, the polynucleotide of claim 5, the vector of claim 6, and the complex of claim 7.
The kit for detecting a target nucleic acid according to claim 8, further comprising at least one of guide RNA or labeled ssDNA capable of binding complementary to the target nucleic acid.
A method for detecting a target nucleic acid comprising treating a sample with at least one of the Cpf1 mutant protein of any one of claims 1 to 4, the polynucleotide of claim 5, the vector of claim 6, and the complex of claim 7.
The method of claim 10, further comprising treating the sample with at least one of guide RNA or labeled ssDNA capable of binding complementary to the target nucleic acid.