Polypeptides with the ability to form complexes with novel guide RNAs

Abstract

Provided are: a novel heat-resistant Cas nuclease belonging to class 2 and type V; and a method for producing the same.

Description

Technical Field

[0001] The present invention relates to a polypeptide having the ability to form a complex with guide RNA, particularly a heat-resistant Cas nuclease (CRISPR-associated enzyme).

Background Art

[0002] CRISPR (clustered regularly interspaced short palindromic repeat) is a locus containing short repetitive sequences of several tens of base pairs and functions as a kind of acquired immune system in prokaryotes. In addition, near the CRISPR repeat, there exists a group of CRISPR-associated (cas) genes encoding nuclease and helicase (Non-Patent Document 1).

[0003] Most CRISPR loci have three elements: a cas gene group, a leader sequence, and a repeat-spacer sequence. Also, depending on the types of Cas protein groups constituting the CRISPR-Cas system and the differences in the mechanisms of action, they are roughly classified into two Classes, and each CRISPR-Cas system is further classified into several subtypes. Class 1 is subdivided into Type I, III, and IV, and Class 2 is subdivided into Type II, V, and VI. Among them, Cas12a is classified into Class 2, Type V CRISPR-Cas (Non-Patent Document 2).

[0004] In the bacterium Francisella novicida, the nuclease Cpf1 was discovered (Non-Patent Document 3, Patent Document 1). Initially, it was named Cpf1, but along with the classification of the CRISPR-Cas system, it was newly named Cas12a.

[0005] Many of the reported Cas nucleases are heat-labile. Although heat-resistant Cas9 was reported recently (Non-Patent Document 4), there is no report on heat-resistant Cas12a.

[0006] The CRISPR-Cas9 system is being explored for various applications, including genome editing, transcriptional regulation, imaging, and nucleic acid detection (Non-Patent Document 5). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2016 / 205711 brochure [Non-patent literature]

[0008] [Non-Patent Document 1] Jansen R. et al., Mol Microbiol., Vol. 43, No. 6, pp. 1565-1575, 2002. [Non-Patent Document 2] Kira S. Makarova et al., Review Nat Rev Microbiol., Vol. 13, No. 11, pp. 722-736, 2015. [Non-Patent Document 3] Bernd Zetsche et al., Cell, Vol. 163, No. 3, pp. 759-771, 2015. [Non-Patent Document 4] ST. Schmidt et al., Proc Natl Acad Sci US A., Vol. 116, No. 46, pp. 23100-23105, 2019. [Non-Patent Document 5] A. Mazhar et al., Nature Communications, Vol. 9, No. 1, p. 1911, 2018. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] However, further development of Cas nucleases is still desired. The object of the present invention is to provide a novel heat-resistant Cas nuclease. [Means for solving the problem]

[0010] The inventors of this invention diligently conducted research to develop a heat-resistant Cas nuclease and, surprisingly, discovered that nucleic acids encoding a heat-resistant Cas nuclease exist in a group of microorganisms collected from a hot spring sample in Oita Prefecture. Thus, the present invention was completed.

[0011] In other words, the present invention is [1] Amino acid sequence of Sequence ID No. 1, An amino acid sequence having 1 to 10 amino acid mutations in the amino acid sequence of SEQ ID NO: 1, where the mutations are substitutions, deletions, insertions and / or additions, and An amino acid sequence having more than 50% identity with the sequence of Sequence ID No. 1, Isolated polypeptides having the ability to form complexes with guide RNA, comprising a sequence selected from the group consisting of; [2] The polypeptide described in [1] further possessing nuclease activity; [3] The polypeptide described in [1] or [2], which is a polypeptide classified as class 2 type V CRISPR / Cas; [4] A polypeptide according to any one of [1] to [3] that has the activity to cleave double-stranded DNA and single-stranded DNA at 55 to 65°C; [5][1] A polypeptide comprising an amino acid sequence resulting from amino acid substitution, deletion, insertion, and / or addition to the amino acid sequence of the polypeptide described above, having the ability to form a complex with guide RNA, and lacking nuclease activity; [6](1) A polypeptide having an amino acid sequence that includes a substitution from aspartic acid to alanine at the 945th amino acid residue in the amino acid sequence of Sequence ID No. 1, having the ability to form a complex with guide RNA, and lacking nuclease activity, or (2) A polypeptide having the ability to form a complex with guide RNA, containing the amino acid sequence of SEQ ID NO: 6, and lacking nuclease activity. The polypeptide described in [5]; An RNA-polypeptide complex comprising the polypeptide according to any one of [1] to [5] and guide RNA; The RNA-polypeptide complex according to [7], wherein the polypeptide and the guide RNA are irreversibly crosslinked; An isolated nucleic acid encoding the polypeptide according to any one of [1] to [6] in [9]; An expression vector comprising the nucleic acid according to [9]; A cell transformed with the expression vector according to

[10] ; and

[12] A composition comprising the following (a) and (b): (a) The polypeptide according to any one of [1] to [6]; (b) Guide RNA; It relates to.

Effect of the Invention

[0012] The present invention provides a novel heat-resistant Cas nuclease and a method for producing the same. According to the present invention, a Cas nuclease Cas12a belonging to Class 2, Type V (hereinafter referred to as the polypeptide of the present invention) is provided. Such heat-resistant Cas12a has not been known and has been found for the first time by the present invention.

Brief Description of the Drawings

[0013] [Figure 1] The results of measuring the target double-stranded DNA cleavage activity using the polypeptide of the present invention are shown. [Figure 2] The results of measuring the dissociation of the complex (RNP) and the target double-stranded DNA are shown. [Figure 3] The results of measuring the heat resistance of the polypeptide of the present invention are shown. [Figure 4] The results of measuring the optimum temperature of the polypeptide of the present invention are shown. [Figure 5] The results of measuring the length of the target double-stranded DNA of the polypeptide of the present invention are shown. [Figure 6] The results of measuring the single-stranded DNA cleavage activity of the polypeptide of the present invention are shown. [Figure 7]The results of the analysis of the PAM sequence recognized by the polypeptide of the present invention are shown. [Figure 8] The results of measuring targeted double-strand DNA cleavage activity using polypeptides lacking the nuclease activity of the present invention are shown. [Figure 9] The results of measuring targeted single-strand DNA cleavage activity using polypeptides lacking the nuclease activity of the present invention are shown. [Figure 10] The results of specific detection of target genes using the polypeptide of the present invention are shown. [Modes for carrying out the invention]

[0014] The details are explained below.

[0015] 1. Polypeptide of the present invention (1) Polypeptide of the present invention A first embodiment of the present invention relates to a polypeptide having the ability to form a complex with a guide RNA.

[0016] An example of the polypeptide of the present invention is a protein containing the amino acid sequence described in Sequence ID No. 1. This amino acid sequence was classified as Cas12a based on its cluster structure, but BLAST search results showed it to be a novel amino acid sequence with low identity to known Cas nucleases. Furthermore, there were no regions with particularly high identity. The polypeptide with the highest homology was Cpf1 (GenBank: PJE64038.1), with an identity of approximately 26%.

[0017] Furthermore, polypeptides of the present invention also include those having an amino acid sequence that is at least 50% identical to SEQ ID NO: 1, for example, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more, and that have the ability to form a complex with guide RNA. Polypeptides of the present invention also include those having an amino acid sequence in which 1 to 10 amino acid residues, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues are substituted, deleted, inserted, and / or added in SEQ ID NO: 1, and that have the ability to form a complex with guide RNA.

[0018] While proteins containing the amino acid sequence described in Sequence ID No. 1 possess nuclease activity, the polypeptide of the present invention is not particularly limited in terms of whether or not it has nuclease activity, as long as it has the ability to form a complex with guide RNA.

[0019] In this invention, "heat resistance" refers to the property of being able to retain enzyme activity even after heat treatment. For example, if an enzyme can retain 50% or more of its activity, such as the ability to form a complex with guide RNA, after being treated at a temperature of 55°C or higher, 65°C or higher, or 75°C or higher for 5 minutes, it can be said to "have heat resistance."

[0020] While known Cas12a compounds are heat-sensitive, the polypeptide of the present invention exhibits activity even under temperature conditions of 40°C or higher, for example, 45°C or higher, 55°C or higher, 65°C or higher, or 75°C or higher. Furthermore, the polypeptide of the present invention exhibits the highest activity in the high-temperature range of 55-65°C. Even after being kept warm in this high-temperature range, it maintains the same activity as before the reaction.

[0021] In one aspect of the present invention, the polypeptide of the present invention has the activity to form a complex with guide RNA to cleave a specific site on a target double-stranded DNA, and the activity to form a complex with guide RNA to nonspecifically cleave any site on a single-stranded DNA that is not a target nucleic acid. The cleavage of the target double-stranded DNA occurs when the polypeptide of the present invention, which has nuclease activity, and the guide RNA act on it. On the other hand, the cleavage of the single-stranded DNA occurs following the cleavage of the target double-stranded DNA, with the polypeptide of the present invention acting alone.

[0022] The polypeptide-guide RNA complex of the present invention (hereinafter sometimes referred to as RNP) is extremely stable compared to known Cas9-guide RNA complexes, etc. While these known complexes are easily dissociated, the polypeptide-guide RNA complex of the present invention remains formed even under temperature conditions of 40°C or higher, for example, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, or 70°C or higher.

[0023] Furthermore, the polypeptide of the present invention and the guide RNA may be covalently linked. There are no limitations on the method of covalent linking, and it can be carried out by methods commonly used in the art. A linker may be used during covalent linking. For example, the polypeptide of the present invention and the guide RNA may be irreversibly crosslinked.

[0024] Furthermore, in another aspect of the present invention, the polypeptide may lack nuclease activity. Such polypeptides can be used for the isolation and purification of nucleic acid fragments having a specific base sequence by utilizing their ability to recognize specific base sequences through complex with guide RNA, for example, in combination with immunoprecipitation technology.

[0025] (2) Guide RNA Guide RNA is the RNA that guides the Cas nuclease protein to the target DNA sequence in the CRISPR-Cas system, and is the nucleic acid that determines the DNA sequence specificity of the CRISPR-Cas system. Guide RNA and Cas nuclease form a complex, recognize the PAM (protospacer adjacent motif) sequence and bind to the target DNA, and then the guide RNA anneals with the target DNA sequence, and the Cas nuclease cleaves the target DNA sequence.

[0026] The guide RNA to which Cas12a binds consists solely of crRNA (CRISPR RNA) having a sequence complementary to the target DNA sequence to be cleaved. In contrast, the guide RNAs of other Cas nucleases, such as Cas9, consist of two RNA molecules: crRNA and tracrRNA (trans-activating crRNA), which serves as the foundation for forming a complex with the Cas nuclease. Single guide RNA (sgRNA), which artificially combines these two RNA molecules into a single RNA molecule, is also widely used. In this respect, Cas12a differs from other Cas nucleases such as Cas9.

[0027] In the present invention, the guide RNA sequence is preferably designed to be the 5' (upstream) or 3' (downstream) base sequence of the PAM sequence present in the target DNA sequence to be cleaved. The design of the guide RNA sequence is not limited in any way to the present invention, but known online tools such as CHOPCHOP (https: / / chopchop.cbu.uib.no / ), CRISPRdirect (https: / / crispr.dbcls.jp / ), and CRISPR Design Tool (https: / / horizondiscovery.com / products / tools / CRISPR-Design-Tool / ) can be suitably used.

[0028] There are no limitations on the length of the guide RNA. The guide RNA sequence should be a sequence that can anneal to the target DNA sequence while in a complex with the polypeptide of the present invention. Alternatively, it should be a sequence that can cleave the target DNA sequence while in a complex with the polypeptide of the present invention having nuclease activity. The length of the guide RNA does not limit the present invention in any way, but for example, 17mer or more, 20mer or more, or 22mer or more is preferred. Furthermore, the length of the guide RNA does not limit the present invention in any way, but for example, 100mer or less, 80mer or less, or 60mer or less is preferred.

[0029] (3) PAM sequence PAM sequences are sequences used by Cas nucleases to recognize target DNA sequences. PAM sequences differ depending on the type of Cas nuclease and the species from which they originate. For example, Cpf1 from Acidaminococcus sp. (sometimes abbreviated as AsCpf1) or Cpf1 from Lachnospiraceae bacterium (sometimes abbreviated as LbCpf1) recognize T-rich sequences such as 5'-TTTV-3', 5'-TTCA-3', 5'-TCTA-3', and 5'-CTTA-3' (T. Yamano et al., Molecular Cell, Vol. 67, No. 4, pp. 633-645, e3, 2017). However, it is thought that these Cpf1s cannot recognize sequences containing two or more Cs as PAM sequences. On the other hand, Cas9 recognizes G-rich sequences such as 5'-NGG-3' and 5'-NGGRR(T)-3'. Note that V means not T (A, C, or G), N means (A, C, G, or T), and R means (A or G).

[0030] The PAM sequence of the polypeptide of the present invention can be determined by known methods, such as those described in the examples below. The PAM sequence of the polypeptide of the present invention having the amino acid sequence of SEQ ID NO: 1 is YYN. Unlike AsCpf1 and LbCpf1, which are also classified as Cas12a, the polypeptide of the present invention can recognize sequences containing two Cs as PAM sequences. Note that Y is (C or T).

[0031] When acting on target DNA, the polypeptide and guide RNA of the present invention may be prepared as separate molecules so that they form a complex when mixed, or they may be mixed beforehand to form a complex. Alternatively, the polypeptide and guide RNA of the present invention may be covalently bonded together and used as a single molecule. The binding mode of the polypeptide and guide RNA of the present invention is not limited to either, as long as they can bind to a target DNA sequence having a sequence complementary to the guide RNA. In this invention, the molecule formed by covalently bonding the polypeptide and guide RNA of the present invention is also included in the complex of the present invention.

[0032] 2. Nucleic acids encoding the polypeptide of the present invention A second embodiment of the present invention relates to a nucleic acid encoding the polypeptide of the present invention. Specifically, it is a nucleic acid encoding the polypeptide of the present invention.

[0033] An example of a nucleic acid encoding the polypeptide of the present invention is a nucleic acid encoding the amino acid sequence described in SEQ ID NO: 1. Another example is a nucleic acid containing the base sequence of SEQ ID NO: 2. However, as long as the nucleic acid encoding the polypeptide of the present invention is used, there are no limitations on the base sequence of the nucleic acid encoding the polypeptide of the present invention; for example, the nucleic acid encoding the amino acid sequence described in SEQ ID NO: 1 may contain codons different from those in SEQ ID NO: 2. When producing the polypeptide of the present invention using the nucleic acid of the present invention, a nucleic acid designed by selecting codons appropriate to the host used (codon optimization) may be used. Such codon optimization can be performed by methods commonly used in the art.

[0034] 3. Expression vector containing nucleic acid encoding the polypeptide of the present invention A third embodiment of the present invention relates to an expression vector comprising a nucleic acid encoding the polypeptide of the present invention.

[0035] The expression vector of the present invention preferably includes a nucleic acid encoding the polypeptide of the present invention, and also includes an expression regulatory sequence operably linked to the nucleic acid.

[0036] The expression vector carrying the nucleic acid encoding the polypeptide of the present invention may be any expression vector commonly used in this art, and is not particularly limited. Vectors capable of autonomous replication in host cells or vectors that can be incorporated into host chromosomes can be used. In other words, any expression vector compatible with the host should be used.

[0037] For example, plasmid vectors, phage vectors, viral vectors, etc., can be used as expression vectors carrying the nucleic acid encoding the polypeptide of the present invention. These vectors are well known to those skilled in the art, and many are commercially available. These known vectors and their modifications can be used in the present invention.

[0038] The promoter to be incorporated into the expression vector of the present invention can be selected according to the host. For example, in E. coli, promoters derived from E. coli or phages, such as the trp promoter, lac promoter, PL promoter, and PR promoter, or their modifiers, can be used, but the invention is not limited to those mentioned above. Furthermore, an expression system combining a phage-derived promoter and an RNA polymerase gene (e.g., the pET expression system) may also be used.

[0039] To facilitate the purification of the expressed polypeptide, the expression vector of the present invention may further contain a nucleic acid encoding an affinity tag.

[0040] The nucleic acid encoding the affinity tag is inserted into the vector so that a fusion protein of the polypeptide of the present invention and the affinity tag is expressed. The nucleic acid encoding the tag may be attached to either the 5' end and / or the 3' end of the nucleic acid encoding the polypeptide of the present invention, and should be attached to a position that does not impair the expression of the fusion protein, the function of the polypeptide of the present invention, or the tag function. The tag may be one that can be removed during or after the purification of the expressed polypeptide. These affinity tags are well known to those skilled in the art and are often incorporated into commercially available vectors. These known affinity tags and their modifications can be used in the present invention.

[0041] The expression vector of the present invention may include one or more expression regulatory sequences in addition to the promoter. The expression regulatory sequences are not particularly limited, but examples include genes involved in promoter control, ribosome binding sequences, polyadenylation signals, transcription termination sequences (transcriptional terminators), enhancers, etc. Furthermore, the expression vector of the present invention may also include genes encoding replication origins or markers used for selecting transformants (drug resistance genes, fluorescent markers, luminescence markers, etc.), and nucleotide sequences for improving translation efficiency.

[0042] 4. Cells transformed with the expression vector of the present invention A fourth embodiment of the present invention relates to cells transformed with the above-mentioned expression vector.

[0043] The cells (hosts) to be transformed with the vector expressing the polypeptide of the present invention are not particularly limited, as long as they are hosts commonly used in the art. For example, bacteria (E. coli, Bacillus subtilis, etc.), yeast, filamentous fungi, insect cells, eukaryotic cells, and animal cells (mammalian cells including human cells, etc.) can be used. These hosts are well known to those skilled in the art, and many are commercially available. These known hosts and their variants can be used in the present invention.

[0044] The method for introducing the expression vector into the host is not particularly limited as long as it can introduce the expression vector into the host. For example, methods using calcium ions, electroporation, spheroplasty, lithium acetate, calcium phosphate, lipofection, etc., can be used. These introduction methods are well known to those skilled in the art, and instruments and kits for them are commercially available.

[0045] The polypeptide of the present invention can be obtained from the culture by culturing a transformant obtained by introducing an expression vector into a host. The culture conditions are not particularly limited as long as they are suitable for the expression vector, host, etc. used. Depending on the type of host or expression vector used, necessary inducing substances may be added at an appropriate time. After culturing, the culture medium is centrifuged, the obtained cells are washed, and then a lysate containing the polypeptide of the present invention can be obtained by ultrasonic disruption, freeze-thaw treatment, lysis enzyme treatment, etc. Since the lysate contains many impurities, it is desirable to purify the polypeptide of the present invention by appropriately combining purification methods used in this art, such as ammonium sulfate precipitation, anion exchange column, cation exchange column, gel filtration column, affinity chromatography column, dialysis, etc. These culture and purification methods are well known to those skilled in the art, and instruments and kits for this purpose are commercially available. A method for producing the polypeptide of the present invention using the above transformant is also one aspect of the present invention.

[0046] 5. Composition or kit of the present invention A fifth embodiment of the present invention relates to a composition or kit comprising the polypeptide of the present invention.

[0047] One embodiment of the composition of the present invention is a nucleic acid cleavage composition. The composition comprises the polypeptide of the present invention having nuclease activity, as well as guide RNA, buffering components, sterile water, etc. Furthermore, it may further contain components necessary for other nucleic acid cleavage reactions, such as divalent metal salts.

[0048] One embodiment of the kit of the present invention is a kit for nucleic acid cleavage. The kit of the present invention is, for example, a kit containing the composition of the present invention. That is, the kit of the present invention contains, in addition to the polypeptide of the present invention having nuclease activity, components necessary for the nucleic acid cleavage reaction, such as guide RNA, divalent metal salt, buffering component, sterile water, etc. Another embodiment is a kit for the purification, separation, and detection of nucleic acid fragments having a specific base sequence, which contains, for example, the polypeptide of the present invention that does not have nuclease activity, guide RNA, a solid support to which an antibody capable of specifically recognizing the polypeptide of the present invention is conjugated, a buffering component, sterile water, etc.

[0049] Furthermore, the composition or kit of the present invention may contain components necessary for a PCR reaction system, such as a PCR primer set for amplifying a target nucleic acid, reverse transcriptase, heat-stable DNA polymerase, and inorganic pyrophosphatase. It may also contain components necessary for detection, such as a labeled reporter nucleic acid.

[0050] 6. Method for cleaving target nucleic acids using the polypeptide of the present invention A sixth embodiment of the present invention relates to a method for cleaving a target nucleic acid using the polypeptide of the present invention having nuclease activity.

[0051] (1) Methods for cutting double-stranded DNA The polypeptide of the present invention, possessing nuclease activity, can be used in conjunction with a guide RNA to specifically cleave a particular site on a target double-stranded DNA. The polypeptide of the present invention may be provided in the form of a complex with the guide RNA, or the polypeptide of the present invention and the guide RNA may be provided as separate molecules to form a complex in situ.

[0052] As described above, the polypeptide of the present invention exhibits heat resistance, showing activity even under temperature conditions of 40°C or higher, for example, 45°C or higher, 50°C or higher, 55°C or higher, 65°C or higher, or 75°C or higher. Therefore, it can be used in reaction systems where known Cas nucleases would be inactivated and thus could not be used. Furthermore, the ability to react at high temperatures makes it possible to dissociate the secondary structure of nucleic acids, thereby improving the cleavage efficiency of target nucleic acids.

[0053] (2) Method for cutting single-stranded DNA The polypeptide-guide RNA complex of the present invention can nonspecifically cleave single-stranded DNA, separate from the cleavage of double-stranded DNA. Therefore, when used with guide RNA, the polypeptide of the present invention nonspecifically cleaves single-stranded DNA, separate from the cleavage of double-stranded DNA. Consequently, the polypeptide-guide RNA complex of the present invention can be used, for example, in a detection system using a probe having a fluorescent dye and a quenching group in the presence of target double-stranded DNA.

[0054] 7. Introduction of the polypeptide of the present invention into target cells A seventh embodiment of the present invention relates to the introduction of the polypeptide of the present invention into target cells.

[0055] The polypeptide and guide RNA complex of the present invention is extremely stable. This allows for repeated introduction of the complex into target cells via electroporation, maintaining high efficiency in the introduction process. Furthermore, even when using other introduction methods, the complex can be introduced into cells with high efficiency.

[0056] Furthermore, due to its high stability, even if inhibitory substances (such as culture medium components or sample-derived components) are present in the solution during cell introduction, the complex can be taken up by the cells while maintaining its formation. In other words, the step of strictly washing the target cells becomes unnecessary. This reduces the number of steps and the amount of reagents used, preventing contamination and allowing for the maintenance of high cell proliferation and viability. These are features not found in known Cas nucleases.

[0057] For introduction into target cells, a vector containing a nucleic acid encoding the polypeptide of the present invention and a nucleic acid encoding a guide RNA may be used. While the vector is not limited to the present invention, plasmid vectors or viral vectors are preferably used. These introduction methods are well known to those skilled in the art, and instruments and kits for this purpose are commercially available.

[0058] 8. Genome editing The eighth embodiment of the present invention relates to genome editing.

[0059] By introducing a complex of the polypeptide and guide RNA of the present invention, which has nuclease activity, into target cells, genome editing of target cells is possible. Genome editing can be performed using base substitution, knock-in, or knock-out. In addition to the method of introducing the complex into cells, methods may also be used to introduce nucleic acids encoding the polypeptide and guide RNA of the present invention into cells and express both within the cells, to introduce nucleic acids encoding the polypeptide and guide RNA into cells, or to introduce the polypeptide and nucleic acids encoding the guide RNA into cells. When introducing nucleic acids encoding the polypeptide and guide RNA into cells, it goes without saying that nucleic acid constructs should be designed so that each is expressed within the cells.

[0060] 9. Use in target nucleic acid detection Nine embodiments of the present invention relate to applications for target nucleic acid detection. The polypeptide of the present invention can be used in conjunction with a guide RNA to detect target nucleic acids. The polypeptide of the present invention may have nuclease activity or may lack nuclease activity.

[0061] Known Cas nucleases are heat-sensitive, meaning that to incorporate them into a target nucleic acid detection system, they must be added after the reaction in which the target nucleic acid has been amplified. This means that the container lid would have to be opened during the detection process, posing a risk of contamination.

[0062] On the other hand, the polypeptide of the present invention is heat-resistant, and for example, when the product amplified by PCR is used as the target nucleic acid, it can be added in the initial step of mixing the reagents for PCR. This eliminates the risk of contamination. Furthermore, the ability to react at high temperatures makes it possible to dissociate the secondary structure of the nucleic acid, thereby improving the cleavage efficiency of the target nucleic acid.

[0063] The composition for detecting target nucleic acids includes, in addition to the polypeptide of the present invention, guide RNA, buffering components, sterile water, a PCR primer set for amplifying the target nucleic acid, reverse transcriptase, DNA polymerase, labeled reporter nucleic acid, inorganic pyrophosphatase, etc. If the polypeptide of the present invention further has nuclease activity, it may further contain components necessary for nucleic acid cleavage reactions, such as divalent metal salts.

[0064] 10. Use in NGS library creation A tenth embodiment of the present invention relates to its use in the preparation of NGS libraries.

[0065] The polypeptide of the present invention can be used in the preparation of libraries for next-generation sequencing (NGS) for the degradation and removal of nucleic acids that are not the target of analysis, as well as for the enrichment and recovery of target nucleic acids.

[0066] In NGS data analysis, reducing noise data, and consequently reducing the nucleic acids that cause noise, is a crucial issue. The polypeptide of the present invention is useful in reducing these noise-causing nucleic acids.

[0067] 11. Polypeptides of the present invention lacking nuclease activity An eleventh embodiment of the present invention relates to a polypeptide of the present invention lacking nuclease activity.

[0068] A mutant of the polypeptide of the present invention lacking nuclease activity (hereinafter sometimes referred to as "dead Cas") is a mutant that, although lacking nuclease activity, retains the ability to bind to target double-stranded DNA and can be used as a protein domain that specifically recognizes DNA sequences.

[0069] The aforementioned mutants can be produced by introducing an amino acid substitution mutation into the nuclease domain of the polypeptide of the present invention, thereby eliminating nuclease activity. In the amino acid sequence of Sequence ID No. 1, the RuvC-I domain is located at positions 920-977, the RuvC-II domain at positions 996-1105, and the RuvC-III domain at positions 1305-1353. Techniques well known to those skilled in the art can be used to introduce such mutations. The above mutation is selected from substitution, deletion, insertion, or addition of one or more amino acid residues (for example, 1 to 10 amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues), or a combination thereof, and is not limited as long as nuclease activity is eliminated.

[0070] Because this dead Cas lacks nuclease activity, it is expected to have different applications than the wild type. While this invention is not limited in any way, it can be used for the following applications, for example. (1) Dead Cas is bound to the promoter region, suppressing transcription through steric hindrance (CRISPRi; CRISPR interference). (2) A fusion of dead Cas and a transcription activator is bound to the promoter region to activate transcription (CRISPRa; CRISPR activation). (3) The combination of dead Cas and a demethylating agent (small molecule compound, enzyme, etc.) demethylates the promoter region and activates transcription. (4) Epigenome editing (histone acetylation, histone demethylation, etc.) is performed using a fusion of dead Cas and histone modification domain. (5) Use dead Cas labeled with a fluorescent dye to perform imaging of a specific DNA region. (6) A fusion of dead Cas and an epitope tag is used to purify specific sequences on the chromosome (a technique similar to ChIP).

[0071] The dead Cas of the present invention means a polypeptide having an amino acid sequence in which a mutation occurs in the amino acid sequence of a polypeptide having the ability to form a complex with a guide RNA, selected from (a) to (c) below, such that the nuclease activity of the polypeptide is lost. (a) Amino acid sequence of Sequence ID No. 1, (b) an amino acid sequence having 1 to 10 amino acid mutations in the amino acid sequence of SEQ ID NO: 1, wherein the mutations are substitutions, deletions, insertions and / or additions, and (c) An amino acid sequence having 50% or more identity with the sequence of Sequence ID No. 1.

[0072] Without limiting the present invention in any way, examples of dead Cass of the present invention include polypeptides that have the ability to form complexes with guide RNA and lack nuclease activity, comprising an amino acid sequence in which the 945th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine, and which have the ability to form complexes with guide RNA. Examples of such polypeptides include polypeptides comprising the amino acid sequence shown in SEQ ID NO: 6. Furthermore, polypeptides that have an amino acid sequence in which the amino acid sequence of SEQ ID NO: 6, in which the 945th aspartic acid in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine, comprises substitutions, deletions, insertions and / or additions of other amino acid residues (preferably up to 10), and which have the ability to form complexes with guide RNA are also included as dead Cass of the present invention.

[0073] Similarly, by substituting glutamic acid at position 1030, arginine at position 1255, or aspartic acid at position 1305 in the amino acid sequence of Sequence ID No. 1 with alanine, polypeptides that can form complexes with guide RNA and lack nuclease activity can be produced. Furthermore, multiple amino acid substitutions may be combined as needed.

[0074] The nucleic acid encoding the dead Cas polypeptide of the present invention is a nucleic acid having a base sequence encoding the above polypeptide, and one embodiment thereof is an example of a nucleic acid encoding the amino acid sequence described in SEQ ID NO: 6. Another example is a nucleic acid containing the base sequence of SEQ ID NO: 7. However, as long as it encodes the dead Cas polypeptide of the present invention, there are no limitations on the base sequence of the nucleic acid encoding the dead Cas polypeptide of the present invention; for example, the nucleic acid encoding the amino acid sequence described in SEQ ID NO: 6 may contain codons different from those in SEQ ID NO: 7. When producing the dead Cas polypeptide of the present invention using the nucleic acid of the present invention, a nucleic acid designed by selecting codons appropriate to the host used (codon optimization) may be used as appropriate. Such codon optimization can be performed by methods commonly used in the art. [Examples]

[0075] The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to these examples.

[0076] Example 1. Metagenomic analysis of geothermal water (1) Metagenome analysis Metagenomic analysis was performed on the microbial community contained in hydrothermal fluid samples from Kamado Jigoku in Beppu City, Oita Prefecture. First, DNA samples extracted from the samples were physically fragmented into several hundred bp fragments using an acoustic stabilizer Covaris (Covaris Corporation), and then size-sorted using AMPure® XP (Beckman Coulter). A library was prepared from these fragments using the Thru PLEX® DNA-Seq Kit (Takara Bio USA), and used for sequencing analysis. MiSeq (Illumina Corporation) was used for sequencing analysis, with a 250 bp paired-end sequencing setting. After assembly of the sequence analysis results, a novel gene (SEQ ID NO: 2) was found in a contig in the library. This novel gene encoded a polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0077] (2) Class and type estimation A BLAST search of the amino acid sequence described in Sequence ID No. 1 found that the closest sequence was type V CRISPR-associated protein Cpf1 [Candidatus Ryanbacteria bacterium CG10_big_fil_rev_8_21_14_0_10_43_42] (GenBank: PJE64038.1), a Cas12a variant. However, the degree of identity was only 25.97%.

[0078] Furthermore, since homologs of the cas4, cas1, and cas2 genes are located in the downstream region of the novel gene of SEQ ID NO: 2, we determined that the novel gene of SEQ ID NO: 2 is a CRISPR-associated protein belonging to class 2, type V. Even further downstream, homologs of the CRISPR1, CRISPR2, CRISPR3, and CRISPR4 genes were found.

[0079] Example 2. Expression of the polypeptide of the present invention (1) Construction of expression plasmid To perform functional analysis of a polypeptide having the amino acid sequence of Sequence ID No. 1, we constructed a forced expression system in E. coli.

[0080] The artificially synthesized nucleic acid of Sequence ID No. 2 was inserted into the in-fusion cloning site of the expression vector pET6xHN-N (Takara Bio USA) according to the instructions provided with the kit. The resulting plasmid was used to transform E. coli BL21 DE3 strain, and clones that became ampicillin resistant were selected. From these clones, the plasmid pET-Cas12a-N for polypeptide expression of the present invention was obtained.

[0081] Similarly, the artificially synthesized nucleic acid of Sequence ID No. 2 was inserted into the in-fusion cloning site of the expression vector pET6xHN-C (Takara Bio USA). This yielded the plasmid pET-Cas12a-C for polypeptide expression of the present invention.

[0082] The expression vector pET6xHN-N has a His tag attached to the N-terminus of the expressed protein, while pET6xHN-C has a His tag attached to the C-terminus of the expressed protein.

[0083] (2) Expression of the polypeptide of the present invention Single colonies of transformants possessing either pET-Cas12a-N or pET-Cas12a-C were inoculated into 2 mL of LB medium containing 100 μg / mL ampicillin (hereinafter referred to as LB-Amp medium) and incubated overnight at 37°C with shaking. 500 μL of this culture was added to 50 mL of LB-Amp medium and incubated overnight at 37°C with shaking. Subsequently, 1 mM IPTG was added to the culture and further induction culture was performed overnight at 25°C. The culture was subjected to centrifugation at 6000xg for 10 minutes, and bacterial cells were collected from the culture.

[0084] (3) Purification of the polypeptide of the present invention The bacterial cells obtained by the above procedure were suspended in 3 mL of a mixed solution of 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol, and 0.5 mM PMSF (phenylmethylsulfonyl fluoride) (hereinafter referred to as Buffer S). Ultrasonic disruption was performed using an ultrasonic disruptor (Sonic&Materials) at 4°C for 10 minutes, and it was confirmed that the suspension became clear.

[0085] The resulting suspension after sonication was heated at 95°C for 10 minutes to denature the heat-labile proteins. Then, it was centrifuged at 9500xg for 30 minutes at 4°C, and the supernatant was collected. Imidazole was added to the supernatant to a final concentration of 50 mM (hereinafter referred to as the crude extract).

[0086] The Ni Sepharose FF column was equilibrated by sequentially adding three times the amount of sterile water as the resin, three times the amount of 200 mM NiCl2, three times the amount of sterile water, and three times the amount of Buffer A (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol, and 50 mM imidazole).

[0087] The obtained crude extract was subjected to an equilibrated column. The column was washed with four times the volume of Buffer A as the crude extract. Subsequently, concentration gradient elution was performed using Buffer A containing 50 mM to 300 mM imidazole to elute the adsorbed material from the column. The fraction containing the polypeptide of the present invention was dialyzed against Buffer B (50 mM Tris-HCl pH 7.5, 500 mM NaCl, and 5% glycerol).

[0088] Next, a hydroxyapatite column (CHT Ceramic Hydroxyapatite Type I 80 μm) was subjected to a 20 mM KPB (dipotassium hydrogen phosphate and potassium dihydrogen phosphate) / Buffer B mixture in an amount 10 times the amount of resin, and equilibrated. The resulting dialysate was then subjected to the hydroxyapatite column. The column was washed with 5 times the amount of 20 mM KPB / Buffer B compared to the dialysate. Subsequently, concentration gradient elution was performed using Buffer B containing 20 mM to 300 mM KPB to elute the adsorbed material from the hydroxyapatite column. The fraction containing the polypeptide of the present invention was dialyzed against Buffer B. The dialysate was concentrated by ultrafiltration and further dialyzed against Pack Buffer (20 mM HEPES-KOH pH 7.5, 150 mM KCl, 10% glycerol, and 1 mM DTT) to obtain the purified polypeptide of the present invention.

[0089] The purified polypeptide of the present invention was confirmed to be free from contamination by host-derived nucleases. Measurements were performed for RNase activity (substrates: 16S rRNA and 23S rRNA), DNase activity (substrate: λ-DNA), endonuclease activity (substrate: pBR322 plasmid), and exonuclease activity (substrate: λ-HindIII digest). No activity was detected in any of these tests. The reaction mixture consisted of 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1 mg / ml BSA (all final concentrations).

[0090] The following tests used the purified polypeptide of the present invention. Hereafter, the expressed protein with the His tag at the N-terminus was referred to as Cas12a-N, and the expressed protein with the His tag at the C-terminus was referred to as Cas12a-C.

[0091] Example 3. Activity Measurement 18-1000 ng of the purified polypeptide (Cas12a-N or Cas12a-C) and 2 pmol of guide RNA (SEQ ID NO: 3) were mixed and heated at 37°C for 5 minutes. Then, 100 ng of substrate double-stranded DNA (SEQ ID NO: 4) was mixed in, and the reaction was carried out in the following order: (1) heated at 37°C for 1 hour, (2) heated at 80°C for 5 minutes, and (3) kept at 4°C after the reaction. The reaction solution used was composed of 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1 mg / ml BSA (all final concentrations). The volume of the mixture after mixing was 20 μl. In each of the following examples, the same composition and volume of reaction solution were used. Next, a portion of the reaction solution was subjected to agarose gel electrophoresis to check for the presence or absence of substrate DNA cleavage.

[0092] Here, the double-stranded DNA substrate is a portion of the human DNMT1 gene (Gene ID: 1786). Specifically, it is the nucleotide sequence corresponding to positions 60927-61566 of RefSeqGene ID: NG_028016.3. In each of the following examples, the same region was used as the double-stranded DNA substrate.

[0093] The results are shown in Figure 1. In the reaction solution containing the polypeptide of the present invention, substrate DNA was cleaved and short-chain DNA was generated, confirming that cleavage of the substrate double-stranded DNA occurs in the presence of the polypeptide of the present invention and guide RNA.

[0094] Example 4. Dissociation of the polypeptide, gRNA, and substrate DNA of the present invention. 450 ng of the purified polypeptide (Cas12a-N or Cas12a-C) and 2 pmol of guide RNA were mixed and heated at 37°C for 5 minutes, after which the mixture was kept at 4°C. 100 ng of substrate double-stranded DNA (SEQ ID NO: 4) was mixed into the reaction mixture and heated at 37°C for 20 minutes. After the reaction, EDTA was added to a final concentration of 16.7 mM, and the mixture was divided into (1) a system heated at 95°C for 5 minutes and (2) a system without heating. The dissociation of the polypeptide, gRNA, and substrate DNA of the present invention was tested. The mixture was kept at 4°C after the reaction. Subsequently, a portion of the reaction mixture was subjected to agarose gel electrophoresis.

[0095] The results are shown in Figure 2. In the unheated system (lanes 2 and 4), bands of cleaved nucleic acids were observed around 600 base and around 250 base, the same as in lane 1 (which did not contain the polypeptide and gRNA of the present invention). On the other hand, in the heated system (lanes 3 and 5), in addition to those two bands, a band of cleaved nucleic acids (nucleic acids hybridized with gRNA) was observed around 350 base. The band around 600 base in lanes 2 to 4 represents unreacted substrate. In other words, it was suggested that the complex consisting of the polypeptide, gRNA, and the cleaved substrate DNA dissociated after heat treatment at 95°C following the addition of EDTA.

[0096] Furthermore, these results indicate that the polypeptide-guide RNA complex of the present invention is highly stable and remains bound to the target double-stranded DNA in its complex form until EDTA is added and high-temperature treatment is performed.

[0097] Example 5. Heat resistance 450 ng of the purified polypeptide (Cas12a-N or Cas12a-C) and 2 pmol of guide RNA (SEQ ID NO: 3) were mixed and heated at various temperatures (37, 45, 55, 65, 75, 85, and 95°C) for 5 minutes. Next, 100 ng of substrate double-stranded DNA (SEQ ID NO: 4) was added to the heated mixture and heated at 37°C for 1 hour. Subsequently, EDTA was added to a final concentration of 16.7 mM and heated at 95°C for 5 minutes. A portion of the reaction mixture was subjected to agarose gel electrophoresis to check for cleavage of the substrate DNA.

[0098] The results are shown in Figure 3. From Figure 3, it can be seen that the polypeptide of the present invention remained active even after treatment at 75°C for 5 minutes.

[0099] Example 6. Optimal temperature 450 ng of the purified polypeptide (Cas12a-N or Cas12a-C) and 2 pmol of guide RNA (SEQ ID NO: 3) were mixed and heated at 37°C for 5 minutes. Next, the heated mixture was added to a solution containing 100 ng of substrate double-stranded DNA (SEQ ID NO: 4), which had been previously heated at various temperatures (37, 45, 55, 65, 75, 85, and 95°C) for 20 minutes, and the mixture was maintained at the same temperature for 20 minutes. Subsequently, EDTA was added to a final concentration of 16.7 mM, and the mixture was heated at 95°C for 5 minutes. A portion of the reaction mixture was then subjected to agarose gel electrophoresis.

[0100] The results are shown in Figure 4. From Figure 4, the optimal temperature for the polypeptide of the present invention was 55-65°C. However, this result is influenced by the temperature range in which the substrate DNA can maintain its double helix, and the optimal temperature for the polypeptide of the present invention itself may be even higher.

[0101] Example 7. Length of guide RNA 450 ng of the purified polypeptide (Cas12a-N or Cas12a-C) and 2 pmol of guide RNA with different target recognition sequence lengths were mixed and heated at 37°C for 5 minutes. Then, a solution containing 100 ng of substrate double-stranded DNA (SEQ ID NO: 4), which had been preheated to 37°C, was added to the heated mixture and heated at 65°C for 20 minutes. Subsequently, EDTA was added to a final concentration of 16.7 mM, and the mixture was heated at 95°C for 5 minutes to inactivate the polypeptide. A portion of the reaction mixture was then subjected to agarose gel electrophoresis.

[0102] The guide RNA used was the one with the nucleotide sequence of Sequence ID No. 3 (corresponding to 23b in Figure 5), as well as eight other guide RNAs (corresponding to 22b-15b in Figure 5) that were created by shortening the 3' end of the aforementioned sequence by one nucleotide at a time.

[0103] The results are shown in Figure 5. From Figure 5, it can be seen that the polypeptide of the present invention was able to cleave the target double-stranded DNA when a guide RNA having a target recognition sequence chain length of 17 bases or more was used.

[0104] Example 8. Single-stranded DNA cleavage 900 ng of the purified polypeptide (Cas12a-N or Cas12a-C), 4 pmol of guide RNA (SEQ ID NO: 3), 200 ng of double-stranded DNA (SEQ ID NO: 4), and 5 pmol of single-stranded DNA (SEQ ID NO: 5) were mixed and heated at 65°C for 60 minutes. The target single-stranded DNA was bound to the 5' end with the fluorescent dye FAM and to the 3' end with the quenching group BHQ1. The fluorescence generated when the single-stranded DNA was cleaved was measured using a Thermal Cycler Dice® Real Time System III (Takara Bio Inc.).

[0105] The results are shown in Figure 6. From Figure 6, it can be seen that the polypeptide of the present invention was able to cleave single-stranded DNA.

[0106] Example 9. PAM array A PAM library containing the sequence "NNNNN" in the 5' upstream region of the target human DNMT1 gene was constructed as a plasmid. This library was cleaved under the same conditions as in Example 7. An adapter for an MGI sequencer was attached to the resulting cleavage product, and the PAM sequences contained therein were sequenced using the sequencer (MGI). Sequences from the uncleaved PAM library were also sequenced using the same sequencer and used for standardization of the obtained reads.

[0107] The results are shown in Figure 7. From Figure 7, the PAM sequence was YYN. Here, Y is C or T, and N is A, C, G, or T.

[0108] Example 10. Preparation of dead Cas polypeptide (1) Construction of mutant plasmids A mutant (SEQ ID NO: 7) was artificially synthesized in which the base gat (encoding aspartic acid) at positions 2833-2835 of SEQ ID NO: 2 was replaced with gcc (encoding alanine).

[0109] The nucleic acid of sequence number 7 was inserted into the in-fusion cloning site of the expression vector pET6xHN-N (Takara Bio USA) according to the instructions provided with the kit. The resulting plasmid was used to transform E. coli BL21 DE3 strain, and clones that became ampicillin resistant were selected. From these clones, the plasmid pET-Cas12a(D945A)-C for polypeptide expression of the present invention was obtained.

[0110] Furthermore, the expression vector pET-Cas12a(D945A)-C is configured so that a His tag is attached to the C-terminus of the expressed protein.

[0111] (2) Expression and purification of mutant polypeptides A purified dead Cas polypeptide was obtained using the same method as in Examples 2(2) to (3) above. The amino acid sequence of the dead Cas polypeptide is shown in SEQ ID NO: 6.

[0112] The following tests used the purified dead Cas polypeptide. Hereafter, this dead Cas polypeptide was referred to as Cas12a(D945A)-C.

[0113] Example 11. Loss of nuclease activity of dead Cas polypeptide (1) 450 ng of the purified Cas12a(D945A)-C and 2 pmol of guide RNA (SEQ ID NO: 3) were mixed and heated at 37°C for 5 minutes. After cooling to 4°C, 100 ng of double-stranded DNA (SEQ ID NO: 4) was added and heated at 65°C for 60 minutes. Subsequently, EDTA was added to a final concentration of 16.7 mM and heated at 95°C for 5 minutes. A portion of the reaction mixture was then subjected to agarose gel electrophoresis.

[0114] The results are shown in Figure 8. From Figure 8, it was confirmed that the dead Cas polypeptide of the present invention has lost its double-strand DNA cleavage activity.

[0115] Example 12. Loss of nuclease activity of dead Cas polypeptide (2) 900 ng of the purified Cas12a(D945A)-C, 4 pmol of guide RNA (SEQ ID NO: 3), 200 ng of double-stranded DNA (SEQ ID NO: 4), and 5 pmol of target single-stranded DNA (SEQ ID NO: 5) were mixed and heated at 65°C for 60 minutes. The target single-stranded DNA had a fluorescent dye FAM bound to its 5' end and a quenching group BHQ1 bound to its 3' end. The fluorescence produced when the target single-stranded DNA was cleaved was measured using a Thermal Cycler Dice® Real Time System III (Takara Bio Inc.). In parallel with this, the same measurement as in Example 8 was performed.

[0116] The results are shown in Figure 9. From Figure 9, it was confirmed that the dead Cas polypeptide of the present invention has lost its double-strand DNA cleavage activity and single-strand DNA cleavage activity.

[0117] Example 13. Specific detection of target genes (1) Two-step method Target λRNA (0, 1, 5, 10, 50, or 100 copies, respectively), 5.2 μM λ primer set 4, 1.4 mM dNTPs each, 65 U of Bca DNA polymerase (Takara Bio), 1 U of Reverse Transcriptase XL (AMV) (Takara Bio), and buffer were mixed and heated at 63°C for 30 minutes (RT-LAMP method). Here, λ primer set 4 is a mixture of FIP primer, BIP primer, LOOP-F primer, LOOP-B primer, F3 primer, and B3 primer.

[0118] Separately, 600 ng of purified polypeptide Cas12a-C (prepared in Example 2), 4 pmol of guide RNA (SEQ ID NO: 8), 5 pmol of reporter nucleic acid (single-stranded DNA) (SEQ ID NO: 9), and a buffer were mixed to prepare reaction solution A.

[0119] Reaction solution A was added to the amplification product of the RT-LAMP method described above, and the mixture was heated at 65°C for 60 minutes. The reporter nucleic acid had a fluorescent dye ROX bound to its 5' end and a quenching group BHQ1 bound to its 3' end. The fluorescence produced when the reporter nucleic acid was cleaved was measured using a Thermal Cycler Dice® Real Time System III (Takara Bio Inc.).

[0120] Furthermore, a system in which 500 ng of double-stranded DNA (SEQ ID NO: 4) was added instead of the target λRNA was used as a positive control. In addition, a system without purified polypeptide Cas12a-C was used as a negative control.

[0121] The results are shown in Figure 10. From Figure 10, it was possible to detect more than 10 copies of the target nucleic acid.

[0122] (2) One-step method 150 ng of purified polypeptide Cas12a-C (prepared in Example 2) and 4 pmol of guide RNA (SEQ ID NO: 8) were mixed and heated at 37°C for 5 minutes to prepare RNP.

[0123] Separately, reaction solution B was prepared by mixing 5.2 μM λ primer set 4, 1.4 mM each of dNTPs, 65 U of Bca DNA polymerase (Takara Bio), 1 U of Reverse Transcriptase XL (AMV) (Takara Bio), 2 U of inorganic pyrophosphatase (PPase) (Takara Bio), 5 pmol of reporter nucleic acid (single-stranded DNA) (sequence: CCCCCC), and buffer.

[0124] 14 μL of reaction solution B was mixed with target λRNA (0, 1, 5, 10, 50, or 100 copies, respectively) and 4 μL of RNP, and heated at 65°C for 60 minutes. The reporter nucleic acid was bound to the 5' end with the fluorescent dye FAM and to the 3' end with the quenching group BHQ1. The fluorescence produced when the reporter nucleic acid was cleaved was measured using a Thermal Cycler Dice® Real Time System III (Takara Bio Inc.).

[0125] As a result, we were able to detect 100 copies of the target nucleic acid. [Industrial applicability]

[0126] The present invention provides a heat-resistant Cas nuclease (polypeptide of the present invention). This heat-resistant Cas nuclease is useful in a wide range of fields, including genetic engineering, biology, medicine, and agriculture. [Sequence Listing Free Text]

[0127] SEQ ID NO: 1: The amino acid sequence of thermostable Cas12a SEQ ID NO: 2: The nucleotide sequence of thermostable Cas12a SEQ ID NO: 3: The nucleotide sequence of guide RNA SEQ ID NO: 4: The nucleotide sequence of target double strand DNA (human DNMT1) SEQ ID NO: 5: The nucleotide sequence of target single strand DNA SEQ ID NO: 6: The amino acid sequence of thermostable dead Cas12a SEQ ID NO: 7: The nucleotide sequence of thermostable dead Cas12a SEQ ID NO: 8: The nucleotide sequence of guide RNA SEQ ID NO: 9: The nucleotide sequence of reporter nucleic acid

Claims

1. The amino acid sequence of sequence number 1, An amino acid sequence having 1 to 10 amino acid mutations in the amino acid sequence of SEQ ID NO: 1, wherein the mutations are substitutions, deletions, insertions and / or additions, and Amino acid sequence having more than 90% identity with sequence number 1 A polypeptide classified as class 2 type V CRISPR / Cas, which contains a sequence selected from the group consisting of the following, has the ability to form a complex with a guide RNA, and possesses nuclease activity.

2. The polypeptide according to claim 1, having the activity to cleave double-stranded DNA and single-stranded DNA at 55 to 65°C.

3. (1) The amino acid sequence of Sequence ID No. 6, (2) An amino acid sequence in which the 945th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is replaced with an alanine substitution, and (3) An amino acid sequence having the amino acid substitutions described in (2) and having 90% or more identity with the sequence of Sequence ID No. 1 A polypeptide classified as class 2 type V CRISPR / Cas, comprising a sequence selected from the group consisting of the following, having the ability to form a complex with guide RNA, and lacking nuclease activity.

4. An RNA-polypeptide complex comprising the polypeptide and guide RNA according to any one of claims 1 to 3.

5. The RNA-polypeptide complex according to claim 4, wherein the polypeptide and the guide RNA are irreversibly crosslinked.

6. An isolated nucleic acid encoding the polypeptide according to any one of claims 1 to 3.

7. An expression vector comprising the nucleic acid described in claim 6.

8. Cells transformed with the expression vector according to claim 7.

9. A composition comprising the following (a) and (b): (a) The polypeptide according to any one of claims 1 to 3; (b) Guide RNA.

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