Characterization and application of novel normal-temperature Argonaute protein variant

Abstract

The invention provides characterization and application of a normal-temperature Argonaute protein variant, and particularly provides a normal-temperature Argonaute protein variant, the variant is a non-natural protein, and the variant mutates at the following core amino acid sites of a wild-type normal-temperature Argonaute protein: a threonine (T) site corresponding to the 708th site of HpAgo and / or a lysine (K) site corresponding to the 709th site of HpAgo, a threonine (T) site corresponding to the 708th site of HpAgo and / or a lysine (K) site corresponding to the 708th site of HpAgo and / or a lysine (K) site corresponding to the 709th site of HpAgo. The invention also provides application of the variant in gene editing and nucleic acid detection.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the characterization and application of a novel room-temperature Argonaute protein variant. Background Art

[0002] Ago (Argonaute) proteins are widely distributed in eukaryotes and prokaryotes and possess diverse biological functions. Their ability to cleave target nucleic acids without requiring specific recognition sequences makes them promising candidates for next-generation gene editing tools. eAgo (eukaryotic Ago) shares high structural homology with most pAgo (prokaryotic Ago), but pAgo exhibits greater diversity in the selection of guide and target nucleic acid types. While eAgo cleaves target RNA under the guidance of gRNA (guide RNA), most pAgo utilizes gDNA (guide DNA), or a few gRNAs to target DNA, and relatively few pAgos target RNA. High-temperature pAgo has been reported to cleave dsDNA (double-stranded DNA) because high temperature conditions unwind the dsDNA double helix, allowing it to cleave two ssDNA (single-stranded DNA) substrates separately. However, room-temperature pAgo can only cleave plasmid-derived dsDNA. Therefore, the in vivo application of dsDNA-targeted pAgo for genome editing at room temperature may require additional auxiliary proteins. Compared to DNA, RNA molecules possess more complex and diverse sequences and functions within organisms. Precise detection and targeted editing of RNA molecules have become a pressing need for scientific research and disease diagnosis and treatment. Therefore, the development of RNA nuclease manipulation tools with higher specificity and broader targeting capabilities is crucial. In summary, the binding and cleavage properties of pAgo proteins hold great promise for their in vivo applications. For example, matching pAgo's binding and cleavage properties can prevent certain nonspecific reactions in vivo. Further exploration of the functions and mechanisms of Ago protein's nucleic acid binding specificity, thereby enabling the engineering of target binding specificity, will open up new possibilities for its application in medicine and biotechnology.

[0003] Therefore, there is an urgent need in this field to develop an Ago enzyme variant that specifically binds to target RNA, has high affinity and activity, and can be used for gene editing and nucleic acid detection. Summary of the Invention

[0004] The purpose of the present invention is to provide an Ago enzyme variant that specifically binds to target RNA, has high affinity and activity, and can be used for gene editing and nucleic acid detection.

[0005] In a first aspect of the present invention, a normothermic Argonaute protein variant is provided. The variant is a non-natural protein, and the variant has mutations at the following core amino acid positions of the wild-type normothermic Argonaute protein:

[0006] A threonine (T) site corresponding to position 708 of HpAgo; and / or

[0007] Corresponding to the lysine (K) site at position 709 of HpAgo.

[0008] In another preferred embodiment, the threonine (T) at position 708 of HpAgo is mutated to one or more amino acids selected from the following group: alanine (A), valine (V), leucine (L), isoleucine (I), and arginine (R).

[0009] In another preferred embodiment, the threonine (T) at position 708 of HpAgo is mutated to alanine (A) or arginine (R).

[0010] In another preferred embodiment, the lysine (K) at position 709 of HpAgo is mutated to one or more amino acids selected from the following group: arginine (R), valine (V), leucine (L), and isoleucine (I).

[0011] In another preferred embodiment, the lysine (K) at position 709 of HpAgo is mutated to arginine (R).

[0012] In another preferred embodiment, the room-temperature Argonaute protein variant has the activity of directed cleavage of RNA targets.

[0013] In another preferred example, the mesophilic Argonaute protein is derived from mesophilic halophiles Haloferax_profundi, Haloferax larsenii, Haloferax alexandrinus, Haloferax alexandrinus, Halocatena pleomorpha, Halorussus marinus, Halorubrum sp.48-1-W, Haloarchaeobiussalinus, Halosimplex salinum, Natronomonas gomsonensis, Halogeometricumborinquense, Halogeometricum limi, Halosolutus gelatinilyticus, Halorussus, Halocalculus aciditolerans, Halalkaliarchaeum desulfuricum, Halosimplexpelagicum, Runella slithyformis, Pedobacter nyackensis, Mucilaginibacterpaludis, Pseudooceanicola lipolyticus.

[0014] In another preferred example, the normal temperature Argonaute protein (Ago enzyme) is selected from the following group (Haloferax_profundi) (HpAgo), Haloferax larsenii, Haloferax alexandrinus (HaleAgo), Haloferaxalexandrinus (HaalAgo), Halocatena pleomorpha (HaplAgo), Halorussus marinus (HamaAgo), Halorubrum sp.48-1-W(HaspAgo), Haloarchaeobius salinus(HalosaAgo), Halosimplex salinum, Natronomonas gomsonensis, Halogeometricum borinquense, Halogeometricum limi, Halosolutus gelatinilyticus, Halorussus, Halocalculusaciditolerans, Halalkaliarchaeum desulfuricum, Halosimplex pelagicum, Runellaslithyformis(RslAgo), Pedobacter nyackensis (PnyAgo), Mucilaginibacter paludis (MbpAgo), Pseudooceanicola lipolyticus (PliAgo) and their mutants.

[0015] In another preferred embodiment, the room-temperature Argonaute protein (Ago enzyme) includes Haloferax_profundi (HpAgo) and a room-temperature Argonaute protein (Ago enzyme) having a homology of more than 85% compared with Haloferax_profundi (HpAgo).

[0016] In another preferred embodiment, the HpAgo 708th position is located at the 708th position of HpAgo.

[0017] In another preferred embodiment, the HpAgo 709th site is located at the 709th position of HpAgo.

[0018] In another preferred embodiment, the site at position 708 of HpAgo is located at position 592 (P) of MbpAgo, and is preferably mutated to alanine (A) or arginine (R).

[0019] In another preferred embodiment, the site at position 709 of HpAgo is located at position 593 (Q) of MbpAgo, and is preferably mutated to alanine (A) or arginine (R).

[0020] In another preferred embodiment, the site at position 708 of HpAgo is located at position 591 (T) of PnyAgo, and is preferably mutated to alanine (A) or arginine (R).

[0021] In another preferred embodiment, the site at position 709 of HpAgo is located at position 592 (G) of PnyAgo, and is preferably mutated to alanine (A) or arginine (R).

[0022] In another preferred embodiment, the site at position 708 of HpAgo is located at position 598 (R) of RslAgo, and is preferably mutated to alanine (A).

[0023] In another preferred embodiment, the site at position 709 of HpAgo is located at position 599 (S) of RslAgo, and is preferably mutated to alanine (A) or arginine (R).

[0024] In another preferred embodiment, the site at position 708 of HpAgo is located at position 586 (R) of PliAgo, and is preferably mutated to alanine (A).

[0025] In another preferred embodiment, the site at position 709 of HpAgo is located at position 587 (S) of PliAgo, and is preferably mutated to alanine (A) or arginine (R).

[0026] In another preferred embodiment, the room-temperature Argonaute protein is HpAgo.

[0027] In another preferred embodiment, the amino acid sequence of the room-temperature Argonaute protein (Ago enzyme) is shown in SEQ ID NO.1.

[0028] In another preferred embodiment, the amino acid sequence of the variant is shown as SEQ ID NO. 2-4.

[0029] In another preferred embodiment, the variant is a polypeptide having an amino acid sequence shown in SEQ ID NO.: 2-4, an active fragment thereof, or a conservative variant polypeptide thereof.

[0030] In another preferred embodiment, except for the mutation (such as position 708), the remaining amino acid sequence of the variant is identical or substantially identical to the sequence of the wild-type gene editing protein.

[0031] In another preferred embodiment, the basic identity is that there are at most 50 (preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid differences, wherein the differences include amino acid substitutions, deletions or additions, and the variant has the activity of directed cleavage of RNA targets.

[0032] In another preferred embodiment, the variant has a homology of at least 80% to the wild-type normothermic Argonaute protein, preferably at least 85% or 90%, more preferably at least 95%, and most preferably at least 98% or 99%.

[0033] In another preferred embodiment, the variant is selected from the following group:

[0034] (a) a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs.: 2-4;

[0035] (b) A polypeptide derived from (a) which is formed by replacing, deleting or adding one or more (such as 2, 3, 4 or 5) amino acid residues of the amino acid sequence shown in any one of SEQ ID NOs.: 2-4 and has the activity of directed cleavage of RNA targets.

[0036] In another preferred embodiment, the derived polypeptide has a homology of at least 60% with the sequence shown in any one of SEQ ID NO.: 2-4, preferably at least 70%, more preferably at least 80%, and most preferably at least 90%, such as 95%, 97%, or 99%.

[0037] In another preferred embodiment, the variant is formed by mutation of the wild-type normothermic Argonaute protein.

[0038] The second aspect of the present invention provides a polynucleotide encoding the variant described in the first aspect of the present invention.

[0039] In another preferred embodiment, the polynucleotide is selected from the following group:

[0040] (a) a polynucleotide encoding a polypeptide as shown in any one of SEQ ID NOs. 2-4;

[0041] (b) a polynucleotide whose sequence is shown in any one of SEQ ID NOs. 5-7;

[0042] (c) a polynucleotide having a nucleotide sequence identity of ≥80% (preferably ≥90%, more preferably ≥95%, and most preferably ≥98%) to any one of SEQ ID NOs.: 5-7, and encoding a polypeptide as set forth in any one of SEQ ID NOs.: 2-4;

[0043] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0044] In another preferred embodiment, the polynucleotide further contains auxiliary elements flanking the ORF of the variant selected from the following group: a signal peptide, a secretory peptide, a tag sequence (such as 6His), or a combination thereof.

[0045] In another preferred embodiment, the polynucleotide is selected from the following group: genomic sequence, cDNA sequence, RNA sequence, or a combination thereof.

[0046] In another preferred embodiment, the polynucleotide further comprises a promoter operably linked to the ORF sequence of the variant.

[0047] In another preferred embodiment, the promoter is selected from the following group: a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a strong promoter.

[0048] The third aspect of the present invention provides a vector comprising the polynucleotide described in the second aspect of the present invention.

[0049] In another preferred embodiment, the vector comprises one or more promoters, which are operably linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, replication origin, selectable marker, nucleic acid restriction site, and / or homologous recombination site.

[0050] In another preferred embodiment, the vector includes a plasmid or a viral vector.

[0051] In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

[0052] In another preferred embodiment, the vector includes a cloning vector, a transformation vector, an expression vector, a shuttle vector, an integration vector, and a multifunctional vector.

[0053] The fourth aspect of the present invention provides a host cell, which contains the vector described in the third aspect of the present invention, or the polynucleotide described in the second aspect of the present invention is integrated into its genome.

[0054] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell or a mammalian cell (including human and non-human mammals).

[0055] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0056] In another preferred embodiment, the yeast cell is selected from one or more yeasts of the following sources: Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell includes: Kluyveromyces, more preferably Kluyveromyces marxianus, and / or Kluyveromyces lactis.

[0057] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, yeast cells, or a combination thereof.

[0058] In another preferred embodiment, the host cell expresses a room-temperature Argonaute protein variant.

[0059] A fifth aspect of the present invention provides a method for preparing a room-temperature Argonaute protein variant, the method comprising the steps of:

[0060] (a) culturing the host cell according to the fourth aspect of the present invention under conditions suitable for expression, thereby expressing the normal temperature Argonaute protein variant; and

[0061] (b) isolating the room-temperature Argonaute protein variant.

[0062] The sixth aspect of the present invention provides an enzyme preparation, which includes the room-temperature Argonaute protein variant described in the first aspect of the present invention.

[0063] In another preferred embodiment, the enzyme preparation includes an injection and / or a freeze-dried preparation.

[0064] A seventh aspect of the present invention provides a gene editing system, comprising:

[0065] The room-temperature Argonaute protein variant, or its encoding gene or its expression vector described in the first aspect of the present invention.

[0066] In another preferred embodiment, the system further comprises:

[0067] (a) a guide gDNA pair or a guide gRNA pair or an expression vector thereof, wherein the guide gDNA pair or the guide gRNA pair targets and binds to a target nucleic acid molecule;

[0068] Wherein, the target nucleic acid molecule is target RNA.

[0069] In another preferred embodiment, the target RNA includes single-stranded RNA and double-stranded RNA.

[0070] In another preferred embodiment, the target RNAs each independently include target RNAs derived from a group consisting of plants, animals, microorganisms, viruses, or a combination thereof.

[0071] In another preferred embodiment, the target RNAs are each independently artificially synthesized or naturally occurring RNAs.

[0072] In another preferred embodiment, the target RNAs each independently include wild-type or mutant RNAs.

[0073] In another preferred embodiment, the expression vector includes a plasmid or a viral vector.

[0074] In another preferred embodiment, the gene editing includes in vitro and in vivo gene editing.

[0075] An eighth aspect of the present invention provides a composition comprising:

[0076] The system according to the seventh aspect of the present invention; and

[0077] Pharmaceutically acceptable carrier.

[0078] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0079] In another preferred embodiment, the dosage form of the composition is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0080] In another preferred embodiment, the composition is in the form of a liquid preparation.

[0081] In another preferred embodiment, the composition is in the form of an injection.

[0082] In another preferred embodiment, the composition is a cell preparation.

[0083] In another preferred embodiment, in the composition, the system according to claim 7 accounts for 1-99 wt %, preferably 10-90 wt %, and more preferably 30-70 wt % of the total weight of the composition.

[0084] In another preferred embodiment, the expression vector of the room-temperature Argonaute protein variant and the expression vector of the guide gDNA pair or guide gRNA pair are the same vector or different vectors.

[0085] The ninth aspect of the present invention provides a kit comprising: the room-temperature Argonaute protein variant described in the first aspect of the present invention or the gene editing system described in the seventh aspect of the present invention.

[0086] In another preferred embodiment, the kit further comprises a label or instructions.

[0087] A tenth aspect of the present invention provides a medicine kit comprising:

[0088] A first container, and the gene editing system described in the seventh aspect of the present invention or the composition described in the eighth aspect of the present invention located in the first container, or a drug containing the gene editing system described in the seventh aspect of the present invention or the composition described in the eighth aspect of the present invention.

[0089] In another preferred embodiment, the medicine in the first container is a single preparation containing the gene editing system described in the seventh aspect of the present invention or the composition described in the eighth aspect of the present invention.

[0090] In another preferred embodiment, the dosage form of the drug is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0091] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

[0092] In another preferred embodiment, the kit further comprises instructions.

[0093] The eleventh aspect of the present invention provides a medicine kit comprising:

[0094] (a1) a first container, and the room-temperature Argonaute protein variant according to the first aspect of the present invention, or a gene encoding the same or an expression vector thereof, or a drug containing the room-temperature Argonaute protein variant according to the first aspect of the present invention, or a gene encoding the same or an expression vector thereof, located in the first container;

[0095] (b1) a second container, and a guide gDNA pair or a guide gRNA pair or an expression vector thereof, or a drug containing the guide gDNA pair or the guide gRNA pair or an expression vector thereof, located in the second container.

[0096] In another preferred embodiment, the first container and the second container are different containers.

[0097] In another preferred embodiment, the medicine in the first container is a single-ingredient preparation containing the room-temperature Argonaute protein variant described in the first aspect of the present invention, or its encoding gene or its expression vector.

[0098] In another preferred embodiment, the drug in the second container is a single preparation containing a guide gDNA pair or a guide gRNA pair or an expression vector thereof.

[0099] In another preferred embodiment, the dosage form of the drug is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0100] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

[0101] In another preferred embodiment, the kit further comprises instructions.

[0102] The twelfth aspect of the present invention provides a use of the room-temperature Argonaute protein variant described in the first aspect of the present invention, the gene editing system described in the seventh aspect of the present invention, the composition described in the eighth aspect of the present invention, the kit described in the ninth aspect of the present invention, or the medicine box described in the tenth aspect of the present invention or the eleventh aspect of the present invention, for preparing a reagent or kit for gene editing.

[0103] In another preferred embodiment, the reagent or kit is used for directed cleavage of RNA targets.

[0104] A thirteenth aspect of the present invention provides a method for in vitro gene editing, comprising the steps of:

[0105] Gene editing is performed on cells in the presence of the room-temperature Argonaute protein variant described in the first aspect of the invention, the gene editing system described in the seventh aspect of the invention, the composition described in the eighth aspect of the invention, the kit described in the ninth aspect of the invention, or the drug kit described in the tenth aspect of the invention or the eleventh aspect of the invention.

[0106] In another preferred embodiment, the cell is a prokaryotic cell or a eukaryotic cell.

[0107] In another preferred embodiment, the cells are mammalian cells.

[0108] In another preferred embodiment, the mammalian cell is a non-human mammal, such as a primate, bovine, ovine, porcine, canine, rodent, or lagomorph, such as a monkey, cow, sheep, pig, dog, rabbit, rat, or mouse cell.

[0109] In another preferred embodiment, the cell is a non-mammalian eukaryotic cell such as a cell of a poultry bird (eg, chicken), a vertebrate fish (eg, salmon), or a crustacean (eg, oyster, clam, lobster, shrimp).

[0110] In another preferred embodiment, the cell is a plant cell.

[0111] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0112] In another preferred embodiment, the cells are in vitro cells.

[0113] A fourteenth aspect of the present invention provides a detection system for directly detecting a target nucleic acid molecule, the system comprising:

[0114] (a) Guide gDNA pair or guide gRNA pair;

[0115] (b) the room-temperature Argonaute protein variant according to the first aspect of the present invention, wherein the room-temperature Argonaute protein variant is a room-temperature Argonaute protein variant that uses DNA or RNA as a guide to guide the cleavage of target RNA;

[0116] (c) a fluorescent reporter nucleic acid having a fluorescent group and a quencher group;

[0117] Wherein, the target nucleic acid molecule is target DNA or target RNA.

[0118] In another preferred embodiment, in the detection system, the target DNA is transcribed to form a target RNA, which is recognized and cleaved by the room-temperature Argonaute protein variant.

[0119] In another preferred embodiment, in the detection system, the target RNA is reverse transcribed and transcribed to form a target RNA, which is recognized and cleaved by the room temperature Argonaute protein variant.

[0120] In another preferred embodiment, the room-temperature Argonaute protein variant has the ability to specifically cleave target RNA using DNA or RNA as a guide.

[0121] In another preferred embodiment, the nucleotide sequence of the guide gDNA is shown in SEQ ID NO: 8.

[0122] In another preferred embodiment, the nucleotide sequence of the guide gRNA is shown in SEQ ID NO:9.

[0123] In another preferred embodiment, the nucleotide sequence of the target RNA is shown in SEQ ID NO: 10.

[0124] In another preferred embodiment, the guide gDNA pair includes a primary guide gDNA and a secondary guide gDNA.

[0125] In another preferred embodiment, the guide gRNA pair includes a primary guide gRNA and a secondary guide gRNA.

[0126] In another preferred embodiment, the guide gDNA or gRNA is a single-stranded DNA or single-stranded RNA molecule.

[0127] In another preferred embodiment, the guide gDNA comprises a single-stranded DNA molecule with a 5' end phosphorylated or hydroxylated.

[0128] In another preferred embodiment, the guide gRNA comprises a single-stranded RNA molecule with a 5' end phosphorylated or hydroxylated.

[0129] In another preferred embodiment, the guide gDNA or gRNA is a 5'-phosphorylated single-stranded DNA or single-stranded RNA molecule.

[0130] In another preferred embodiment, the guide gDNA and guide gRNA have complementary fragments to the fluorescent reporter nucleic acid.

[0131] In another preferred embodiment, the length of the guide gDNA is 8-60 nt, preferably 10-30 nt.

[0132] In another preferred embodiment, the length of the guide gRNA is 8-60 nt, preferably 10-30 nt.

[0133] In another preferred embodiment, the first nucleotide at the 5' end of the guide gDNA is a phosphorylated or hydroxylated thymine (T).

[0134] In another preferred embodiment, the first nucleotide at the 5' end of the guide gRNA is phosphorylated or hydroxylated uracil (U).

[0135] In another preferred embodiment, the number of the primary guide gDNAs or gRNAs is one or more pairs.

[0136] In another preferred embodiment, the number of the secondary guide gDNAs or gRNAs is one or more.

[0137] In another preferred embodiment, the primary guide gDNAs or gRNAs are complementary to the target RNA.

[0138] In another preferred embodiment, the detection system further contains (d) reagents required for reverse transcription and transcription.

[0139] In another preferred embodiment, the reagents required for reverse transcription include reverse transcriptase: II reverse transcriptase, RNase Inhibitor, template, dNTP Mix, reverse transcription primer, 5× ProtoScript II Buffer, DTT, reverse transcription reporter, MnCl2, reverse transcription Guide A&B, HpAgo, Nuclease-free Water 0.0.

[0140] In another preferred embodiment, the reagents required for transcription include: Nuclease-free Water, dNTP Buffer Mix, purified product, and T7 RNA Polymerase.

[0141] In another preferred embodiment, the reagents required for reverse transcription further include: dNTP, DTT and reverse transcription reaction buffer.

[0142] In another preferred embodiment, the reverse transcriptase is AMV having RNA-guided DNA polymerase activity.

[0143] In another preferred embodiment, when the fluorescent reporter nucleic acid is cleaved, the cleavage can be detected by urea polyacrylamide electrophoresis.

[0144] In another preferred embodiment, the fluorescent group and the quencher group are independently located at the 5' end or the 3' end of the fluorescent reporter nucleic acid.

[0145] In another preferred embodiment, the fluorescent group and the quencher group are respectively located on both sides of the complementary region between the fluorescent reporter nucleic acid and the guide gDNA or guide gRNA.

[0146] In another preferred embodiment, the fluorescent groups include FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, and Texas Red-X.

[0147] In another preferred embodiment, the quenching group includes: BHQ, TAMRA, DABCYL, and DDQ.

[0148] In another preferred embodiment, the length of the fluorescent reporter nucleic acid is 10-100 nt, preferably 20-70 nt, and more preferably 20-50 nt.

[0149] In another preferred embodiment, the working temperature of the room-temperature Argonaute protein (Ago enzyme) variant is 5-60°C, preferably 10-45°C, and more preferably 25-37°C.

[0150] In another preferred embodiment, the action time of the room temperature Argonaute protein (Ago enzyme) variant is ≥5 minutes, preferably ≥15 minutes, and more preferably ≥30 minutes.

[0151] In another preferred embodiment, the detection system further comprises: (e) divalent metal ions.

[0152] In another preferred embodiment, the divalent metal ions are selected from the following group: Mg2+, Mn2+, Fe2+, Co2+, Cu2+, Ni2+, Zn2+, Ca2+, or a combination thereof.

[0153] In another preferred embodiment, the divalent metal ion is selected from the following group: Co2+, Mn2+, or a combination thereof.

[0154] In another preferred embodiment, the manganese ions are derived from manganese chloride, manganese sulfate and related compounds, preferably manganese chloride.

[0155] In another preferred embodiment, in the detection system, the concentration of the divalent metal ions is 0 mM-8 mM, preferably 1-5 mM, and more preferably 2-3 mM.

[0156] In another preferred embodiment, the Mn 2+ The concentration is 0mM-8mM, preferably 1-5mM, more preferably 2-3mM.

[0157] In another preferred embodiment, the detection system further comprises: (f) a buffer solution.

[0158] In another preferred embodiment, the concentration of NaCl in the buffer solution is 50-500 mM, preferably 100-300 mM, more preferably 200-300 mM.

[0159] In another preferred embodiment, the pH value of the buffer solution is 4-10, preferably 6-8.

[0160] In another preferred embodiment, the detection system further contains a target nucleic acid molecule to be detected.

[0161] In another preferred embodiment, the target nucleic acid molecule or its reverse transcription-transcription product is cleaved by the Ago enzyme to produce a secondary guide gRNA.

[0162] In another preferred embodiment, the length of the secondary guide gRNA is 8-60 nt, preferably 10-30 nt.

[0163] In another preferred embodiment, the sequence of the secondary guide gRNA is complementary to that of the fluorescent reporter nucleic acid.

[0164] In another preferred embodiment, after the secondary guide gRNA binds complementary to the sequence of the fluorescent reporter nucleic acid, it guides the Ago enzyme to cut the fluorescent reporter nucleic acid, thereby generating a detectable signal (such as fluorescence).

[0165] In another preferred embodiment, the concentration of the target nucleic acid molecule to be detected in the detection system is 20-800 nM, preferably 50-600 nM, and more preferably 100-300 nM.

[0166] In another preferred embodiment, in the detection system, the concentration of the room temperature Argonaute protein (Ago enzyme) variant is 80-500 nM, preferably, 100-2000 nM, more preferably, 1000-2000 nM.

[0167] In another preferred embodiment, in the detection system, the concentration of the guide DNA or RNA is 80-5000 nM, preferably 100-4000 nM, and more preferably 500-1000 nM.

[0168] In another preferred embodiment, in the detection system, the molar ratio of the target nucleic acid molecule to be detected, the guide DNA or RNA, and the room-temperature Argonaute protein (Ago enzyme) variant is 1:(0.4-30):(1-10), preferably 1:(1-10):(2-8), and more preferably 1:2-6:2-6.

[0169] In another preferred embodiment, the concentration of the fluorescent reporter nucleic acid is 20-1000 nM, preferably 50-600 nM, and more preferably 100-300 nM.

[0170] In another preferred embodiment, the target RNA includes single-stranded RNA and double-stranded RNA.

[0171] In another preferred embodiment, the target DNA includes single-stranded DNA and double-stranded DNA.

[0172] In another preferred embodiment, the target DNA or RNA each independently includes a target DNA or RNA derived from a plant, an animal, a microorganism, a virus, or a combination thereof.

[0173] In another preferred embodiment, the target DNA or RNA is independently artificially synthesized or naturally occurring DNA or RNA.

[0174] In another preferred embodiment, the target DNA or RNA independently includes wild-type or mutant DNA or RNA.

[0175] In another preferred embodiment, the detection system is a one-step detection system.

[0176] A fifteenth aspect of the present invention provides a kit for directly detecting a target nucleic acid molecule, the kit comprising:

[0177] (i) the detection system according to the fourteenth aspect of the present invention or a reagent for preparing the detection system; and

[0178] (ii) instructions for use, wherein the instructions describe a method for directly detecting a target nucleic acid molecule using the detection system.

[0179] In another preferred embodiment, the kit further comprises reagents required for transcription or reverse transcription-transcription.

[0180] In another preferred embodiment, the kit further comprises a divalent metal ion solution.

[0181] In another preferred embodiment, the kit further comprises a buffer solution.

[0182] In another preferred embodiment, the kit comprises:

[0183] (a) a first container and a guide gDNA or guide gRNA located in the first container;

[0184] (b) a second container and the room-temperature Argonaute protein (Ago enzyme) variant of the first aspect of the present invention located in the second container, wherein the room-temperature Argonaute protein (Ago enzyme) variant is a room-temperature Argonaute protein (Ago enzyme) variant that uses DNA or RNA as a guide to guide the cleavage of target RNA; and

[0185] (c) a third container and a fluorescent reporter nucleic acid located in the third container.

[0186] In another preferred embodiment, the kit further comprises:

[0187] (d) a fourth container and reagents required for performing transcription or reverse transcription-transcription located in the fourth container.

[0188] In another preferred embodiment, the kit further comprises:

[0189] (f) a fifth container and a divalent metal ion solution in the fifth container.

[0190] A sixteenth aspect of the present invention provides a method for directly detecting whether a target nucleic acid molecule is present in a sample, comprising the following steps:

[0191] (a) providing the detection system for detecting a target nucleic acid molecule according to the fourteenth aspect of the present invention; and

[0192] (b) reacting the detection system with a sample to be detected at a certain temperature to form a first reaction solution;

[0193] (c) performing fluorescence detection on the first reaction solution to obtain a fluorescence signal value;

[0194] Among them, if a fluorescence signal value is detected in the first reaction solution, it indicates that a target nucleic acid molecule exists in the sample; and if no fluorescence signal value is detected in the first reaction solution, it indicates that no target nucleic acid molecule exists in the sample, and the target nucleic acid molecule is a target DNA or a target RNA.

[0195] In another preferred embodiment, the sample to be detected is a sample obtained without amplification.

[0196] In another preferred embodiment, the sample to be tested includes nucleic acid from a test sample, wherein the test sample is selected from the following group: blood, cells, serum, saliva, body fluid, plasma, urine, prostatic fluid, bronchial lavage fluid, cerebrospinal fluid, gastric juice, bile, lymph fluid, peritoneal fluid and feces, or a combination thereof.

[0197] In another preferred embodiment, the sample to be detected includes a nucleic acid sample directly lysed by heating, a nucleic acid sample directly treated with a protease, an extracted nucleic acid sample, a nucleic acid sample pre-amplified by PCR, or any sample containing nucleic acid.

[0198] In another preferred embodiment, the method is used to detect whether the nucleic acid at the target site has a SNP, point mutation, deletion, and / or insertion.

[0199] In another preferred embodiment, the step (b) includes the step of transcribing or reverse transcription-transcription the sample to be detected.

[0200] In another preferred embodiment, the fluorescence detection in step (c) is performed using a qPCR instrument, a microplate reader or a fluorescence spectrophotometer.

[0201] In another preferred embodiment, the method is an in vitro method.

[0202] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0203] In another preferred embodiment, the method is a one-step method.

[0204] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0205] Figure 1 It shows that the differences in amino acid distribution near the conserved active site DEDX were analyzed by dividing the known pAgo into three categories: only targeting DNA binding / binding to DNA and RNA / binding to RNA only, and performing structural comparison.

[0206] Figure 2 The conserved motif Y / RKQK that binds to the 5' P-guide was identified by sequence alignment of HpAgo with known pAgos.

[0207] Figure 3 The HpAgo structure and YKQK motif position obtained by Swiss-Modle homology modeling are shown in FIG.

[0208] Figure 4The figure shows the similarity comparison between the HpAgo structure (red) obtained by homology modeling and the known AaAgo (blue) crystal structure. The left figure is the overall picture, in which the stick model represents the R / YKQK motif, and the right figure is an enlarged view of the R / YKQK motif.

[0209] Figure 5 The HpAgo structure, DEDD active site, and T708 mutation site obtained by Swiss-Modle homology modeling are shown in FIG.

[0210] Figure 6 Figure 3 shows the change in the percentage of RNA target cleavage over time for HpAgo wild type, T708A mutant, T708R mutant, and K709R mutant under the same conditions. DETAILED DESCRIPTION

[0211] After extensive and intensive research and extensive screening, the inventors unexpectedly discovered for the first time a mutant protein with significantly improved activity compared to conventional Ago enzymes. Specifically, the mutated room-temperature Ago protein has the activity of directed cleavage of RNA targets, with improved cleavage efficiency. Furthermore, the mutated room-temperature Ago protein of the present invention can specifically bind to a target RNA and can be used for gene editing. This is the basis for the completion of the present invention.

[0212] the term

[0213] In order to make the present invention more easily understood, certain technical and scientific terms are defined below.

[0214] Where clearly defined, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. The scope of the present invention will be limited only by the appended claims.

[0215] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0216] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not.

[0217] As used herein, the term "comprising" or "including" may be open, semi-closed, or closed. In other words, the term also includes "consisting essentially of" or "consisting of."

[0218] "Transduction", "transfection", "transformation" or other terms used in this article refer to the process of transferring exogenous polynucleotides into host cells, transcribing and translating them to produce polypeptide products, including the use of plasmid molecules to introduce exogenous polynucleotides into host cells (such as Escherichia coli).

[0219] As used herein, "gene expression" or "expression" refers to the process of gene transcription, translation, and post-translational modification to produce the RNA or protein product of a gene.

[0220] As used herein, "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and analogs thereof. A polynucleotide may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide as used herein refers to single-stranded and double-stranded molecules interchangeably. Unless otherwise indicated, any polynucleotide described herein includes a double-stranded form and two complementary single strands that are known or predicted to constitute a double-stranded form.

[0221] Conservative amino acid substitutions are known in the art. In some embodiments, potential substituting amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine, and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine, lysine, arginine, and histidine; and / or phenylalanine, tryptophan, and tyrosine; methionine, and cysteine. In addition, the present invention also provides non-conservative amino acid substitutions that allow substitution of amino acids from different groups.

[0222] Those skilled in the art will readily understand the meaning of all parameters, dimensions, materials, and configurations described herein. Actual parameters, dimensions, materials, and / or configurations will depend on the specific application for which the invention is to be used. Those skilled in the art will appreciate that the embodiments or claims are given by way of example only, and that, within the scope of equivalents or claims, the scope of the embodiments of the invention is not limited to the scope specifically described and claimed.

[0223] Definitions and all definitions used herein should be understood to control over dictionary definitions or definitions in documents incorporated by reference.

[0224] All references, patents, and patent applications cited herein are incorporated by reference with respect to the subject matter in which they are cited, in some cases in their entirety.

[0225] It should be understood that for any method described herein comprising more than one step, the order of the steps is not necessarily limited to the order described in the examples.

[0226] As used herein, the terms "detection system of the present invention" and "nucleic acid detection system based on room temperature Argonaute protein variants" are used interchangeably to refer to the detection system described in the fourteenth aspect of the present invention.

[0227] As used herein, the terms "detection method of the present invention" and "nucleic acid detection method based on room temperature Argonaute protein variants" are used interchangeably to refer to the detection method described in the sixteenth aspect of the present invention.

[0228] As used herein, the terms "normal temperature Argonaute protein variant", "gene editing mutant protein", and "Ago enzyme variant" are used interchangeably to refer to the variants described in the first aspect of the present invention.

[0229] As used herein, the term "secondary cleavage" refers to the detection method of the present invention, in the presence of primary guide gDNAs or guide gRNA, the ambient temperature Argonaute protein variant of the present invention reverse-transcribes the target RNA sequence (such as single-stranded, double-stranded RNA) or directly cuts the target RNA (such as single-stranded, double-stranded RNA) to form a new 5' phosphorylated nucleic acid sequence (secondary guide gDNAs or secondary guide gRNAs); then, the secondary guide gDNA or secondary guide gRNA continues to guide the ambient temperature Argonaute protein variant to cut the fluorescent reporter nucleic acid complementary to the secondary guide gDNAs or secondary guide gRNAs under the action of the ambient temperature Argonaute protein variant. This specific cleavage of the target RNA formed by the target nucleic acid sequence or directly cutting the target nucleic acid sequence (first cleavage) and then specifically cutting the fluorescent reporter nucleic acid (second cleavage) is defined as "secondary cleavage". In the present invention, both the first cleavage and the second cleavage are specific cleavages.

[0230] Wild-type gene-editing protein

[0231] As used herein, "wild-type, room-temperature Argonaute protein" refers to a naturally occurring, unmodified room-temperature Argonaute protein, whose nucleotide sequence can be obtained through genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and whose amino acid sequence can be deduced from the nucleotide sequence. The wild-type normal-temperature Argonaute protein is derived from normal-temperature halophilic bacteria Haloferax_profundi, Haloferax larsenii, Haloferax alexandrinus, Haloferax alexandrinus, Halocatena pleomorpha, Halorussus marinus, Halorubrum sp.48-1-W, Haloarchaeobius salinus, Halosimplex salinum, Natronomonasgomsonensis, Halogeometricum borinquense, Halogeometricum limi, Halosolutus gelatinilyticus, Halorussus, Halocalculus aciditolerans, Halalkaliarchaeumdesulfuricum, Halosimplex pelagicum, Runella slithyformis, Pedobacternyackensis, Mucilaginibacter paludis, Pseudooceanicola lipolyticus.

[0232] In a preferred embodiment of the present invention, the room temperature Argonaute protein (Ago enzyme) is selected from the group consisting of Haloferax_profundi (HpAgo), Haloferax larsenii, Haloferax alexandrinus (HaleAgo), Haloferax alexandrinus (HaalAgo), Halocatena pleomorpha (HaplAgo), Halorussus marinus (HamaAgo), Halorubrum sp.48-1-W (HaspAgo), Haloarchaeobiussalinus (HalosaAgo), Halosimplex salinum, Natronomonas gomsonensis, Halogeometricum borinquense, Halogeometricum limi, Halosolutus gelatinilyticus, Halorussus, Halocalculus aciditolerans, Halalkaliarchaeum desulfuricum, Halosimplex pelagicum, Runella slithyformis (RslAgo), Pedobacter nyackensis (PnyAgo), Mucilaginibacter paludis (MbpAgo), Pseudooceanicola lipolyticus (PliAgo) and their mutants.

[0233] In another preferred embodiment of the present invention, the room-temperature Argonaute protein (Ago enzyme) includes Haloferax_profundi (HpAgo) and a room-temperature Argonaute protein (Ago enzyme) having a homology of more than 85% (preferably more than 90%, more preferably more than 95%, more preferably more than 98% or 99%) compared with Haloferax_profundi (HpAgo).

[0234] In another preferred embodiment of the present invention, the wild-type normothermic Argonaute protein (Ago enzyme) is HpAgo, and the amino acid sequence is shown in SEQ ID NO.1.

[0235] Wild-type HpAgo amino acid sequence (SEQ ID NO.1):

[0236] MAVKADIEDGEEIDIALRVTGIDEWEHDAIAQKIQLEDIDNAAIDLTVFHNNE

[0237] VANFEWQIGEWYLLENVVGNEFRGEMQLNPGYDLNVTLLDDPPAAVENDK

[0238] LPGSEPSERPVDLSGERGSSGAAATTSDASDGEEFVSGSEVDGSSRPTADGGG

[0239] KLLHQQPLSDGNYLLQFELGSLPELPVHEYELQATGSGGIDPDDFTNGIEGFT

[0240] AKAANYYQSRIGSPVTTADASRRRIYATEKLHSTISIHGYTVKPVHQGETTLE

[0241] ARSYTNDGPLQEFVKQDVKRAVAGRFEVSGIDSIIEPTPQRTANSGLFEAYRK

[0242] YKCRIRVDADGTVICGVNVAYHLESTFSAAEWVQRGHDIAEVTVEHDTDLY

[0243] DSARTARVKEVIDMDYDDVLDGPGVPMSEYHEKHVEQDVIDSMRAGDPIIA

[0244] DLQYGSGEDSIFPQLLAYCKVIPTFDQLGRVDETFLNVIHNESRMKPEERFNV

[0245] VTSFVDLLGPTPYFGFDPVPQPTNAGYREHQIRNRPNLRFGDGQTGFYGAGG

[0246] LERKGYGVYKAPESFDIIALYPEDEEDDARPYVLSLLNKLADYDASPTAFDR

[0247] DTYELSSEFHYSQHAQKASDYDAALIVVPDADEAAAADYDDPYPEFKRRLG

[0248] QLGVPSQMISVDNLGNDNYRGNICSSLIGKAGGVPWRIDDVPGAVDAFVGL

[0249] DVTYDHATKQHLGAAANVIMADGTILASEAVTKQAGETFDEDDVANVIKH

[0250] VLEIFAEEEGRPPRHVVIHRDGKFYLDIESLISRLDKARDLIQRFDLVEIRKSG

[0251] NPRIAEYDESNSRFDIADKGVAFHVHNGDHSYLTTTGGKEGSPGTPRPLQIVK

[0252] RYGSTDLDTLAEQTYWLSEAHVGSLSRSTRLPITTYYADKCADFAMKGYLT

[0253] KGSVIRGVPYI

[0254] Homeothermic Argonaute protein variants and their encoding nucleic acids

[0255] As used herein, the terms "normal-temperature Argonaute protein variant," "variant of the present invention," "mutant protein of the present invention," and "mutant protein" are used interchangeably to refer to a non-naturally occurring mutant normal-temperature Argonaute protein that has the activity of directed cleavage of an RNA target, and the variant has mutations at the following core amino acid positions of the wild-type normal-temperature Argonaute protein:

[0256] A threonine (T) site corresponding to position 708 of HpAgo; and / or

[0257] Corresponding to the lysine (K) site at position 709 of HpAgo.

[0258] The term "core amino acids" refers to sequences based on a wild-type, normothermic Argonaute protein that are at least 80%, such as 84%, 85%, 90%, 92%, 95%, 98%, or 99%, homologous to the wild-type, normothermic Argonaute protein, at the corresponding positions of the specific amino acids described herein. For example, based on a wild-type, normothermic Argonaute protein, the core amino acids are:

[0259] A threonine (T) site corresponding to position 708 of HpAgo; and / or

[0260] Corresponding to the lysine (K) site at position 709 of HpAgo.

[0261] Furthermore, the mutant protein obtained by mutating the above core amino acids has the activity of directed cleavage of RNA targets.

[0262] Preferably, in the present invention, the core amino acids of the present invention are mutated as follows:

[0263] The threonine (T) at position 708 corresponding to HpAgo is mutated to alanine (A); and / or

[0264] The lysine (K) at position 709 of HpAgo was mutated to arginine (R).

[0265] It should be understood that the amino acid numbering in the mutant proteins of the present invention is based on the wild-type, room-temperature Argonaute protein. When the sequence homology of a specific mutant protein to the wild-type, room-temperature Argonaute protein reaches 80% or more, the amino acid numbering of the mutant protein may be misplaced relative to the amino acid numbering of the wild-type, room-temperature Argonaute protein, such as a misplacement of 1-100 positions toward the N-terminus or C-terminus of the amino acid. Using conventional sequence alignment techniques in the art, those skilled in the art can generally understand that such misplacement is within a reasonable range, and mutant proteins with a homology of 80% (such as 90%, 95%, 98%) and having the same or similar activity of directed cleavage of RNA targets should not be excluded from the scope of the mutant proteins of the present invention due to the misplacement of amino acid numbering.

[0266] The muteins of the present invention are synthetic or recombinant proteins, i.e., they can be the product of chemical synthesis or produced using recombinant technology from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plants). Depending on the host used in the recombinant production protocol, the muteins of the present invention can be glycosylated or non-glycosylated. The muteins of the present invention may or may not include an initial methionine residue.

[0267] The present invention also includes fragments, derivatives and analogs of the mutant protein. As used herein, the terms "fragment", "derivative" and "analog" refer to proteins that substantially retain the same biological function or activity of the mutant protein.

[0268] Mutant protein fragments, derivatives or analogs of the present invention can be (i) mutant proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and the amino acid residues of such substitutions may or may not be encoded by the genetic code, or (ii) mutant proteins with substituent groups in one or more amino acid residues, or (iii) mutant proteins formed by the fusion of a mature mutant protein with another compound (such as a compound that prolongs the half-life of the mutant protein, for example polyethylene glycol), or (iv) mutant proteins formed by the fusion of an additional amino acid sequence to the mutant protein sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence for purifying the mutant protein, or a fusion protein formed with an antigen IgG fragment). According to the teachings of this article, these fragments, derivatives and analogs belong to the well-known scope of those skilled in the art. In the present invention, the conservatively substituted amino acids are preferably produced by amino acid substitution according to Table 1.

[0269] Table I

[0270]

[0271]

[0272] The active mutant protein of the present invention has the activity of directed cleavage of RNA target.

[0273] Preferably, the mutant protein is shown in SEQ ID NO.: 2-4.

[0274] Protein sequence of HpAgo T708A mutant (SEQ ID NO. 2):

[0275] MAVKADIEDGEEIDIALRVTGIDEWEHDAIAQKIQLEDIDNAAIDLTVFHNNEVANFEWQIGEWYLLENVVGNEFRGEMQLNPGYDLNVTLLDDPPAAVENDKLPGSEPSERPVDLSGERGSSGAAATTSDASDGEEFVSGSEVDGSSRPTADGGGKLLHQQPLSDGNYLLQFELGSLPELPVHEYELQATGSGGIDPDDFTNGIEGFTAKAANYYQSRIGSPVTTADASRRRIYATEKLHSTISIHGYTVKPVHQGETTLEARSYTNDGPLQEFVKQDVKRAVAGRFEVSGIDSIIEPTPQRTANSGLFEAYRKYKCRIRVDADGTVICGVNVAYHLESTFSAAEWVQRGHDIAEVTVEHDTDLYDSARTARVKEVIDMDYDDVLDGPGVPMSEYHEKHVEQDVIDSMRAGDPIIADLQYGSGEDSIFPQLLAYCKVIPTFDQLGRVDETFLNVIHNESRMKPEERFNVVTSFVDLLGPTPYFGFDPVPQPTNAGYREHQIRNRPNLRFGDGQTGFYGAGGLERKGYGVYKAPESFDIIALYPEDEEDDARPYVLSLLNKLADYDASPTAFDRDTYELSSEFHYSQHAQKASDYDAALIVVPDADEAAAADYDDPYPEFKRRLGQLGVPSQMISVDNLGNDNYRGNICSSLIGKAGGVPWRIDDVPGAVDAFVGLDVTYDHATKQHLGAAANVIMADGTILASEAVAKQAGETFDEDDVANVIKHVLEIFAEEEGRPPRHVVIHRDGKFYLDIESLISRLDKARDLIQRFDLVEIRKSGNPRIAEYDESNSRFDIADKGVAFHVHNGDHSYLTTTGGKEGSPGTPRPLQIVKRYGSTDLDTLAEQTYWLSEAHVGSLSRSTRLPITTYYADKCADFAMKGYLTKGSVIRGVPYI

[0276] Protein sequence of the HpAgo T708R mutant (SEQ ID NO.3):

[0277] MAVKADIEDGEEIDIALRVTGIDEWEHDAIAQKIQLEDIDNAAIDLTVFHNNEVANFEWQIGEWYLLENVVGNEFRGEMQLNPGYDLNVTLLDDPPAAVENDKLPGSEPSERPVDLSGERGSSGAAATTSDASDGEEFVSGSEVDGSSRPTADGGGKLLHQQPLSDGNYLLQFELGSLPELPVHEYELQATGSGGIDPDDFTNGIEGFTAKAANYYQSRIGSPVTTADASRRRIYATEKLHSTISIHGYTVKPVHQGETTLEARSYTNDGPLQEFVKQDVKRAVAGRFEVSGIDSIIEPTPQRTANSGLFEAYRKYKCRIRVDADGTVICGVNVAYHLESTFSAAEWVQRGHDIAEVTVEHDTDLYDSARTARVKEVIDMDYDDVLDGPGVPMSEYHEKHVEQDVIDSMRAGDPIIADLQYGSGEDSIFPQLLAYCKVIPTFDQLGRVDETFLNVIHNESRMKPEERFNVVTSFVDLLGPTPYFGFDPVPQPTNAGYREHQIRNRPNLRFGDGQTGFYGAGGLERKGYGVYKAPESFDIIALYPEDEEDDARPYVLSLLNKLADYDASPTAFDRDTYELSSEFHYSQHAQKASDYDAALIVVPDADEAAAADYDDPYPEFKRRLGQLGVPSQMISVDNLGNDNYRGNICSSLIGKAGGVPWRIDDVPGAVDAFVGLDVTYDHATKQHLGAAANVIMADGTILASEAVRKQAGETFDEDDVANVIKHVLEIFAEEEGRPPRHVVIHRDGKFYLDIESLISRLDKARDLIQRFDLVEIRKSGNPRIAEYDESNSRFDIADKGVAFHVHNGDHSYLTTTGGKEGSPGTPRPLQIVKRYGSTDLDTLAEQTYWLSEAHVGSLSRSTRLPITTYYADKCADFAMKGYLTKGSVIRGVPYIK

[0278] Protein sequence of the HpAgo K709R mutant (SEQ ID NO.4):

[0279] MAVKADIEDGEEIDIALRVTGIDEWEHDAIAQKIQLEDIDNAAIDLTVFHNNEVANFEWQIGEWYLENVVGNEFRGEMQLNPGYDLNVTLLDDPPPAAVENDKLPGSEPSERPVDLSGERGSSGAAATTSDASDGEEFVSGSEVDGSSRPTADGGGKLLHQQPLSDGNYLLQFELGSLPELPVHEYELQATGSGGIDPDDDFTNGIEGFTAKAANYYQSRIGSPVTTADASRRRIYATEKLHSTISIHGYTVKPVHQGETTLEARSYTNDGPLQEFVKQDVKRAVAGRFEVSGIDSIIEPTPQRTANSGLFEAYRKYKCRIRVDADGTVICGVNVAYHLESTFSAAEWVQRGHDIAEVTVEHDTDLYDSARTARVKEVIDMDYDDVLDGPGVPMSEYHEKHVEQDVIDSMRAGDPIIADLQYGSGEDSIFPQLLAYCKVIPTFDQLGRV DETFLNVIHNESRMKPEERFNVVTSFVDLLGPTPYFGFDPVPQPTNAGYREHQIRNRPNLRFGDGQTGFYGAGGLERKGYGVYKAPESFDIIALYPEDEEDDARPYVLSLLNKLADYDASPTAFDRDTYELSSEFHYSQHAQKASDYDAALIVVPDADEAAAADYDDPYPEFKRRLGQLGVPSQMISVDNLGNDNYRGNICSSLIGKAGGVPWRIDDVPGAVDAFVGLDVTYDHATKQHLGAAANVIMADGTILASEAVTRQAGETFDEDDVANVIKHVLEIFAEEEGRPPRHVVIHRDGKFYLDIESLISRLDKARDLIQRFDLVEIRKSGNPRIAEYDESNSRFDIADKGVAFHVHNGDHSYLTTTGGKEGSPGTPRPLQIVKRYGSTDLDTLAEQTYWLSEAHVGSLSRSTRLPITTYYADKCADFAMKGYLTKGSVIRGVPYIK

[0280] It should be understood that the mutant protein of the present invention generally has a higher homology (identity) than the sequences shown in SEQ ID NO.: 2, SEQ ID NO.: 3, and SEQ ID NO.: 4. Preferably, the mutant protein has a homology of at least 80% with the sequences shown in SEQ ID NO.: 2-4, more preferably at least 85%-90%, more preferably at least 95%, and most preferably at least 98% or 99%.

[0281] In addition, the mutant proteins of the present invention can also be modified. Modifications (usually without altering the primary structure) include: chemical derivatization of the mutant protein in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation during the synthesis and processing of the mutant protein or in further processing steps. Such modifications can be accomplished by exposing the mutant protein to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). Also included are mutant proteins that have been modified to improve their resistance to proteolysis or optimize their solubility.

[0282] The term "polynucleotide encoding a mutant protein" may include a polynucleotide encoding a mutant protein of the present invention, or may also include additional coding and / or non-coding sequences.

[0283] In a preferred embodiment, the sequence of the polynucleotide encoding the mutant protein of the present invention is shown in SEQ ID NO.: 5-7.

[0284] T708A nucleic acid sequence (SEQ ID NO.5)

[0285]

[0286] T708R nucleic acid sequence (SEQ ID NO.6)

[0287] ATGGCGGTTAAAGCGGACATCGAAGATGGTGAAGAAATTGATATCG

[0288] CGCTGCGTGTTACCGGTATCGATGAATGGGAACATGATGCGATTGCCCAG

[0289] AAAATCCAGCTGGAGGACATTGATAACGCGGCGATTGATCTGACCGTTTT

[0290] CCACAACAACGAAGTTGCAAACTTTGAATGGCAGATTGGTGAATGGTACC

[0291] TGCTGGAAAACGTGGTGGGTAACGAATTCCGTGGCGAAATGCAGCTGAA

[0292] CCCAGGTTATGATCTGAACGTAACCCTGCTGGACGATCCGCCGGCTGCGG

[0293] TTGAAAACGATAAACTGCCGGGCTCTGAACCGTCTGAACGTCCGGTTGAC

[0294] CTGAGCGGCGAACGTGGTTCCAGCGGCGCTGCTGCCACCACCTCTGACGC

[0295] ATCTGATGGCGAAGAATTCGTGTCCGGTAGCGAAGTTGATGGTTCTTCTC

[0296] GTCCGACCGCTGACGGTGGTGGTAAACTGCTGCACCAGCAGCCGCTGTCT

[0297] GATGGTAACTACCTGCTGCAGTTCGAACTGGGTAGCCTGCCGGAACTGCC

[0298] GGTGCACGAATACGAACTGCAGGCGACCGGCTCTGGTGGCATCGATCCTG

[0299] ATGATTTCACCAACGGCATCGAAGGCTTTACCGCGAAAGCTGCGAACTAC

[0300] TACCAGTCTCGCATTGGTAGCCCGGTAACCACCGCCGATGCGAGCCGTCG

[0301] CCGTATCTATGCTACCGAAAAACTGCATTCCACCATTAGCATCCACGGCT

[0302] ATACCGTGAAACCGGTGCACCAGGGCGAAACTACCCTGGAAGCTCGTTCT

[0303] TACACCAACGATGGTCCACTGCAGGAATTCGTTAAACAGGACGTTAAACG

[0304] TGCGGTGGCAGGTCGCTTTGAAGTGTCTGGTATCGATTCTATCATCGAAC

[0305] CGACCCCGCAGCGCACCGCTAACAGCGGTCTGTTCGAAGCGTACCGTAAA

[0306] TACAAATGTCGTATCCGTGTCGACGCTGACGGTACCGTTATTTGTGGTGTT

[0307] AACGTGGCGTACCACCTGGAATCTACCTTCTCTGCGGCGGAATGGGTTCA

[0308] GCGCGGTCATGATATCGCAGAAGTGACCGTTGAACACGATACCGATCTGT

[0309] ACGACAGCGCGCGTACCGCGCGCGTTAAAGAAGTTATCGATATGGACTAC

[0310] GATGATGTGTTAGATGGTCCGGGTGTGCCAATGTCTGAATATCACGAAAA

[0311] ACATGTAGAACAGGACGTTATCGATAGCATGCGCGCTGGCGATCCGATCA

[0312] TTGCAGATCTGCAGTACGGCAGTGGTGAGGATTCTATCTTTCCGCAGCTG

[0313] CTTGCTTATTGTAAAGTGATCCCGACCTTCGACCAGCTGGGCCGTGTTGAT

[0314] GAAACCTTCCTGAACGTAATCCACAACGAATCCCGTATGAAACCGGAAG

[0315] AACGCTTCAACGTTGTTACCAGCTTCGTTGACCTGCTCGGCCCGACCCCGT

[0316] ACTTCGGTTTCGACCCGGTGCCGCAGCCGACTAACGCTGGCTATCGCGAA

[0317] CACCAGATCCGTAACCGTCCGAACCTGCGCTTCGGCGACGGCCAGACTGG

[0318] CTTCTATGGTGCGGGTGGTCTGGAACGCAAAGGTTACGGCGTTTACAAGG

[0319] CACCGGAATCTTTCGACATCATCGCGCTGTACCCGGAAGATGAAGAAGAT

[0320] GATGCGCGCCCGTACGTTCTGAGTCTGCTGAACAAACTGGCGGACTACGA

[0321] CGCGAGCCCGACTGCGTTCGACCGTGATACCTATGAACTGTCCTCTGAAT

[0322] TCCACTACAGCCAGCATGCACAGAAAGCATCTGATTATGATGCCGCGCTG

[0323] ATTGTAGTTCCGGACGCTGACGAAGCGGCGGCAGCTGATTACGACGACCC

[0324] GTACCCGGAATTTAAACGTCGCCTGGGCCAGCTGGGCGTACCGTCCCAGA

[0325] TGATCAGCGTTGATAATCTGGGTAACGATAACTACCGCGGCAACATTTGT

[0326] TCCAGCCTGATCGGCAAAGCAGGTGGCGTGCCGTGGCGTATCGATGATGT

[0327] TCCGGGTGCTGTTGATGCCTTCGTGGGCCTGGACGTCACCTACGATCACG

[0328] CTACTAAACAGCACCTGGGCGCGGCGGCGAACGTTATCATGGCTGACGGC

[0329] ACCATTCTGGCTTCCGAAGCAGTGAGAAAACAGGCTGGTGAAACCTTCGA

[0330] CGAAGATGACGTTGCTAACGTTATCAAACACGTGCTGGAAATTTTCGCAG

[0331] AAGAAGAAGGTCGTCCGCCGCGTCACGTTGTAATTCATCGTGATGGCAAA

[0332] TTTTACCTGGATATCGAAAGCCTGATCAGCCGTCTGGATAAAGCGCGTGA

[0333] TCTGATCCAGCGCTTTGATCTGGTTGAAATCCGTAAAAGCGGTAACCCGC

[0334] GTATTGCGGAATATGATGAATCTAACTCTCGTTTCGACATCGCGGATAAA

[0335] GGTGTTGCCTTCCACGTCCACAACGGTGACCATTCCTATCTGACCACCAC

[0336] CGGTGGTAAAGAAGGCTCTCCGGGTACCCCGCGTCCGCTGCAGATCGTTA

[0337] AACGTTACGGTAGCACCGACCTGGATACCCTGGCCGAACAGACCTATTGG

[0338] CTGTCTGAAGCTCACGTTGGTTCTCTGTCTCGTAGCACCCGCCTGCCGATC

[0339] ACCACCTACTACGCTGATAAATGCGCTGATTTCGCTATGAAAGGCTACCT

[0340] GACTAAAGGTAGCGTTATCCGTGGCGTTCCGTACATCAAG

[0341] Nucleic acid sequence of K709R (SEQ ID NO.7)

[0342] ATGGCGGTTAAAGCGGACATCGAAGATGGTGAAGAAATTGATATCGCGCTGCGTGTTACCGGTATCGATGAATGGGAACATGATGCGATTGCCCAGAAAATCCAGCTGGAGGACATTGATAACGCGGCGATTGATCTGACCGTTTTCCACAACAACGAAGTTGCAAACTTTGAATGGCAGATTGGTGAATGGTACCTGCTGGAAAACGTGGTGGGTAACGAATTCCGTGGCGAAATGCAGCTGAACCCAGGTTATGATCTGAACGTAACCCTGCTGGACGATCCGCCGGCTGCGGTTGAAAACGATAAACTGCCGGGCTCTGAACCGTCTGAACGTCCGGTTGACCTGAGCGGCGAACGTGGTTCCAGCGGCGCTGCTGCCACCACCTCTGACGCATCTGATGGCGAAGAATTCGTGTCCGGTAGCGAAGTTGATGGTTCTTCTC

[0343] GTCCGACCGCTGACGGTGGTGGTAAACTGCTGCACCAGCAGCCGCTGTCT

[0344] GATGGTAACTACCTGCTGCAGTTCGAACTGGGTAGCCTGCCGGAACTGCC[[ID=*]]<* [[ID=*]]<*

[0345] GGTGCACGAATACGAACTGCAGGCGACCGGCTCTGGTGGCATCGATCCTG

[0346] It should be noted that the tags and

[0345] in the original seem to have an asterisk added in the provided text which might be an error or special marking not clearly defined. If this is an actual error in the source, the translation is based on the best understanding of the original text as presented.ATGATTTCACCAACGGCATCGAAGGCTTTACCGCGAAAGCTGCGAACTAC

[0347] TACCAGTCTCGCATTGGTAGCCCGGTAACCACCGCCGATGCGAGCCGTCG

[0348] CCGTATCTATGCTACCGAAAAACTGCATTCCACCATTAGCATCCACGGCT

[0349] ATACCGTGAAACCGGTGCACCAGGGCGAAACTACCCTGGAAGCTCGTTCT

[0350] TACACCAACGATGGTCCACTGCAGGAATTCGTTAAACAGGACGTTAAACG

[0351] TGCGGTGGCAGGTCGCTTTGAAGTGTCTGGTATCGATTCTATCATCGAAC

[0352] CGACCCCGCAGCGCACCGCTAACAGCGGTCTGTTCGAAGCGTACCGTAAA

[0353] TACAAATGTCGTATCCGTGTCGACGCTGACGGTACCGTTATTTGTGGTGTT

[0354] AACGTGGCGTACCACCTGGAATCTACCTTCTCTGCGGCGGAATGGGTTCA

[0355] GCGCGGTCATGATATCGCAGAAGTGACCGTTGAACACGATACCGATCTGT

[0356] ACGACAGCGCGCGTACCGCGCGCGTTAAAGAAGTTATCGATATGGACTAC

[0357] GATGATGTGTTAGATGGTCCGGGTGTGCCAATGTCTGAATATCACGAAAA

[0358] ACATGTAGAACAGGACGTTATCGATAGCATGCGCGCTGGCGATCCGATCA

[0359] TTGCAGATCTGCAGTACGGCAGTGGTGAGGATTCTATCTTTCCGCAGCTG

[0360] CTTGCTTATTGTAAAGTGATCCCGACCTTCGACCAGCTGGGCCGTGTTGAT

[0361] GAAACCTTCCTGAACGTAATCCACAACGAATCCCGTATGAAACCGGAAG

[0362] AACGCTTCAACGTTGTTACCAGCTTCGTTGACCTGCTCGGCCCGACCCCGT

[0363] ACTTCGGTTTCGACCCGGTGCCGCAGCCGACTAACGCTGGCTATCGCGAA

[0364] CACCAGATCCGTAACCGTCCGAACCTGCGCTTCGGCGACGGCCAGACTGG

[0365] CTTCTATGGTGCGGGTGGTCTGGAACGCAAAGGTTACGGCGTTTACAAGG

[0366] CACCGGAATCTTTCGACATCATCGCGCTGTACCCGGAAGATGAAGAAGAT

[0367] GATGCGCGCCCGTACGTTCTGAGTCTGCTGAACAAACTGGCGGACTACGA

[0368] CGCGAGCCCGACTGCGTTCGACCGTGATACCTATGAACTGTCCTCTGAAT

[0369] TCCACTACAGCCAGCATGCACAGAAAGCATCTGATTATGATGCCGCGCTG

[0370] ATTGTAGTTCCGGACGCTGACGAAGCGGCGGCAGCTGATTACGACGACCC

[0371] GTACCCGGAATTTAAACGTCGCCTGGGCCAGCTGGGCGTACCGTCCCAGA

[0372] TGATCAGCGTTGATAATCTGGGTAACGATAACTACCGCGGCAACATTTGT

[0373] TCCAGCCTGATCGGCAAAGCAGGTGGCGTGCCGTGGCGTATCGATGATGT

[0374] TCCGGGTGCTGTTGATGCCTTCGTGGGCCTGGACGTCACCTACGATCACG

[0375] CTACTAAACAGCACCTGGGCGCGGCGGCGAACGTTATCATGGCTGACGGC

[0376] ACCATTCTGGCTTCCGAAGCAGTGACTAGACAGGCTGGTGAAACCTTCGA

[0377] CGAAGATGACGTTGCTAACGTTATCAAACACGTGCTGGAAATTTTCGCAG

[0378] AAGAAGAAGGTCGTCCGCCGCGTCACGTTGTAATTCATCGTGATGGCAAA

[0379] TTTTACCTGGATATCGAAAGCCTGATCAGCCGTCTGGATAAAGCGCGTGA

[0380] TCTGATCCAGCGCTTTGATCTGGTTGAAATCCGTAAAAGCGGTAACCCGC

[0381] GTATTGCGGAATATGATGAATCTAACTCTCGTTTCGACATCGCGGATAAA

[0382] GGTGTTGCCTTCCACGTCCACAACGGTGACCATTCCTATCTGACCACCAC

[0383] CGGTGGTAAAGAAGGCTCTCCGGGTACCCCGCGTCCGCTGCAGATCGTTA

[0384] AACGTTACGGTAGCACCGACCTGGATACCCTGGCCGAACAGACCTATTGG

[0385] CTGTCTGAAGCTCACGTTGGTTCTCTGTCTCGTAGCACCCGCCTGCCGATC

[0386] ACCACCTACTACGCTGATAAATGGCTGATTTCGCTATGAAAGGCTACCT

[0387] GACTAAAGGTAGCGTTATCCGTGGCGTTCCGTACATCAAG

[0388] The present invention also relates to variants of the aforementioned polynucleotides, including fragments, analogs, and derivatives encoding polypeptides or muteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide that may contain one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded mutein.

[0389] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%.

[0390] The mutant proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.

[0391] The full-length sequences of the polynucleotides of the present invention can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. For long sequences, two or more PCR amplifications are often required, followed by splicing the fragments amplified in the correct order.

[0392] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0393] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0394] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0395] Methods using PCR techniques to amplify DNA / RNA are preferably used to obtain the polynucleotides of the present invention. In particular, when full-length cDNA is difficult to obtain from a library, the RACE method (RACE - rapid amplification of cDNA ends) is preferably used. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods, such as gel electrophoresis.

[0396] It should be noted that the corresponding sites of position 708 in the amino acid sequence of the homeothermic Argonaute protein (HpAgo) derived from Haloferax profundi in the present invention are all conserved sites in homeothermic Argonaute proteins from other sources. The specific correspondence is shown in Table II.

[0397] Table II

[0398] protein Site HpAgo T708, K709 MbpAgo P592, Q593 PnyAgo T591, G592 RslAgo R598、S599 PliAgo R586、S587

[0399] T708 and K709 of HpAgo are key conserved sites involved in the specificity of targeted binding and will increase activity. Other pAgos also have similar functions at the corresponding sites. Therefore, mutations at the above sites are not only crucial for the study of the mechanism of Ago in directed cleavage of RNA targets, but also further promote the application of RNA-targeted Ago in vivo.

[0400] Expression vectors and host cells

[0401] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the mutant protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0402] The polynucleotide sequences of the present invention can be used to express or produce recombinant mutant proteins using conventional recombinant DNA techniques. Generally, the following steps are involved:

[0403] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a mutant protein of the present invention, or a recombinant expression vector containing the polynucleotide;

[0404] (2) Host cells cultured in a suitable culture medium;

[0405] (3) Isolate and purify proteins from culture medium or cells.

[0406] In the present invention, the polynucleotide sequence encoding the mutant protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it generally contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0407] Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding the mutein of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operatively linked to an appropriate promoter within the expression vector to direct mRNA synthesis. Representative examples of such promoters include the lac or trp promoters of Escherichia coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoter, retroviral LTRs, and other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0408] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0409] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0410] The host cell can be a prokaryotic cell (such as Escherichia coli), a lower eukaryotic cell, or a higher eukaryotic cell, such as a yeast cell, a plant cell, or a mammalian cell (including human and non-human mammals). Representative examples include Escherichia coli, wheat germ cells, insect cells, SF9, HeLa, HEK293, CHO, yeast cells, etc. In a preferred embodiment of the present invention, a yeast cell (such as Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell includes Kluyveromyces, more preferably Kluyveromyces marxianus, and / or Kluyveromyces lactis) is selected as the host cell.

[0411] When the polynucleotides of the present invention are expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting DNA factors, typically about 10 to 300 base pairs in length, that act on promoters to increase gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs on the late replication origin side), the polyoma enhancer on the late replication origin side, and adenovirus enhancers.

[0412] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0413] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0414] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0415] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0416] Guide gDNA pair or guide gRNA pair

[0417] In the detection system and detection method of the present invention, a core component is a guide gDNA pair or a guide gRNA pair.

[0418] In the present invention, the preferred guide gDNAs are oligonucleotides with a length of 8-60 nt, and the first 5' nucleotides are phosphorylated or hydroxylated thymine, cytosine, adenine, or guanine.

[0419] In the present invention, the preferred guide gRNAs are all oligonucleotides with a length of 8-60 nt, and the first 5' nucleotides thereof are all phosphorylated or hydroxylated modified uracil, cytosine, adenine, or guanine.

[0420] Detection system

[0421] As used herein, the terms "detection system of the present invention" and "Argonaute protein-based nucleic acid detection system" are used interchangeably to refer to the detection system for detecting target nucleic acid molecules described in the fourteenth aspect of the present invention. The detection system of the present invention is based on the reaction conditions provided by the present invention that are suitable for the functional activity of the room-temperature Ago enzyme variant of the present invention (particularly the HpAgo enzyme variant).

[0422] In a preferred embodiment, the present invention provides a detection system for detecting a target nucleic acid molecule, comprising:

[0423] (a) Guide ssDNA / ssRNA pair;

[0424] (b) the room-temperature Argonaute protein variant according to the first aspect of the present invention, wherein the room-temperature Argonaute protein variant is a room-temperature Argonaute protein variant that uses DNA or RNA as a guide to guide the cleavage of target RNA;

[0425] (c) Fluorescent reporter nucleic acid, which carries a fluorescent group and a quencher group.

[0426] Wherein, the target nucleic acid molecule is target DNA or RNA.

[0427] In the detection system of the present invention, the working temperature of the nuclease is 10-45°C, preferably 20-37°C, and more preferably 25-37°C.

[0428] Preferably, the guide DNA / RNA is a single-stranded DNA / RNA molecule with a 5'-end phosphorylation. Preferably, the guide DNA / RNA is 8-60 nt in length.

[0429] Preferably, the reporter nucleic acid is single-stranded RNA (ssRNA).In one embodiment, when the reporter nucleic acid is cleaved, the cleavage can be detected by electrophoresis.

[0430] In another embodiment, the fluorescent group and quencher carried by the fluorescent reporter nucleic acid are independently located at the 5' and 3' ends of the fluorescent reporter nucleic acid. Preferably, the fluorescent group and quencher are located on either side of the complementary region between the fluorescent reporter nucleic acid and the guide DNA / RNA. In another preferred embodiment, the fluorescent group includes FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, Texas Red-X, or a combination thereof.

[0431] In another preferred embodiment, the quenching group includes: BHQ, TAMRA, DABCYL, DDQ, or a combination thereof.

[0432] The detection system of the present invention may also include a divalent metal ion. The divalent metal ion is selected from the group consisting of Mn2+, Co2+, or a combination thereof. Preferably, Mn2+ is present. In the detection system, the concentration of the divalent metal ion is 0 mM to 8 mM, preferably 1 to 5 mM, and even more preferably 2 to 3 mM.

[0433] Detection method

[0434] As used herein, the terms "the detection method of the present invention" and "the nucleic acid detection method based on Argonaute protein" are used interchangeably to refer to the detection method described in the sixteenth aspect of the present invention.

[0435] In the present invention, a method for directly detecting whether a target molecule is present in a sample is provided, comprising the following steps:

[0436] (a) providing the detection system for detecting a target nucleic acid molecule according to the fourteenth aspect of the present invention; and

[0437] (b) reacting the detection system with a sample to be detected at a certain temperature to form a first reaction solution;

[0438] (c) performing fluorescence detection on the first reaction solution to obtain a fluorescence signal value;

[0439] Among them, if a fluorescence signal value is detected in the first reaction solution, it indicates that a target nucleic acid molecule exists in the sample; and if no fluorescence signal value is detected in the first reaction solution, it indicates that no target nucleic acid molecule exists in the sample, and the target nucleic acid molecule is a target DNA or a target RNA.

[0440] In a preferred embodiment, the detection includes qualitative detection and quantitative detection.

[0441] In one embodiment, the detection in step (c) may include: using electrophoresis to determine the length of the reporter nucleic acid to determine whether the reporter nucleic acid has been cleaved. If a shorter nucleic acid product is produced, this indicates that the reporter nucleic acid has been cleaved by Ago in the detection system.

[0442] In another embodiment, if the reporter nucleic acid in the detection system carries a fluorescent group and / or a quencher group, then the detection in step (c) includes: detecting using a microplate reader or a fluorescence spectrophotometer.

[0443] In one embodiment of the invention, the method is an in vitro method.

[0444] In another embodiment, the method is non-diagnostic and non-therapeutic.

[0445] Reagent test kit

[0446] The present invention provides a kit for directly detecting a target nucleic acid molecule, the kit comprising:

[0447] (i) the detection system according to the fourteenth aspect of the present invention or a reagent for preparing the detection system; and

[0448] (ii) instructions for use, wherein the instructions describe a method for directly detecting a target nucleic acid molecule using the detection system.

[0449] Typically, the kit includes:

[0450] (a) a first container and a guide gDNA or guide gRNA located in the first container;

[0451] (b) a second container and the room-temperature Argonaute protein (Ago enzyme) variant according to the first aspect of the present invention located in the second container, wherein the room-temperature Argonaute protein (Ago enzyme) variant is a room-temperature Argonaute protein (Ago enzyme) variant that uses DNA or RNA as a guide to guide the cleavage of target RNA; and

[0452] (c) a third container and a fluorescent reporter nucleic acid located in the third container.

[0453] In another preferred embodiment, the kit further comprises:

[0454] (d) a fourth container and reagents required for performing transcription or reverse transcription-transcription located in the fourth container.

[0455] In another preferred embodiment, the kit further comprises:

[0456] (f) a fifth container and a divalent metal ion solution in the fifth container.

[0457] One-step detection

[0458] The "one-step assay" is a simple and rapid nucleic acid detection solution based on the high compatibility of reverse transcriptase and HpAgo variants, such as room-temperature Ago variants, with reaction temperatures and reaction systems. It combines the reverse transcription-transcription reaction and detection reaction in a single PCR reaction tube for isothermal detection. This eliminates the need for temperature changes, centrifugation, pipetting, and tube changes, significantly simplifying the detection apparatus and process and significantly reducing the potential for contamination from opening the tube. This facilitates automated, high-volume sample processing and facilitates widespread adoption of home molecular diagnostics.

[0459] The main advantages of the present invention include:

[0460] (1) Through extensive screening, the present invention unexpectedly obtained for the first time a mutant protein with significantly improved activity compared to conventional Ago enzymes. Specifically, the mutated room-temperature Ago protein has the activity of directed cleavage of RNA targets with higher cleavage efficiency, which is more practical for subsequent gene editing and detection applications.

[0461] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0462] Unless otherwise specified, the reagents and materials used in the examples of the present invention are commercially available products.

[0463] Example 1 Definition of mutation sites

[0464] HpAgo that binds to ssDNA and ssRNA was classified with other known pAgo based on cleavage and nucleic acid binding specificity: binding to DNA / binding to RNA / binding to DNA and RNA and sequence alignment was performed, and the differences in amino acid distribution near the cleavage active site were analyzed, such as Figure 1 As shown in AD, it was found that the T708 site of HpAgo was significantly different from that of the other two types of pAgo, which suggests that it may be involved in the specific binding of HpAgo nucleic acids and thus affect the cleavage activity.

[0465] Example 2 Protein sequence of wild-type HpAgo

[0466] MAVKADIEDGEEIDIALRVTGIDEWEHDAIAQKIQLEDIDNAAIDLTVFHNNEVANFEWQIGEWYLLENVVGNEFRGEMQLNPGYDLNVTLLDDPPAAVENDKLPGSEPSERPVDLSGERGSSGAAATTSDASDGEEFVSGSEVDGSSRPTADGGGKLLHQQPLSDGNYLLQFELGSLPELPVHEYELQATGSGGIDPDDFTNGIEGFTAKAANYYQSRIGSPVTTADASRRRIYATEKLHSTISIHGYTVKPVHQGETTLEARSYTNDGPLQEFVKQDVKRAVAGRFEVSGIDSIIEPTPQRTANSGLFEAYRKYKCRIRVDADGTVICGVNVAYHLESTFSAAEWVQRGHDIAEVTVEHDTDLYDSARTARVKEVIDMDYDDVLDGPGVPMSEYHEKHVEQDVIDSMRAGDPIIADLQYGSGEDSIFPQLLAYCKVIPTFDQLGRVDETFLNVIHNESRMKPEERFNVVTSFVDLLGPTPYFGFDPVPQPTNAGYREHQIRNRPNLRFGDGQTGFYGAGGLERKGYGVYKAPESFDIIALYPEDEEDDARPYVLSLLNKLADYDASPTAFDRDTYELSSEFHYSQHAQKASDYDAALIVVPDADEAAAADYDDPYPEFKRRLGQLGVPSQMISVDNLGNDNYRGNICSSLIGKAGGVPWRIDDVPGAVDAFVGLDVTYDHATKQHLGAAANVIMADGTILASEAVTKQAGETFDEDDVANVIKHVLEIFAEEEGRPPRHVVIHRDGKFYLDIESLISRLDKARDLIQRFDLVEIRKSGNPRIAEYDESNSRFDIADKGVAFHVHNGDHSYLTTTGGKEGSPGTPRPLQIVKRYGSTDLDTLAEQTYWLSEAHVGSLSRSTRLPITTYYADKCADFAMKGYLTKGSVIRGVPYI (SEQ ID NO.1)

[0467] Protein sequence of the HpAgo T708A mutant

[0468] MAVKADIEDGEEIDIALRVTGIDEWEHDAIAQKIQLEDIDNAAIDLTVFHNNEVANFEWQIGEWYLLENVVGNEFRGEMQLNPGYDLNVTLLDDPPAAVENDKLPGSEPSERPVDLSGERGSSGAAATTSDASDGEEFVSGSEVDGSSRPTADGGGKLLHQQPLSDGNYLLQFELGSLPELPVHEYELQATGSGGIDPDDFTNGIEGFTAKAANYYQSRIGSPVTTADASRRRIYATEKLHSTISIHGYTVKPVHQGETTLEARSYTNDGPLQEFVKQDVKRAVAGRFEVSGIDSIIEPTPQRTANSGLFEAYRKYKCRIRVDADGTVICGVNVAYHLESTFSAAEWVQRGHDIAEVTVEHDTDLYDSARTARVKEVIDMDYDDVLDGPGVPMSEYHEKHVEQDVIDSMRAGDPIIADLQYGSGEDSIFPQLLAYCKVIPTFDQLGRVDETFLNVIHNESRMKPEERFNVVTSFVDLLGPTPYFGFDPVPQPTNAGYREHQIRNRPNLRFGDGQTGFYGAGGLERKGYGVYKAPESFDIIALYPEDEEDDARPYVLSLLNKLADYDASPTAFDRDTYELSSEFHYSQHAQKASDYDAALIVVPDADEAAAADYDDPYPEFKRRLGQLGVPSQMISVDNLGNDNYRGNICSSLIGKAGGVPWRIDDVPGAVDAFVGLDVTYDHATKQHLGAAANVIMADGTILASEAVAKQAGETFDEDDVANVIKHVLEIFAEEEGRPPRHVVIHRDGKFYLDIESLISRLDKARDLIQRFDLVEIRKSGNPRIAEYDESNSRFDIADKGVAFHVHNGDHSYLTTTGGKEGSPGTPRPLQIVKRYGSTDLDTLAEQTYWLSEAHVGSLSRSTRLPITTYYADKCADFAMKGYLTKGSVIRGVPYI (SEQ ID NO.2)

[0469] Protein sequence of the HpAgo T708R mutant

[0470] MAVKADIEDGEEIDIALRVTGIDEWEHDAIAQKIQLEDIDNAAIDLTVFHNNEVANFEWQIGEWYLLENVVGNEFRGEMQLNPGYDLNVTLLDDPPAAVENDKLPGSEPSERPVDLSGERGSSGAAATTSDASDGEEFVSGSEVDGSSRPTADGGGKLLHQQPLSDGNYLLQFELGSLPELPVHEYELQATGSGGIDPDDFTNGIEGFTAKAANYYQSRIGSPVTTADASRRRIYATEKLHSTISIHGYTVKPVHQGETTLEARSYTNDGPLQEFVKQDVKRAVAGRFEVSGIDSIIEPTPQRTANSGLFEAYRKYKCRIRVDADGTVICGVNVAYHLESTFSAAEWVQRGHDIAEVTVEHDTDLYDSARTARVKEVIDMDYDDVLDGPGVPMSEYHEKHVEQDVIDSMRAGDPIIADLQYGSGEDSIFPQLLAYCKVIPTFDQLGRVDETFLNVIHNESRMKPEERFNVVTSFVDLLGPTPYFGFDPVPQPTNAGYREHQIRNRPNLRFGDGQTGFYGAGGLERKGYGVYKAPESFDIIALYPEDEEDDARPYVLSLLNKLADYDASPTAFDRDTYELSSEFHYSQHAQKASDYDAALIVVPDADEAAAADYDDPYPEFKRRLGQLGVPSQMISVDNLGNDNYRGNICSSLIGKAGGVPWRIDDVPGAVDAFVGLDVTYDHATKQHLGAAANVIMADGTILASEAVRKQAGETFDEDDVANVIKHVLEIFAEEEGRPPRHVVIHRDGKFYLDIESLISRLDKARDLIQRFDLVEIRKSGNPRIAEYDESNSRFDIADKGVAFHVHNGDHSYLTTTGGKEGSPGTPRPLQIVKRYGSTDLDTLAEQTYWLSEAHVGSLSRSTRLPITTYYADKCADFAMKGYLTKGSVIRGVPYI (SEQ ID NO.3)

[0471] Protein sequence of the HpAgo K709R mutant

[0472] MAVKADIEDGEEIDIALRVTGIDEWEHDAIAQKIQLEDIDNAAIDLTVFHNNEVANFEWQIGEWYLLENVVGNEFRGEMQLNPGYDLNVTLLDDPPAAVENDKLPGSEPSERPVDLSGERGSSGAAATTSDASDGEEFVSGSEVDGSSRPTADGGGKLLHQQPLSDGNYLLQFELGSLPELPVHEYELQATGSGGIDPDDFTNGIEGFTAKAANYYQSRIGSPVTTADASRRRIYATEKLHSTISIHGYTVKPVHQGETTLEARSYTNDGPLQEFVKQDVKRAVAGRFEVSGIDSIIEPTPQRTANSGLFEAYRKYKCRIRVDADGTVICGVNVAYHLESTFSAAEWVQRGHDIAEVTVEHDTDLYDSARTARVKEVIDMDYDDVLDGPGVPMSEYHEKHVEQDVIDSMRAGDPIIADLQYGSGEDSIFPQLLAYCKVIPTFDQLGRVDETFLNVIHNESRMKPEERFNVVTSFVDLLGPTPYFGFDPVPQPTNAGYREHQIRNRPNLRFGDGQTGFYGAGGLERKGYGVYKAPESFDIIALYPEDEEDDARPYVLSLLNKLADYDASPTAFDRDTYELSSEFHYSQHAQKASDYDAALIVVPDADEAAAADYDDPYPEFKRRLGQLGVPSQMISVDNLGNDNYRGNICSSLIGKAGGVPWRIDDVPGAVDAFVGLDVTYDHATKQHLGAAANVIMADGTILASEAVTRQAGETFDEDDVANVIKHVLEIFAEEEGRPPRHVVIHRDGKFYLDIESLISRLDKARDLIQRFDLVEIRKSGNPRIAEYDESNSRFDIADKGVAFHVHNGDHSYLTTTGGKEGSPGTPRPLQIVKRYGSTDLDTLAEQTYWLSEAHVGSLSRSTRLPITTYYADKCADFAMKGYLTKGSVIRGVPYI(SEQ ID NO.4)

[0473] The plasmid pET28a-HpAgo (purchased from Sangon Biotech, Shanghai) based on wild-type HpAgo was used to construct the HpAgoT708A mutant by whole plasmid PCR. The mutant primers are as follows:

[0474] F:5'-GCAGTGGCTAAACAGGCTGGTGAAACCT-3'

[0475] R:5'-GTTTAGCCACTGCTTCGGAAGCCAG-3'

[0476] Whole plasmid PCR 50ul system:

[0477]

[0478] Reaction procedure:

[0479]

[0480] Example 3: Evaluation of the cleavage efficiency of HpAgo wild type and T708A mutant on RNA targets by time course analysis

[0481] Based on the structural and bioinformatics studies of pAgo, it was found that the MID domain of most pAgos has a set of conserved amino acid residues that can be used to anchor the 5' end of the guide. For example, the four conserved amino acid residues Y463, K467, ​​Q478 and K506 in the MID domain of RsAgo interact with divalent ions (Mg 2+ or Mn 2+ ) bind to form a hydrogen bond network to anchor the 5'P of the guide. The recently reported PliAgo differs from other pAgo in its conserved amino acid sequence in the MID domain. It interacts synergistically with the 5'P of the guide through four residues: R467, H498, S501, and Y530, without the involvement of metal ions.

[0482] HpAgo was compared with known Ago sequences, such as Figure 2 As shown in Figure 2, it was found that the MID of HpAgo also contains conserved amino acid motifs (Y617, K621, Q632, K655), which is similar to most known pAgos and can interact with the 5'P-guide. Further structural prediction of HpAgo using the Alphafold 2Colab online website confirmed the presence of four conserved motifs in the MID domain of HpAgo, which is consistent with the results of multiple sequence alignment. The structural model of HpAgo is shown as follows: Figure 3 HpAgo is also shown to have six domains, including N, L1, PAZ, L2, MID and PIWI domains, which are highlighted in purple, yellow, pink, gray, orange and green, respectively. Since AaAgo is known to cleave ssRNA using 5'P-gDNA / RNA at medium temperature (50°C), the predicted structure of HpAgo was compared with the crystallized structure of AaAgo, as shown in Figure 2. Figure 4 As shown, the RMSD is Comparison revealed that despite significant differences in the overall structures between the two proteins, their MID binding pockets are similar, which may lead to their similar cleavage properties at different temperatures.

[0483] The cleavage activity of Ago protein is related to many key sites. At present, the conserved catalytic quadruplex DEDX of Ago protein has been widely reported, while other key sites related to catalytic activity have been reported less. Figure 5 As shown, there are key sites 708T and 709K predicted to be related to HpAgo cleavage activity near the DEDD cleavage quadruplex of the PIWI domain in HpAgo.

[0484] To quantitatively analyze the differences in cleavage activity between HpAgo and the T708A, T708K, and K709R mutants, the percentage of cleaved RNA targets was measured at different reaction times under identical conditions. The cleavage activity differences between HpAgo, T708A, T708K, and K709R mutants were analyzed using a 5'-FAM-labeled target nucleic acid / fluorescent reporter and a complementary 5'P-labeled guide. The pH of the reaction was adjusted to 7.5 by adding MnCl2 to a final concentration of 2.5 mM in 1× reaction buffer (20 mM Tris-HCl, pH 7.5, 250 mM NaCl). The purified protein, guide nucleic acid, and target nucleic acid / fluorescent reporter were mixed at a ratio of 4:4:1 (800 nM HpAgo: 800 nM guide: 200 nM target nucleic acid / fluorescent reporter) and added to the reaction system, which was then incubated at 37°C for various times. The reaction was terminated with 2× loading buffer (95% formamide, 0.5 mM EDTA, 0.025% bromophenol blue, and 0.025% xylene cyanol FF). The terminated samples were analyzed by 16% urea-polyacrylamide gel electrophoresis and stained with GelRed (Invitrogen). Nucleic acid bands were visualized using a GelImage System (Tanon-3500BR). The grayscale values ​​of the nucleic acid gel images were quantitatively calculated and analyzed using Image J and GraphPad Prism 8.0 software, and bar graphs were prepared.

[0485] The sequence used is as follows:

[0486] 5'P-DNA guide with 3'FAM 5'-TGAGGTAGTAGGTTGTATAGT-3'(SEQ ID NO.8)

[0487] 5'P-RNA guide with 3'FAM 5'-UGAGGUAGUAGGUUGUAUAGU-3'

[0488] (SEQ ID NO.9)

[0489] 5'FAM-ssRNA target / fluorescent reporter nucleic acid 5'-AUAUACUAUACAACCUACUACCUCGUAUAAAUUUUUAAAUAAAUA-3'

[0490] (SEQ ID NO.10)

[0491] The results show that if Figure 6 As shown in Table 1, under the same shearing conditions and shearing system, the shearing activity and shearing efficiency of the T708A, T708R, and K709R mutants on RNA targets were improved.

[0492] Table 1

[0493]

[0494] MbpAgo, PnyAgo, RslAgo, and PliAgo also possess the ability to cleave single-stranded RNA targets at room temperature. Sequence alignment and structural analysis revealed that MbpAgo, PnyAgo, RslAgo, and PliAgo, at positions corresponding to HpAgo 708, such as positions 592, 591, and 598, are also located near the conserved catalytic quadruplex DEDX, sharing the same function as positions 708T and 709K in HpAgo. Therefore, similar amino acid mutations should have similar effects.

[0495] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A room-temperature Argonaute protein variant, characterized in that, The variant is a non-natural protein, and the variant is mutated at the following core amino acid sites of the wild-type room-temperature Argonaute protein: the threonine (T) site corresponding to the 708th position of HpAgo; and / or the lysine (K) site corresponding to the 709th position of HpAgo.

2. A polynucleotide, characterized in that, The polynucleotide encodes the variant according to claim 1.

3. A carrier, characterized in that, The vector contains the polynucleotide according to claim 2.

4. A host cell, characterized in that, The host cell contains the vector according to claim 3, or the polynucleotide according to claim 2 is integrated into its genome.

5. A method for preparing a room-temperature Argonaute protein variant, characterized in that, The method includes the steps of: (a) culturing the host cell according to claim 4 under conditions suitable for expression, so as to express the room-temperature Argonaute protein variant; and (b) isolating the room-temperature Argonaute protein variant.

6. An enzyme preparation, characterized in that, The enzyme preparation includes the room-temperature Argonaute protein variant according to claim 1.

7. A gene editing system, characterized in that, Including: the room-temperature Argonaute protein variant according to claim 1, or its encoding gene or its expression vector.

8. A composition, characterized in that, Including: the system according to claim 7; and a pharmaceutically acceptable carrier.

9. A kit, characterized in that, Including: the room-temperature Argonaute protein variant according to claim 1 or the gene editing system according to claim 7.

10. A medicine box, characterized in that, Including: a first container, and the gene editing system according to claim 7 or the composition according to claim 8 located in the first container, or a drug containing the gene editing system according to claim 7 or the composition according to claim 8.

11. A medicine box, characterized in that, Including: (a1) a first container, and the room-temperature Argonaute protein variant according to claim 1, or its encoding gene or its expression vector, or a drug containing the room-temperature Argonaute protein variant according to claim 1, or its encoding gene or its expression vector located in the first container; (b1) a second container, and a guide gDNA pair or a guide gRNA pair or its expression vector located in the second container, or a drug containing a guide gDNA pair or a guide gRNA pair or its expression vector.

12. Use of the Argonaute protein variant at room temperature according to claim 1, the gene editing system according to claim 7, the composition according to claim 8, the kit according to claim 9, or the cartridge according to claim 10 or claim 11, characterized in that For preparing a reagent or kit for gene editing.

13. A method for gene editing in vitro, characterized in that, Including the steps of: performing gene editing on cells in the presence of the room-temperature Argonaute protein variant according to claim 1, the gene editing system according to claim 7, the composition according to claim 8, the kit according to claim 9, or the kit according to claim 10 or claim 11.

14. A detection system for directly detecting a target nucleic acid molecule, characterized in that, The system includes: (a) a guide gDNA pair or a guide gRNA pair; (b) the room-temperature Argonaute protein variant according to claim 1, which is a room-temperature Argonaute protein variant that guides the cleavage of a target RNA with DNA or RNA as a guide; (c) a fluorescent reporter nucleic acid, which carries a fluorophore and a quenching group; wherein the target nucleic acid molecule is a target DNA or a target RNA.

15. A kit for directly detecting a target nucleic acid molecule, characterized in that, The kit includes: (i) the detection system according to claim 14 or a reagent for formulating the detection system; and (ii) Instruction Manual, which describes the method for directly detecting the target nucleic acid molecule using the detection system described above.

16. A method for directly detecting the presence of a target nucleic acid molecule in a sample, characterized in that, (a) comprising the following steps: (a) providing the detection system for detecting the target nucleic acid molecule as described in claim 14; and (b) reacting the detection system with the sample to be detected at a certain temperature to form a first reaction solution; (c) performing fluorescence detection on the first reaction solution to obtain a fluorescence signal value; wherein, if a fluorescence signal value is detected in the first reaction solution, it indicates that the target nucleic acid molecule exists in the sample; if no fluorescence signal value is detected in the first reaction solution, it indicates that the target nucleic acid molecule does not exist in the sample, and the target nucleic acid molecule is the target DNA or target RNA.

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