TtdAgo mutant protein with room-temperature target nucleic acid cleavage activity and its application

Abstract

The present invention provides TtdAgo mutant proteins with room-temperature target nucleic acid cleavage activity and their applications, belonging to the field of programmable nuclease technology. Relative to the wild-type TtdAgo protein, the TtdAgo mutant proteins have mutations at amino acids 527 and / or 561 and / or 593 and / or 599. Compared to the wild-type TtdAgo protein, the TtdAgo mutant proteins provided by the present invention not only have significantly improved activity at room temperature but are also capable of cleaving target RNA, effectively expanding the scope of application of the pAgo protein.

Description

Technical Field

[0001] The present invention relates to the technical field of programmable nucleases, and in particular to a TtdAgo mutant protein having target nucleic acid cleavage activity at room temperature, and a preparation method and application thereof. Background Art

[0002] Argonaute (Ago) proteins are an emerging class of programmable nucleases that utilize small DNA or RNA guides (gDNA or gRNA) to cleave complementary target nucleic acids (target DNA and / or target RNA). Unlike the widely used Cas nucleases, Ago nucleases' catalytic activity is independent of specific motifs (such as PAM and PFS) within the target molecule. Furthermore, they can utilize DNA or RNA as guides, offering potential for the development of various Ago-based biotechnologies.

[0003] TtdAgo is a Thermococcus thioreducens TtdAgo is a prokaryotic Ago protein that can cleave target DNA under the guidance of 5'OH-gDNA and 5'P-gDNA, and its activity is highest in the temperature range of 70-80°C. However, these characteristics of TtdAgo limit its application at room temperature and its ability to be used as an RNA manipulation tool. Summary of the Invention

[0004] In view of the technical problems existing in the background technology, the present invention provides a TtdAgo mutant protein with room-temperature target nucleic acid cleavage activity, aiming to solve the limitations of wild-type TtdAgo in catalytic activity temperature and catalytic substrates, so as to expand the scope of use of TtdAgo and lay the foundation for its application in molecular diagnosis, molecular cloning, gene editing and other fields.

[0005] In the first aspect, the present invention provides a TtdAgo mutant protein having room temperature target nucleic acid cleavage activity, wherein the TtdAgo mutant protein has an amino acid mutation at position 527 and / or position 561 and / or position 593 and / or position 599 relative to the sequence shown in SEQ ID NO.1 (i.e., the amino acid sequence of the wild-type TtdAgo protein).

[0006] In the sequence shown in SEQ ID NO.1, the amino acid at position 527 is histidine (His, H), the amino acid at position 561 is tyrosine (Tyr, Y), the amino acid at position 593 is lysine (Lys, K), and the amino acid at position 599 is glutamic acid (Glu, E). Specifically, in the TtdAgo mutant protein, an amino acid mutation at position 527 refers to a mutation of histidine to any amino acid other than histidine, an amino acid mutation at position 561 refers to a mutation of tyrosine to any amino acid other than tyrosine, an amino acid mutation at position 593 refers to a mutation of lysine to any amino acid other than lysine, and an amino acid mutation at position 599 refers to a mutation of glutamic acid to any amino acid other than glutamic acid.

[0007] Compared with the wild-type TtdAgo protein, the TtdAgo mutant protein provided by the present invention has better target nucleic acid cleavage activity at 30~90°C, especially better activity at lower temperatures, and has the ability to efficiently cleave target nucleic acids at room temperature.

[0008] Preferably, the TtdAgo mutant protein is mutated to glycine (Gly, G) at positions 527, 561, 593, and 599, respectively or simultaneously, relative to the sequence shown in SEQ ID NO. 1. Specifically, the TtdAgo mutant can be selected from the following:

[0009] TtdAgo_H527G, in which position 527 of the mutant is mutated to glycine relative to the sequence shown in SEQ ID NO. 1;

[0010] TtdAgo_Y561G, relative to the sequence shown in SEQ ID NO. 1, this mutant has position 561 mutated to glycine;

[0011] TtdAgo_K593G, in which position 593 of the mutant is mutated to glycine relative to the sequence shown in SEQ ID NO. 1;

[0012] TtdAgo_E599G, relative to the sequence shown in SEQ ID NO. 1, this mutant has position 593 mutated to glycine;

[0013] TtdAgo_H527G / Y561G / K593G / E599G, relative to the sequence shown in SEQ ID NO.1, positions 527, 561, 593 and 599 of this mutant are simultaneously mutated to glycine.

[0014] Among the mutants described above, TtdAgo_H527G, TtdAgo_Y561G, TtdAgo_K593G, and TtdAgo_E599G were all capable of cleaving target DNA under the guidance of gDNA and exhibited superior activity at 37°C. Furthermore, compared to single amino acid mutations, the combined mutant TtdAgo_H527G / Y561G / K593G / E599G exhibited further enhanced activity at 37°C, enabling it to cleave target DNA and RNA under the guidance of gDNA, as well as target RNA under the guidance of 5'P-gRNA.

[0015] In a second aspect, the present invention provides a biomaterial related to a TtdAgo mutant protein, comprising:

[0016] (b1) a nucleic acid molecule encoding a TtdAgo mutant protein;

[0017] (b2) An expression cassette, vector or transformant comprising the nucleic acid molecule of (b1).

[0018] As for nucleic acid molecules, any nucleic acid molecule can be used as long as it can express the TtdAgo mutant protein in the expression system. For example, in one embodiment of the present invention, a nucleic acid molecule encoding TtdAgo_H527G / Y561G / K593G / E599G is provided, the nucleotide sequence of which is shown in SEQ ID NO. 3, and the amino acid sequence of the mutant protein encoded by it is shown in SEQ ID NO. 2.

[0019] The vector can be a pET plasmid. For example, in one embodiment of the present invention, a nucleic acid molecule encoding a TtdAgo mutant protein is ligated into pET-28a to obtain a recombinant vector. The transformant can be obtained by transforming a host cell with the recombinant vector. The host cell can be a conventional host cell in the art, but must be able to satisfy the requirements of stable self-replication of the recombinant vector and effective expression of the nucleic acid molecule encoding the TtdAgo mutant protein carried by the recombinant vector.

[0020] In a third aspect, the present invention provides a nucleic acid cleavage system comprising at least:

[0021] guide nucleic acid;

[0022] and TtdAgo mutant proteins.

[0023] Preferably, the guide nucleic acid is 11-25 nt in length, more preferably 13-19 nt in length, and most preferably 15 nt in length.

[0024] Preferably, the guide nucleic acid is selected from 5'P-gRNA, 5'P-gDNA or 5'OH-gDNA.

[0025] In a fourth aspect, the present invention provides the use of a TtdAgo mutant protein or nucleic acid cleavage system for specifically cleaving a target nucleic acid. Specifically, a reaction system comprising a guide nucleic acid, a divalent metal cation, a TtdAgo mutant protein, and a target nucleic acid is constructed to perform a cleavage reaction, wherein the target nucleic acid and the guide nucleic acid sequence are complementary to each other, and the TtdAgo mutant protein specifically cleaves the target nucleic acid under the guidance of the guide nucleic acid.

[0026] In the above applications, the target nucleic acid includes target RNA and / or target DNA. The target RNA may have no higher-order structure or a higher-order structure, or may be double-stranded RNA, in vitro transcribed RNA, viral genomic RNA, mRNA, or other intracellular RNA. The target DNA may be single-stranded DNA or double-stranded DNA.

[0027] In the above applications, complementary pairing specifically refers to the following two situations:

[0028] The target nucleic acid has a nucleotide sequence that is completely complementary to the guide nucleic acid sequence; or

[0029] The target nucleic acid has a nucleotide sequence that has single or multiple base mismatches with the guide nucleic acid sequence, wherein the number of mismatches may be 1, 2, 3, 4 or 5, and the mismatches are usually isolated or continuous.

[0030] Preferably, in the above application, the divalent metal cation is Mn 2+ and / or Mg 2+ , where Mn 2+ Better.

[0031] Based on the ability of TtdAgo mutant protein to specifically cut target nucleic acids, it can be developed for use in gene editing, nucleic acid detection and other fields.

[0032] In a fifth aspect, the present invention provides a kit comprising the TtdAgo mutant protein provided by the present invention; depending on the purpose of detection, it may also comprise a guide nucleic acid with a specific sequence, or a buffer for supporting the catalytic activity of the TtdAgo mutant protein, etc.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) Compared with wild-type TtdAgo, which can only effectively cut target DNA using guide DNA at higher temperatures, the TtdAgo mutant protein provided by the present invention improves its cutting activity at medium and low temperatures, expands the temperature range of use, and retains its heat resistance, which is beneficial to the storage and transportation of the enzyme.

[0035] (2) Compared with other TtdAgo mutant proteins, TtdAgo_H527G / Y561G / K593G / E599G can not only effectively cut target DNA using guide DNA at medium and low temperatures, but also effectively cut target RNA using guide DNA and guide RNA, further expanding the types of guide nucleic acids and cutting targets of TtdAgo.

[0036] (3) The present invention provides nucleic acid expression vectors encoding TtdAgo mutant proteins, especially the mutant TtdAgo_H527G / Y561G / K593G / E599G, as well as compositions, kits and methods for cutting and editing target nucleic acids in a sequence-specific manner, laying the foundation for the application of various TtdAgo mutant proteins in many fields of biotechnology, such as molecular diagnosis, molecular cloning, and gene editing.

[0037] (4) The TtdAgo mutant protein can cut target DNA using guide DNA at 37°C, laying the foundation for its in vivo genome editing. At the same time, the mutant TtdAgo_H527G / Y561G / K593G / E599G can cut target RNA using guide DNA at 37°C, laying the foundation for its in vivo RNA targeting; TtdAgo_H527G / Y561G / K593G / E599G can also cut target RNA using 5'P-gRNA at 37°C, laying the foundation for its in vivo RNA targeting by co-expression of protein and guide nucleic acid.

[0038] (5) Each TtdAgo mutant protein strictly depends on the complementary pairing of the guide and target to exert its cleavage activity, and there is no non-specific "incidental cleavage" activity of CRISPR-related proteins, which has better specificity. Moreover, the pAgo complex formed by the TtdAgo mutant protein and the guide nucleic acid does not rely on a special motif near the target site to recognize and bind to the target, making the guide nucleic acid design convenient without considering site restrictions. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] To more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0040] Figure 1 This is a diagram showing the SDS-PAGE analysis results of the TtdAgo protein and some of its mutant proteins in Example 1;

[0041] Figure 2Comparison of the cleavage activities of TtdAgo and its mutants at 37°C in Example 2;

[0042] Figure 3 This is a urea / polyacrylamide gel assay result of the products obtained by cleaving single-stranded target DNA or target RNA using four guide nucleic acids using TtdAgo_H527G / Y561G / K593G / E599G in Example 2;

[0043] Figure 4 This is a graph showing the urea / polyacrylamide gel assay results of the products obtained by cleaving single-stranded target DNA or target RNA by TtdAgo_H527G / Y561G / K593G / E599G at different temperatures in Example 3;

[0044] Figure 5 This is a urea / polyacrylamide gel assay result of the products obtained by cleaving single-stranded target DNA or target RNA by TtdAgo_H527G / Y561G / K593G / E599G under different metal cation conditions in Example 4;

[0045] Figure 6 This is a graph showing the urea / polyacrylamide gel assay results of products obtained by cleaving single-stranded target DNA or target RNA under the guidance of guide nucleic acids of different lengths using TtdAgo_H527G / Y561G / K593G / E599G in Example 5;

[0046] Figure 7 This is a urea / polyacrylamide gel assay result of the products obtained by cleaving single-stranded target DNA or target RNA by TtdAgo_H527G / Y561G / K593G / E599G under the guidance of guide nucleic acids with different 5' terminal nucleotides in Example 6;

[0047] Figure 8 This is a urea / polyacrylamide gel assay result of the products obtained by cleaving single-stranded target DNA or target RNA under different single-base mismatch conditions in Example 7 by TtdAgo_H527G / Y561G / K593G / E599G. DETAILED DESCRIPTION

[0048] The following embodiments of the technical solution of the present invention will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only examples and are not intended to limit the scope of protection of the present invention.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs; the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. In the present specification and claims, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions; the term "and / or" is merely a description of an association relationship between associated objects, indicating that three relationships may exist. For example, A and / or B can represent three situations: the existence of A alone, the existence of A and B at the same time, and the existence of B alone.

[0050] TtdAgo is a Thermococcus thioreducens Previous studies have shown that the prokaryotic Ago protein only cleaves target DNA under the guidance of a guide DNA. Furthermore, while it exhibits activity over a wide temperature range, its activity is extremely low at low and medium temperatures. To address the limitations of wild-type TtdAgo's catalytic activity temperature and catalytic substrates, the present invention provides a modification strategy.

[0051] By elucidating the structure of the ternary complex of TtdAgo bound to a guide nucleic acid and a target nucleic acid, the inventors discovered that target addition induces dimerization. Structural analysis and biochemical experiments further clarified the catalytic mechanism. Like most Ago proteins, TtdAgo has a bilobed architecture. The PAZ lobe (N-terminal domain, linker L1, and PAZ domain) is connected to the PIWI lobe (MID and PIWI domain) via linker L2. The MID and PAZ domains anchor the 5' and 3' ends of the guide strand, respectively. The active site within the PIWI domain is structurally similar to that of RNase H, and the DEDH catalytic quadruplex confers TtdAgo endonuclease activity. Enzymes from diverse organisms typically maintain similar catalytic rates at their respective physiological temperatures, and sequence changes associated with these adaptive differences are often located in surface-exposed regions distal to the substrate binding site, leaving the active site structurally undisturbed.

[0052] Based on this research, the present invention successfully screened TtdAgo mutants with improved activity at low and medium temperatures by mutating the wild-type TtdAgo protein's PIWI domain at amino acids located more than 20 Å away from the active site and substrate binding. Specifically, the active site refers to the DEDH catalytic quadruplex of the wild-type TtdAgo protein.

[0053] Specifically, the TtdAgo mutant protein provided by the present invention, which has good activity under medium and low temperature conditions, has mutations at positions 527 and / or 561 and / or 593 and / or 599, respectively or simultaneously, relative to wild-type TtdAgo (amino acid sequence shown in SEQ ID NO. 1). More preferably, the TtdAgo mutant protein has mutations at positions 527 and / or 561 and / or 593 and / or 599, respectively or simultaneously, relative to wild-type TtdAgo. The presence of surface-exposed glycine residues can help increase structural flexibility and enzyme activity by acting locally near the catalytic site or through an allosteric mechanism.

[0054] Compared with the wild-type TtdAgo protein, the TtdAgo mutant protein provided by the present invention has better target nucleic acid cleavage activity at medium and low temperatures, and can cut target DNA and RNA, expanding the scope of application of the TtdAgo protein and making it have the potential to be used in intracellular RNA and genome editing.

[0055] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications were used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0056] Example 1

[0057] This example provides a method for screening and preparing TtdAgo mutant proteins, comprising the following steps:

[0058] (1) Screening of TtdAgo mutation sites based on structure.

[0059] The structure of the TtdAgo ternary complex was loaded into the PyMOL software, and the mutation sites were designed based on the following three principles: first, the side chain of the mutation site should be highly exposed on the surface, away from the binding substrate and active site (i.e., >20Å); second, the mutation site should be located in the PIWI domain; and third, the amino acid should be mutated to glycine.

[0060] A gene fragment encoding TtdAgo as shown in SEQ ID NO. 1 was synthesized and ligated into pET28a using conventional methods to obtain a pET28a-TtdAgo plasmid.

[0061] TtdAgo mutants were constructed based on the screened mutation sites, including: TtdAgo_H527G, which mutated histidine at position 527 to glycine, TtdAgo_K528G, which mutated lysine at position 528 to glycine, TtdAgo_Q559G, which mutated glutamine at position 559 to glycine, TtdAgo_Y561G, which mutated tyrosine at position 561 to glycine, TtdAgo_K593G, which mutated lysine at position 593 to glycine, TtdAgo_D597G, which mutated aspartic acid at position 597 to glycine, TtdAgo_E599G, which mutated glutamate at position 599 to glycine, TtdAgo_V625G, which mutated valine at position 625 to glycine, and TtdAgo_D684G, which mutated asparagine at position 684 to glycine.

[0062] Further combined mutations were performed on TtdAgo, such as simultaneously mutating histidine at position 527, tyrosine at position 561, lysine at position 593, and glutamic acid at position 599 to glycine, to obtain TtdAgo_H527G / Y561G / K593G / E599G. The amino acid sequence and nucleotide sequence of TtdAgo_H527G / Y561G / K593G / E599G are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0063] Primers were designed for different sites, PCR was performed using the pET28a-TtdAgo plasmid as a template, and the mutant plasmids were obtained by T5 recombination cloning.

[0064] (2) Expression and purification of TtdAgo and its mutants.

[0065] Protein expression was performed in Escherichia coli using IPTG induction. The protein was purified using Ni column affinity chromatography and heparin affinity chromatography, and the purification results were analyzed by SDS-PAGE. The eluted target protein of high purity was concentrated by ultrafiltration using Amicon 50K ultrafiltration tubes. The concentrated protein was aliquoted, rapidly frozen in liquid nitrogen, and stored at -80°C.

[0066] The results of SDS-PAGE analysis of TtdAgo and some of its mutants are shown in Figure 1 As shown, from Figure 1 It can be seen that the purified TtdAgo and its mutants have a purity of more than 90%.

[0067] Example 2

[0068] This example tested the cleavage activity of various TtdAgo mutants at room temperature, including the following:

[0069] (1) Detection of TtdAgo and its single amino acid mutants.

[0070] Activity was verified at 37°C, and a target DNA cleavage experiment was performed using 5'P-gDNA.

[0071] To an enzyme-free PCR tube, add the guide nucleic acid to a final concentration of 1 μM, 1 μM TtdAgo or mutant protein, and 1× enzyme digestion buffer (10 mM HEPES pH 7.5, 100 mM NaCl, 5% glycerol, 5 mM MnCl₂) and incubate at 37°C for 10 minutes. Then, add 200 nM 5'-end FAM-labeled target DNA or RNA and incubate at 37°C for 30 minutes for target cleavage. The reaction is terminated by mixing the sample with 2× RNA loading dye (95% formamide, 18 mM EDTA, 0.025% SDS, and 0.025% bromophenol blue) and heating at 95°C for 5 minutes. Cleavage products are detected by electrophoresis on a 20% denaturing polyacrylamide gel and visualized using a gel imaging system. Three replicates were performed, and cleavage ratios were analyzed using ImageJ and Prism 8 (GraphPad) software.

[0072] Comparison of the cleavage activities of wild-type TtdAgo and its mutants Figure 2 As shown, from Figure 2 It can be seen that TtdAgo_H527G, TtdAgo_Y561G, TtdAgo_K593G, and TtdAgo_E599G have higher activities at 37°C.

[0073] (2) The activity of TtdAgo_H527G / Y561G / K593G / E599G was verified at 37°C, and four guide nucleic acids (5'OH-gDNA, 5'P-gDNA, 5'OH-gRNA, 5'P-gRNA) were used to perform target DNA and RNA cutting experiments.

[0074] The types of guides that can be used for TtdAgo_H527G / Y561G / K593G / E599G and the types of target nucleic acids that can be cleaved are as follows: Figure 3 As shown, from Figure 3 It can be seen that TtdAgo_H527G / Y561G / K593G / E599G can cleave complementary target DNA and RNA using 5'OH-gDNA and 5'P-gDNA at 37°C, and cleave complementary target RNA using 5'P-gRNA.

[0075] The sequences of the guide nucleic acid and target nucleic acid used in this example are shown in Table 1.

[0076] Table 1 Sequence information of guide nucleic acid and target nucleic acid

[0077]

[0078] Example 3

[0079] Taking TtdAgo_H527G / Y561G / K593G / E599G as an example, this example demonstrates the effect of reaction temperature on the cleavage activity of TtdAgo mutant protein.

[0080] Referring to Example 2, the mutant TtdAgo_H527G / Y561G / K593G / E599G was subjected to 5'P-gDNA guided target DNA and RNA cleavage experiments at different reaction temperatures. Figure 4 As shown, from Figure 4 It can be seen that the mutant TtdAgo_H527G / Y561G / K593G / E599G has catalytic activity at 30~90℃ and has better cleavage efficiency at 50~65℃.

[0081] Example 4

[0082] Taking the mutant TtdAgo_H527G / Y561G / K593G / E599G as an example, this example explores the effect of the type of divalent metal ions in the cleavage system on the cleavage activity of the mutant.

[0083] The experimental process was similar to that of Example 2, except that the guide nucleic acid was 18 nt in length, and the MnCl2 in the enzyme cleavage reaction buffer was replaced with FeCl2, CoCl2, NiCl2, CuCl2, ZnCl2, CaCl2, and MgCl2 at the same concentrations for 5' P-gDNA-guided target DNA and target RNA cleavage experiments.

[0084] Detection was performed using 20% ​​urea / polyacrylamide gel electrophoresis. Figure 5 The results showed that TtdAgo_H527G / Y561G / K593G / E599G can use Mg for the cleavage of target DNA guided by 5'P-gDNA. 2+ and Mn 2+ As a divalent metal ion, and in the presence of Mn 2+ The activity is best in the enzyme digestion buffer; for the cleavage of the target RNA guided by 5'P-gDNA, TtdAgo_H527G / Y561G / K593G / E599G can also use Mg 2+ and Mn 2+ As a divalent metal ion, and in the presence of Mn 2+ The activity is best in the enzyme digestion buffer. Therefore, the optimal divalent metal ion for TtdAgo_H527G / Y561G / K593G / E599G is Mn 2+ .

[0085] Example 5

[0086] Taking the mutant TtdAgo_H527G / Y561G / K593G / E599G as an example, this case explored the optimal length of the guide nucleic acid.

[0087] Based on the guide nucleic acid and target sequences described in Example 2, gDNAs ranging in length from 11 to 25 nt were synthesized. The 25-nt gDNA sequence is shown in SEQ ID NO. 8, and the 11-21-nt gDNA sequences are identical to the first 11, 13, 15, 17, 18, 19, and 21 bases of this sequence, respectively. These guide nucleic acids share the same 5'-end sequence, thus resulting in the same cleavage site and cleavage product size.

[0088] The synthesized guide nucleic acids of different lengths were phosphorylated at the 5' end, and 5' P-gDNA of different lengths were used to perform target DNA and RNA cleavage experiments. The experimental process was referred to Example 2, and the test results were as follows: Figure 6 The results showed that TtdAgo_H527G / Y561G / K593G / E599G can cleave target DNA using 13-25 nt 5'P-gDNA, and exhibits the highest enzymatic activity under the guidance of a 15 nt guide nucleic acid. TtdAgo_H527G / Y561G / K593G / E599G can cleave target RNA under the guidance of 11-25 nt 5'P-gDNA, and exhibits higher enzymatic activity under the guidance of a 15 nt guide nucleic acid.

[0089] Example 6

[0090] Taking the mutant TtdAgo_H527G / Y561G / K593G / E599G as an example, this case explored the effect of the 5'-terminal nucleotide of the guide nucleic acid on the enzyme cleavage activity of the mutant.

[0091] Referring to the guide nucleic acid sequences in Example 2, a series of 5'-terminally phosphorylated DNA guide nucleic acids were designed and synthesized. These guide nucleic acids had different 5'-terminal nucleotides (A, T, G, C) but otherwise identical sequences. Correspondingly, a series of target DNA and RNA complementary to each guide nucleic acid were also synthesized. The sequences of the corresponding target nucleic acids are shown in Table 2.

[0092] Table 2 Sequence information of different targets

[0093]

[0094] Referring to Example 2, the activity of the mutant TtdAgo_H527G / Y561G / K593G / E599G in cleaving the corresponding target DNA and RNA was tested under the guidance of 5'P-gDNA with different 5' terminal nucleotides, and 20% urea / polyacrylamide gel electrophoresis was used for detection, and the detection results were as follows: Figure 7 The results showed that when TtdAgo_H527G / Y561G / K593G / E599G cleaved complementary target DNA and RNA under the guidance of 5'P-gDNA, no obvious preference for the 5'-terminal nucleotide was observed, and the cleavage activities mediated by guide nucleic acids containing 5'-A, 5'-T, 5'-G, and 5'-C were comparable.

[0095] Example 7

[0096] Taking the mutant TtdAgo_H527G / Y561G / K593G / E599G as an example, this example studies the effect of single base mispairing at different sites on the enzyme cleavage activity of the mutant.

[0097] First, referring to the guide nucleic acid and target sequences described in Example 2, a series of gDNAs were designed and synthesized that differed from the original guide nucleic acid at various positions by a single base. These guide nucleic acids exhibited single-base mismatches with the target DNA or RNA. Specifically, the sequences of guide nucleic acids with single-base mismatches at various positions are shown in Table 3.

[0098] Table 3 Mismatch information of different guide nucleic acids

[0099]

[0100] Then, these guide nucleic acids were phosphorylated at the 5' end, and the target DNA and RNA were cleaved using 5'P-gDNA as described in Example 2. The cleavage products were detected by 20% urea / polyacrylamide gel electrophoresis.

[0101] Test results such as Figure 8As shown, the results showed that when the target DNA was cut under the guidance of 5'P-gDNA, a single base mismatch at position 7 in the seed region of the guide nucleic acid reduced the enzymatic activity of TtdAgo_H527G / Y561G / K593G / E599G. In contrast, single base mismatches in the 5' anchor region, central region, and 3' supplementary region of the guide nucleic acid did not significantly reduce its cleavage activity. In addition, a single base mismatch at position 4 in the central region of the guide nucleic acid increased the enzyme cleavage activity. When the target RNA was cut under the guidance of 5'P-gDNA, single base mismatches at positions 6 and 7 in the seed region of the guide nucleic acid reduced the enzymatic activity of TtdAgo_H527G / Y561G / K593G / E599G. In contrast, single base mismatches in the 5' anchor region, central region, and 3' supplementary region of the guide nucleic acid did not significantly reduce its cleavage activity. In fact, mismatches at some sites increased the cleavage activity.

[0102] In summary, the TtdAgo mutant protein provided by the present invention not only has better cleavage activity at room temperature than the wild-type TtdAgo protein, but also can cleave target RNA, effectively expanding the scope of use of the pAgo protein; the present invention also explores the effects of guide nucleic acid design, divalent metal cations, etc. on the cleavage activity of the TtdAgo mutant protein, laying the foundation for the application of TtdAgo mutant proteins in many fields of biotechnology such as molecular diagnosis, molecular cloning, RNA and genome editing.

[0103] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the present invention, other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A TtdAgo mutant protein having room temperature target nucleic acid cleavage activity, characterized in that: The TtdAgo mutant protein is selected from TtdAgo_H527G / Y561G / K593G / E599G, TtdAgo_H527G, TtdAgo_Y561G, TtdAgo_K593G and TtdAgo_E599G; In the TtdAgo_H527G / Y561G / K593G / E599G, relative to the sequence shown in SEQ ID NO. 1, the amino acid at position 527 is mutated from histidine to glycine, the amino acid at position 561 is mutated from tyrosine to glycine, the amino acid at position 593 is mutated from lysine to glycine, and the amino acid at position 599 is mutated from glutamic acid to glycine; The amino acid at position 527 of TtdAgo_H527G is mutated from histidine to glycine relative to the sequence shown in SEQ ID NO.1; The TtdAgo_Y561G is mutated from tyrosine to glycine in the sequence shown in SEQ ID NO.1; The amino acid at position 593 of TtdAgo_K593G is mutated from lysine to glycine relative to the sequence shown in SEQ ID NO.1; In the TtdAgo_E599G, relative to the sequence shown in SEQ ID NO.1, the amino acid at position 599 is mutated from glutamic acid to glycine.

2. A nucleic acid molecule encoding the TtdAgo mutant protein according to claim 1.

3. An expression cassette, vector or transformant containing the nucleic acid molecule according to claim 2.

4. A nucleic acid cleavage system, characterized in that: The nucleic acid cleavage system comprises: guide nucleic acid; The TtdAgo mutant protein according to claim 1.

5. Use of the TtdAgo mutant protein according to claim 1 or the nucleic acid cleavage system according to claim 4 in specifically cleaving a target nucleic acid.

6. The use according to claim 5, characterized in that A reaction system containing a guide nucleic acid, a divalent metal cation, a TtdAgo mutant protein and a target nucleic acid is constructed to perform a cleavage reaction; wherein the target nucleic acid is complementary to the guide nucleic acid sequence, and the TtdAgo mutant protein specifically cleaves the target nucleic acid under the guidance of the guide nucleic acid.

7. The use according to claim 6, characterized in that The divalent metal cation is Mn 2+ and / or Mg 2+ .

8. A kit, characterized in that Comprising the TtdAgo mutant protein according to claim 1.

Images