TtdAgo mutant protein with normal-temperature target nucleic acid cleavage activity and application of TtdAgo mutant protein

Abstract

The invention provides a TtdAgo mutant protein with normal-temperature target nucleic acid cleavage activity and application thereof, and belongs to the technical field of programmable nuclease, and the TtdAgo mutant protein is mutated on the 527 site and / or the 561 site and / or the 593 site and / or the 599 site amino acid relative to a wild type TtdAgo protein. Compared with a wild type TtdAgo protein, the TtdAgo mutant protein provided by the invention not only has obviously improved activity at normal temperature, but also can cut target RNA (Ribonucleic Acid), so that the application range of the pAgo protein is effectively expanded.

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 use small DNA or RNA guide nucleic acids (gDNA or gRNA) to cut complementary target nucleic acids (target DNA and / or target RNA). Unlike the widely used Cas nucleases, the catalytic activity of Ago nucleases does not depend on specific motifs (such as PAM and PFS) in the target molecule, and can use DNA or RNA as guide nucleic acids, providing potential for the development of various Ago-based biotechnologies.

[0003] TtdAgo is a genus derived from thermophilic archaea Thermococcus thioreducens TtdAgo is a prokaryotic Ago protein that can cut 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, the amino acid mutation at position 527 refers to the mutation of histidine to any other amino acid except histidine, the amino acid mutation at position 561 refers to the mutation of tyrosine to any other amino acid except tyrosine, the amino acid mutation at position 593 refers to the mutation of lysine to any other amino acid except lysine, and the amino acid mutation at position 599 refers to the mutation of glutamic acid to any other amino acid except 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: TtdAgo_H527G, in which position 527 of the mutant is mutated to glycine relative to the sequence shown in SEQ ID NO.1; TtdAgo_Y561G, relative to the sequence shown in SEQ ID NO.1, the 561st position of this mutant is mutated to glycine; TtdAgo_K593G, in which position 593 of the mutant is mutated to glycine relative to the sequence shown in SEQ ID NO.1; TtdAgo_E599G, relative to the sequence shown in SEQ ID NO.1, the 593rd position of this mutant is mutated to glycine; TtdAgo_H527G / Y561G / K593G / E599G, relative to the sequence shown in SEQ ID NO.1, the 527th, 561st, 593rd and 599th positions of this mutant are simultaneously mutated to glycine.

[0009] Among the above mutants, TtdAgo_H527G, TtdAgo_Y561G, TtdAgo_K593G and TtdAgo_E599G can all cleave target DNA under the guidance of gDNA, and have better activity at 37°C. Moreover, compared with single amino acid mutations, the combined mutant TtdAgo_H527G / Y561G / K593G / E599G has further improved activity at 37°C, and can not only cleave target DNA and RNA under the guidance of gDNA, but also cleave target RNA under the guidance of 5'P-gRNA.

[0010] In a second aspect, the present invention provides a biomaterial related to a TtdAgo mutant protein, comprising: (b1) a nucleic acid molecule encoding a TtdAgo mutant protein; (b2) An expression cassette, vector or transformant comprising the nucleic acid molecule of (b1).

[0011] For nucleic acid molecules, as long as the TtdAgo mutant protein can be expressed in the expression system, it is sufficient. 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.

[0012] For the vector, it can be a pET plasmid. For example, in one embodiment of the present invention, a nucleic acid molecule encoding a TtdAgo mutant protein is connected to pET-28a to obtain a recombinant vector. For the transformant, it can be obtained by transforming a host cell with a recombinant vector. The host cell can be a conventional host cell in the art, but it must be able to satisfy the stable self-replication of the recombinant vector, and the nucleic acid molecule encoding the TtdAgo mutant protein carried can be effectively expressed.

[0013] In a third aspect, the present invention provides a nucleic acid cleavage system, which at least comprises: Guide nucleic acid; and TtdAgo mutant proteins.

[0014] Preferably, the length of the guide nucleic acid is 11-25 nt, wherein the length is more preferably 13-19 nt, and the best length is 15 nt.

[0015] Preferably, the guide nucleic acid is selected from 5'P-gRNA, 5'P-gDNA or 5'OH-gDNA.

[0016] In a fourth aspect, the present invention provides an application of a TtdAgo mutant protein or a nucleic acid cleavage system in the specific cleavage of a target nucleic acid. Specifically, 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.

[0017] In the above applications, the target nucleic acid includes target RNA and / or target DNA. The target RNA has no higher-order structure, or has a higher-order structure, or is double-stranded RNA, or is RNA transcribed in vitro, or is viral genomic RNA, or is mRNA, or other RNA in the cell. The target DNA is single-stranded DNA or double-stranded DNA.

[0018] In the above applications, complementary pairing specifically refers to the following two situations: The target nucleic acid has a nucleotide sequence that is completely complementary to the guide nucleic acid sequence; or, The target nucleic acid has a nucleotide sequence that has a 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 may be continuous.

[0019] Preferably, in the above application, the divalent metal cation is Mn 2+ and / or Mg 2+ , where Mn 2+ Better.

[0020] 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.

[0021] 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.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) Compared with the wild-type TtdAgo, which can only effectively cut the target DNA using the 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.

[0023] (2) Compared with other TtdAgo mutant proteins, TtdAgo_H527G / Y561G / K593G / E599G can not only effectively cut target DNA using guide DNA at 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.

[0024] (3) The present invention provides nucleic acid expression vectors encoding TtdAgo mutant proteins, especially 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 a foundation for the application of various TtdAgo mutant proteins in many fields of biotechnology, such as molecular diagnosis, molecular cloning, gene editing, etc.

[0025] (4) The mutant TtdAgo protein can cut the target DNA using the 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 the target RNA using the guide DNA at 37°C, laying the foundation for its in vivo RNA targeting; TtdAgo_H527G / Y561G / K593G / E599G can also cut the 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.

[0026] (5) Each TtdAgo mutant protein strictly depends on the complementary pairing of the guide and the target to exert its cleavage activity. There is no non-specific "incidental cleavage" activity of CRISPR-related proteins, and the specificity is better. In addition, the pAgo complex formed by the TtdAgo mutant protein and the guide nucleic acid does not rely on special motifs near the target site to recognize and bind to the target. The guide nucleic acid is easy to design without considering site restrictions. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 This is a diagram showing the results of SDS-PAGE identification and analysis of the TtdAgo protein and some of its mutant proteins in Example 1; Figure 2 Comparison of the cleavage activities of TtdAgo and its mutants at 37°C in Example 2; Figure 3 This is a urea / polyacrylamide gel detection result of the products obtained by cleaving single-stranded target DNA or target RNA using four guide nucleic acids in TtdAgo_H527G / Y561G / K593G / E599G in Example 2; Figure 4 This is a urea / polyacrylamide gel detection result of the products obtained by cleaving single-stranded target DNA or target RNA by TtdAgo_H527G / Y561G / K593G / E599G under different temperature conditions in Example 3; Figure 5 This is a urea / polyacrylamide gel detection result of the product obtained by cleaving single-stranded target DNA or target RNA by TtdAgo_H527G / Y561G / K593G / E599G under different metal cation conditions in Example 4; Figure 6 This is a urea / polyacrylamide gel detection 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 of different lengths in Example 5; Figure 7 This is a urea / polyacrylamide gel detection result of the product obtained by cleaving the 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; Figure 8 This is a graph showing the urea / polyacrylamide gel detection results of the products obtained by TtdAgo_H527G / Y561G / K593G / E599G cutting single-stranded target DNA or target RNA under different single-base mismatch conditions in Example 7. DETAILED DESCRIPTION

[0029] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In the present specification and claims, the terms "including" and "having" and any of their variations 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 may represent: A exists alone, A and B exist at the same time, and B exists alone.

[0031] TtdAgo is a genus derived from thermophilic archaea Thermococcus thioreducens The prokaryotic Ago protein, previous studies have shown that it only has the activity of cutting target DNA under the guidance of guide DNA, and although it has activity in a wide temperature range, its activity is extremely low at medium and low temperatures. In order to solve the limitations of the catalytic activity temperature and catalytic substrate of wild-type TtdAgo, the present invention provides a modification strategy.

[0032] The inventors analyzed the structure of the ternary complex of TtdAgo binding to the guide nucleic acid and the target nucleic acid, and found that the addition of the target induced the formation of its dimerization, and further analyzed its catalytic mechanism through structural analysis and biochemical experiments. Like most Ago proteins, TtdAgo adopts a two-leaf architecture, with the PAZ lobe (N-terminal domain, linker L1 and PAZ domain) connected to the PIWI lobe (MID and PIWI domain) through the linker L2, where the MID and PAZ domains anchor the 5' and 3' ends of the guide chain respectively; the active center in the PIWI domain is structurally similar to the active center of RNase H, and the DEDH catalytic quadruplex gives TtdAgo nuclease endonuclease activity. In general, enzymes from organisms in different environments maintain similar catalytic rates at their respective physiological temperatures, and sequence changes associated with these adaptive differences are usually located in surface-exposed areas away from the substrate binding site, leaving the enzyme's active site structurally undisturbed.

[0033] Based on the above research, the present invention successfully screened TtdAgo mutant proteins that have good activity under medium and low temperature conditions by mutating the amino acids in the PIWI domain of the wild-type TtdAgo protein that are 20Å away from the active site and the binding substrate. Among them, the active site specifically refers to the DEDH catalytic quadruplex of the wild-type TtdAgo protein.

[0034] Specifically, the TtdAgo mutant protein provided by the present invention having 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 the wild-type TtdAgo (amino acid sequence as 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 the 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 by an allosteric mechanism.

[0035] Compared with the wild-type TtdAgo protein, the TtdAgo mutant protein provided by the present invention has better target nucleic acid cleavage activity at both medium and low temperatures, and can cleave target DNA and RNA, thereby expanding the scope of application of the TtdAgo protein and making it potentially applicable to intracellular RNA and genome editing.

[0036] 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. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0037] Example 1 This example provides a method for screening and preparing TtdAgo mutant proteins, comprising the following steps: (1) Screening of mutation sites of TtdAgo based on structure.

[0038] 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 is located in the PIWI domain; third, the amino acid is mutated to glycine.

[0039] A gene fragment encoding TtdAgo as shown in SEQ ID NO. 1 was synthesized and ligated to pET28a using conventional methods to obtain a pET28a-TtdAgo plasmid.

[0040] TtdAgo mutants were constructed based on the screened mutation sites, including: TtdAgo_H527G, in which the histidine at position 527 was mutated to glycine, TtdAgo_K528G, in which the lysine at position 528 was mutated to glycine, TtdAgo_Q559G, in which the glutamine at position 559 was mutated to glycine, TtdAgo_Y561G, in which the tyrosine at position 561 was mutated to glycine, TtdAgo_K593G, in which the lysine at position 593 was mutated to glycine, TtdAgo_D597G, in which the aspartic acid at position 597 was mutated to glycine, TtdAgo_E599G, in which the glutamate at position 599 was mutated to glycine, TtdAgo_V625G, in which the valine at position 625 was mutated to glycine, and TtdAgo_D684G, in which the asparagine at position 684 was mutated to glycine.

[0041] Further, TtdAgo was subjected to combined mutations, 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.

[0042] Primers were designed for different sites, PCR was performed using pET28a-TtdAgo plasmid as a template, and each mutant plasmid was obtained by T5 recombination cloning.

[0043] (2) Expression and purification of TtdAgo and its mutants.

[0044] E. coli was used and IPTG was used to induce protein expression. The protein was purified by Ni column affinity chromatography and heparin affinity chromatography, and the purification results were analyzed by SDS-PAGE. The eluted target protein with higher purity was selected and concentrated by ultrafiltration using Amicon 50K ultrafiltration tube. The concentrated protein was stored in aliquots, quickly frozen with liquid nitrogen, and stored in a -80℃ refrigerator.

[0045] 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%.

[0046] Example 2 This example tests the cleavage activity of various TtdAgo mutants at room temperature, including the following: (1) Detection of TtdAgo and single amino acid mutants.

[0047] Activity was verified at 37°C and a cleavage experiment of the target DNA was performed using 5'P-gDNA.

[0048] Guide nucleic acid at 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) were added to a PCR tube without enzymes and incubated at 37°C for 10 minutes. Then, 200 nM 5'-end FAM-labeled target DNA or RNA was added and reacted at 37°C for 30 minutes for target cleavage. The reaction was terminated by mixing the sample with 2× RNA loading dye (95% formamide, 18 mM EDTA and 0.025% SDS and 0.025% bromophenol blue) and heating at 95°C for 5 minutes. The cleavage products were detected by 20% denaturing polyacrylamide gel electrophoresis and the results were visualized using a gel imaging system. The cleavage ratio was analyzed in triplicate using ImageJ and Prism 8 (GraphPad) software.

[0049] 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.

[0050] (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 cutting experiments on target DNA and RNA.

[0051] The types of guides and target nucleic acids that can be used for TtdAgo_H527G / Y561G / K593G / E599G are as follows: Figure 3 As shown, from Figure 3 It can be seen that TtdAgo_H527G / Y561G / K593G / E599G can cut complementary target DNA and RNA using 5'OH-gDNA and 5'P-gDNA at 37°C, and cut complementary target RNA using 5'P-gRNA.

[0052] The sequences of the guide nucleic acid and target nucleic acid used in this example are shown in Table 1.

[0053] Table 1 Sequence information of guide nucleic acid and target nucleic acid

[0054] Example 3 Taking TtdAgo_H527G / Y561G / K593G / E599G as an example, this example shows the effect of reaction temperature on the cleavage activity of TtdAgo mutant protein.

[0055] 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 cutting efficiency at 50~65℃.

[0056] Example 4 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.

[0057] The experimental process was referred to Example 2, but different from Example 2, the length of the guide nucleic acid was 18 nt, and the MnCl2 in the enzyme cleavage reaction buffer was replaced by FeCl2, CoCl2, NiCl2, CuCl2, ZnCl2, CaCl2 and MgCl2 of the same concentration to carry out 5'P-gDNA-guided target DNA and target RNA cleavage experiments.

[0058] The results were detected by 20% urea / polyacrylamide gel electrophoresis. Figure 5 The results show that for 5'P-gDNA-guided target DNA cleavage, TtdAgo_H527G / Y561G / K593G / E599G can use Mg 2+ and Mn 2+ As a divalent metal ion, and in the presence of Mn 2+ The activity is best in the digestion buffer; for 5'P-gDNA-guided target RNA cleavage, 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 was best in the digestion buffer. Therefore, the optimal divalent metal ion for TtdAgo_H527G / Y561G / K593G / E599G is Mn 2+ .

[0059] Example 5 Taking the mutant TtdAgo_H527G / Y561G / K593G / E599G as an example, this case explored the optimal length of the guide nucleic acid.

[0060] According to the guide nucleic acid sequence and target sequence in Example 2, gDNA with a length of 11 to 25 nt was synthesized; wherein the sequence of the 25 nt gDNA is shown in SEQ ID NO. 8, and the sequence of the 11 to 21 nt gDNA is respectively consistent with the first 11, 13, 15, 17, 18, 19, and 21 base sequences of the sequence. The 5' end sequences of these guide nucleic acids are the same, so the cleavage site and the size of the cleavage product should be the same.

[0061] 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 use 13~25nt 5'P-gDNA to cut the target DNA, and it showed the highest enzyme activity under the guidance of 15nt guide nucleic acid; TtdAgo_H527G / Y561G / K593G / E599G can cut the target RNA under the guidance of 11~25nt 5'P-gDNA, and it showed higher enzyme activity under the guidance of 15nt guide nucleic acid.

[0062] Example 6 Taking the mutant TtdAgo_H527G / Y561G / K593G / E599G as an example, this example explored the effect of the 5' terminal nucleotide of the guide nucleic acid on the enzyme cleavage activity of the mutant.

[0063] Referring to the guide nucleic acid sequences in Example 2, a series of 5' terminal phosphorylated DNA guide nucleic acids were designed and synthesized, which had different 5' terminal nucleotides (A, T, G, C) but the same other sequences. Correspondingly, a series of target DNA and RNA complementary to various guide nucleic acids were also synthesized, and the sequences of the corresponding target nucleic acids are shown in Table 2.

[0064] Table 2 Sequence information of different targets

[0065] Referring to Example 2, the activity of mutant TtdAgo_H527G / Y561G / K593G / E599G in cutting corresponding target DNA and RNA was tested under the guidance of 5'P-gDNA with different 5' terminal nucleotides, and detected by 20% urea / polyacrylamide gel electrophoresis, and the test results were as follows: Figure 7The 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'-end nucleotide was observed, and the cleavage activities mediated by guide nucleic acids containing 5'-A, 5'-T, 5'-G and 5'-C were comparable.

[0066] Example 7 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.

[0067] First, referring to the guide nucleic acid sequence and target sequence of Example 2, a series of gDNAs with single base differences at different sites from the original guide nucleic acid were designed and synthesized, and single base mismatches would occur between these guide nucleic acids and the target DNA or target RNA. Specifically, the sequences of guide nucleic acids with single base mismatches at different positions are shown in Table 3.

[0068] Table 3 Mismatch information of different guide nucleic acids

[0069] Then, the 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, and the cleavage products were detected using 20% ​​urea / polyacrylamide gel electrophoresis.

[0070] Test results such as Figure 8 As shown, the results show that: when the target DNA is cut under the guidance of 5'P-gDNA, the single base mismatch at the 7th site of the seed region of the guide nucleic acid will reduce the enzyme cleavage activity of TtdAgo_H527G / Y561G / K593G / E599G. On the contrary, the single base mismatch at the 5' anchor region, central region and 3' supplementary region of the guide nucleic acid does not significantly reduce its cleavage activity, and the single base mismatch at the 4th site of the central region of the guide nucleic acid also improves the enzyme cleavage activity; when the target RNA is cut under the guidance of 5'P-gDNA, the single base mismatch at the 6th and 7th sites of the seed region of the guide nucleic acid will reduce the enzyme cleavage activity of TtdAgo_H527G / Y561G / K593G / E599G. On the contrary, the single base mismatch at the 5' anchor region, central region and 3' supplementary region of the guide nucleic acid does not significantly reduce its cleavage activity. On the contrary, the mismatch at some sites also improves the cleavage activity.

[0071] 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 the target RNA, effectively expanding the scope of use of the pAgo protein; the present invention also explores the influence 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 the TtdAgo mutant protein in many fields of biotechnology such as molecular diagnosis, molecular cloning, RNA and genome editing.

[0072] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect 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 main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining 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 has amino acid mutations at positions 527 and / or 561 and / or 593 and / or 599 relative to the sequence shown in SEQ ID NO.

1.

2. The TtdAgo mutant protein according to claim 1, characterized in that The TtdAgo mutant is selected from TtdAgo_H527G / Y561G / K593G / E599G, TtdAgo_H527G, TtdAgo_Y561G, TtdAgo_K593G and TtdAgo_E599G.

3. A nucleic acid molecule encoding the gene of claim 1 or 2.

4. An expression cassette, vector or transformant containing the nucleic acid molecule of claim 3.

5. A nucleic acid cleavage system, characterized in that: The nucleic acid cleavage system comprises: Guide nucleic acid; The TtdAgo mutant protein as described in claim 1.

6. The nucleic acid cleavage system according to claim 5, characterized in that The guide nucleic acid has a length of 11 to 25 nt, specifically 5'P-gRNA, 5'P-gDNA or 5'OH-gDNA.

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

8. The use according to claim 7, 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.

9. The use according to claim 8, characterized in that: The divalent metal cation is Mn 2+ and / or Mg 2+ .

10. A kit, characterized in that: Comprising the TtdAgo mutant protein according to claim 1 or 2.

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