A highly active transposase and its application

Abstract

The present invention belongs to the fields of molecular biology and biomedicine, and particularly relates to a highly active transposase and its applications. The amino acid sequence of the highly active transposase is shown as SEQ ID NO:2, and the coding nucleotide sequence of this amino acid sequence is shown as SEQ ID NO:3. The highly active transposase based on this amino acid sequence can significantly improve the gene transfer activity of transposons when used in a transposition system. The highly active transposase and the nucleotide sequence encoding this highly active enzyme can be used to construct a gene transfer system, and to prepare or serve as drugs, preparations or tools for genomic research, gene therapy, cell therapy, or the induction and / or differentiation of pluripotent stem cells, etc.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and biomedicine, and particularly relates to a highly active transposase and its applications. Background Art

[0002] DNA transposons are mobile DNA sequences that can transpose from one location in the genome to another through a series of processes such as cleavage and reintegration. The PiggyBac (PB) transposon is a DNA transposon isolated from the Trichoplusia ni TN368 cell line, which can specifically insert into the "TTAA" target site. With the help of transposase, the PB transposon can precisely excise the target gene from the host without causing chromosomal rearrangement in the host. The PB transposon has no potential viral genetic toxicity, can carry relatively long foreign gene fragments (up to 150 kb), and has strong remodeling ability. Transgenesis mediated by PB transposase has the characteristics of high integration efficiency, stable integration, long-term expression, single-copy integration, locatable insertion sites, and simple operation, and is often used in multiple fields such as transgenic mouse production, genetic manipulation of mouse embryonic stem cells, gene mutagenesis and other gene operations, and induction of pluripotent stem cells.

[0003] The transposition activity of PB transposase is the highest among existing mammalian DNA transposons and has broad application prospects. Many studies at home and abroad have used the PB transposon system as a gene editing method for transgenesis and gene mutation in various organisms, including insect cells, protists, plants, and vertebrates. In 2003, Tomita fused human type III collagen with enhanced green fluorescent protein EGFP and used the PB transposon to integrate it into the silkworm silk protein gene, obtaining transgenic silkworms that could stably express human collagen. In 2005, Balu inserted human dihydrofolate reductase (hDHFR) into the Plasmodium genome through the PB transposon system. In 2014, Eric T obtained a stable transgenic line in which the PB transposon could perform in vivo transposition. In 2005, Sheng Ding efficiently introduced foreign gene fragments into human cells and mouse cell lines cultured in vitro through the PB transposon and made them stably express, and cultivated transgenic fluorescent mice with stable traits, demonstrating the possibility that the PB transposon system can be used as an effective operation tool for studying the gene functions of other vertebrates.

[0004] The DNA transposon system consists of two parts: a transposon with inverted repeat sequences (IRs) at both ends that can carry the target DNA fragment, and a transposase that can catalyze the "cut and paste" of the transposon. The transposase first binds to the IRs sequences on both sides of the transposon, then precisely and seamlessly removes the transposon from the host DNA site, and finally integrates the DNA fragment into a new site. The establishment of an efficient transposon system can achieve targeted knockout of target genes or targeted introduction of target genes, providing an effective vector tool for gene editing in mammalian cells. The transposition efficiency of the transposon system determines the efficiency of gene editing, and a large part of the transposition efficiency depends on the expression level of the transposase. Therefore, increasing the transposase activity is the key technical point for increasing the transposition efficiency of the transposon.

[0005] The transposition activity of the transposase is affected by factors such as binding sites, active sites, and structure. At present, the crystal structure of the transposase has not been clearly resolved, but some domains are considered important structures, and experiments have shown that the activity of the transposase can be affected by any non-specific amino acid.

[0006] A hyperactive piggyBac transposase for mammalian applications (PNAS|January 25,2011|vol.108|no.4|1531–1536) discloses a hyperactive piggyBac transposase (hereinafter referred to as the existing hyperactive transposase hyPBase, as shown in SEQ ID NO:1) with a transposition efficiency 10 times that of mPBase (wild-type piggyBac transposase optimized for mammalian codons) and with the following site amino acid mutations: I30V, G165S, S103P, M282V, S509G, N570S, and N538K.

[0007] PiggyBac transposon variants and methods of use (US9670503B2) and PiggyBac transposon variants and their use methods (CN102421902A) are re-applications based on the priority of US Provisional Application No. 61 / 155206. Both disclose that on the basis of the integration-deficient PiggyBac mutant, mutations are continued to select mutants with higher integrase activity than the integration-deficient PiggyBac mutant, and on the basis of the wild-type PiggyBac normal body, mutations are selected to obtain mutants with higher integration activity than the wild-type PiggyBac normal body.

[0008] Although the enzyme activity of existing PiggyBac transposase mutants has been improved relative to wild-type PiggyBac transposase, it still cannot meet the higher and more stringent requirements for enzyme activity. Therefore, it is still necessary to study PiggyBac transposase with high enzyme activity. Summary of the Invention

[0009] The present invention provides a new highly active transposase, which exhibits extremely high transposition activity in cells such as Escherichia coli, insect cells, yeast cells, and mammalian cells. Compared with the existing highly active transposase hyPBase, it has a broad spectrum of applicable host cells, and also has high transposition activity in mammalian cells, especially high transposition activity in human cells, providing new clues and basis for the exploration of transposase, especially the exploration of transposase in human cells.

[0010] The present invention also provides the amino acid sequences and peptide segments that are the basis of the new highly active transposase of the present invention, as well as the nucleotide sequences encoding the amino acid sequences, peptide segments, and proteins of the highly active transposase of the present invention, and nucleic acids, nucleic acid constructs, recombinant vectors, and host cells based on the nucleotide sequences, and gene transfer systems and applications based on the above peptide segments, proteins, nucleic acids, nucleic acid constructs, recombinant vectors, and host cell components.

[0011] In the present invention, the isoleucine at position 92, valine at position 119, and glutamine at position 601 in the amino acid sequence of the existing highly active transposase hyPBase (shown in SEQ ID NO: 1) are simultaneously mutated to asparagine, alanine, and arginine respectively, resulting in a target mutant amino acid sequence, as shown in SEQ ID NO: 2. In CHO cells, compared with the transposition efficiency (30.9%) of the existing highly active transposase hyPBase after codon optimization and addition of a nuclear localization signal system, the transposition efficiency (51.7%) of the target highly active enzyme bz-hyPBase generated based on the amino acid sequence of SEQ ID NO: 2 is increased by nearly 21%; in PBMC cells, compared with the transposition efficiency (9.81%) of the existing highly active transposase hyPBase after codon optimization and addition of a nuclear localization signal system, the transposition efficiency (19.4%) of the target highly active bz-hyPBase enzyme generated based on the amino acid sequence of SEQ ID NO: 2 is increased by nearly 10%. This shows that the target highly active enzyme based on the mutant amino acid sequence of the present invention exhibits more excellent transposition activity compared with the existing highly active transposase hyPBase, especially high transposition activity in mammalian cells and human cells. Therefore, the present invention provides a new highly active transposase, which contains one or more amino acid sequences shown in SEQ ID NO: 2, and this highly active transposase exhibits extremely high transposition activity in Escherichia coli, insect cells, yeast cells, and mammalian cells, especially meeting the high transposition activity requirements of mammalian and human cells.

[0012] Amino acid sequence of hyPBase (SEQ ID NO: 1):

[0013] MGPAAKRVKLDGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNR I LTLPQRTIRGKNKHCWSTSKPTRRSR VSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMC Q SCF*

[0014] Target mutant amino acid sequence (SEQ ID NO:2):

[0015] MGPAAKRVKLDGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNR N LTLPQRTIRGKNKHCWSTSKPTRRSR ASALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMC R SCF*

[0016] The mutant amino acid sequences obtained by performing the above-mentioned amino acid mutations on positions 92, 119, and 601 of the amino acid sequence of the existing highly active transposase hyPBase (shown in SEQ ID NO: 1) individually or at any two positions, and the enzyme formed based on one or more of these mutant amino acid sequences also has the same or similar transposition efficiency as the target highly active transposase bz-hyPBase described in the embodiments of the present invention or the existing hyPBase. It also belongs to the mutant amino acid sequences of the new highly active transposase to be protected by the present invention. The enzyme formed based on this mutant amino acid sequence also belongs to the new highly active transposase to be protected by the present invention.

[0017] As described above, the mutant amino acid sequences obtained by performing the above amino acid mutations on positions 92, 119, and 601 of the amino acid sequence of the existing highly active transposase hyPBase (shown in SEQ ID NO: 1) individually, at any two positions, or at all three positions, and then performing one or more amino acid deletions, substitutions, insertions, or additions to obtain amino acid sequences that still maintain or improve enzyme activity also belong to the alternative solutions with the same or similar technical effects of the technical solution of the present invention and are within the protection scope of the present invention. Similarly, the mutant amino acid sequences of the new highly active transposase to be protected by the present invention, and the enzymes formed based on one or more of these mutant amino acid sequences also belong to the new highly active transposase to be protected by the present invention.

[0018] As described above, the mutant amino acid sequences obtained by performing the above amino acid mutations on positions 92, 119, and 601 of the amino acid sequence of the existing highly active transposase hyPBase (shown in SEQ ID NO: 1) individually, at any two positions, or at all three positions, also contain the amino acid sequences of functional proteins. Adding functional proteins to the new highly active transposase can improve or increase the functions of the new highly active transposase, such as the amino acid sequences of nuclear localization signals, the amino acid sequences expressing EGFP green fluorescent protein, the amino acid sequences of tag proteins, or the amino acid sequences of antibodies. These functional proteins can improve the transposition activity of the new highly active transposase. For example, nuclear localization signals can assist in improving the transposition activity of transposases; or can enhance the transposition monitoring function of highly active transposases, such as EGFP green fluorescent protein or tag proteins facilitating the qualitative and / or quantitative monitoring of the transposition activity of transposases; or can add new functions to the new highly active transposase, such as antibodies adding additional immune activity.

[0019] The present invention also protects the mutant amino acid sequences obtained by performing the above amino acid mutations on positions 92, 119, and 601 of the amino acid sequence of the existing highly active transposase hyPBase (shown in SEQ ID NO: 1) individually, at any two positions, or at all three positions, and the derived amino acid sequences that still maintain or improve enzyme activity obtained by performing one or more amino acid deletions, substitutions, insertions, or additions on the basis of the mutant amino acid sequences. The chain compounds formed by peptide bonds through amino acid dehydration condensation, namely peptide segments. The number of the above mutant amino acids or the above derived amino acid sequences contained in the peptide segment can be one or more. The peptide segment is also connected to the peptide segments of functional proteins formed by peptide bonds through amino acid dehydration condensation of the amino acid sequences of functional proteins, such as the peptide segments of nuclear localization signals, the peptide segments expressing EGFP green fluorescent protein, the peptide segments of tag proteins, or the peptide segments of antibodies.

[0020] The present invention relates to a new highly active transposase, which is based on the mutant amino acid sequences obtained by performing amino acid mutations at positions 92, 119, and 601 of the amino acid sequence of the existing highly active transposase hyPBase (shown in SEQ ID NO: 1) alone, any two positions, or all three positions as described above, peptide segments formed based on the mutant amino acid sequences, and derivative amino acid sequences obtained by performing one or more amino acid deletions, substitutions, insertions, or additions on the basis of the mutant amino acid sequences and still maintaining or enhancing enzyme activity, and peptide segments formed based on the derivative amino acid sequences. The number of the above-mentioned mutant amino acid sequences, derivative amino acid sequences, and peptide segments formed based on the mutant amino acid sequences and derivative amino acid sequences is one or more.

[0021] The mutant nucleotide sequences encoding the new highly active transposase, peptide segments, and their amino acid sequences of the present invention mentioned above, nucleotide sequences complementary to, hybridizing with, or overlapping with the mutant nucleotide sequences, or nucleotide sequences obtained by performing base substitutions, deletions, or additions on the mutant nucleotide sequences and having the nucleotide sequences encoding the new highly active transposase, or nucleotide sequences having at least 80% homology with the mutant nucleotide sequences, preferably at least 90% homology with the mutant nucleotide sequences, and most preferably at least 96% homology with the mutant nucleotide sequences, all belong to the mutant nucleotide sequences encoding the new highly active transposase, peptide segments, and their amino acid sequences of the present invention to be protected, and the number thereof can be one or multiple repeated copies. Specifically as follows:

[0022] The nucleotide sequence encoding the amino acid sequence of the existing highly active enzyme hyPBase (shown in SEQ ID NO: 1) is optimized with human codons to obtain a human codon-optimized nucleotide sequence. Based on the human codon-optimized nucleotide sequence (SEQ ID NO: 4), base mutations are performed at the following positions: the base T at position 276 is mutated to base C, the base T at position 356 is mutated to base C, the base G at position 900 is mutated to base A, and the base A at position 1802 is mutated to base G; to obtain a mutant nucleotide sequence encoding the amino acid sequence of the new highly active transposase bz-hyPBase of the present invention (shown in SEQ ID NO: 2), as shown in SEQ ID NO: 3.

[0023] Nucleotide sequence of the existing highly active enzyme hyPBase with human codon optimization (SEQ ID NO: 4): atgggccctgctgccaagagggtcaagttggacggcagcagcctggacgacgagcacatcctgagcgccctgctgcagagcgacgacgagctggtgggcgaggacagcgacagcgaggtgagcgaccacgtgagcgaggacgacgtgcagagcgacaccgaggaggccttcatcgacgaggtgcacgaggtgcagcccaccagcagcggcagcgagatcctggacgagcagaacgtgatcgagcagcccggcagcagcctggccagcaaccgcaa t

[0024] Mutated nucleotide sequence (SEQ ID NO: 3):

[0025]

[0026] Alternatively, perform base substitution, deletion, or addition operations on the mutant nucleotide sequence (shown in SEQ ID NO: 3) and have a nucleotide sequence encoding a new highly active transposase bz-hyPBase;

[0027] Alternatively, according to the base complementary pairing principle, a nucleotide sequence complementary to the mutant nucleotide sequence (shown in SEQ ID NO: 3), and then perform base substitution, deletion, or addition operations on it and have a nucleotide sequence of a new highly active transposase bz-hyPBase;

[0028] Alternatively, a nucleotide sequence that overlaps with the mutant nucleotide sequence (shown in SEQ ID NO: 3) and has a nucleotide sequence encoding a new highly active transposase bz-hyPBase;

[0029] Alternatively, a nucleotide sequence that hybridizes with the mutant nucleotide sequence (shown in SEQ ID NO: 3) and has a nucleotide sequence encoding a new highly active transposase bz-hyPBase;

[0030] Alternatively, a nucleotide sequence that has more than 80% homology with the mutant nucleotide sequence (shown in SEQ ID NO: 3) and has a nucleotide sequence encoding a new highly active transposase bz-hyPBase; specifically, preferably a nucleotide sequence that has more than 90% homology with the mutant nucleotide sequence (shown in SEQ ID NO: 3) and has a nucleotide sequence encoding a new highly active transposase bz-hyPBase; more preferably a nucleotide sequence that has more than 96% homology with the mutant nucleotide sequence (shown in SEQ ID NO: 3) and has a nucleotide sequence encoding a new highly active transposase bz-hyPBase;

[0031] All belong to the mutant nucleotide sequences encoding the new highly active transposase bz-hyPBase, or its peptide segment, or its amino acid sequence to be protected by the present invention.

[0032] When a functional protein is further connected to the new highly active transposase of the present invention, the mutant nucleotide sequence encoding it also contains a nucleotide sequence encoding the functional protein, such as a nucleotide sequence encoding a nuclear localization signal, a nucleotide sequence expressing EGFP green fluorescent protein, a nucleotide sequence encoding a tag protein peptide segment, or a nucleotide sequence encoding an antibody, etc.

[0033] The present invention also provides a nucleic acid polymerized from the above mutant nucleotide sequences encoding the new highly active transposase, or its peptide segment, or its amino acid sequence of the present invention. When a functional protein is connected to the new highly active transposase of the present invention, this nucleic acid also contains a nucleotide sequence encoding a functional protein (nuclear localization signal, EGFP green fluorescent protein, tag protein, or antibody).

[0034] The present invention also provides a nucleic acid construct, which is operably linked to one or more regulatory sequences that direct the expression encoding of a target sequence in a host cell. The expression encoding includes any steps involved in the production of a protein or polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion. The nucleic acid construct further contains a mutant nucleotide sequence encoding the novel highly active transposase of the present invention, or a peptide segment thereof, or an amino acid sequence thereof, or a nucleic acid polymerized from the mutant nucleotide sequence.

[0035] The present invention also provides a recombinant vector, which contains a mutant nucleotide sequence encoding the novel highly active transposase of the present invention, or a peptide segment thereof, or an amino acid sequence thereof, or a nucleic acid polymerized from the mutant nucleotide sequence, or the above-mentioned nucleic acid construct. The recombinant vector includes a recombinant cloning vector, a recombinant eukaryotic expression vector, or a recombinant viral vector. The recombinant cloning vector includes pRS vector, T vector, pUC vector, etc. The recombinant eukaryotic expression vector includes pEGFP, pCMVp-NEO-BAN, pSV2, etc. The recombinant viral vector includes a recombinant adenovirus vector, a lentiviral vector, etc.

[0036] The present invention also provides a host cell, which contains a mutant nucleotide sequence encoding the novel highly active transposase of the present invention, or a peptide segment thereof, or an amino acid sequence thereof, or a nucleic acid polymerized from the mutant nucleotide sequence, or the above-mentioned nucleic acid construct, or the above-mentioned recombinant vector. The host cell includes Escherichia coli cells, insect cells, yeast cells, mammalian cells, etc.

[0037] The novel highly active transposase provided by the present invention for improving the transposition activity of a transposon system, or a peptide segment constituting the novel highly active transposase, or a nucleic acid construct encoding the novel highly active transposase, or a recombinant vector encoding the novel highly active transposase, or a host cell (such as Escherichia coli cells, insect cells, yeast cells, mammalian cells, etc.) containing the novel highly active transposase and / or a nucleic acid construct encoding the novel highly active transposase and / or a recombinant vector encoding the novel highly active transposase can stably and efficiently integrate a foreign gene into the genome of the host cell at a specific site, and achieve long-term and stable expression without affecting the stable expression of the original genes of the host. It can be used to construct a new gene transfer system, and can also be used to prepare or serve as a drug and / or preparation for genomic research, gene therapy, cell therapy, or induction and / or differentiation of pluripotent stem cells, and can also be used to prepare or serve as a tool for genomic research, gene therapy, cell therapy, or induction and / or differentiation of pluripotent stem cells.

[0038] A gene transfer system comprising the novel highly active transposase of the present invention, or a nucleic acid construct encoding the novel highly active transposase, or a recombinant vector encoding the novel highly active transposase, or a host cell containing the novel highly active transposase and / or a nucleic acid construct encoding the novel highly active transposase and / or a recombinant vector encoding the novel highly active transposase.

[0039] In this gene transfer system, it further contains a transposon gene, and the nucleic acid or nucleic acid construct encoding the novel highly active transposase is integrated with the transposon gene; or the nucleic acid or nucleic acid construct encoding the novel highly active transposase is independent of the transposon gene; or the nucleic acid or nucleic acid construct encoding the novel highly active transposase is located on the same recombinant vector as the transposon gene; or the nucleic acid or nucleic acid construct encoding the novel highly active transposase is located on different recombinant vectors from the transposon gene; or the transposon gene is integrated into the nucleic acid construct encoding the novel highly active transposase; or the transposon gene is integrated into the recombinant vector encoding the novel highly active transposase; or the transposon gene is independent of the recombinant vector encoding the novel highly active transposase; or the transposon gene is transferred into a host cell containing the novel highly active transposase and / or a nucleic acid construct encoding the novel highly active transposase and / or a recombinant vector encoding the novel highly active transposase; or the transposon gene is located outside the host cell containing the novel highly active transposase and / or a nucleic acid construct encoding the novel highly active transposase and / or a recombinant vector encoding the novel highly active transposase.

[0040] A drug and / or preparation for genomic research, gene therapy, cell therapy, or induction and / or differentiation of pluripotent stem cells, comprising the novel highly active transposase of the present invention, or a nucleic acid construct encoding the novel highly active transposase, or a recombinant vector encoding the novel highly active transposase, or a host cell containing the novel highly active transposase and / or a nucleic acid construct encoding the novel highly active transposase and / or a recombinant vector encoding the novel highly active transposase, or the above gene transfer system.

[0041] In the drug for genomic research, gene therapy, cell therapy, or induction and / or differentiation of pluripotent stem cells, it further contains pharmaceutically acceptable excipients, can be prepared into any pharmaceutically feasible dosage form, and can also be supplemented with adjuvant therapeutic components.

[0042] A tool for genomic research, gene therapy, cell therapy, or induction and / or differentiation of pluripotent stem cells, comprising the novel highly active transposase of the present invention, or a nucleic acid construct encoding the novel highly active transposase, or a recombinant vector encoding the novel highly active transposase, or a host cell containing the novel highly active transposase and / or a nucleic acid construct encoding the novel highly active transposase and / or a recombinant vector encoding the novel highly active transposase, or the above gene transfer system. Description of the Drawings

[0043] Figure 1 It is the vector map of PRS316-URA-PBase in step (3) of Example 1.

[0044] Figure 2 It is the schematic diagram of the process of multiple cumulative error-prone PCR mutations of the transposase in step (3) of Example 1 (upper figure), and the schematic diagram of the process of transforming the error-prone PCR-recovered transposase fragment and the linearized vector into the ura-deficient yeast strain according to a molar ratio of 10:1 (lower figure).

[0045] Figure 3 It is the schematic diagram of the mutant library and the process of screening for highly efficient transposase in step (3) of Example 1.

[0046] Figure 4 It is the plasmid map of PRS316-URA-PBase and the working principle diagram of the plasmid in Example 2 (A), the intuitive diagram of the transposition situation of WT PBase, hyPBase, optimized hyPBase, and bz-hyPBase in yeast (B), the statistical chart of the transposition situation of WT PBase, hyPBase, optimized hyPBase, and bz-hyPBase in yeast (C), and the statistical column histogram of the transposition situation of WT PBase, hyPBase, optimized hyPBase, and bz-hyPBase in yeast (D).

[0047] Figure 5 It is the schematic diagram of the structure of the ploxP-bz-HyPB plasmid in Example 3.

[0048] Figure 6 It is the schematic diagram of the structure of the pSAD-EGFP plasmid in Example 3.

[0049] Figure 7 It is the comparison chart of the efficiency of editing the CHO cell genome using the optimized hyPBase and bz-hyPBase transposases in Example 3. It can be seen that the transposition efficiency of bz-hyPBase in CHO cells is significantly increased.

[0050] Figure 8 It is the comparison chart of the efficiency of preparing CAR T cells using the optimized hyPBase and bz-hyPBase transposases in Example 4. It can be seen that the transposition efficiency of bz-hyPBase in multiple donors of PBMC is significantly increased. Detailed implementation manners

[0051] The present invention will be described more clearly in conjunction with the accompanying drawings of the specification and specific embodiments. The specific embodiments are only used to explain the present invention and are not limited thereby. The experimental method conditions in the embodiments are conventional experimental method conditions unless otherwise specified; reagents and the like are carried out according to the manufacturer's instructions without special instructions.

[0052] Example 1 Obtaining of highly active bz-hyPBase mutants

[0053] Based on the original sequence of the existing highly active piggybac transposase (abbreviated as hyPBase, amino acid sequence shown in SEQ ID NO: 1), we made the following changes to obtain the sequence information of the protected baize piggyBac transposase (abbreviated as bz-hyPBase):

[0054] (1) Based on the human codon usage preference, we optimized the codons of the existing highly active piggybac transposase to obtain the nucleotide sequence shown in SEQ ID NO: 4 to improve the expression level of the transposase;

[0055] (2) A human c-myc nuclear localization signal was added after the start codon to improve the integration efficiency of foreign genes in host cells;

[0056] (3) The nucleotide sequence shown in SEQ ID NO: 4 was randomly mutated by the following method to obtain a mutant with a transposition efficiency significantly better than that of the existing highly active piggybac transposase. We named it bz-hyPBase (amino acid sequence shown in SEQ ID NO: 2, nucleotide sequence shown in SEQ ID NO: 3). The specific steps are as follows:

[0057] a. Construction of the screening reporter vector

[0058] The resistance gene G418 was inserted between the 5’ IR and 3’ IR of the transposon element by gene synthesis to form the transposon G418-IR. The transposon was inserted into the TTAA site of the URA3 gene by PCR recombination, and the transposase with an inducible promoter was inserted into the multiple cloning sites of PRS316. Finally, the screening reporter vector PRS316-URA-PBase was constructed. The specific operations are as follows:

[0059] (1) Using primers pURA-F (SEQ ID NO: 5: aagccgctaaaggcattatccgcc) and pURA-R (SEQ ID NO: 6: aactgtgccctccatggaaaaatcagtc), PCR was performed on the template PRS316 to obtain the linearized fragment 1 of the plasmid PRS316.

[0060] (2) Using primers pURA-IR-F (SEQ ID NO:7) and pURA-IR-R (SEQ ID NO:8), perform PCR on the synthesized transposon G418-IR to obtain linearized fragment 2 of the transposon with a homologous sequence to PRS316.

[0061] pURA-IR-F (SEQ ID NO:7):

[0062] gactgatttttccatggagggcacagttaaccctagaaagatagtctgcgtaaaattgacgcatgcgac

[0063] pURA-IR-R (SEQ ID NO:8):

[0064] ggcggataatgcctttagcggcttaaccctagaaagataatcatattgtg

[0065] (3) Ligate fragment 1 and fragment 2 using NEBuilder homologous recombination enzyme to construct plasmid PRS316-URA.

[0066] (4) Synthesize the GALS-inducible promoter gene PB transposase, and clone it into vector PRS316-URA using SacI and EcoRI, finally generating plasmid PRS316-URA-PBase. The map of the PRS316-URA-PBase vector is as Figure 1 shown.

[0067] b. Construction of mutant library

[0068] Design PCR primers outside the open reading frame (ORF) of the transposase: GR-F (SEQ ID NO:9: taatcagcgaagcgatga) and GR-R (SEQ ID NO:10: cagcatgcctgctattgtcttcc). There are about 50 bp of homologous sequences at both ends of the transposase ORF on the PRS-URA-PBase vector. Use the error-prone PCR kit of clonth to mutate the transposase, and the number of mutations can be accumulated by recycling the PCR fragment as a template multiple times ( Figure 2 as shown in the above flow chart), and finally obtain the transposase fragment with point mutations. Screen the reporter vector PRS316-URA-PBase and linearize it using XbaI and EcoRI, and remove the original unmutated transposase. Transform the PCR-recovered transposase fragment and the linearized vector into the ura-deficient yeast strain according to a molar ratio of 10:1 ( Figure 2The following flow chart and Figure 3 as shown, the yeast will utilize its own homologous recombination repair mechanism to enable the exogenous target fragment to be replaced into the DNA plasmid with a nick through the homologous arms, thereby automatically assembling into a complete plasmid with the target fragment in the yeast cell. Through this method, one-step cloning of DNA fragments into yeast strains can be achieved, and at the same time, the high-frequency repetition phenomenon of mutants during the process of constructing plasmids in Escherichia coli and then transferring them into yeast is reduced. Through this method, the clones obtained on the plate after transformation are mutants, and a certain number of mutant libraries can be obtained by picking single clones.

[0069] c. Screening process for highly efficient transposase

[0070] As Figure 3 shown, the screening process is divided into two screenings. The first screening conducts a large-scale screening of all mutants to screen out mutants with significantly higher transposition efficiency than the unmutated control group. The second screening is carried out among the yeasts obtained from the first screening. By calculating the exact transposition efficiency, mutants with increased transposition efficiency in yeast are obtained, namely bz-hyPBase (amino acid sequence of SEQ ID NO: 2, nucleotide sequence of SEQ ID NO: 3).

[0071] First screening: Pick single clones from the transformed mutant library into 96-well plates and YPD medium containing G418 antibiotic for activation. After 24 hours of activation, use a replicator to transfer them and inoculate them into YPD medium containing 2% galactose for induction. After 24 hours of induction, dilute the bacterial solution to 10-2 or 10-3 (determined according to the growth of yeast), take 10 μl and spot it onto ura-deficient solid medium. After culturing for 48 hours, observe the growth of mutants and compare them with the unmutated clones. Screen out the clones with significantly increased transposition efficiency for the second screening.

[0072] Second screening: Activate the suspected mutants obtained from the first screening for 24 hours. After activation, adjust the OD600 values to be the same. Inoculate them into YPD medium containing 2% galactose at a ratio of 1:100 and induce for 24 hours. After induction, adjust the OD600 values to be the same again, and dilute them serially to 10-2, 10-3, 10-4. Take 20 μl of the dilutions to 10-2 and 10-3 and spread them on ura-deficient solid medium for culturing for 24 hours. Count the number of colonies. The colonies growing on the ura-deficient solid medium are the clones that have undergone transposition. At the same time, take 20 μl of the dilutions to 10-3 and 10-4 and spread them on YPD complete solid medium for a control in parallel. The colonies growing are the total number of yeasts. Transposase transposition efficiency = the number of clones that have undergone transposition / the total number of clones = (the number of colonies in the ura-deficient medium * dilution factor) / (the number of colonies in the YPD medium * dilution factor) * 100%. By this method, high-throughput screening can be achieved. One single-person operation can achieve a throughput screening of 96 - 960 mutants, greatly increasing the probability of obtaining highly active transposase.

[0073] Through the above calculation, we can obtain the accurate transposition efficiency of the mutants. We will select the strains with increased transposition efficiency for mutation site analysis. Inoculate the yeasts in the initially activated 96-well plate for scale-up culture, extract the yeast plasmids, and send them to the company for sequencing analysis. By comparing with the original sequence, the mutation sites of the mutants can be obtained.

[0074] The amino acid sequence of hyPBase (SEQ ID NO:1):

[0075] MGPAAKRVKLDGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNR I LTLPQRTIRGKNKHCWSTSKPTRRSR VSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMC Q SCF*

[0076] Amino acid sequence of bz - hyPBase (SEQ ID NO:2):

[0077] MGPAAKRVKLDGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNR N LTLPQRTIRGKNKHCWSTSKPTRRSR ASALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMC R SCF*

[0078] The isoleucine at position 92, valine at position 119, and glutamine at position 601 in the amino acid sequence of the existing highly active transposase hyPBase (shown in SEQ ID NO: 1) are mutated to asparagine, alanine, and arginine respectively, resulting in the amino acid sequence of bz-hyPBase shown in SEQ ID NO: 2.

[0079] The nucleotide sequence of human codon-optimized hyPBase (SEQ ID NO: 4):

[0080]

[0081] Nucleotide sequence of bz-hyPBase (SEQ ID NO: 3):

[0082]

[0083] The nucleotide sequence of the existing highly active enzyme hyPBase is optimized with human codons to obtain a human-codon-optimized nucleotide sequence. Based on the human-codon-optimized nucleotide sequence (SEQ ID NO: 4), base mutations are made at the following sites: the base T at position 276 is mutated to base C, the base T at position 356 is mutated to base C, the base G at position 900 is mutated to base A, and the base A at position 1802 is mutated to base G; thus, a mutated nucleotide sequence encoding the novel highly active transposase bz-hyPBase of the present invention as shown in SEQ ID NO: 3 is obtained.

[0084] Example 2 bz-hyPBase has a higher transposition efficiency in yeast

[0085] We inserted a transposon carrying the G418 resistance gene into the URA3 gene on the yeast plasmid PRS316 to disrupt the expression of the URA gene, and cloned the transposase with an inducible promoter into PRS316 at the same time to generate the plasmid PRS316-URA-Pbase. Plasmids carrying different transposases WT PBase, hyPBase, optimized hyPBase, and bz-hyPBase were prepared in parallel. This plasmid was transformed into the ura-deficient Saccharomyces cerevisiae BJ2168, which cannot survive in the ura-deficient medium. The transposase is expressed under the regulation of the inducer galactose, promoting the transposition of the transposon. When the transposon transposes, the URA gene is normally expressed, and the transposed clones resume normal growth in the ura-deficient medium. By counting the number of transposed clones in a certain number of yeasts, the transposition efficiency of the transposase in Saccharomyces cerevisiae can be calculated. We compared the transposition efficiencies of the wild-type piggybac transposase WT PBase, the existing highly active piggybac transposase hyPBase, the transposase optimized hyPBase with codon optimization and addition of a nuclear localization signal, and bz-hyPBase by this method. Figure 4 The experimental results show that the transposition efficiency of bz-hyPBase is 3 times that of hyPBase, proving that bz-hyPBase has a higher transposition efficiency in yeast.

[0086] WT PBase is a plasmid carrying a piggybac transposase optimized with mammalian codons, hyPBase is a plasmid carrying an existing highly active piggybac transposase (obtained by mutating 7 amino acid sites of the WTPBase described in the background art), optimized hyPBase is a plasmid carrying a transposase obtained by optimizing the existing highly active piggybac transposase with human codons and adding a nuclear localization signal system, and bz-hyPBase is a plasmid carrying a new highly active transposase screened in the present invention (i.e., the transposase obtained by mutating three amino acid sites described in the examples of the present invention on optimized hyPBase).

[0087] Example 3: bz-hyPBase has a higher gene editing efficiency in CHO cells

[0088] We cloned optimized hyPBase and bz-hyPBase into a mammalian cell expression vector to generate plasmids ploxP-optimized hyPBase (with the same structure as Figure 5 , only replacing the transposase in Figure 5 with optimized hyPBase instead of bz-hyPBase) and ploxP-bz-HyPB ( Figure 5 ), to express the transposase. A human c-myc nuclear localization signal is connected behind the promoters of optimized hyPBase and bz-hyPBase. The transposon carrying the EGFP gene was cloned into the vector pSAD-EGFP ( Figure 6 ) to express green fluorescent protein. The two plasmids expressing the transposase and the transposon were co-electroporated into CHO cells. The transposon carrying EGFP will be inserted into the genome under the action of the transposase to stably express green fluorescent protein. After two passages of culture, on the 7th and 14th days, flow cytometry was used to count the cells expressing green fluorescent protein. The more cells that can express fluorescent protein, the higher the transposition efficiency of the transposase. From the Figure 7 statistical results, the transposition activity of bz-hyPBase is significantly better than that of hyPBase.

[0089] Example 4: bz-hyPBase has a higher gene editing efficiency in T cells

[0090] We co-electroporated the ploxP-optimized hyPBase and ploxP-bz-HyPB plasmids in Example 3 into peripheral blood mononuclear cells (PBMCs) for T cell genome editing preparation. The transposon carrying the EGFP green fluorescent protein gene edits the T cell genome under the action of the transposase, and the editing efficiency of T cells can reflect the strength of the transposase activity. We used PBMCs from 3 different healthy donors for multiple groups of experiments. On the 5th day, flow cytometry was used to detect the gene editing efficiency. The higher the EGFP positive rate, the higher the transposase activity. The experimental results are as Figure 8 shown that in PBMCs from different donors, the transposition activity of bz-hyPBase is superior to that of optimized hyPBase.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that several improvements can be made without departing from the principle of the present invention, and these improvements should also be regarded as the protection scope of the present invention. Sequence Listing <110> Shanghai Institute of Cellular Therapy Shanghai Cellular Therapy Group Co., Ltd. <120> A Highly Active Transposase and Its Application <130> 199908Z1 <150> CN 201911227263.5 <151> 2019-12-04 <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 604 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Ser Ser Leu Asp 1 5 10 15 Asp Glu His Ile Leu Ser Ala Leu Leu Gln Ser Asp Asp Glu Leu Val 20 25 30 Gly Glu Asp Ser Asp Ser Glu Val Ser Asp His Val Ser Glu Asp Asp 35 40 45 Val Gln Ser Asp Thr Glu Glu Ala Phe Ile Asp Glu Val His Glu Val 50 55 60 Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu Asp Glu Gln Asn Val Ile 65 70 75 80 Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn Arg Ile Leu Thr Leu Pro 85 90 95 Gln Arg Thr Ile Arg Gly Lys Asn Lys His Cys Trp Ser Thr Ser Lys 100 105 110 Pro Thr Arg Arg Ser Arg Val Ser Ala Leu Asn Ile Val Arg Ser Gln 115 120 125 Arg Gly Pro Thr Arg Met Cys Arg Asn Ile Tyr Asp Pro Leu Leu Cys 130 135 140 Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile Ser Glu Ile Val Lys Trp 145 150 155 160 Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg Glu Ser Met Thr Ser Ala 165 170 175 Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile Tyr Ala Phe Phe Gly Ile 180 185 190 Leu Val Met Thr Ala Val Arg Lys Asp Asn His Met Ser Thr Asp Asp 195 200 205 Leu Phe Asp Arg Ser Leu Ser Met Val Tyr Val Ser Val Met Ser Arg 210 215 220 Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu Arg Met Asp Asp Lys Ser 225 230 235 240 Ile Arg Pro Thr Leu Arg Glu Asn Asp Val Phe Thr Pro Val Arg Lys 245 250 255 Ile Trp Asp Leu Phe Ile His Gln Cys Ile Gln Asn Tyr Thr Pro Gly 260 265 270 Ala His Leu Thr Ile Asp Glu Gln Leu Leu Gly Phe Arg Gly Arg Cys 275 280 285 Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro Ser Lys Tyr Gly Ile Lys 290 295 300 Ile Leu Met Met Cys Asp Ser Gly Thr Lys Tyr Met Ile Asn Gly Met 305 310 315 320 Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn Gly Val Pro Leu Gly Glu 325 330 335 Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val His Gly Ser Cys Arg Asn 340 345 350 Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile Pro Leu Ala Lys Asn Leu 355 360 365 Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val Gly Thr Val Arg Ser Asn 370 375 380 Lys Arg Glu Ile Pro Glu Val Leu Lys Asn Ser Arg Ser Arg Pro Val 385 390 395 400 Gly Thr Ser Met Phe Cys Phe Asp Gly Pro Leu Thr Leu Val Ser Tyr 405 410 415 Lys Pro Lys Pro Ala Lys Met Val Tyr Leu Leu Ser Ser Cys Asp Glu 420 425 430 Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys Pro Gln Met Val Met Tyr 435 440 445 Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr Leu Asp Gln Met Cys Ser 450 455 460 Val Met Thr Cys Ser Arg Lys Thr Asn Arg Trp Pro Met Ala Leu Leu 465 470 475 480 Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn Ser Phe Ile Ile Tyr Ser 485 490 495 His Asn Val Ser Ser Lys Gly Glu Lys Val Gln Ser Arg Lys Lys Phe 500 505 510 Met Arg Asn Leu Tyr Met Gly Leu Thr Ser Ser Phe Met Arg Lys Arg 515 520 525 Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu Arg Asp Asn Ile Ser Asn 530 535 540 Ile Leu Pro Lys Glu Val Pro Gly Thr Ser Asp Asp Ser Thr Glu Glu 545 550 555 560 Pro Val Met Lys Lys Arg Thr Tyr Cys Thr Tyr Cys Pro Ser Lys Ile 565 570 575 Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys Cys Lys Lys Val Ile Cys 580 585 590 Arg Glu His Asn Ile Asp Met Cys Gln Ser Cys Phe 595 600 <210> 2 <211> 604 <212> PRT <213> Artificial Sequence <400> 2 Met Gly Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Ser Ser Leu Asp 1 5 10 15 Asp Glu His Ile Leu Ser Ala Leu Leu Gln Ser Asp Asp Glu Leu Val 20 25 30 Gly Glu Asp Ser Asp Ser Glu Val Ser Asp His Val Ser Glu Asp Asp 35 40 45 Val Gln Ser Asp Thr Glu Glu Ala Phe Ile Asp Glu Val His Glu Val 50 55 60 Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu Asp Glu Gln Asn Val Ile 65 70 75 80 Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn Arg Asn Leu Thr Leu Pro 85 90 95 Gln Arg Thr Ile Arg Gly Lys Asn Lys His Cys Trp Ser Thr Ser Lys 100 105 110 Pro Thr Arg Arg Ser Arg Ala Ser Ala Leu Asn Ile Val Arg Ser Gln 115 120 125 Arg Gly Pro Thr Arg Met Cys Arg Asn Ile Tyr Asp Pro Leu Leu Cys 130 135 140 Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile Ser Glu Ile Val Lys Trp 145 150 155 160 Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg Glu Ser Met Thr Ser Ala 165 170 175 Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile Tyr Ala Phe Phe Gly Ile 180 185 190 Leu Val Met Thr Ala Val Arg Lys Asp Asn His Met Ser Thr Asp Asp 195 200 205 Leu Phe Asp Arg Ser Leu Ser Met Val Tyr Val Ser Val Met Ser Arg 210 215 220 Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu Arg Met Asp Asp Lys Ser 225 230 235 240 Ile Arg Pro Thr Leu Arg Glu Asn Asp Val Phe Thr Pro Val Arg Lys 245 250 255 Ile Trp Asp Leu Phe Ile His Gln Cys Ile Gln Asn Tyr Thr Pro Gly 260 265 270 Ala His Leu Thr Ile Asp Glu Gln Leu Leu Gly Phe Arg Gly Arg Cys 275 280 285 Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro Ser Lys Tyr Gly Ile Lys 290 295 300 Ile Leu Met Met Cys Asp Ser Gly Thr Lys Tyr Met Ile Asn Gly Met 305 310 315 320 Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn Gly Val Pro Leu Gly Glu 325 330 335 Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val His Gly Ser Cys Arg Asn 340 345 350 Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile Pro Leu Ala Lys Asn Leu 355 360 365 Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val Gly Thr Val Arg Ser Asn 370 375 380 Lys Arg Glu Ile Pro Glu Val Leu Lys Asn Ser Arg Ser Arg Pro Val 385 390 395 400 Gly Thr Ser Met Phe Cys Phe Asp Gly Pro Leu Thr Leu Val Ser Tyr 405 410 415 Lys Pro Lys Pro Ala Lys Met Val Tyr Leu Leu Ser Ser Cys Asp Glu 420 425 430 Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys Pro Gln Met Val Met Tyr 435 440 445 Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr Leu Asp Gln Met Cys Ser 450 455 460 Val Met Thr Cys Ser Arg Lys Thr Asn Arg Trp Pro Met Ala Leu Leu 465 470 475 480 Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn Ser Phe Ile Ile Tyr Ser 485 490 495 His Asn Val Ser Ser Lys Gly Glu Lys Val Gln Ser Arg Lys Lys Phe 500 505 510 Met Arg Asn Leu Tyr Met Gly Leu Thr Ser Ser Phe Met Arg Lys Arg 515 520 525 Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu Arg Asp Asn Ile Ser Asn 530 535 540 Ile Leu Pro Lys Glu Val Pro Gly Thr Ser Asp Asp Ser Thr Glu Glu 545 550 555 560 Pro Val Met Lys Lys Arg Thr Tyr Cys Thr Tyr Cys Pro Ser Lys Ile 565 570 575 Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys Cys Lys Lys Val Ile Cys 580 585 590 Arg Glu His Asn Ile Asp Met Cys Arg Ser Cys Phe 595 600 <210> 3 <211> 1815 <212> DNA <213> Artificial Sequence <400> 3 atgggccctg ctgccaagag ggtcaagttg gacggcagca gcctggacga cgagcacatc 60 ctgagcgccc tgctgcagag cgacgacgag ctggtgggcg aggacagcga cagcgaggtg 120 agcgaccacg tgagcgagga cgacgtgcag agcgacaccg aggaggcctt catcgacgag 180 gtgcacgagg tgcagcccac cagcagcggc agcgagatcc tggacgagca gaacgtgatc 240 gagcagcccg gcagcagcct ggccagcaac cgcaacctga ccctgcccca gcgcaccatc 300 cgcggcaaga acaagcactg ctggagcacc agcaagccca cccgccgcag ccgcgccagc 360 gccctgaaca tcgtgcgcag ccagcgcggc cccacccgca tgtgccgcaa catctacgac 420 cccctgctgt gcttcaagct gttcttcacc gacgagatca tcagcgagat cgtgaagtgg 480 accaacgccg agatcagcct gaagcgccgc gagagcatga ccagcgccac cttccgcgac 540 accaacgagg acgagatcta cgccttcttc ggcatcctgg tgatgaccgc cgtgcgcaag 600 gacaaccaca tgagcaccga cgacctgttc gaccgcagcc tgagcatggt gtacgtgagc 660 gtgatgagcc gcgaccgctt cgacttcctg atccgctgcc tgcgcatgga cgacaagagc 720 atccgcccca ccctgcgcga gaacgacgtg ttcacccccg tgcgcaagat ctgggacctg 780 ttcatccacc agtgcatcca gaactacacc cccggcgccc acctgaccat cgacgagcag 840 ctgctgggct tccgcggccg ctgccccttc cgcgtgtaca tccccaacaa gcccagcaaa 900 tacggcatca agatcctgat gatgtgcgac agcggcacca agtacatgat caacggcatg 960 ccctacctgg gccgcggcac ccagaccaac ggcgtgcccc tgggcgagta ctacgtgaag 1020 gagctgagca agcccgtgca cggcagctgc cgcaacatca cctgcgacaa ctggttcacc 1080 agcatccccc tggccaagaa cctgctgcag gagccctaca agctgaccat cgtgggcacc 1140 gtgcgcagca acaagcgcga gatccccgag gtgctgaaga acagccgcag ccgccccgtg 1200 ggcaccagca tgttctgctt cgacggcccc ctgaccctgg tgagctacaa gcccaagccc 1260 gccaagatgg tgtacctgct gagcagctgc gacgaggacg ccagcatcaa cgagagcacc 1320 ggcaagcccc agatggtgat gtactacaac cagaccaagg gcggcgtgga caccctggac 1380 cagatgtgca gcgtgatgac ctgcagccgc aagaccaacc gctggcccat ggccctgctg 1440 tacggcatga tcaacatcgc ctgcatcaac agcttcatca tctacagcca caacgtgagc 1500 agcaagggcg agaaggtgca gagccgcaag aagttcatgc gcaacctgta catgggcctg 1560 accagcagct tcatgcgcaa gcgcctggag gcccccaccc tgaagcgcta cctgcgcgac 1620 aacatcagca acatcctgcc caaggaggtg cccggcacca gcgacgacag caccgaggag 1680 cccgtgatga agaagcgcac ctactgcacc tactgcccca gcaagatccg ccgcaaggcc 1740 agcgccagct gcaagaagtg caagaaggtg atctgccgcg agcacaacat cgacatgtgc 1800 cggagctgct tctaa 1815 <210> 4 <211> 1815 <212> DNA <213> Artificial Sequence <400> 4 atgggccctg ctgccaagag ggtcaagttg gacggcagca gcctggacga cgagcacatc 60 ctgagcgccc tgctgcagag cgacgacgag ctggtgggcg aggacagcga cagcgaggtg 120 agcgaccacg tgagcgagga cgacgtgcag agcgacaccg aggaggcctt catcgacgag 180 gtgcacgagg tgcagcccac cagcagcggc agcgagatcc tggacgagca gaacgtgatc 240 gagcagcccg gcagcagcct ggccagcaac cgcaatctga ccctgcccca gcgcaccatc 300 cgcggcaaga acaagcactg ctggagcacc agcaagccca cccgccgcag ccgcgtcagc 360 gccctgaaca tcgtgcgcag ccagcgcggc cccacccgca tgtgccgcaa catctacgac 420 cccctgctgt gcttcaagct gttcttcacc gacgagatca tcagcgagat cgtgaagtgg 480 accaacgccg agatcagcct gaagcgccgc gagagcatga ccagcgccac cttccgcgac 540 accaacgagg acgagatcta cgccttcttc ggcatcctgg tgatgaccgc cgtgcgcaag 600 gacaaccaca tgagcaccga cgacctgttc gaccgcagcc tgagcatggt gtacgtgagc 660 gtgatgagcc gcgaccgctt cgacttcctg atccgctgcc tgcgcatgga cgacaagagc 720 atccgcccca ccctgcgcga gaacgacgtg ttcacccccg tgcgcaagat ctgggacctg 780[[ID=u11]] ttcatccacc agtgcatcca gaactacacc cccggcgccc acctgaccat cgacgagcag 840 ctgctgggct tccgcggccg ctgccccttc cgcgtgtaca tccccaacaa gcccagcaag 900 tacggcatca agatcctgat gatgtgcgac agcggcacca agtacatgat caacggcatg 960 ccctacctgg gccgcggcac ccagaccaac ggcgtgcccc tgggcgagta ctacgtgaag 102o gagctgagca agcccgtgca cggcagctgc cgcaacatca cctgcgacaa ctggttcacc 1080 agcatccccc tggccaagaa cctgctgcag gagccctaca agctgaccat cgtgggcacc 1140 It should be noted that there may be an error in the "ccctacctgg gccgcggcac ccagaccaac ggcgtgcccc tgggcgagta ctacgtgaag 102o" in the original text, where "102o" might be a typo and is translated as "1020" in the above content.gtgcgcagca acaagcgcga gatccccgag gtgctgaaga acagccgcag ccgccccgtg 1200 ggcaccagca tgttctgctt cgacggcccc ctgaccctgg tgagctacaa gcccaagccc 1260 gccaagatgg tgtacctgct gagcagctgc gacgaggacg ccagcatcaa cgagagcacc 1320 ggcaagcccc agatggtgat gtactacaac cagaccaagg gcggcgtgga caccctggac 1380 cagatgtgca gcgtgatgac ctgcagccgc aagaccaacc gctggcccat ggccctgctg 1440 tacggcatga tcaacatcgc ctgcatcaac agcttcatca tctacagcca caacgtgagc 1500 agcaagggcg agaaggtgca gagccgcaag aagttcatgc gcaacctgta catgggcctg 1560 accagcagct tcatgcgcaa gcgcctggag gcccccaccc tgaagcgcta cctgcgcgac 1620 aacatcagca acatcctgcc caaggaggtg cccggcacca gcgacgacag caccgaggag 1680 cccgtgatga agaagcgcac ctactgcacc tactgcccca gcaagatccg ccgcaaggcc 1740 agcgccagct gcaagaagtg caagaaggtg atctgccgcg agcacaacat cgacatgtgc 1800 cagagctgct tctaa 1815 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <400> 5 aagccgctaa aggcattatc cgcc 24 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <400> 6 aactgtgccc tccatggaaa aatcagtc 28 <210> 7 <211> 69 <212> DNA <213> Artificial Sequence <400> 7 gactgatttt tccatggagg gcacagttaa ccctagaaag atagtctgcg taaaattgac 60 gcatgcgac 69 <210> 8 <211> 50 <212> DNA <213> Artificial Sequence <400> d ggcggataat gcctttagcg gcttaaccct agaaagataa tcatattgtg 50 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <400> 9 taatcagcga agcgatga 18 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <400> 10 cagcatgcct gctattgtct tcc 23

Claims

1. A highly active transposase, characterized in that, Its amino acid sequence is: the amino acid sequence with transposase activity obtained by mutating the isoleucine at position 92, the valine at position 119, and the glutamine at position 601 in the amino acid sequence shown in SEQ ID NO: 1 to asparagine, alanine, and arginine, respectively.

2. The highly active transposase according to claim 1, characterized in that, It also contains the amino acid sequence of a functional protein, and the amino acid sequence of the functional protein is the amino acid sequence of a nuclear localization signal, a tag protein amino acid sequence, or an antibody amino acid sequence.

3. The highly active transposase according to claim 2, wherein The amino acid sequence of the functional protein is the amino acid sequence expressing the EGFP green fluorescent protein.

4. A peptide segment containing one or more highly active transposases according to any one of claims 1-3.

5. A protein containing one or more highly active transposases according to any one of claims 1-3 or one or more peptide segments according to claim 4, having transposase activity.

6. A nucleic acid encoding the highly active transposase according to any one of claims 1-3, the peptide segment according to claim 4, or the protein according to claim 5.

7. The nucleic acid according to claim 6, which further contains a nucleotide sequence encoding a functional protein.

8. The nucleic acid according to claim 7, wherein The nucleotide sequence encoding the functional protein is the nucleotide sequence encoding a nuclear localization signal, the nucleotide sequence encoding a tag protein peptide segment, or the nucleotide sequence encoding an antibody.

9. The nucleic acid according to claim 7, wherein The nucleotide sequence encoding the functional protein is the nucleotide sequence expressing the EGFP green fluorescent protein.

10. A nucleic acid construct encoding the highly active transposase according to any one of claims 1-3, the peptide segment according to claim 4, or the protein according to claim 5.

11. The nucleic acid construct according to claim 10, containing the nucleic acid according to any one of claims 6-9.

12. A recombinant vector containing the nucleic acid according to any one of claims 6-9, or the nucleic acid construct according to any one of claims 10-11.

13. The recombinant vector according to claim 12, wherein, The recombinant vector is a recombinant cloning vector.

14. The recombinant vector according to claim 12, characterized in that, The recombinant vector is a recombinant eukaryotic expression vector or a recombinant viral vector.

15. The recombinant vector according to claim 13, wherein The recombinant cloning vector is a pRS vector, a T vector, or a pUC vector.

16. The recombinant vector according to claim 14, wherein The recombinant eukaryotic expression vector is pEGFP, pCMVp-NEO-BAN, or pSV2.

17. The recombinant vector according to claim 14, characterized in that, The recombinant viral vector is a recombinant adenovirus vector or a lentiviral vector.

18. A host cell containing the nucleic acid construct according to claim 10 or 11, or the recombinant vector according to any one of claims 12-17.

19. The host cell according to claim 18, characterized in that, The host cell is an Escherichia coli cell, an insect cell, a yeast cell, or a mammalian cell.

20. A gene transfer system, characterized in that, ​ 21. A gene transfer system according to claim 20, wherein, It also contains a transposon gene, and the nucleic acid according to any one of claims 6-9 or the nucleic acid construct according to any one of claims 10-11 is integrated with the transposon gene; or the nucleic acid according to any one of claims 6-9 or the nucleic acid construct according to any one of claims 10-11 is relatively independent of the transposon gene.

22. The gene transfer system according to claim 21, wherein, The nucleic acid according to any one of claims 6-9 or the nucleic acid construct according to any one of claims 10-11 and the transposon gene are located on the same recombinant vector.

23. The gene transfer system according to claim 21, wherein The nucleic acid according to any one of claims 6-9 or the nucleic acid construct according to any one of claims 10-11 and the transposon gene are located on different recombinant vectors.

24. The gene transfer system according to claim 21, wherein, The transposon gene is integrated into the nucleic acid construct according to any one of claims 10-11.

25. The gene transfer system according to claim 21, wherein, The transposon gene is integrated into the recombinant vector according to any one of claims 12-17.

26. The gene transfer system according to claim 21, wherein The transposon gene is transferred into the host cell according to claim 18 or 19.

27. The gene transfer system according to claim 21, wherein The transposon gene is located outside the host cell according to claim 18 or 19.

28. Use of the peptide segment according to claim 4, or the protein according to claim 5, or the nucleic acid according to any one of claims 6-9, or the nucleic acid construct according to any one of claims 10-11, or the recombinant vector according to any one of claims 12-17, or the host cell according to claim 18 or 19, or the gene transfer system according to any one of claims 20-27 in any one of the following: (1) Preparing a preparation for gene editing; (2) Preparing a tool for gene editing.

29. The use according to claim 28, characterized in that, The host cell is an Escherichia coli cell, an insect cell, a yeast cell or a mammalian cell.

30. A preparation for gene editing, containing the peptide segment according to claim 4, or the protein according to claim 5, or the nucleic acid according to any one of claims 6-9, or the nucleic acid construct according to any one of claims 10-11, or the recombinant vector according to any one of claims 12-17, or the host cell according to claim 18 or 19, or the gene transfer system according to any one of claims 20-27.

31. A tool for gene editing, containing the peptide segment according to claim 4, or the protein according to claim 5, or the nucleic acid according to any one of claims 6-9, or the nucleic acid construct according to any one of claims 10-11, or the recombinant vector according to any one of claims 12-17, or the host cell according to claim 18 or 19, or the gene transfer system according to any one of claims 20-27.

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