Transposase, transposon, transposon system and application thereof

Abstract

Provided are a transposase, a transposon, a transposon system and applications thereof, the transposase is a mutant transposase or a fusion transposase, the mutant transposase contains amino acid mutation compared with a wild-type PS transposase as shown in SEQ ID NO: 1, the fusion transposase is obtained by fusing a functional polypeptide on the wild-type PS transposase or a mutant thereof, and the transposon has an amino acid mutation compared with the wild-type PS transposon. The transposon system contains nucleotide mutation and / or TIR multi-copy series connection and / or TA multi-copy series connection, and the transposon system contains transposase or polynucleotide encoding the transposase, and transposon or PS transposon. According to the JL transposase, the JL transposon and the JL transposon system containing the JL transposase and the JL transposon which are obtained by engineering modification of the wild type PS transposase through the means, the transposon gene integration efficiency is remarkably improved.

Description

A transposase, a transposon, a transposon system and applications thereof This application claims priority to Chinese Patent Application No. 202310081106.8 filed on February 8, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field The present invention relates to the field of genetic engineering, and in particular to an engineered transposase, a transposon, a transposon system and applications thereof. Background Art Transposable genetic elements, also called transposons, are segments of DNA that can move from one genomic location to another within a single cell. Transposons can be divided into two major categories based on their transposition mechanism: (1) for elements known as retrotransposons, transposition can occur by reverse transcription of an RNA intermediate, and (2) for DNA transposons, transposition can occur by direct transposition of DNA flanked by inverted terminal repeats (TIR or ITR). Active transposons encode one or more proteins required for transposition, and naturally active DNA transposons carry a transposase gene. Transposons have been widely used as gene transfer vectors in the fields of transgenic, gene capture and gene therapy, and have achieved good application results. Transposons can be used as natural DNA transfer vectors, which can efficiently carry exogenous genes into the genome. Their function is similar to that of viral vectors, and the transposition process can be controlled by adjusting the supply of transposase. Therefore, the use of transposons is divided into transposon donor vectors and transposase auxiliary vectors. DNA fragments of interest, such as fluorescent marker genes GFP / RFP, gene capture cassettes, therapeutic gene construction, etc., can be cloned between the two sides of the TIR as transposon donor vector plasmids. The transposase vector contains components such as promoters, transposase gene expression cassettes, PolyA, etc., and the transposase can also be in the form of mRNA or protein. Transposon donor vectors and transposases are co-transfected into cells or injected into recipients to achieve stable gene insertion in target cells. Tc1 / Mariner transposons are the most widely distributed DNA transposon superfamily in nature, including bacteria, invertebrates and vertebrates. Among vertebrates, Tc1 / Mariner transposons are most widely distributed in bony fish. Through bioinformatics and experimental verification, 16 Tc1 / Mariner superfamily transposon members with natural transposition activity have been reported, namely Tc1, Tc3, Minos, Mos1, Bari3, Fot1, impala, Famar1, Osmar5, ISY100, Mboumar-9, Passport, Tana1, Thm3, SB, ZB, PS transposon. Prior patent applications CN110257425A and CN112159822A disclosed a PS transposon with a unique transposition catalytic domain "D35D", which belongs to the pogo transposon superfamily and can exhibit certain transposition activity and cleavage activity in mammalian cells. Although the above-mentioned PS transposon system has certain transgenic application value, because the PS transposase sequence is a wild-type natural amino acid sequence, its transposition efficiency is relatively low. In some types of cell or gene therapy, PS has no significant advantages over the high-efficiency transposons such as Piggybac (PB) or Sleeping Beauty (SB) in this technical field. As we all know, naturally active transposons may cause genome instability. Therefore, during evolution, transposons will accumulate mutations, gradually reduce or even lose their activity. For example, most of the Tc1 / Mariner superfamily transposons found in higher animals (such as mammals) lose their transposition activity due to defects in the transposase open reading frame (such as mutations, frameshifts, insertions, deletions, or the presence of stop codons). In view of the above reasons, the Sleeping Beauty (SB) transposon and PiggyBac transposon, which are currently the most widely used in clinical gene therapy, are often highly efficient mutant transposase / transposon systems that have undergone bioinformatics molecular reconstruction and / or engineering transformation. In view of the above reasons, the PS transposon system still needs amino acid / DNA mutation and / or engineering modification to obtain a more efficient and more practical transposase and / or transposon system. Summary of the invention In one aspect, the present disclosure provides a transposase (JL transposase), It is a mutant transposase or a fusion transposase, The mutant transposase comprises an amino acid mutation compared to the wild-type PS transposase as shown in SEQ ID NO: 1, wherein any one or more amino acids in the double-stranded DNA binding and oligomerization domain of the wild-type PS transposase are mutated into positively charged amino acids; and / or the amino acid mutation comprises one or more mutations selected from the group consisting of TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59RK -59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R , T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T3 50S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159 H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V;; The fusion transposase is a wild-type PS transposase or a mutant thereof fused with a functional polypeptide, wherein the functional polypeptide is: a DNA sequence-specific or non-specific binding domain, and / or a cell nucleus localization signal domain. In one or more embodiments, the positively charged amino acid is selected from histidine, lysine or arginine, and / or the amino acid mutation is selected from one or more mutations of the following group: TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57 -58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR , E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K. In one or more embodiments, the mutant comprises one or more mutations selected from the group consisting of: compared to the wild-type PS transposase as shown in SEQ ID NO: 1,Including one or more mutations selected from the following group: TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R , T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N1 93S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M , E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H2 15E, H215Q, I237V, Q69R, L100I, K96R, Q162R, A271K, R372K, H20Y, TYCR 150-153KFVI, N232K, A363N, A377C, Q346M, A17V, N105G, Q76E, I354V, VS133-134IA, S253K, V370I, SV297-298TE, EH286-286DS, A352S, ANS24 1-243VHE, S297T, A79I, T147S, I178M, I276V, Q331E, R257E, T339S, A79 V, H104L, V74L, S107E, Q142E, R375K, S243E, R195K, Q22E, A328Q, G111A , R110K, I365V, VTE380-382SNA, R190K, RA372-373KS, K73L, K121R, R102K, C152V, IY276-277VW, V292I, K73E, H104M, I239V, K42E, K356Q, T150 K, G316S, IY259-260VW, V53M, E108D, V53L, DKV72-74EEL, M280F, S85G, N63K, V91L, K251L, H124N, K308S, N10D, H7R, G112S, H7K, TA327-328KE. , In one or more embodiments, the mutant comprises one or more mutations selected from the group consisting of TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V , N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L 228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H21 5E, H215Q, I237V, Q69R, L100I, K96R, Q162R, A271K, R372K, H20Y, TYCR150-15 3KFVI, N232K, A363N, A377C, Q346M, A17V, N105G, Q76E, I354V, VS133-134IA, S253K, V370I, SV297-298TE, EH286-286DS, A352S, ANS241-243VHE, S297T, A7 9I, T147S, I178M, I276V, Q331E, R257E, T339S, A79V, H104L, V74L, S107E, Q14 2E, R375K, S243E, R195K, Q22E, A328Q, G111A, R110K, I365V, VTE380-382SNA. In one or more embodiments, the DNA sequence-specific or non-specific binding domain comprises a leucine zipper domain, a CRISPR / Cas domain, a TALE domain, a zinc finger domain, an AAV Rep DNA binding domain, or any combination thereof. Preferably, the amino acid sequence of the leucine zipper domain is as shown in any one of SEQ ID NOs: 28-31. In one or more embodiments, the nuclear localization signal domain comprises SV40 NLS, C-myc NLS, TAF1 NLS, TP53 NLS, STAT3 NLS or any combination thereof. In one or more embodiments, the functional polypeptide and the PS transposase protein may or may not contain a linker. Yet another aspect of the present disclosure provides a polynucleotide encoding the transposase of any embodiment herein. In one or more embodiments, the polynucleotide is DNA or messenger RNA. In another aspect, the present disclosure provides a transposon that can be recognized by the transposase of any of the above embodiments, wherein the transposon has any one, two or three features selected from the following (1) to (3): (1) The transposon has a mutant terminal inverted repeat sequence, and the mutant terminal inverted repeat sequence has 1-5 nucleotide mutations compared with the wild-type PS transposon terminal inverted repeat sequence shown in SEQ ID NO: 2 or 3; (2) The transposon has two or more terminal inverted repeat sequences at at least one end; (3) The transposon has two or more repeats of TA sequences outside the terminal inverted repeat sequence at at least one end. In one or more embodiments, the mutant terminal inverted repeat sequence is as shown in any one of SEQ ID NOs: 4-27, preferably as shown in any one of SEQ ID NOs: 4-6, or is a nucleotide sequence containing any combination of nucleotide mutations in SEQ ID NOs: 4-27, or is their reverse complementary sequences. In one or more embodiments, the transposon has two terminal inverted repeat sequences at each end, and includes four TA sequence repeats outside the terminal inverted repeat sequences at each end. In some embodiments, the transposon comprises a combination of DNA elements located between two terminal inverted repeat sequences, and the combination of DNA elements includes but is not limited to sequence elements well known to those skilled in the art such as promoters, enhancers, expression genes, 5-UTRs, 3-UTRs, etc. In some embodiments, either of the two terminal inverted repeat sequences (both reverse complementary) comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 2 or 3. In one or more embodiments, the transposon includes or does not include a target gene (or foreign gene) expression cassette located between the terminal inverted repeat sequences. The target gene expression cassette may include gene function elements such as a promoter, a target gene, and a polyA signal sequence. In one or more embodiments, the gene of interest encodes a protein selected from a cell receptor, an immune checkpoint protein, a cytokine, a T cell receptor, a B cell receptor, a chimeric antigen receptor, and any combination thereof. Yet another aspect of the present disclosure provides a transposon system, comprising the transposase according to any embodiment herein or a polynucleotide encoding the transposase; and a transposon, wherein the transposon is the transposon according to any embodiment herein or a PS transposon. In one or more embodiments, the polynucleotide encoding the transposase is mRNA, preferably, the mRNA is chemically modified. In one or more embodiments, the transposon is present in a DNA vector. Preferably, the DNA vector is selected from conventional circular DNA plasmids, linear DNA plasmids, or DNA forms that do not contain antibiotics or / and replicon DNA sequences such as minicircle plasmids, nanoplasmids, doggybone linear miniplasmids, etc. Preferably, the DNA vector is an antibiotic-free miniplasmid. In one or more embodiments, the DNA vector is a microcarrier, the backbone sequence of which is within 600 bp in length, and has reduced and / or no CpG DNA motif. In one or more embodiments, the polynucleotide encoding the transposase is a DNA, and the polynucleotide and the transposon are present in the same DNA vector or in different DNA vectors. In some embodiments, the polynucleotide molecule comprising the sequence encoding the JL transposase and / or the transposon TIR is present in a viral vector, including but not limited to AAV, lentivirus, retrovirus, adenovirus, etc. In yet another aspect, the present disclosure provides a host cell comprising or capable of producing the transposase according to any embodiment herein, the polynucleotide according to any embodiment herein, the transposon according to any embodiment herein, or the transposon system according to any embodiment herein. In yet another aspect, the present disclosure provides a method for preparing a cell, comprising the step of introducing the transposase according to any embodiment of the present invention, the polynucleotide according to any embodiment of the present invention, the transposon according to any embodiment of the present invention, or the transposon system according to any embodiment of the present invention into the cell. In some embodiments, the introducing comprises contacting the cell with the polynucleotide. In some embodiments, the introducing comprises contacting the cell with a DNA vector containing the transposon. In some embodiments, the introducing comprises contacting the cell with an mRNA encoding the transposase and a plasmid vector containing the transposon. In some embodiments, the introducing comprises contacting the cell with a plasmid vector containing the polynucleotide and the transposon. In some embodiments, the introducing comprises contacting the cell with a plasmid vector containing the polynucleotide and a plasmid vector containing the transposon. In some embodiments, the introducing comprises contacting the cell with the transposase. In some embodiments, the cell is a primary cell isolated from a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a patient with a disease. In some embodiments, the subject has been diagnosed with cancer or a tumor. In some embodiments, the cell is separated from the blood of the subject. In some embodiments, the cell comprises a primary immune cell. In some embodiments, the cell comprises a primary leukocyte. In some embodiments, the cell comprises a primary T cell. In some embodiments, the primary T cell comprises a γδT cell, a helper T cell, a memory T cell, a natural killer T cell, an effector T cell, or any combination thereof. In some embodiments, the primary immune cell comprises a CD3+ cell. In some embodiments, the cell comprises a stem cell. In some embodiments, the stem cell is selected from: an embryonic stem cell, a hematopoietic stem cell, an epidermal stem cell, an epithelial stem cell, a bronchial alveolar stem cell, a mammary stem cell, a mesenchymal stem cell, an intestinal stem cell, an endothelial stem cell, a neural stem cell, an olfactory adult stem cell, a neural crest stem cell, a testicular cell, and any combination thereof. In some embodiments, the stem cell comprises an induced pluripotent stem cell. In some embodiments, the cell is from an animal, such as a vertebrate or an invertebrate, preferably a mammal, and further preferably a human. In some embodiments, the cell is an immune cell, preferably a T cell. In some embodiments, the introducing comprises transfecting the cell by electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposomes, lipid nanoparticles (LNP), microparticle bombardment, fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection, or any combination thereof. In some embodiments, the introducing comprises electroporating the cells. In some embodiments, the introduction comprises contacting the cell with mRNA encoding the transposase and a plasmid containing the transposon. Preferably, the mRNA is used at a dosage of 1×10 7 Cells 1-30 μg, The dosage of the plasmid was 1×10 7 The most preferred dosage of the mRNA is 0.1-5 μg per 1×10 7 The concentration of the plasmid used is 1-5 μg per 1×10 7 Cells 0.1-2μg. Another aspect of the present disclosure provides a transgenic animal or transgenic cell produced using the transposase of any embodiment herein, the polynucleotide of any embodiment herein, the transposon of any embodiment herein, or the transposon system of any embodiment herein, wherein the transgenic expression element includes a gene expression cassette and transposon TIR sequence elements on both sides. Yet another aspect of the present disclosure provides a kit comprising: the transposase according to any embodiment herein, the polynucleotide according to any embodiment herein, the transposon according to any embodiment herein, or the transposon system according to any embodiment herein, or the cell according to any embodiment herein. Yet another aspect of the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and cells prepared by the method of any embodiment herein. In yet another aspect, the present disclosure provides a transposase according to any embodiment of the present invention, a polynucleotide according to any embodiment of the present invention, a transposon according to any embodiment of the present invention, a transposon system according to any embodiment of the present invention, a cell according to any embodiment of the present invention, or a use of a cell according to any embodiment of the present invention, which is selected from any one of the following (1)-(6): (1) Application in the preparation of drugs or reagents for integrating the target gene expression cassette into the host cell genome; (2) Application in the preparation of tools for integrating target gene expression cassettes into the host cell genome; (3) Application in the preparation of transgenic animals and transgenic cells; (4) Application in the preparation of drugs or preparations for genome research, gene therapy, cell therapy, or stem cell induction and differentiation after induction; (5) Application in the preparation of tools for genome research, gene therapy, cell therapy, or stem cell induction and post-induced differentiation; (6) Application in the preparation of kits, engineered immune cells or pharmaceutical compositions. Another aspect of the present disclosure provides a method of treatment, comprising: (a) introducing a transposon as described herein and a transposase as described herein that recognizes the transposon into a cell, thereby generating a genetically modified cell; (b) administering the genetically modified cell to a patient in need of the treatment. In some embodiments, the genetically modified cell comprises a transgene introduced by the transposon. In some embodiments, the patient has been diagnosed with cancer or a tumor. In some embodiments, the administration comprises infusing the genetically modified cell into a blood vessel of the patient. Beneficial effects brought by the technical solution of the present invention 1) Engineering and transforming wild-type PS transposase by mutation and / or fusion protein polypeptide to obtain JL transposase significantly improves the efficiency of transposon gene integration and has greater application value in the transgenic industry. 2) The engineered JL transposase in the form of mRNA is used in cell therapy and transgenics, which reduces the expression time of the transposase in cells and further improves safety. 3) The functional sequence structure of transposon DNA was optimized by means of TIR sequence mutation, TIR sequence multi-copy concatenation, and TA cleavage site sequence multi-copy concatenation, further significantly improving the efficiency of JL transposase-mediated transposase gene integration. 4) Using a microcarrier with a DNA backbone sequence length limited to less than 600 bp and reduced and / or free of CpG DNA motifs as a transposon expression vector further improves the efficiency of JL transposase-mediated gene transposition. BRIEF DESCRIPTION OF THE DRAWINGS FIG1 is a schematic diagram and a three-dimensional structure diagram of PS transposase; FIG2 is a schematic diagram of the structure of a JL transposase according to an embodiment of the present invention (a schematic diagram of the structure of a fusion JL transposase comprising a leucine zipper domain and a c-myc NLS domain); Fig. 3 is a schematic diagram of the structure of a leucine zipper dimer; FIG4 is a schematic diagram of the structure of a JL transposon comprising 2 times TIR sequences and 2 times cleavage site sequences; FIG5 is a map of wild-type PS transposase expression plasmid; Fig. 6 is a schematic diagram of PPS-DT-GFP (ta) plasmid; FIG7 is a schematic diagram of the structure of PS transposase fused with a functional polypeptide; FIG8 is a fluorescent photograph showing that the transposition efficiency of PS transposase is improved by fusing a functional polypeptide; FIG9 is a schematic diagram of n-fold sequence duplication multi-copy design of the transposon TIR element DNA sequence and its flanking TA shear recognition sequence in the plasmid expression vector PPS-DT-GFP (ta); Figure 10 is a fluorescence photograph of CHO cells in Example 2; Figure 11 is the PBMC cell test results in Example 2, the left figure is the positive rate, and the right figure is the fluorescence expression intensity; FIG12 shows statistics of several positively charged amino acid mutants with improved integration efficiency (positive rate) relative to wild-type PS transposase (three replicates); Figure 13 shows the EGFP expression on day 14 in Example 4; FIG14 shows the positive rate and eGFP expression level of the mutant and the control group in Example 4; Figure 15 is a schematic diagram of the plasmid structure of pPG-PGK-NEO (PSTIR0) in Example 5; FIG16 is the results of the TIR DNA motif mutation screening evaluation; FIG17 is a result of the evaluation of multiple copies of tandem screening of motif mutation TIR DNA motifs; FIG18 is the evaluation results of Example 6, the upper figure shows the positive rate of transposition integration in different groups, and the lower figure shows the expression level of transposition integration eGFP; FIG19 is the evaluation results of Example 7, and each small figure is CAR positivity rate, expression level, total cell number, total number of CAR-positive cells, and change in total number of CAR-positive cells; FIG. 20 is a schematic diagram of the pCpGfree MCS-0637 empty miniplasmid, and its corresponding nucleotide sequence is shown in SEQ ID NO: 59. FIG. 21 is a map of the pCpGfree MCS-43009 empty vector miniplasmid, and its corresponding nucleotide sequence is shown in SEQ ID NO: 60. DETAILED DESCRIPTION Because the wild-type PS transposase or PS transposon system has the disadvantage of low transposition activity, it is impossible to achieve the expected optimal effect in the actual application scenario of transgenic or cell therapy, so the present invention optimizes the transposase and / or transposon by engineering methods to improve the overall activity of the PS transposon system. In the present invention, the engineered PS transposase, PS transposon, and PS transposon system are respectively referred to as JL transposase, JL transposon, and JL transposon system. Compared with viral transduction of immune cells (such as T lymphocytes), delivery of transgenes by DNA transposons has multiple advantages: ease of use, potential for delivery of large gene fragments, rapid clinical application and low production cost, long-term and high-level stable expression of transgenes, significantly less mutagenicity, non-carcinogenicity and reversibility compared to retroviruses. The in vitro genetic modification of non-transformed primary human T lymphocytes based on non-viral vector-based gene transfer delivery systems is extremely difficult, and it needs to be based on a high level of transposition efficiency to be achieved. The mature cases reported so far are limited to Sleeping Beauty transposon, PiggyBac transposon, and TcBuster transposon. The present invention improves the transposition efficiency mediated by JL transposase to make it suitable for clinical application in cellular immunotherapy. Prior patent application CN110257425B (the entire contents of which are incorporated herein by reference) demonstrates that the PS transposon is an effective non-viral tool for integrating transgenes into cell chromosomes, but the transposition efficiency of the PS transposon system still cannot meet the needs for cell or gene therapy, and there are safety risks in the use of DNA transfection to express the transposase. The use of transposase-encoding DNA leads to prolonged expression of the transposase protein in the target cells, and the lack of control over the time and kinetics of transposase exposure brings about the risk of continuous and uncontrolled re-transposition, which raises safety concerns about the conversion of adverse therapeutic cell products. To ensure transposase clearance and avoid the input of abnormal or unstable cell products, the engineered T cells in the ongoing trial are cultured for 2-4 weeks after CAR gene delivery, which reduces cell adaptability and therapeutic effects. Therefore, there is an urgent need to improve the controllability and safety of the transposase / transposon system, which is also a key requirement for general cell and gene therapy. In order to control this Transposase exposure risk. Some embodiments of the present invention use a method of engineering highly active JL transposase based on mRNA expression, which shortens the time of protein expression and reduces the cytotoxicity of cells such as immune cells, hematopoietic stem cells and progenitor cells (HSPCs). DNA transposons can transpose via a non-replicative "cut and paste" mechanism. This requires recognition of two terminal inverted repeats by a catalytic enzyme, the transposase, which cuts its target, thereby releasing the DNA transposon from its donor template. After excision, the DNA transposon can then integrate into a recipient DNA cut by the same transposase. In some of their natural configurations, DNA transposons are flanked by two terminal inverted repeats and may contain genes encoding transposases that catalyze transposition. The genome editing application based on DNA transposon includes a binary system containing a transposase component and a transposon component, wherein the transposase component is a transposase or its encoding nucleic acid (mRNA or DNA), and the transposon component is a transposon DNA containing a target gene flanked by terminal inverted repeat sequences and for insertion into the host genome. The transposon component and the transposase component are co-delivered into the target cell to achieve a transposition integration effect that relies on a cutting and pasting mechanism. Various devices, systems and methods related to the collaborative method of using engineered high-activity JL transposase for cell gene integration, especially enhancing gene transfer into human hematopoietic and / or immune system cells are discussed herein. The present disclosure relates to improved engineered high-activity JL transposase, transposon system, transposon vector sequence, transposase delivery method and transposon delivery method, etc. In one embodiment, this study identifies specific mutation sites for preparing high-activity JL transposase. In another embodiment, a high-activity JL transposase of a fusion polypeptide is described, wherein examples of fusion polypeptides include leucine zipper polypeptides, and / or nuclear localization signal domains. In another embodiment, an improved method of expressing a high-activity JL transposase by chemically modified in vitro transcribed mRNA is described. In another embodiment, an improved method of optimizing transposon TIR DNA elements for mutation and / or multi-copy tandem, and / or multi-copy tandem of TIR flanking shearing site TA elements is described. In another embodiment, a method for delivering a JL transposon vector using a DNA microcarrier is described, wherein the DNA backbone sequence of the microcarrier does not contain an antibiotic expression cassette and is preferably limited to a length of less than 600 bp, and the DNA motifs containing CpG are reduced and / or not contained. Reducing the size of the expression vector further improves the efficiency of JL transposase-mediated gene transposition, and reducing and / or not containing CpG DNA motifs can reduce the cellular immunotoxicity induced by the DNA vector and improve the transposition efficiency. The above embodiments can improve the efficiency of gene transfer of transposons to various target cells alone or in combination. JL transposase One aspect of the present disclosure provides a JL transposase comprising amino acid mutations and / or fused polypeptides compared to a PS transposase. In some embodiments, the JL transposase is a mutant transposase comprising an amino acid mutation compared to the PS transposase. In some embodiments, the JL transposase may comprise one or more amino acid substitutions compared to the wild-type PS transposase (SEQ ID NO: 1). The JL transposase may comprise an amino acid sequence having at least 70% sequence identity to the full-length sequence of the wild-type PS transposase (SEQ ID NO: 1). In some embodiments, the JL transposase may comprise an amino acid sequence having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the full-length sequence of the wild-type PS transposase (SEQ ID NO: 1). In some embodiments, one or more amino acid substitution mutations of the JL transposase are positively charged amino acids, such as histidine, lysine or arginine, compared to SEQ ID NO: 1. The JL transposase may comprise an amino acid sequence having at least one amino acid different from the full-length sequence of the wild-type PS transposase (SEQ ID NO: 1). In some embodiments, the JL transposase may comprise an amino acid sequence having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more amino acids different from the full-length sequence of the wild-type PS transposase (SEQ ID NO: 1). The JL transposase may comprise one or more amino acid substitutions in any of the double-stranded DNA binding and oligomerization domains (two HTH structures, amino acids 1-129), the DDE catalytic domain, and the interdomain region between the domains of the PS transposase, or any combination thereof (as shown in FIG. 1 ). The CENPB-type HTH domain is a DNA binding, helix-turn-helix (HTH) domain of about 70-75 amino acids that is present in eukaryotic centromere proteins and transposases. This domain is named after the mammalian major centromere autoantigen B or centromere protein B (CENP-B), which is the basic centromere component of chromosomes. The N-terminus of CENP-B contains two DNA binding HTH domains, which bind to the adjacent DNA major groove. The structure of the CENPB type HTH domain consists of three alpha helices. The second and third helices connected by turns include a helix-turn-helix pattern. Helix 3 is called a recognition helix because it binds to the DNA major groove in other HTHs. Preferably, the JL transposase can be replaced with positively charged amino acids such as histidine (H), lysine (K) or arginine (R) in the double-stranded DNA binding and oligomerization domain of the PS transposase. In some cases, the JL transposase can contain one or more amino acid substitutions in the double-stranded DNA binding and oligomerization domain, the DDE catalytic domain, the interdomain region between the domains, or a combination thereof. In some embodiments, the JL transposase comprises an amino acid sequence having a mutation at one or more of the following positions: 7, 8, 16, 20, 22, 24, 32, 35, 42, 45, 46, 47, 51, 55, 57, 58, 59, 69, 73, 74, 76, 79, 84, 95, 98, 123, 129, 136, 159, 165, 178, 187, 193, 199, 213, 215, 228 compared to the full-length SEQ ID NO: 1, wherein the position numbers are the position numbers of SEQ ID NO: 1. In some embodiments, the one or more amino acid mutations are to histidine, lysine, or arginine. In some embodiments, the JL transposase comprises an amino acid sequence having a mutation at one or more of the following positions: 8, 16, 22, 24, 32, 35, 45, 46, 47, 51, 55, 57, 58, 59, 69, 73, 84, 95, 98, 123, 129, 136, 150, 159, 165, 187, 193, 199, 213, 215, 228, 237, 284, 335, 359, 368 compared to the full-length SEQ ID NO: 1, wherein the position numbers are the position numbers of SEQ ID NO: 1. In some embodiments, the one or more amino acid mutations are to histidine, lysine, or arginine. In some embodiments, the JL transposase of the present invention is a JL transposase having any one or any combination of the following mutations compared to SEQ ID NO: 1: TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK (ie, containing both T57R and Q58K), TQ57-58RK\T129K (ie, containing both T57R, Q58K, T129K), TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59KKA\I98K\T129K , R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q5 8K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K , T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V3 59L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V. The numerical value indicates the position of the mutation, and the letters before and after the numerical value indicate the amino acid residues before and after the mutation. In some embodiments, the JL transposase of the present invention is a JL transposase having any one or any combination of the following mutations compared to SEQ ID NO: 1: H7K, H7R, A8S, K16R, H20Y, Q22K, Q22E, H24R, E32K, I35V, K42E, K45R, Y46Q, E47K, R51K, K55R, TQ57-58RK (i.e., T57R\Q58K), TQS57-59KKA (i.e., T57K\Q5 8K\S59A), Q69E, Q69R, K73R, K73E, V74L, Q76E, A79I, A84L, M95L, I98K, R123H, T129K, T12 9R, T129Q, Q136K, K159H, H165D, I178M, T187K, N193S, N199H, N213D, H215Q, H215E, L228M. In some embodiments, the JL transposase of the present invention is a JL transposase having any one or any combination of the following mutations compared to SEQ ID NO: 1: A8S, K16R, Q22K, H24R, E32K, I35V, K45R, Y46Q, K55R, TQ57-58RK, TQS57-59KKA, Q69E, K73R, A84L, M95L, I98K, R123H, T129K, T129R, Q136K, K159H, H165D, T187K, or H215Q. In some embodiments, the JL transposase of the present invention has a substitution mutation at positions 57-58 compared to SEQ ID NO: 1. In some embodiments, in addition to the substitution mutation at positions 57-58, the JL transposase of the present invention also has a substitution mutation at one or more positions selected from 8, 16, 22, 24, 32, 35, 45, 46, 47, 51, 55, 59, 69, 73, 84, 95, 98, 123, 129, 136, 150, 159, 165, 187, 193, 199, 213, 215, 228, 237, 284, 335, 359, 368, wherein the positions are numbered as SEQ ID NO: 1. Preferably, the substitution mutation at position 57 is R or K, and the substitution mutation at position 58 is R or K. Further preferably, the JL transposase of the present invention also has one or more substitution mutations selected from T129R, T129K, I98K, E32K, R123H, Q136K, K16R, E47K, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, and I237V. In some embodiments, the JL transposase of the present invention has a substitution mutation at least at positions 57-59 compared to SEQ ID NO: 1. In some embodiments, in addition to the substitution mutation at positions 57-59, the JL transposase of the present invention also has a substitution mutation at one or more positions selected from 8, 16, 22, 24, 32, 35, 45, 46, 47, 51, 55, 69, 73, 84, 95, 98, 123, 129, 136, 150, 159, 165, 187, 193, 199, 213, 215, 228, 237, 284, 335, 359, 368, and the position numbers are the position numbers of SEQ ID NO: 1. Preferably, the substitution mutation at positions 57-59 is KKA. Further preferably, the JL transposase of the present invention also has one or more substitution mutations selected from T129R, T129K, I98K, E32K, R123H, Q136K, K16R, E47K, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, and I237V. In some embodiments, the JL transposase of the present invention has substitution mutations at positions 57-58 and 129 compared to SEQ ID NO: 1. In some embodiments, in addition to the substitution mutations at positions 57-58 and 129, the JL transposase of the present invention also has substitution mutations at one or more positions selected from 8, 16, 22, 24, 32, 35, 45, 46, 47, 51, 55, 59, 69, 73, 84, 95, 98, 123, 136, 150, 159, 165, 187, 193, 199, 213, 215, 228, 237, 284, 335, 359, 368, the position numbers being the position numbers of SEQ ID NO: 1. Preferably, the substitution mutation at position 57 is R or K, the substitution mutation at position 58 is R or K, and the substitution mutation at position 129 is K or R. Further, the JL transposase of the present invention also has one or more substitution mutations selected from I98K, E32K, R123H, Q136K, K16R, E47K, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, and I237V. In some embodiments, the JL transposase of the present invention has a substitution mutation at least at position 98 compared to SEQ ID NO: 1. In some embodiments, in addition to the substitution mutation at position 98, the JL transposase of the present invention has a substitution mutation at position 8, 16, 22, 24, 32, 35, 45, 46, 47, 51, 55, 57, 58, 59, 69, 73, 84, There is a substitution mutation at one or more positions of 95, 123, 129, 136, 150, 159, 165, 187, 193, 199, 213, 215, 228, 237, 284, 335, 359, 368, and the position numbers are the position numbers of SEQ ID NO: 1. Preferably, the substitution mutation at position 98 is K. Furthermore, the JL transposase of the present invention also has a member selected from TQS57-59KKA, T129R, T129K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S One or more substitution mutations of 59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, and I237V. In some embodiments, compared with SEQ ID NO: 1, the JL transposase of the present invention has a substitution mutation at least at position 129. In some embodiments, in addition to the substitution mutation at position 129, the JL transposase of the present invention also has a substitution mutation at one or more positions selected from 8, 16, 22, 24, 32, 35, 45, 46, 47, 51, 55, 57, 58, 59, 69, 73, 84, 95, 98, 123, 136, 150, 159, 165, 187, 193, 199, 213, 215, 228, 237, 284, 335, 359, 368, and the position numbers are the position numbers of SEQ ID NO: 1. Preferably, the substitution mutation at position 129 is K or R. Furthermore, the JL transposase of the present invention also has a member selected from TQS57-59KKA, I98K, TQ57-58RK, E32K, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, One or more substitution mutations of M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V. Exemplary JL transposases can comprise one or more amino acid substitutions from Table 1. Sometimes, a JL transposase can comprise at least one amino acid substitution from Table 1. A JL transposase can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, or more amino acid substitutions from Table 1. Preferably, the JL transposase comprises one or more amino acid replacements or replacement combinations selected from the group consisting of H24R, E32K, TQS57-59KKA, TQ57-58RK, I98K, T129K, T187K, N193S, TQ57-58RK\T129K, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\T129K, TQS57-59KKA\I98K TQS57-59KKA\I98K\T129K, E32K\T57R\Q58R or E32K\T129K. The inventors have also found that PS transposase or its mutants can be fused with other functional polypeptides or protein domains without affecting or even improving the transposase or transposon gene integration function. The JL transposase of the scheme is a fusion transposase, which comprises PS transposase or a mutant thereof, and a functional polypeptide linked to the PS transposase or a mutant thereof. In the present invention, there is no limitation on the PS transposase mutant in the fusion transposase. The present inventors have found that, when a functional polypeptide (especially the leucine zipper domain described below) is fused, any mutation of the PS transposase can obtain a higher gene integration function than the wild-type PS transposase. Herein, a "mutant" of a polypeptide sequence includes an amino acid sequence having one or more amino acid residues inserted, replaced and / or deleted compared to a reference sequence. In some embodiments, the "mutant" described herein includes a mutant having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the sequence used as a comparison. The sequence identity between the two aligned sequences can be calculated using, for example, NCBI's BLASTp. Alternatively, in some embodiments, compared to the sequence used as a comparison, the mutant described herein has one or more (such as within 20, within 15, within 10, within 8, within 5 or within 3, such as 1-20, 1-10, etc.) amino acid residues inserted, replaced or deleted. In some preferred embodiments, in the fusion transposase, the PS transposase mutant has 1-5 (e.g., 1, 2, 3, 4, 5) amino acid mutations, such as substitutions, compared to the PS transposase. In some preferred embodiments, in the fusion transposase, the PS transposase mutant has 1-5 (e.g., 1, 2, 3, 4, 5) amino acid mutations, such as substitutions, compared to the PS transposase. NO: Compared with the wild-type PS transposase shown in 1, it includes one or more mutations selected from the following group: TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N21 3D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V, Q6 9R, L100I, K96R, Q162R, A271K, R372K, H20Y, TYCR150-153KFVI, N232K, A363N, A377C, Q346M, A17V, N105G, Q76E, I354V, VS133-134IA, S 253K, V370I, SV297-298TE, EH286-286DS, A352S, ANS241-243VHE, S297T, A79I, T147S, I178M, I276V, Q331E, R257E, T339S, A79V, H104L , V74L, S107E, Q142E, R375K, S243E, R195K, Q22E, A328Q, G111A, R110K, I365V, VTE380-382SNA, R190K, RA372-373KS, K73L, K121R, R102 K, C152V, IY276-277VW, V292I, K73E, H104M, I239V, K42E, K356Q, T150K, G316S, IY259-260VW, V53M, E108D, V53L, DKV72-74EEL, M280F,S85G, N63K, V91L, K251L, H124N, K308S, N10D, H7R, G112S, H7K, TA327-328KE, preferably, including one or more mutations selected from the group consisting of TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57- 58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58 R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H16 5D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V, Q69R, L1 00I, K96R, Q162R, A271K, R372K, H20Y, TYCR150-153KFVI, N232K, A363N, A377C, Q346M, A17V, N105G, Q76E, I354V, VS133-1 34IA, S253K, V370I, SV297-298TE, EH286-286DS, A352S, ANS241-243VHE, S297T, A79I, T147S, I178M, I276V, Q331E, R257E, T339S, A79V, H104L, V74L, S107E, Q142E, R375K, S243E, R195K, Q22E, A328Q, G111A, R110K, I365V, VTE380-382SNA, more preferably,comprising one or more mutations selected from the group consisting of TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ5 7-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V. , In a preferred embodiment, the mutant of PS transposase is a mutant transposase as described in any embodiment herein. The "functional polypeptide" described herein refers to a polypeptide having its own function, such as a DNA sequence-specific or non-specific binding domain and / or a nuclear localization signal domain (NLS). The DNA sequence-specific or non-specific binding domain refers to a domain that functions based on DNA sequence specificity or non-specificity, such as a leucine zipper domain, a CRISPR / Cas domain, a TALE domain, a zinc finger domain, an AAV Rep DNA binding domain, or any combination thereof. The nuclear localization signal domain includes SV40 NLS, C-myc NLS, TAF1 NLS, TP53 NLS, STAT3 NLS, or any combination thereof. The amino acid sequences of these nuclear localization signal domains can be found in Chinese patent application CN202211150935.9, etc. In some embodiments, the leucine zipper domain is derived from the Basic_leucine-zipper_C protein family of IPR020983 in the InterPro protein database (https: / / www.ebi.ac.uk / interpro / ). family, the bZIP protein family of IPR004827, and the bZIP_sf protein family of IPR046347. In some embodiments, the leucine zipper domain is selected from C / EBPα (also referred to herein as C-EBPO or C / EBPO, whose amino acid sequence is shown in SEQ ID NO: 28, and the nucleotide sequence encoding it is shown in SEQ ID NO: 34), LZIP (whose amino acid sequence is shown in SEQ ID NO: 29, and the nucleotide sequence encoding it is shown in SEQ ID NO: 35), CREPT (whose amino acid sequence is shown in SEQ ID NO: 30, and the nucleotide sequence encoding it is shown in SEQ ID NO: 36), TEF (whose amino acid sequence is shown in SEQ ID NO: 31, and the nucleotide sequence encoding it is shown in SEQ ID NO: 37). In some embodiments, the DNA sequence-specific or non-specific binding domain may be located at the C-terminus or N-terminus of the transposase. In some embodiments, the nuclear localization signal domain may be located at the C-terminus or N-terminus of the transposase, preferably the N-terminus. In some embodiments, the DNA sequence-specific or non-specific binding domain and the nuclear localization signal domain may be located at the same end or different ends of the transposase. When located at the same end of the transposase, from the N-terminus to the C-terminus, it may be a DNA sequence-specific or non-specific binding domain-nuclear localization signal domain, or it may be a nuclear localization signal domain-DNA sequence-specific or non-specific binding domain. When located at different ends of the transposase, the DNA sequence-specific or non-specific binding domain and the nuclear localization signal domain may be located at the N-terminus and C-terminus of the transposase, or at the C-terminus and N-terminus, respectively. In an exemplary structure, as shown in Figure 2, when the C-myc NLS domain is present, from the N-terminus to the C-terminus are a leucine zipper domain, a C-myc NLS domain, and a transposase. By adding a leucine zipper domain, homodimerization can be mediated, thereby improving the transposition efficiency. In the fusion transposase, the transposase, the DNA sequence-specific or non-specific binding domain, and the nuclear localization signal domain can be directly connected or separated by a linker. Typically, the linker comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 50 amino acids. Exemplarily, the sequence of the linker is (GGGS) n or A(EAAAK) n A, n is a positive integer greater than 0, such as 1, 2, 3, 4 or 5; preferably, the sequence of the linker is GGGS. The sequence of the transposase described herein may be a modified polypeptide sequence. Modifications (usually without changing the primary structure) include: chemical derivatization of the polypeptide in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties. It should be understood that in gene cloning operations, it is often necessary to design suitable restriction sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed amino acid sequence, which does not affect the activity of the target sequence. In order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable regions in the protein of the recombinant protein, for example, including but not limited to suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Therefore, the amino terminus or carboxyl terminus of the transposase of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used herein. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6. These tags can be used to purify proteins. The JL transposase of the present invention has higher activity than the wild-type PS transposase. As used herein, "higher activity" may refer to increased transposition efficiency compared to the wild-type PS transposase having the amino acid sequence of SEQ ID NO: 1. Transposition efficiency can be measured according to the percentage of successful transposition events occurring in the host cell colony, and this percentage is normalized with the amount of the transposon and transposase introduced into this host cell colony.In many cases, when comparing the transposition efficiency of two or more transposases, for the transfection of host cells under the same or similar transfection conditions, the same transposon construct is paired with each of the two or more transposases.The amount of the transposition event in the host cell can be checked by various methods.For example, the transposon construct can be designed to contain the reporter gene between the terminal inverted repeats, and the transfected cells that this reporter gene is positive can be counted as the cells in which the successful transposition event occurs, which can draw the estimated value of the amount of the transposition event.In some embodiments, when comparing the transposition efficiency of two or more different transposons, the same transposase can be paired with every different transposon for the transfection of host cells under the same or similar transfection conditions.Similar methods can be used to measure transposition efficiency.Other methods well known to those skilled in the art can also be implemented to compare transposition efficiency. Polynucleotide molecule encoding JL transposase The present invention provides polynucleotide molecules encoding the JL transposase of the present invention. The present invention also provides a complementary sequence of the coding sequence of the transposase. The polynucleotide can be a recombinant nucleic acid molecule or a synthetic one; it can include DNA, RNA and PNA (peptide nucleic acid) and can be a hybrid thereof. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The present invention also includes degenerate variants of the polynucleotide sequence encoding the JL transposase, i.e., nucleotide sequences encoding the same amino acid sequence but different nucleotide sequences. In some embodiments, the polynucleotide molecule comprises a DNA encoding the JL transposase. In some embodiments, the polynucleotide is present in a nucleic acid construct such as a DNA vector. The vector can be a cloning vector or an expression vector. The expression vector comprises an expression cassette for the transposase, which is a nucleic acid construct that contains Promoter, transposase coding sequence and PolyA tailing signal sequence. The expression vector also generally contains other elements commonly contained in the vector, such as multiple cloning sites, resistance genes, replication initiation sites, etc. The nucleic acid construct may also contain other elements required for expression, including but not limited to enhancers, etc. The DNA vector includes conventional circular DNA plasmids, linear DNA plasmids, and also includes DNA forms such as minicircle plasmids, nanoplasmids, doggybone linear microplasmids that do not contain antibiotics and / or replicon DNA sequences. In some embodiments, the polynucleotide is present in a viral vector, including but not limited to AAV, slow virus, retrovirus, adenovirus, etc. In some embodiments, the DNA vector is a DNA microcarrier, the DNA backbone sequence of the microcarrier does not contain an antibiotic expression frame and is preferably limited to a length of 600bp, and / or does not contain a CpG DNA motif. In some embodiments, the polynucleotide comprises a messenger RNA (mRNA) encoding the JL transposase. In some embodiments, the mRNA is chemically modified, such as pseudouridine (ψ), N1-methyl pseudouridine (m1ψ), 5-methylcytosine nucleoside (m5C), 5-methoxyuridine (5moU), etc. The present invention also relates to polynucleotides that hybridize to the above-mentioned polynucleotide sequence and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, and more preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide. The polynucleotide molecules can be prepared by conventional methods in the art, and the corresponding vectors can be constructed, usually by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequences by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a long sequence can be obtained by first synthesizing multiple small fragments and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein. Alternatively, the sequences of the various parts of the fusion transposase can be obtained as above and then connected to obtain the full length of the CAR. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombinant methods. This is usually cloned into a vector, then transferred into cells, and then the relevant sequence is isolated from the host cell after propagation by conventional methods. Recombinant vectors can be constructed using methods well known to those skilled in the art, see, for example, Sambrook et al., Ausubel (1989) or other standard textbooks. The vector containing the nucleic acid molecule of the present invention can be transferred to the host cell by well-known methods, which varies according to the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook et al. In addition, the protein encoding the present invention (or its fragment, or its derivative) can be obtained entirely by chemical synthesis. DNA sequence. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis. The present invention also relates to nucleic acid constructs comprising the nucleic acid sequences of the polynucleotide molecules described herein, and one or more regulatory sequences operably linked to these sequences. The nucleic acid constructs of the present invention can be manipulated in a variety of ways to ensure expression of the transposase. The nucleic acid constructs can be manipulated prior to insertion into a vector depending on the expression vector or requirements. The regulatory sequences and manipulations required to express proteins via DNA or mRNA are known in the art. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art. In certain embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector. Expression of the polynucleotide sequence of the present invention is usually achieved by operably connecting the polynucleotide sequence of the present invention to an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Integration vectors contain components that integrate the target sequence into the cell genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors usually contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) usually include one or more of the following nucleotide sequences: promoters, one or more enhancer sequences, replication origins, transcription termination sequences, complete intron sequences containing donor and acceptor splice sites, sequences encoding leader sequences for polypeptide secretion, ribosome binding sites, polyadenylation sequences, multiple linker regions for inserting nucleic acids encoding antibodies to be expressed, and selectable marker elements. The type of vector is not limited, for example, plasmid, phagemid, phage derivative, animal virus and cosmid, which can be changed according to the host cell to be introduced. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses. To assess expression of the transposase, the vector introduced into the cells may also contain either or both a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected by the viral vector. JL Transposon Compared with the wild-type PS transposon, the JL transposon of the present disclosure comprises any one, two or three of the following features: (1) a nucleotide mutation in at least one terminal inverted repeat sequence (i.e., a mutant terminal inverted repeat sequence); (2) a repeated terminal inverted repeat sequence at least at one end; and (3) a repeated TA sequence outside the terminal inverted repeat sequence at at least one end. In some embodiments, the mutant terminal inverted repeat sequence has 1-5, preferably 1-4, more preferably 1-3 nucleotide mutations compared to SEQ ID NO: 2 (wild-type PS transposon 5'TIR) or SEQ ID NO: 3 (wild-type PS transposon 3'TIR); and / or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 or 3. In some embodiments, the mutant terminal inverted repeat sequence is selected from but not limited to any one of SEQ ID NO: 4-27, or contains any combination of nucleotide mutations of SEQ ID NO: 4-27 compared to wild-type PS TIR (referred to as a combined mutant sequence); or is the reverse complementary sequence of SEQ ID NO: 4-27 or the combined mutant sequence. Preferably, the mutant terminal inverted repeat sequence is selected from SEQ ID NO: 4-6 and the reverse complementary sequence thereof. It should be understood that one of the terminal inverted repeat sequences at both ends of the transposon may have a nucleotide mutation, or multiple or all of them may have a nucleotide mutation, and the nucleotide mutations of each terminal inverted repeat sequence may be the same or different. In some embodiments, the JL transposon terminal inverted repeat sequences are each independently selected from SEQ ID NOs: 3-27 or sequences that are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NOs: 3-27, or their reverse complements. In one or more embodiments, the transposon DNA sequence has multiple terminal inverted repeats (i.e., multiple copies of terminal inverted repeats in series) at one or both ends, which can be expressed as (TIR)n, n≥2, for example, 2, 3, 4, etc. The multiple includes but is not limited to 2 times, 3 times or 4 times, etc. The number of copies of the terminal inverted repeats at both ends may be the same or different, for example, each end independently has 1, 2, 3, 4 or more segments of repeated terminal inverted repeats, and both ends are not 1 segment of terminal inverted repeats at the same time. The repeated terminal inverted repeats may or may not contain the above-mentioned nucleotide mutations compared to the wild-type terminal inverted repeats. In some embodiments, the transposon DNA includes 2 times of terminal inverted repeats at both ends, and the structural schematic diagram is shown in Figure 4, wherein 5'PST refers to the TIR at the 5' end, 3'PST refers to the TIR at the 3' end, 5'PST5'PST and 3'PST3'PST refer to 2 times of terminal inverted repeats. The terminal inverted repeat sequence of JL transposon is short. By increasing the number of terminal inverted repeat sequences and extending the sequence length that can be recognized by JL transposase, the transposition efficiency can be improved. In one or more embodiments, the terminal inverted repeat sequences at both ends of the transposon DNA sequence have a cleavage site sequence for a transposase, and the cleavage site sequence is TA (nucleotide sequence). The terminal inverted repeat sequence may or may not contain the above-mentioned nucleotide mutation compared to the wild-type terminal inverted repeat sequence. In one or more embodiments, one or both ends of the transposon DNA have a multi-copy cleavage site sequence, expressed as (TA)m, m≥2, and the multi-copy value m is preferably 2, 3 or 4. The number of TA repeats at both ends can be the same or different, for example, each end independently has 1, 2, 3, 4 or more repeated TA sequences. In some embodiments, the transposon DNA includes 2 times of cleavage site sequences at both ends, and the structural schematic diagram is shown in Figure 4. Wherein TA refers to the cleavage site sequence, and TATA refers to 2 times of the cleavage site sequence. In some embodiments, the transposon DNA includes 4 times of the cleavage site sequence, i.e., TATATATA. By increasing the number of cleavage site sequences and increasing the cleavage sites of the JL transposase, the transposition efficiency can be improved. In one or more embodiments, the JL transposon includes at the left end (TA) m (TIR)n , including (TIR) ​​on the right side p (TA) q , wherein m, n, p, q are integers, m≥1, preferably 2-4, n≥1, preferably 2-4, p≥1, preferably 2-4, q≥1, preferably 2-4, TIR can be a wild-type PS transposon TIR or a mutant TIR, and when TIR is a wild-type TIR, m, n, p, q are not 1 at the same time (i.e., at least one of m, n, p, q is ≥2). In addition, when TIR is repeated, each repeated TIR can be independently selected from any sequence of the wild-type TIR sequence and its mutant. In a preferred embodiment, m=2-4, preferably 4, n=2, p=2-4, preferably 4, and q is 2. In a more preferred embodiment, TIR is a mutant TIR, m=2-4, preferably 4, and n=2. In one or more embodiments, the JL transposon DNA sequence preferably includes (TA) 2 (TIR1) 2 , (TA) 3 (TIR1) 2 , or (TA) 4 (TIR1) 2 , wherein TIR1 is a mutant TIR, most preferably (TA) 4 (TIR1) 2 The nucleotide sequence is shown in SEQ ID NO: 32. In one or more embodiments, one or both ends of the JL transposon DNA sequence contain (TA) 4 (TIR1) 2 . The JL transposon may comprise a combination of DNA elements located between the terminal inverted repeats, such as a target gene expression cassette. The combination of DNA elements includes, but is not limited to, sequence elements known to those skilled in the art such as promoters, enhancers, target genes (expressed genes), 5-UTRs, 3-UTRs, etc. As used throughout this article, the term "exogenous gene" or "target gene" refers to a nucleic acid or protein sequence that is not naturally associated with a target gene or a host cell that is an object of introduction, including a naturally occurring nucleic acid sequence located in a non-naturally occurring genomic location, or a non-naturally occurring multiple copy of a naturally occurring nucleic acid (e.g., a DNA sequence). In some embodiments, the DNA element combination includes an expressed gene. Accordingly, the DNA element combination may also include a promoter selected from, but not limited to, CMV, EFS, MND, EF1α, CAGC, PGK, UBC, U6, H1, and Cumate. In some embodiments, the expressed gene encoding is selected from a protein selected from a cell receptor, an immune checkpoint protein, a cytokine, and any combination thereof. In some embodiments, the expressed gene encoding is selected from a protein selected from a cell receptor, an immune checkpoint protein, a cytokine, a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor, and any combination thereof. In some embodiments, the expressed gene encodes a CAR. The CAR may include an antigen binding domain, a hinge region, a transmembrane region, and an intracellular signaling region in sequence. The hinge region, transmembrane region, and intracellular signaling region known in the art for constructing a CAR may be used to construct the CAR of the present invention. Typically, the antigen binding domain is capable of binding to the tumor with moderate affinity. The membrane antigen widely expressed by cells, the polypeptide is usually inserted with an antigen epitope, and the insertion position is selected from any one, two or three of the following three positions: the N-terminus of the polypeptide, between the polypeptide and the hinge region, and inside the polypeptide. The antigen binding domain can be a natural polypeptide or an artificially synthesized polypeptide. The chimeric antigen receptor can be directed against one or more of the following antigens: CD19, CD20, CEA, GD2, FR, PSMA, PMEL, CA9, CD171 / L1-CAM, IL-13RL1, MART-1, ERBB2, NY-ESO-1, AFP, MUC1, CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, IL-11R / , EGP-2, EGP-40, FBP, GD3, PSCA, FSA, PSA, HMGA2, LeY, EpCAM, MSLN, IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC, AFU, EBV-VCA, POA and PROGRP. In some embodiments, the antigen binding domain may comprise an antibody, an antibody mimetic, a protein scaffold, or a fragment thereof. In certain embodiments, the antibody is a chimeric antibody, a recombinant antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is affinity-tuned. Non-limiting examples of antibodies of the present invention include single-chain variable fragments (scFv), VHH, single-domain antibodies (sdAB), small modular immunopharmaceuticals (SMIP) molecules, or nanobodies. In certain embodiments, VHH is of the camelid family. Alternatively or in addition, in certain embodiments, VHH is humanized. Non-limiting examples of antibody fragments of the present invention include complementary determining regions, variable regions, heavy chains, light chains, or any combination thereof. Non-limiting examples of antibody mimetics of the present invention include: affibodies, Afflilin molecules, affimers, Affitin molecules, alphabodies, anticalins, and Avimer molecules, DARPins, Fynomers, Kunitz domain peptides, or monobodies. Non-limiting examples of protein scaffolds of the present invention include Centyrin. In some embodiments, CAR suitable for the present invention can refer to CN 202111681582.0, the entire contents of which are incorporated herein by reference. In some embodiments, the expressed gene encodes an immune checkpoint inhibitor, such as a PD-1 antibody, a CTLA-4 antibody, etc. Non-limiting examples of the antibody include a single-chain variable fragment (scFv), a VHH, a single domain antibody (sdAB), a small modular immunopharmaceutical (SMIP) molecule, or a nanobody. In some embodiments, the PD-1 antibodies suitable for use in the present invention can be referenced to CN 202111681582.0, the entire contents of which are incorporated herein by reference. In some embodiments, the expressed genes encode CAR and PD-1 antibodies as described in CN202111681582.0. Transposons are usually provided in the form of nucleic acid constructs, particularly DNA vectors. In this regard, any DNA vector that is convenient for introducing transposons into cells can be used, including but not limited to conventional circular DNA plasmids, linear DNA plasmids, minicircle plasmids, nanoplasmids, Doggybone, etc., which do not contain antibiotics or / and replicon DNA. The DNA form of the sequence, etc. In some embodiments, the DNA vector is a DNA microcarrier, the DNA backbone sequence of the microcarrier does not contain an antibiotic expression frame and the length is preferably limited to 600bp, and / or does not contain a CpG DNA motif. In some embodiments, the DNA vector is an anti-microplasmid, that is, a microplasmid without an antibiotic resistance gene (a microplasmid without an antibiotic expression frame), also known as a tiny plasmid (tiny or tiniplasmid). The anti-microplasmid suitable for the present invention can refer to the Chinese patent application entitled "Anti-microplasmid and its preparation method and application" filed by Shanghai Jiliang Pharmaceutical Engineering Co., Ltd. on January 13, 2023 (application number: 202310072956.1), and this application incorporates its entire contents into this article by reference. In some embodiments, the anti-microplasmid comprises a nucleotide sequence encoding an antitoxin protein and a replicon; the amino acid sequence of the antitoxin protein comprises the following sequence: (1) the amino acid sequence as shown in SEQ ID NO: 49, or an amino acid sequence having one or more mutations of E24D, I35V, V43I compared to SEQ ID NO: 49; or (2) the amino acid sequence as shown in SEQ ID NO: 50, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A, N54K compared to SEQ ID NO: 50; the length of the replicon is ≤ 800 bp, preferably ≤ 600 bp or ≤ 300 bp. In some embodiments, the amino acid sequence of the antitoxin protein is as shown in any one of SEQ ID NOs: 49-55. In some embodiments, the replicon is selected from ColE1, ColE2, pMB1, pSC101, RSF, R6K, pUC57, RK2 and p15A; preferably R6K or pUC57. In some embodiments, the length of the plasmid backbone of the anti-microplasmid is ≤1000bp, preferably ≤900bp, ≤800bp or ≤600bp. In some embodiments, the nucleotide sequence encoding the antitoxin protein does not contain a CpG motif, preferably, the nucleotide sequence encoding the antitoxin protein is as shown in SEQ ID NO: 56 or SEQ ID NO: 57. In some embodiments, the nucleotide sequence of the replicon does not contain a CpG motif. In a preferred embodiment, the length of the backbone sequence of the anti-microplasmid is ≤600bp, and the replicon is a R6K replicon without a CpG motif. The nucleotide sequence of the R6K replicon without a CpG motif is, for example, as shown in SEQ ID NO: 58. In some embodiments, the nucleotide sequence without anti-microplasmid (empty vector) is shown in SEQ ID NO: 59 or 60; the map structure is shown in Figure 20 or 21. Transposon system The present invention also relates to a system for genome editing (transposon system), which comprises a transposase and a transposon that can be recognized by the transposase. In some embodiments, a transposon system is provided, which comprises a JL transposase or a polynucleotide encoding it, and a transposon that can be recognized by the transposase. In some embodiments, a transposon system is provided, which comprises a JL transposon and a transposase that can recognize the transposon or a polynucleotide encoding it. In some embodiments, the transposon system provided is a JL transposon system, which comprises: (1) a JL transposase as described herein, or a polynucleotide encoding it, and (2) a JL transposon or a wild-type PS transposon as described herein. In some embodiments, the transposon terminal inverted repeat sequences of the transposon system are each independently selected from SEQ ID NOs: 2-27 or a sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NOs: 2-27, or their reverse complements. In some embodiments, the transposon may be present in an expression vector. In many cases, the expression vector may be a DNA plasmid, including but not limited to conventional circular DNA plasmids, linear DNA plasmids, minicircle plasmids, nanoplasmids, Doggybone, etc., which do not contain antibiotics or / and replicon DNA sequences. Sometimes, the expression vector may be a minicircle vector. As used herein, the term "minicircle vector" may refer to a minicircle plasmid derivative that does not contain most (if not all) prokaryotic vector parts (e.g., control sequences or non-functional sequences of prokaryotic origin). In some cases, the toxicity to cells produced by transfection or electroporation may be mitigated by using a "minicircle" as described herein. In some embodiments, the expression vector is a DNA microcarrier, the DNA backbone sequence of which does not contain an antibiotic expression cassette and is preferably limited in length to within 600 bp, and / or does not contain a CpG DNA motif. In some embodiments, the expression vector is an anti-microplasmid of any of the above embodiments. In some embodiments, the polynucleotide encoding the transposase of the transposon system may be DNA, which may be present in an expression vector, and may be present in the same expression vector or in a different expression vector as the transposon. The expression vector may be as described in any embodiment herein. In some embodiments, the polynucleotide encoding the transposase of the transposon system may be a messenger RNA (mRNA). mRNA can be produced by many methods well known to those of ordinary skill in the art, such as but not limited to in vivo transcription and RNA purification, in vitro transcription and de novo synthesis. In many cases, mRNA can be chemically modified. Chemically modified mRNA can be more resistant to degradation than unmodified or natural mRNA, or can be degraded faster. In many cases, chemical modification of mRNA can enable mRNA to be translated with higher efficiency. Chemical modification of mRNA can be performed using known techniques available to those skilled in the art or by commercial suppliers. The JL transposase, JL transposon or JL transposon system disclosed in the present invention has significantly improved integration efficiency and transgene expression compared to wild-type PS transposase, PS transposon or PS transposase system. The JL transposase disclosed in the present invention can be applied to stable transfection cells, cell line development, genome modification, gene therapy, cell therapy, transgenic animals and other aspects in the form of virus, plasmid DNA, mRNA or protein. Host cells As used herein, "host cell" refers to a prokaryotic or eukaryotic cell that is capable of replicating a vector and / or expressing an exogenous gene encoded by a vector when expressing an exogenous nucleic acid sequence. A host cell can serve as a recipient of a vector or mRNA. A host cell can be "transfected" or "transformed," which refers to the process by which an exogenous nucleic acid is transfected or transduced into a host cell. Transformed cells include the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells often refer to cells into which an exogenous nucleic acid sequence, such as a vector or mRNA, has been introduced. Thus, recombinant cells are distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acid. Herein, host cells include cells that carry and / or produce transposases or their coding sequences, transposons or transposon systems described herein. Specifically, the present invention provides cells that carry the transposases and / or their coding sequences described herein. The cells may also contain a nucleic acid sequence encoding a transposon that can be recognized by the transposase. The type of cell is not limited, as long as it can express the transposase described herein or can use the transposase described herein to achieve transposition of the transposon. In some embodiments, the cell is a primary cell isolated from a subject. The subject is a healthy subject or has been diagnosed with a disease (e.g., cancer or tumor). In some embodiments, the cells are isolated from the blood of the subject, such as PBMC or cells derived therefrom. The cells may comprise primary immune cells, such as primary leukocytes. In some embodiments, the cells comprise primary T cells. The primary T cells comprise γδT cells, helper T cells, memory T cells, natural killer T cells, effector T cells, or any combination thereof. In some embodiments, the primary immune cells comprise CD3+ cells. In some embodiments, the cell comprises a stem cell. The stem cell is selected from the group consisting of embryonic stem cells, hematopoietic stem cells, epidermal stem cells, epithelial stem cells, bronchial alveolar stem cells, mammary stem cells, mesenchymal stem cells, intestinal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, testicular cells, and any combination thereof. The stem cell comprises an induced pluripotent stem cell. The transposase of the present invention or its coding sequence, transposon or transposon system can be introduced into the cell of interest. Herein, the method of introduction includes transfection of the cell by means of electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposomes, lipid nanoparticles (LNP), microparticle bombardment, fugene, direct sonic loading, cell extrusion, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection or any combination thereof. In certain embodiments, introduction is by electroporation. The obtained transformant can be cultured by conventional methods to express the JL transposase of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. In the embodiment of using an inducible promoter to express the transposase, after the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time. The polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography technology and a combination of these methods. method The present invention also relates to a method for preparing a cell, comprising the step of introducing the JL transposon system described herein into the cell. In some embodiments, the introducing comprises contacting the cell with a polynucleotide encoding the JL transposase. The polynucleotide comprises DNA or messenger RNA (mRNA) encoding the JL transposase. In some embodiments, the introducing comprises contacting the cell with the JL transposase, preferably providing the transposase protein by adding the JL transposase directly to a culture medium containing the cell (preferably to a cell culture medium of cells of a target organism). In direct contact of the JL transposase with the target cell according to the present invention, no reagent, carrier or method that alters the penetration of the protein across the cell membrane may be used. In some embodiments, the introduction includes contacting the cell with a DNA vector containing the transposon. In some embodiments, the DNA vector includes a microcarrier plasmid, preferably the DNA backbone sequence of the microcarrier does not contain an antibiotic expression frame and is preferably limited to 600bp in length, and / or does not contain a CpG DNA motif. In some embodiments, the introduction includes contacting the cell with a plasmid vector containing the transposon and a polynucleotide encoding the JL transposase. In some embodiments, the introduction includes contacting the cell with an mRNA encoding the transposase and a plasmid vector containing the transposon. In some embodiments, the introduction includes contacting the cell with a plasmid vector containing the polynucleotide and the transposon. In some embodiments, the introduction includes contacting the cell with a plasmid vector containing the polynucleotide and the transposon. In some embodiments, the introduction includes contacting the cell with a plasmid vector containing the polynucleotide and a plasmid vector containing the transposon. In some embodiments, the transposon system described herein can be delivered to cells by viruses, including retroviruses (such as lentiviruses, etc.), adenoviruses, adeno-associated viruses (AAV), herpes viruses, etc. The transposon and transposase genes can be contained together on the same recombinant viral genome; one infection can deliver the two parts of the transposon system, so that the expression of the transposase can guide the transposon to cleave from the recombinant viral genome and then insert into the cell chromosome. In another example, the transposase and the transposon can be delivered separately by a combination of viruses and / or non-viral systems (such as lipid-containing reagents). In these cases, the transposon and / or the transposase gene can be delivered by a recombinant virus. In each case, the expressed transposase gene guides the transposon to be liberated from its carrier DNA (viral genome) and inserted into the chromosomal DNA. The JL transposase of the present invention is very small and is particularly suitable for delivery using AAV. The present invention also provides a method for genetically engineering cells using a transposase, wherein the method involves allowing the transposase to penetrate the cell membrane but does not include a protein transfection step, and in particular, the method does not include the use of a protein transfection reagent or procedure to introduce the transposase protein into the cell. The method comprises introducing the transposase into the cell membrane and the cell membrane to be transfected with the transposase. The method of the present invention comprises the step of introducing the transposase protein without using any vector, reagent or method that changes the penetration of the protein across the cell membrane. The term "protein transfection" in the context of the present invention should be understood to broadly refer to any method or reagent that is sufficient to introduce a protein that cannot effectively enter a target cell into the target cell. Common protein transfection systems and reagents include commercial protein transfection reagents such as PULSin TM 、ProteoJuice TM 、Xfect TM and Pierce TM Protein transfection reagent (ThermoFisher), TransPass TM , and methods such as protein electroporation. The present invention also provides transgenic animals and transgenic cells obtained by the above method. Also provided herein is a method of treatment, comprising: (a) introducing a transposon system into a cell, thereby generating a genetically modified cell comprising a transgene introduced by the transposon; (b) administering the genetically modified cell to a patient in need of the treatment. In some embodiments, the patient has been diagnosed with cancer or a tumor. In some embodiments, the administration comprises infusing the genetically modified cell into a blood vessel of the patient. The present invention also relates to the use of the JL transposase described herein, its coding sequence (DNA or RNA) or nucleic acid construct, cell, or genome editing system in the preparation of products, such as gene editing kits, engineered immune cells or pharmaceutical compositions. The immune cells include γδT cells, helper T cells, memory T cells, natural killer T cells, effector T cells, etc. The scope of the present invention also encompasses a kit comprising the JL transposase described herein, its coding sequence (DNA or RNA) or nucleic acid construct, cell, or genome editing system. The kit also comprises one or more of a nucleic acid encoding a transposon recognizable by the JL transposase or a nucleic acid construct thereof (e.g., a DNA vector comprising the transposon), a host cell, a cell culture medium suitable for the host cell, a cytokine, and instructions for use. Pharmaceutical compositions and administration The transposase, nucleic acid molecule, transposon, transposon system and cell of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as related cytokines or cell groups. Therefore, the present invention also provides a pharmaceutical composition comprising a transposase, nucleic acid construct, gene editing system or cell prepared by the method described herein and a pharmaceutically acceptable excipient. In the present invention, "pharmaceutically acceptable excipients" are pharmaceutically or food-acceptable carriers, solvents, suspending agents or excipients used to deliver the transposase, nucleic acid molecule, transposon, transposon system or cell of the present invention to animals or humans. In this article, pharmaceutically acceptable excipients are non-toxic to the recipient of the composition at the dosage and concentration used. They may include various types of carriers or excipients commonly used in the treatment of proteins, nucleic acids or cells known in the art. Exemplary excipients may be liquid or solid, including but not limited to: pH adjusters, surface Surfactant, carbohydrate, adjuvant, antioxidant, chelating agent, ionic strength enhancer, preservative, carrier, glidant, sweetener, dye / colorant, flavor enhancer, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier. In some embodiments, pharmaceutically acceptable excipients may include one or more inactive ingredients, including but not limited to: stabilizer, preservative, additive, adjuvant, spray, compressed air or other suitable gas, or other suitable inactive ingredients used with pharmacodynamic compounds. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition, A.R. Genrmo, 1990, Mack Publishing Company. The optimal pharmaceutical composition can be determined by the expected route of administration, delivery mode and required dosage. The pharmaceutical composition of the present invention can be selected for parenteral delivery, for inhalation or delivery through the digestive tract (such as oral), for example, for intravenous infusion delivery. The preparation of the composition is within the technology of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing immune cells, particularly immune cells (e.g., T cells), in sustained or controlled release delivery formulations. The pharmaceutical composition of the present invention can also be administered in a manner suitable for the disease to be treated (or prevented). Pharmaceutical compositions for in vivo administration are usually provided in the form of sterile preparations. Sterilization is achieved by filtering through a sterile filtration membrane. Compositions for parenteral administration can be stored in lyophilized form or in solution (e.g., frozen preparations). Parenteral compositions are usually placed in containers with sterile access holes, such as intravenous solution strips or vials with stoppers that can be pierced by hypodermic needles. Once the pharmaceutical composition is formulated, it is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal, frozen material or in a dehydrated or lyophilized powder. The pharmaceutical formulation (e.g., a frozen preparation) can be stored in a ready-to-use form or in a form that is further formulated before administration. For example, suitable for delivering the pharmaceutical composition described herein can be a frozen preparation, which can withstand long-distance transportation without damaging cells. In addition to the cells themselves, the frozen preparation generally includes components such as cell freezing solution and human serum albumin (HSA). Before administration (e.g., intravenous infusion), the frozen pharmaceutical composition needs to be stored at low temperatures (e.g., placed in liquid nitrogen). After thawing, the frozen preparation can be directly or formulated as an infusion composition for infusion to the patient. Those skilled in the art are aware of the components and concentrations of conventional freezing solutions. For example, the freezing solution or infusion composition may also contain dimethyl sulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride or magnesium chloride, etc., and its concentration may be determined by those skilled in the art (e.g., experienced physicians) according to conditions such as cells, diseases, and patients. In some embodiments of the invention, the genetically modified cells of the invention or compositions thereof may be combined with other therapies known in the art. "Patient", "subject", "individual" and the like are used interchangeably herein and refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof. application The subject matter presented herein can be widely applied to applications related to genome editing, such as the Preparation, gene therapy, cell therapy, etc. A non-limiting example relates to the generation of genetically modified cells for research and clinical applications. For example, as described above, genetically modified T cells can be prepared using the subject matter provided herein, which can be applied to help people resist a variety of diseases, such as, but not limited to, cancer and infectious diseases. A specific example includes using the method provided herein to produce genetically modified primary leukocytes, and applying the genetically modified primary leukocytes to patients in need. The generation of genetically modified primary leukocytes can include introducing a transposon system into leukocytes, thereby producing genetically modified leukocytes. In many cases, the transposon may include a transgene. The transgene can be a cell receptor, an immune checkpoint protein, a cytokine, an antibody, and any combination thereof. Sometimes, a cell receptor may include but is not limited to a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof. In some other cases, the transposon system is designed to delete or modify endogenous genes, such as cytokines, immune checkpoint proteins, oncogenes, or any combination thereof. The genetic modification of primary leukocytes can be designed to promote immunity to infectious pathogens or cancer cells that make patients in a sick state. Another non-limiting example relates to the generation of genetically modified organisms for agriculture, food production, medicine and pharmacy. The species range that can be genetically modified is very wide, including but not limited to plants and animals. Genetically modified organisms, such as genetically modified crops or livestock, can be modified in some aspects of their physiological properties. The example in edible crops includes resistance to certain pests, diseases or environmental conditions, the reduction of corruption, or resistance to chemical treatment (such as resistance to herbicides), or improves the nutritional profile of crops. The example in non-edible crops includes the production of medicaments, biofuels and other industrially useful commodities, and is used for bioremediation. The example in livestock includes the resistance to certain parasites, the generation of certain nutrients, the increase of growth rate and the increase of milk production. Partial embodiments of the present invention 1. A transposase, which is a mutant transposase or a fusion transposase, The mutant transposase comprises an amino acid mutation compared to the wild-type PS transposase as shown in SEQ ID NO: 1, wherein any one or more amino acids in the double-stranded DNA binding and oligomerization domain of the wild-type PS transposase are mutated into positively charged amino acids; and / or the amino acid mutation comprises one or more mutations selected from the group consisting of TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129R 29K, TQS57-59KKA\I98K, TQS57-59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N213 D. H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V; The fusion transposase is a wild-type PS transposase or a mutant thereof fused with a functional polypeptide, wherein the functional polypeptide is: a DNA sequence-specific or non-specific binding domain, and / or a cell nucleus localization signal domain. 2. The transposase according to item 1, wherein the mutant comprises one or more mutations selected from the group consisting of TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K 32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I 35V, N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R , K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I28 4L, K45R, H215E, H215Q, I237V, Q69R, L100I, K96R, Q162R, A271K, R372K, H20Y, TYCR150-153KFVI, N232K, A363N, A377C, Q346M, A17V, N105G, Q76E, I354V, VS133-134IA, S253K, V370I, SV297-298TE, EH286-286DS, A352S , ANS241-243VHE, S297T, A79I, T147S, I178M, I276V, Q331E, R257E, T339 S, A79V, H104L, V74L, S107E, Q142E, R375K, S243E, R195K, Q22E, A328Q, G 111A, R110K, I365V, VTE380-382SNA, R190K, RA372-373KS, K73L, K121R, R102K, C152V, IY276-277VW, V292I, K73E, H104M, I239V, K42E, K356Q, T1 50K, G316S, IY259-260VW, V53M, E108D, V53L, DKV72-74EEL, M280F, S85G , N63K, V91L, K251L, H124N, K308S, N10D, H7R, G112S, H7K, TA327-328KE. 3. The transposase according to item 1 or 2, wherein the mutant comprises one or more mutations selected from the group consisting of TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ5 7-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59KKA\I98K\ T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K , Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N2 13D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V, Q69R, L100I, K96R, Q162R, A 271K, R372K, H20Y, TYCR150-153KFVI, N232K, A363N, A377C, Q346M, A17V, N105G, Q76E, I354V, VS133-134IA, S253K, V370I, SV297-298TE, EH286-286DS, A352S, ANS241-243VHE, S297T, A79I, T147S, I178M, I276V , Q331E, R257E, T339S, A79V, H104L, V74L, S107E, Q142E, R375K, S243E, R195K, Q22E, A328Q, G111A, R110K, I365V, VTE380-382SNA. 4. The transposase according to any one of items 1 to 3, characterized in that the DNA sequence-specific or non-specific binding domain comprises a leucine zipper domain, a CRISPR / Cas domain, a TALE domain, a zinc finger domain, an AAV Rep DNA binding domain or any combination thereof, preferably, the amino acid sequence of the leucine zipper domain is shown in any one of SEQ ID NOs: 28-31, and / or The nuclear localization signal domain includes SV40 NLS, C-myc NLS, TAF1 NLS, TP53 NLS, STAT3 NLS or any combination thereof, and / or The functional polypeptide and the wild-type PS transposase or its mutant may or may not contain a linker. 5. A polynucleotide encoding the transposase according to any one of items 1 to 4; preferably, the polynucleotide is DNA or messenger RNA. 6. A transposon that can be recognized by the transposase according to any one of items 1 to 4, wherein the transposon has any one, two or three characteristics selected from the following (1) to (3): (1) The transposon has a mutant terminal inverted repeat sequence, and the mutant terminal inverted repeat sequence has 1-5 nucleotide mutations compared with the wild-type PS transposon terminal inverted repeat sequence shown in SEQ ID NO: 2 or 3; (2) The transposon has two or more terminal inverted repeat sequences at at least one end; (3) The transposon has two or more repeats of TA sequences outside the terminal inverted repeat sequence at at least one end. 7. The transposon according to item 6, wherein the mutant terminal inverted repeat sequence is as shown in any one of SEQ ID NOs: 4-27, preferably as shown in any one of SEQ ID NOs: 4-6, or is a nucleotide sequence containing any combination of nucleotide mutations in SEQ ID NOs: 4-27, or is their reverse complementary sequences. 8. The transposon according to item 6 or 7, wherein the transposon has two terminal inverted repeat sequences at each end, and includes four TA sequence repeats outside the terminal inverted repeat sequences at each end. 9. A transposon as described in any one of items 6-8, wherein the transposon comprises a target gene expression frame located between terminal inverted repeat sequences, and preferably, the target gene encodes a protein selected from cell receptors, immune checkpoint proteins, cytokines, T cell receptors, B cell receptors, chimeric antigen receptors and any combination thereof. 10. A transposon system, comprising the transposase described in any one of items 1 to 4 or the polynucleotide encoding the transposase described in item 5; and a transposon, wherein the transposon is a PS transposon or a polynucleotide encoding the transposase described in any one of items 6 to 9. The transposon described. 11. In the transposon system as described in Item 10, the polynucleotide encoding the transposase is mRNA. Preferably, the mRNA is chemically modified, including but not limited to pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine nucleoside (m5C), 5-methoxyuridine (5moU), etc. 12. The transposon system according to item 10 or 11, wherein the polynucleotide encoding the transposase and / or the transposon are present in a DNA vector, preferably, the DNA vector is a plasmid vector or a minicircle DNA vector, preferably a non-antimicrobial plasmid vector; and / or the DNA vector is a minicarrier, the backbone sequence of which is within 600 bp in length, and has reduced and / or no CpG DNA motif; or The polynucleotide encoding the transposase and / or the transposon are present in a viral vector. Preferably, the viral vector is selected from an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a herpes simplex viral vector or a vaccinia viral vector. 13. The transposon system according to item 12, wherein the polynucleotide encoding the transposase and the transposon are present in the same vector or in different vectors. 14. A host cell comprising or capable of producing the transposase according to any one of items 1 to 4, the polynucleotide according to item 5, the transposon according to any one of items 6 to 9, or the transposon system according to any one of items 10 to 13. 15. A method for preparing a cell, comprising: introducing the transposase according to any one of items 1 to 4, the polynucleotide according to item 5, the transposon according to any one of items 6 to 9, or the transposon system according to any one of items 10 to 13 into the cell, Preferably, The introducing comprises contacting the cell with the polynucleotide, and / or The introducing comprises contacting the cell with a DNA vector containing the transposon, and / or The introducing comprises contacting the cell with mRNA encoding the transposase and a plasmid vector containing the transposon, and / or The introducing comprises contacting the cell with a plasmid vector containing the polynucleotide and the transposon, and / or The introducing comprises contacting the cell with a plasmid vector containing the polynucleotide and a plasmid vector containing the transposon, and / or The introducing comprises contacting the cell with the transposase. 16. The method according to item 15, characterized in that the cells are from animals, such as vertebrates or invertebrates, preferably mammals, and more preferably humans; and / or the cells are immune cells, preferably T cells. 17. The method of item 15 or 16, characterized in that the introduction comprises electroporation, microscopy The cells are transfected by injection, calcium phosphate precipitation, cationic polymers, dendrimers, liposomes, lipid nanoparticles, microparticle bombardment, fugene, direct sonication, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection, virus-mediated transformation, or any combination thereof. 18. The method of item 16 or 17, wherein the introducing comprises electroporating the cells. 19. The method of item 18, characterized in that the introduction comprises contacting the cell with mRNA encoding the transposase and a plasmid containing the transposon, preferably, the plasmid is a microcarrier, the backbone sequence length of which is within 600 bp, with reduced and / or no CpG DNA motif, and / or the plasmid is a non-antimicrobial plasmid. 20. A kit comprising: the transposase according to any one of items 1 to 4, the polynucleotide according to item 5, the transposon according to any one of items 6 to 9, or the transposon system according to any one of items 10 to 13, or the cell according to item 14. 21. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and cells prepared by the method according to any one of items 15 to 19. 22. Use of the transposase according to any one of items 1 to 4, the polynucleotide according to item 5, the transposon according to any one of items 6 to 9, the transposon system according to any one of items 10 to 13, the cell according to item 14, or the pharmaceutical composition according to item 21, which is selected from any one of the following (1) to (6): (1) Application in the preparation of drugs or reagents for integrating the target gene expression cassette into the host cell genome; (2) Application in the preparation of tools for integrating target gene expression cassettes into the host cell genome; (3) Application in the preparation of transgenic animals and transgenic cells; (4) Application in the preparation of drugs or preparations for genome research, gene therapy, cell therapy, or stem cell induction and differentiation after induction; (5) Application in the preparation of tools for genome research, gene therapy, cell therapy, or stem cell induction and post-induced differentiation; (6) Application in the preparation of kits, engineered immune cells or pharmaceutical compositions. The present invention is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should never be construed as being limited to the following examples, but should be construed as including any and all variations that become apparent due to the teachings provided herein. The methods and reagents used in the examples, unless otherwise stated, are conventional methods and reagents in the art. Example This article adopts the following method. (1) Site-directed mutagenesis for preparation of JL mutants High-Fidelity DNA Polymerase (New England BioLabs) was used for all site-directed mutagenesis. For single-point mutations, rolling circle PCR was performed for mutagenesis, followed by digestion with DpnI restriction endonuclease, and 5 μL of the digestion reaction product was transformed into TOP10 E. coli competent cells. For combined mutations, multiple PCR reactions were used for mutagenesis, and the PCR product was purified using an agarose gel DNA recovery kit (Tianmo Bio) and purified using Hieff Plus Multi One Step Cloning Kit (YEASEN) was used to perform 10 μL ligation reaction on the purified PCR product, and 5 μL of the ligation reaction product was transformed in TOP10 E. coli competent cells. For each mutant, three single clones were selected for culture, and half of each was taken for sequencing identification and bacterial preservation. For mutants with correct identification sequences, the NucleoBond Xtra MiDi EF Plasmid Preparation Kit (MACHEREY-NAGEL) was used to prepare plasmids for transfection. The supercoiling ratio and endotoxin content of the plasmid samples were randomly inspected and identified. (2) Transposition efficiency detection in CHO cells On the day of electroporation, CHO-K1 cells were digested with trypsin containing 0.25% EDTA, collected and centrifuged, and resuspended in 1× DPBS for counting. 1.5-2.5×10 6 Cells were centrifuged and the supernatant was removed. According to the instructions of Lonza 2B electroporation reagent, 100ul of electroporation solution was taken, 4μg of transposase plasmid and 4μg of transposon plasmid were mixed with electroporation reagent, and then added to the cell pellet after centrifugation. After resuspending, it was added to the electroporation cup, placed in the Lonza 2B electroporator, and the electroporation program was set to H-014. After electroporation, the cells were transferred to a six-well plate with complete culture medium for culture. Fluorescence photography was performed on D5-D13 days after electroporation, and the transposition efficiency of transposase in CHO-K1 cells was detected by flow cytometry. (3) PBMC (peripheral blood mononuclear cell) cell transposition efficiency detection On the day of electroporation, the frozen PBMCs were thawed and counted, and 1×10 7 Cells were centrifuged and the supernatant was removed. According to the instructions of Lonza 2B electroporation reagent, 100ul of electroporation solution was taken, 4μg of transposase plasmid and 4μg of transposon plasmid were mixed with electroporation reagent, and then added to the cell pellet after centrifugation. After resuspending, it was added to the electroporation cup, placed in the Lonza 2B electroporator, and the electroporation program was set to U-014. After electroporation, the cells were transferred to a six-well plate with complete culture medium (AIM-V+2% FBS) and supplemented with 500U / ml of IL-2. The plate was transferred on the 5th day after electroporation, and fluorescence photography, counting, and flow cytometry were performed on D5-D13 days to detect the transposition efficiency of transposase in PBMC cells. The transposon plasmid in Example 1-6 contains an eGFP (also referred to herein as GFP, EGFP) fluorescent protein expression cassette. For fluorescence photography analysis, GFP fluorescence was captured using an Olympus inverted fluorescence microscope at D5, D9, and D13 with an exposure time of 500 ms and a shooting magnification of 100×. The transposition efficiency of the transposase in the cells was evaluated by the number of GFP fluorescent cells and the fluorescence intensity. For flow cytometric analysis, stable integration of the gene was assessed by measuring GFP fluorescence in cells grown without drug selection. Transfected cells were harvested at the indicated time points, washed once with 1× DPBS, and resuspended in 500ul DPBS and added to flow tubes or 96-well plates for on-line detection. Cells were analyzed using Cytek's SpectroFlo and GFP expression was assessed using the FITC channel. (4) Screening of transposase mutants in CHO cells On the day of electroporation, CHO-K1 cells were digested with trypsin containing 0.25% EDTA, collected and centrifuged, and resuspended in 1× DPBS for counting. 1.5-2.5×10 6Cells were centrifuged and the supernatant was removed. According to the instructions of Lonza2B electroporation reagent, 100ul of electroporation solution was taken, 4μg of different transposase mutant plasmids and 4μg of transposon plasmids (for example: PPS-DT-GFP(ta)) were mixed with electroporation reagent and added to the cell pellet after centrifugation. After resuspending, the mixture was added to the electroporation cup, placed in the Lonza 2B electroporator, and the electroporation program was set to H-014. After electroporation, the cells were transferred to a six-well plate with complete culture medium for culture. Fluorescence photography was performed on D3-D14 days after electroporation, and the transfected cells were harvested, washed once with 1×DPBS, and resuspended with 500ul DPBS and added to a flow tube or 96-well plate for on-line detection. Cells were analyzed using Cytek's SpectroFlo, and GFP expression was evaluated using the FITC channel. Different transposase mutants were screened and evaluated based on the final transposition efficiency. (5) Screening of transposase mutants in PBMC cells On the day of electroporation, the frozen PBMCs were thawed and counted, and 1×10 7 Cells were centrifuged and the supernatant was removed. According to the instructions of Lonza 2B electroporation reagent, 100ul of electroporation solution was taken, 4μg of transposase plasmid and 4μg of transposon plasmid (e.g., PPS-DT-GFP(ta)) were mixed with electroporation reagent and added to the cell pellet after centrifugation. After resuspending, the cells were added to the electroporation cup, placed in the Lonza 2B electroporator, and the electroporation program was set to U-014. After electroporation, the cells were transferred to a six-well plate with complete medium (AIM-V+2% FBS) and supplemented with 500U / ml of IL-2. The plates were transferred on the 5th day after electroporation, and fluorescent photos were taken on D3-D14 days, counted, and transfected cells were harvested, washed once with 1×DPBS, resuspended with 500ul DPBS, and added to the flow tube or 96-well plate for on-machine detection. Cells were analyzed using Cytek's SpectroFlo, and GFP expression was evaluated using the FITC channel. Different transposase mutants were screened and evaluated based on their final transposition efficiency. Example 1: Fusion of functional polypeptides to improve the transposition efficiency of PS transposase Purpose of the test: When transposase exerts its transposition activity, it is accompanied by the dimerization process of protein and DNA complex. The efficiency of this dimerization may be reduced due to the accumulated mutations of natural transposase, thereby affecting the transposition efficiency. As shown in Figure 3, leucine zipper is a structural motif in DNA-binding proteins, which often contains regular leucine residues. When the hydrophobic surfaces of two amphiphilic α-helices from the same or different polypeptide chains interact to form a coiled coil region, a leucine zipper dimer is formed. This experiment uses the fact that leucine zipper polypeptides also have similar DNA binding and dimerization characteristics to enhance the dimerization efficiency of PS transposase to achieve the purpose of improving transposition efficiency. Experimental methods: In this embodiment, pLoxP-mPS is a wild-type PS transposase expression plasmid (the plasmid map is shown in FIG5 , and the sequence is shown in SEQ ID NO: 33). The amino acid sequence of the wild-type PS transposase is shown in SEQ ID NO: 1. The amino acid sequences of the fusion polypeptides C / EBPO, LZIP, and TEF are shown in SEQ ID NOs: 28, 29, and 31, respectively, and the nucleotide sequences are shown in SEQ ID NOs: 34, 35, and 37, respectively. The structure of the transposon plasmid PPS-DT-GFP (ta) is shown in FIG6 , and its nucleotide sequence is shown in SEQ ID NO: 38. Different types of leucine zipper domains and / or C-myc NLS polypeptides were fused to the N-terminus or C-terminus of the natural PS transposase polypeptide in the pLoxP-mPS plasmid, and the schematic diagram of the structure is shown in Figure 7. The transposition efficiency of the fusion PS transposase was determined according to the CHO cell transposition efficiency detection method described in this article, and eGFP expression was evaluated by fluorescence photography and flow cytometry to verify the effect of the leucine zipper domain on the natural PS transposase. Test results Fluorescence photos and flow cytometry results are shown in Figure 8 and Table 1. Among them, ploxp-MPS refers to a transposase plasmid without a leucine zipper domain, ploxp-mps-TEF1, ploxp-mps-LZIP1, and ploxp-mps-C / EBPO1 refer to leucine zipper domains at the N-terminus of the transposase, ploxp-mps-C / EBPO2 and ploxp-mps-LZIP2 refer to leucine zipper domains at the C-terminus of the transposase, and NTC refers to a control group. The mps in the plasmid name is sometimes also written as mPS or MPS. In this specification and its accompanying drawings, the English letters in the names of some plasmids are not case-sensitive. It can be seen from the results that compared with the wild-type PS transposase group of 7.17%, the N-terminal fusion of LZIP, TEF, and C / EBPO leucine zipper domains can greatly improve the transposition efficiency, among which the C / EBPO leucine zipper domain has the highest improvement, and the positive rate after 14 days of electroporation reaches 40.01%. Table 1. Effect of fusion peptide on PS transposition efficiency Example 2: Optimizing the DNA sequence of the transposon TIR element can further improve the transposition efficiency of the fusion polypeptide PS transposase Experimental purpose: To optimize the DNA sequence of the TIR element and its flanking TA shear recognition sequence and repeat multiple copies in tandem to further improve the transposition efficiency of PS transposase. Experimental method: As shown in Figure 9, the transposon TIR element DNA sequence and its flanking TA shear recognition sequence in the plasmid expression vector PPS-DT-GFP (ta) were designed with n-fold sequence repeats and multiple copies (the TIR and TA at both ends were designed the same in this embodiment), and then the CHO / PBMC cell transposition efficiency detection method described in this article was followed to evaluate eGFP expression by fluorescence photography and flow cytometry to verify whether the TIR element DNA sequence optimization can continue to improve the transposition efficiency of the PS transposase of the fusion polypeptide. This experiment only used the PS transposase of the fusion polypeptide pLoxP-mPS-C / EBPO1 for testing, in which PST1 and PST2 in the transposon plasmids PO-PST1-DCGEGFP and PO-PST2-DCGEGFP represent (TIR) 1 (TA) 2 and (TIR) 2 (TA) 2, which is derived from the PPS-DT-GFP(ta) plasmid by modifying the TIR functional sequence as shown in Figure 9. The sequence of PO-PST2-DCGEGFP is shown in SEQ ID NO: 61. ploxp-hTZB+pZB-dCGEGFP refers to the ZB transposon system (see CN105018523A), referred to as ZB; pLoxP-fHyPB+pC23S-dCGEGFP refers to the efficient hyPB transposon system (see CN105154473A), referred to as PB. In the CHO cell test, 3 replicate wells were set for each group, and fluorescence photography and flow cytometry were used for detection on the 3rd to 14th day after transfection; in the PBMC cell test, 6 replicate wells were set for each group, and fluorescence photography and flow cytometry were used for detection on the 5th, 9th and 13th day after transfection. The positive rate and expression level of eGFP expression in the cells can reflect whether the transposition efficiency is improved. Test results The results in CHO cells are shown in Figure 10 and Table 2. The results show that based on the N-terminal leucine zipper C / EBPO fusion PS transposase, optimizing the TIR element DNA motif and / or the TA motif repeat copy tandem can further improve the transposition efficiency of the transposon system, and the eGFP fluorescence intensity is stronger. Table 2 Transposition effect of optimized TIR element DNA motif in CHO cells (positive rate) The results in PBMC cells are shown in Figure 11. The results show that optimizing the TIR element DNA motif and / or the TA motif in tandem with repeated copies can further improve the transposition efficiency of the transposon system, and the eGFP fluorescence intensity is stronger, indicating that this improvement can obtain repeated results in primary immune cells and has a synergistic effect with the fusion PS transposase. Compared with PB and ZB plasmids, the cell positivity of pLoxP-mPS-C / EBPO1+PO-PST2-dCGEGFP was slightly higher than that of the PB group, but the MFI was slightly lower than that of the PB plasmid. The above results indicate that the transposition efficiency of the JL transposon system with multiple copies of optimized TIR motifs and TA motifs tandem is comparable to that of the PB transposon system after the N-terminal leucine zipper C / EBPO fusion PS transposase. Example 3: Screening for PS transposase mutants with efficient gene integration in CHO cells Implementation purpose: The transposition efficiency of different PS transposase mutants in CHO cells was tested and transposase mutants with efficient transposition integration were identified. Implementation Methods The electroporation plasmids of the verification system of this embodiment include pLoxP-mPS-C-EBPO1 and its mutant plasmids containing a wild-type PS transposase expression frame, and a transposon vector plasmid PPS-DT-GFP (ta) containing an eGFP fluorescent protein expression frame. The sequence of the vector pLoxP-mPS-C-EBPO1 containing a wild-type PS transposase is shown in SEQ ID NO: 39. According to the sequence of SEQ ID NO: 39, the codons of the amino acids at the corresponding mutation positions are replaced to obtain different vector sequences of engineered PS transposases. To test the transposition efficiency of different engineered PS transposase mutants, the constructed engineered transposase or transposon was transfected into CHO cells at a certain mass ratio using electroporation according to method 4. CHO cells were transfected with fused wild-type PS transposase and unfused wild-type PS transposase (PS transposase) as control transposases. Cells were grown in complete medium (without drug selection), and eGFP expression was evaluated by fluorescence photography and flow cytometry on days 3-14 after transfection. Implementation Results According to the experimental results of CHO cells, a series of fusion PS transposase mutants with improved transposition positive rates compared with fusion wild-type PS transposase and unfusion wild-type PS transposase were screened, as shown in Table 3, Table 4 and Figure 12. Table 3 Efficiency improvement of dominant mutants compared to wild-type PS transposase 14 days after transfection The C-EBPO1 in the table is mPS-C-EBPO1 fused with wild-type PS transposase. Among these mutants, A8S, I35V, Y46Q, TQS57-59KKA, M95L, I98K, R123H, Q136K, K16R, Q22K, E47K, K55R, TQ57-58RK, T129R, T129K, Q136K, etc. have significantly improved transposition activity. In particular, when the amino acids in the HTH structure of the double-stranded DNA binding and oligomerization domain of PS transposase are mutated to positively charged amino acids such as histidine (H), lysine (K) or arginine (R) (such as K16R, E32K, T57R, TQ57-58RK, TQS57-59KKA, I98K, R123H, T129K, T129R, etc.), the transposition efficiency increases more significantly. In addition, although the positive rate of some fusion PS transposase mutants was not improved compared with the fusion wild-type PS transposase (mPS-C-EBPO1), it was significantly improved compared with the wild-type PS (mPS), as shown in Table 4. Table 4 Efficiency improvement of some mutant and wild-type PS transposases compared with fusion wild-type PS transposase 14 days after transfection Example 4: Validation of PS transposase mutants with efficient transposable gene integration in PBMC cells Implementation purpose: The three dominant mutants in CHO cells will be further verified in primary immune cell PBMCs to verify whether the mutants also have significant improvement effects in different types or primary cells. Experimental steps: The electroporated plasmids of the verification system of this embodiment include pLoxP-mPS-C-EBPO1 and its mutant plasmids containing a fused wild-type PS transposase expression frame, and a transposon vector plasmid PPS-DT-GFP(ta) containing an eGFP fluorescent protein expression frame. To test the transposition efficiency of the superior engineered PS transposase mutants, the constructed engineered transposase or transposon was transfected into PBMC cells at a certain mass ratio using electroporation according to method 5. PBMC cells were transfected with fused wild-type PS transposase and PiggyBac (hyPB) transposase as control transposases. Cells were grown in complete medium (without drug selection), and eGFP expression was evaluated by fluorescence photography and flow cytometry on days 3-14 after transfection. Experimental results: Samples were taken on the 7th and 14th days after electroporation, and the expression of EGFP was detected by flow cytometry. The analyzed data were samples on the 14th day. As shown in Figure 13, the cell data of the three groups of PBMCs, PS18, PS114, and PS144, whether fused with wild-type PS or ploxp-fHyPB as reference, showed a certain degree of improvement in the positive rate and fluorescence intensity. The dominant PS transposase mutants 18, 114, and 144 can basically be maintained at a level of 40%, and the expression level is doubled compared with the fusion wild-type PS transposase. A\B\C in Figure 14 show the positive rate and eGFP expression level of the mutant and the control group, respectively; the increase in the positive rate and eGFP expression level of the PS mutant relative to the fused wild-type PS transposase; and the increase in the positive rate and eGFP expression level of the PS mutant relative to the efficient hyPB transposase. Example 5: Evaluation of the efficiency of the JL transposition system containing TIR DNA motif mutations and / or multiple copies, fused mutant PS transposase Implementation purpose: 1. Mutate the PS transposon TIR DNA motif and evaluate and screen the dominant mutant DNA motif in Hela cells; 2. The screened advantageous mutant TIR DNA motifs were grouped in the form of (TIR)n(TA)n, and combined with fusion mutant PS transposases to finally evaluate and screen the optimal JL transposition system. Test method: The initial transposon integration plasmid pPG-PGK-NEO (PSTIR0) was constructed, and the plasmid sequence was shown in SEQ ID NO: 40. The nucleotide mutations of TIR were performed in sequence to construct pPG-PGK-NEO (PSTIR1), pPG-PGK-NEO (PSTIR2), and pPG-PGK-NEO (PSTIR3). The above plasmid pPG-PGK-NEO (PSTIR1) was expressed as 1TA-1TIR1, and multi-copy tandem transposon DNA functional elements were constructed in the form of (TIR)n(TA)n: 1TA-2TIR1, 2TA-1TIR1, 2TA-2TIR1, 2TA-2TIR1, 4TA-1TIR1, and 4TA-2TIR1. The TIR and TA at both ends of this embodiment were designed in the same way. The transposase plasmid used in this example is pLoxP-mPS-C-EBPO1 in which the amino acid of the PS transposase is mutated to a PS18 mutant sequence, that is, the transposase is a PS18 mutant with C-EBPO fused to the N-terminus. Table 6 Hela cells were cultured in high-glucose medium DMEM (Gibco, USA) containing 10% fetal bovine serum and 0.1 mg / mL penicillin-streptomycin in a 37°C, 5% CO2 cell culture incubator. 5 The cells were plated on a six-well plate at a density of 1:5, with three replicate wells set up for each group. The cells were mixed with Transfectamine 5000 transfection reagent (AAT Bioquest, USA) at a ratio of 1:5, and transfected according to the instructions. 24 hours after cell transfection, 1% of the cells were inoculated into each well and cultured in a 10 cm dish. When the cells grew to 10%, the culture medium was replaced with 600 ng / μL G418 selection medium. After that, the selection medium was replaced every three days. After about 2 weeks, when the cells in the control group were completely dead, Giemsa staining was performed on each group to ImageJ software was used for counting and GraphPad software was used for statistical analysis. Test results: As shown in Figure 16, this example proves that the transposon can still achieve the transposition integration function by designing TIR DNA motif nucleotide mutations, among which TIR1 mutation can improve the transposition efficiency. As shown in Figure 17, the gene integration transposition efficiency of the transposition system can be further improved by combining multiple copies of the advantageously screened TIR1 DNA motif functional element in series. Example 6: Evaluation of transposition integration efficiency of engineered JL transposase mRNA and sequence-optimized transposon TIR functional sequences on PBMCs 1. Test purpose: Verify the effect of the engineered JL transposon system on integrating plasmids using JL transposase mRNA in combination with transposons of different plasmid types. 2. Test methods: The fusion PS18 transposase mutant in Example 5 was selected to construct a DNA transcription template for mRNA synthesis, and the PS18 mRNA was synthesized and purified according to the Chinese patent CN115197327A "RNA modified chimeric protein and its application". The sequence of the fusion PS18 transposase mutant PS18 mRNA is shown in SEQ ID NO: 41. Different plasmid types of transposon integration plasmids were constructed, wherein pTini-DPS (CES) -dCGEGFP is an anti-microplasmid without antitoxin (also referred to as microplasmid or metoplasmid in the examples), its replicon is R6K, and contains the coding sequence of antitoxin protein (the amino acid sequence of antitoxin protein is shown in SEQ ID NO: 52), the sequence is shown in SEQ ID NO: 42, and pTini-DPS (CES) -dCGEGFP contains the above (TIR) 2 (TA) 2 , which differs from pO-PST2-dCGEGFP in that the standard STD plasmid is replaced by a non-antimicrobial plasmid; the sequence of pO-PST2-dCGEGFP is shown in Example 2 above, and PC23S-dCGEGFP is a standard STD plasmid containing the PB transposon TIR, and the sequence is shown in SEQ ID NO: 43. To test the transposition efficiency of the dominant fusion PS transposase mutant mRNA, the engineered transposase or transposon constructed was transfected into PBMC cells at a certain mass ratio using electroporation according to method 5. WT PS transposase and PiggyBac (hyPB) transposase were used as control transposases to transfect PBMC cells. Cells were grown in complete medium (without drug selection), and eGFP expression was evaluated by fluorescence photography and flow cytometry on days 3-14 after transfection. The grouping and plasmid dosage are shown in Table 7 below, and the mRNA dosage is 20 μg. Table 7 Grouping and plasmid dosage Test results As shown in Figure 18, the JL transposase mRNA + JL transposon miniplasmid group showed the best transposition efficiency and integrated gene expression level. The JL transposase mRNA + JL transposon miniplasmid group was better than the JL transposase mRNA + standard STD plasmid group; the JL transposase mRNA was better than the control group PB mRNA group overall. This shows that the new JL transposase in the form of mRNA combined with the miniplasmid transposon plasmid can further improve the overall efficiency of the JL transposon. Example 7: Engineering JL transposon system for the preparation and efficiency evaluation of CAR-T immune cells Purpose of the test Verify the transposition integration efficiency and transgene expression of the JL transposon system in the preparation of CAR-T immune cells. Test methods The plasmids used in this experiment and their grouping are shown in Table 8 below. The corresponding sequences of plasmids pC23S-1444-8E, pC24S-1194, pTini-PS(DTS)-1444-8E, pTini-PS(CIT)-1194, and pC23S-EGFP-8E are shown in SEQ ID NOs: 44-48, respectively. The JL transposon in the plasmid contains (TIR) 1 (TA) 2 The mRNA used in this experiment is the same as that in Example 6. Specifically, the CAR-T is the MSLN CAR-T of the autocrine PD-1 nanobody, as described in patent CN202111681582.0. The CAR-T used in this example is the mesothelin nanobody MSLN3 containing the autocrine PD1 nanobody 1194nla in Example 6 of patent CN202111681582.0.

[1444] CAR-T cells (abbreviated as "1444+1194nla" or "1444-1194"). A PB control group was set up. According to method 5, the engineered transposase or transposon constructed was transfected into PBMC cells at a certain mass ratio by electroporation. PiggyBac (hyPB) transposase was used as a control transposase to transfect PBMC cells. Cells were grown in complete medium (without drug selection), and CAR gene expression was evaluated by fluorescence photography and flow cytometry on days 3-14 after transfection, and antibody secretion expression levels were detected by ELISA. The grouping and plasmid dosage are shown in the following table, and the mRNA dosage is 20 μg. Table 8 Plasmids and grouping Test results As shown in Figure 19, from the perspective of CAR positivity rate, expression level, total number of positive cells and absolute number indicators, JL transposase showed higher efficiency, among which the positivity rate and expression level increased in a dose-dependent manner. Under the premise of the same transgenic effect, when the anti-free microplasmid was used as the donor DNA, the dosage of JL transposase and transposon plasmid was much lower than that of the classic hyPB system; Table 9 shows the expression amount of PD-1 nanoantibody on days 3-9. It can be seen from Table 9 that the use of the JL transposon system of the present invention combined with the anti-free microcarrier (Tai plasmid) can reduce the amount of plasmid used while greatly improving the expression level of the autocrine antibody gene compared with the PB transposon system. Table 9 Autocrine PD-1 Nanobody Expression Levels of D3-D9 In summary, the new JL transposon system (especially the one with the antibiotic-free microplasmid as the vector) showed good performance in the preparation of immune cell CAR-T, and the overall effect far exceeded the current gold standard piggybac transposon system. Both the gene integration transposition efficiency and the transgene expression level were much higher than those of the PB control group. When the antibiotic-free microplasmid was used as the transposon donor DNA, the new and efficient JL transposon system had advantages in both efficiency and dosage. sequence of this article

Claims

1. A transposase, which is a mutant transposase or a fusion transposase, The mutant transposase comprises an amino acid mutation compared to the wild-type PS transposase as shown in SEQ ID NO: 1, wherein any one or more amino acids in the double-stranded DNA binding and oligomerization domain of the wild-type PS transposase are mutated into positively charged amino acids; and / or the amino acid mutation comprises one or more mutations selected from the group consisting of TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59RK 7-59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58 R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T 350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K15 9H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V; The fusion transposase is a wild-type PS transposase or a mutant thereof fused with a functional polypeptide, wherein the functional polypeptide is: a DNA sequence-specific or non-specific binding domain, and / or a cell nucleus localization signal domain.

2. The transposase of claim 1, wherein the mutant comprises one or more mutations selected from the group consisting of TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59KKA\I98 K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K , I35V, N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H2 15K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V, Q69R, L100I, K96R, Q162R, A271K, R372K, H20Y, TYCR150-153K FVI, N232K, A363N, A377C, Q346M, A17V, N105G, Q76E, I354V, VS133-134IA, S253K, V370I, SV297-298TE, EH286-286DS, A3 52S, ANS241-243VHE, S297T, A79I, T147S, I178M, I276V, Q331E, R257E, T339S, A79V, H104L, V74L, S107E, Q142E, R375K, S 243E, R195K, Q22E, A328Q, G111A, R110K, I365V, VTE380-382SNA, R190K, RA372-373KS, K73L, K121R, R102K, C152V, IY276 - 277VW、V292I、K73E、H104M、I239V、 K42E、K356Q、T150K、G316S、IY259-260VW、V53M、E108D、V53L、DKV72-74EEL、M280F、S85G、N63K、V91L、K251L、H124N、K308S、N10D、H7R、G112S、H7K、TA327-328KE。 3. The transposase according to claim 1 or 2, wherein the mutant comprises one or more mutations selected from the group consisting of TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS 57-59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, M9 5L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E33 5S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V, Q69R, L100I, K96R, Q162R, A 271K, R372K, H20Y, TYCR150-153KFVI, N232K, A363N, A377C, Q346M, A17V, N105G, Q76E, I354V, VS133-134IA, S253 K, V370I, SV297-298TE, EH286-286DS, A352S, ANS241-243VHE, S297T, A79I, T147S, I178M, I276V, Q331E, R257E, T 339S, A79V, H104L, V74L, S107E, Q142E, R375K, S243E, R195K, Q22E, A328Q, G111A, R110K, I365V, VTE380-382SNA.

4. The transposase according to any one of claims 1 to 3, characterized in that The DNA sequence-specific or non-specific binding domain comprises a leucine zipper domain, a CRISPR / Cas domain, a TALE domain, a zinc finger domain, an AAV Rep DNA binding domain or any combination thereof, preferably, the amino acid sequence of the leucine zipper domain is as shown in any one of SEQ ID NOs: 28-31, and / or The nuclear localization signal domain includes SV40 NLS, C-myc NLS, TAF1 NLS, TP53 NLS, STAT3 NLS or any combination thereof, and / or The functional polypeptide and the wild-type PS transposase or its mutant may or may not contain a linker.

5. A polynucleotide encoding the transposase according to any one of claims 1 to 4; preferably, the polynucleotide is DNA or messenger RNA.

6. A transposon that can be recognized by the transposase according to any one of claims 1 to 4, wherein the transposon has any one, two or three characteristics selected from the following (1) to (3): (1) The transposon has a mutant terminal inverted repeat sequence, and the mutant terminal inverted repeat sequence has 1-5 nucleotide mutations compared with the wild-type PS transposon terminal inverted repeat sequence shown in SEQ ID NO: 2 or 3; (2) The transposon has two or more terminal inverted repeat sequences at at least one end; (3) The transposon has two or more repeats of TA sequences outside the terminal inverted repeat sequence at at least one end.

7. The transposon according to claim 6, wherein the mutant terminal inverted repeat sequence is as shown in any one of SEQ ID NOs: 4-27, preferably as shown in any one of SEQ ID NOs: 4-6, or is a nucleotide sequence containing any combination of nucleotide mutations in SEQ ID NOs: 4-27, or is their reverse complementary sequences.

8. The transposon according to claim 6 or 7, wherein the transposon has two terminal inverted repeat sequences at both ends, and each includes four TA sequence repeats outside the terminal inverted repeat sequences at both ends.

9. The transposon according to any one of claims 6 to 8, comprising a target gene expression cassette located between terminal inverted repeat sequences, preferably, the target gene encodes a protein selected from a cell receptor, an immune checkpoint protein, a cytokine, a T cell receptor, a B cell receptor, a chimeric antigen receptor, and any combination thereof.

10. A transposon system, comprising the transposase according to any one of claims 1 to 4 or the polynucleotide encoding the transposase according to claim 5; and a transposon, wherein the transposon is a PS transposon or the transposon according to any one of claims 6 to 9.

11. The transposon system as described in claim 10, wherein the polynucleotide encoding the transposase is mRNA, and preferably, the mRNA is chemically modified, including but not limited to pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine nucleoside (m5C), 5-methoxyuridine (5moU) and the like.

12. The transposon system according to claim 10 or 11, wherein the polynucleotide encoding the transposase and / or the transposon are present in a DNA vector, preferably, the DNA vector is a plasmid vector or a minicircle DNA vector, preferably a non-antimicrobial plasmid vector; and / or the DNA vector is a microcarrier, the backbone sequence of which is within 600 bp in length, and has reduced and / or no CpG DNA motif; or The polynucleotide encoding the transposase and / or the transposon are present in a viral vector. Preferably, the viral vector is selected from an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a herpes simplex viral vector or a vaccinia viral vector. 13 . The transposon system according to claim 12 , wherein the polynucleotide encoding the transposase and the transposon are present in the same vector or in different vectors.

14. A host cell comprising or capable of producing the transposase according to any one of claims 1 to 4, the polynucleotide according to claim 5, the transposon according to any one of claims 6 to 9, or the transposon system according to any one of claims 10 to 13.

15. A method for preparing a cell, comprising: The step of introducing the transposase according to any one of claims 1 to 4, the polynucleotide according to claim 5, the transposon according to any one of claims 6 to 9, or the transposon system according to any one of claims 10 to 13 into a cell, Preferably, The introducing comprises contacting the cell with the polynucleotide, and / or The introducing comprises contacting the cell with a DNA vector containing the transposon, and / or The introducing comprises contacting the cell with mRNA encoding the transposase and a plasmid vector containing the transposon, and / or The introducing comprises contacting the cell with a plasmid vector containing the polynucleotide and the transposon, and / or The introducing comprises contacting the cell with a plasmid vector containing the polynucleotide and a plasmid vector containing the transposon, and / or The introducing comprises contacting the cell with the transposase.

16. The method according to claim 15, characterized in that The cell is from an animal, such as a vertebrate or an invertebrate, preferably a mammal, and more preferably a human; and / or the cell is an immune cell, preferably a T cell.

17. The method according to claim 15 or 16, characterized in that The introducing comprises transfecting the cell by means of electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposomes, lipid nanoparticles, microparticle bombardment, fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection, virus-mediated transformation, or any combination thereof.

18. The method according to claim 16 or 17, characterized in that Said introducing comprises electroporating said cells.

19. The method according to claim 18, characterized in that The introduction comprises contacting the cell with mRNA encoding the transposase and a plasmid containing the transposon. Preferably, the plasmid is a microcarrier, the backbone sequence of which is within 600 bp in length, with reduced and / or no CpG DNA motif, and / or the plasmid is a non-antimicroplasmid.

20. A kit comprising: the transposase according to any one of claims 1 to 4, the polynucleotide according to claim 5, the transposon according to any one of claims 6 to 9, or the transposon system according to any one of claims 10 to 13, or the cell according to claim 14.

21. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and cells prepared by the method according to any one of claims 15 to 19.

22. Use of the transposase according to any one of claims 1 to 4, the polynucleotide according to claim 5, the transposon according to any one of claims 6 to 9, the transposon system according to any one of claims 10 to 13, the cell according to claim 14, or the pharmaceutical composition according to claim 21, which is selected from any one of the following (1) to (6): (1) Application in the preparation of drugs or reagents for integrating the target gene expression cassette into the host cell genome; (2) Application in the preparation of tools for integrating target gene expression cassettes into the host cell genome; (3) Application in the preparation of transgenic animals and transgenic cells; (4) Application in the preparation of drugs or preparations for genome research, gene therapy, cell therapy, or stem cell induction and differentiation after induction; (5) Application in the preparation of tools for genome research, gene therapy, cell therapy, or stem cell induction and post-induced differentiation; (6) Application in the preparation of kits, engineered immune cells or pharmaceutical compositions.