Caspobase transposase-based gene fixed-point insertion tool and application

Abstract

The invention relates to a gene fixed-point insertion tool based on caspoase transposase and application of the gene fixed-point insertion tool. The gene fixed point insertion tool is composed of caspoase transposase, a 1Donor sequence and an insertion target pTarget. The 1Donor sequence is composed of any target sequence with 8 bp homologous arms at two ends, and the insertion target is composed of a leader-TSD target sequence. According to the invention, the caspoase transposase is utilized to efficiently and specifically insert a target gene which is 8000 bp long into a target site at a fixed point, and meanwhile, the insertion direction of the target gene can be selectively controlled. In addition, the caspoase transposase provided by the invention can realize gene fixed-point insertion in two forms of protein and plasmid. The tool is applied to realizing fixed-point insertion of a target gene in a prokaryotic or eukaryotic cell, and is suitable for genetic information transformation and endowing a new function to the cell.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a gene site-directed insertion tool based on casposase transposase and its application. Background Art

[0002] Site-directed gene insertion technology is an effective means to transform cellular genetic information to give it new functions, achieve precise gene repair and targeted gene therapy, and has been widely used in many fields such as life sciences, crop improvement, clinical treatment and drug development (Chen X et al., “Recent advances in CRISPR-Cas9-based genome insertion technologies.” Mol Ther Nucleic Acids, 2024, 35(1):102138.). Existing site-directed gene insertion tools such as nucleases, transposases and recombinases have defects such as poor insertion site specificity, low efficiency, reliance on double-stranded DNA breaks and easy mutation, inability to achieve continuous gene insertion at adjacent sites, and large and complex functional execution elements that are not conducive to cell delivery (Allen A. G et al., “A highly efficient transgene knock-in technology inclinically relevant cell types.” Nature Biotechnology, 2024, 3:458-469.). Therefore, exploring and developing highly specific, efficient and easily delivered gene insertion tools is a hot topic and difficulty that needs to be broken through in the field of gene editing.

[0003] Currently, the main gene site-directed insertion tools include nucleases, transposases, and recombinases. Most of these tools have defects such as poor insertion site specificity, low efficiency, and large functional elements, which restrict their further application and promotion (Chen X et al., “Recent advances in CRISPR-Cas9-based genome insertion technologies.” Mol Ther Nucleic Acids, 2024, 35(1): 102138). Gene site-directed insertion mediated by nucleases such as Cas9 is achieved by cutting specific genomic target sites to induce double-strand breaks, and then completing the site-directed insertion of the target gene through repair methods such as homology-directed repair (HDR). However, this method has defects such as low insertion efficiency and the fact that DNA double-strand breaks are prone to cause chromosomal translocations, rearrangements and other variations (Pandey S et al., “Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing.” Nat Biomed Eng, 2025, 9(1): 22-39). Piggy Bac (PB) Sleeping beautyAlthough transposases such as (SB) transposase can catalyze the transposition reaction of long gene fragments, their transposition site specificity is poor and cannot achieve precise gene site insertion (Miskey C et al., “ Engineered Sleeping Beauty transposase redirects transposon integration away from genes.” Nucleic Acids Research, 2022, 50(5):2807-2825.). Recent studies have found that the CRISPR-associated transposase (CAST) system can partially improve the transposition site specificity and achieve site-specific gene insertion by combining Cas12k or Cascade with sgRNA to form a complex. However, gene insertion mediated by this type of transposase has transposition exclusivity and cannot achieve continuous site-specific insertion of genes at adjacent sites. In addition, the system components are complex, and site-specific gene insertion requires the coordinated action of a "huge" protein complex composed of several proteins, which further limits its application (Liu J et al., "Integration of therapeutic cargo into the human genome with programmable type VK CAST." Nat Commun, 2025, 16(1):2427.). Gene insertion mediated by recombinase systems such as Cre / loxP is relatively cumbersome, requiring the introduction of a pair of recombinase recognition sites (~30-50 bp) in the insertion sequence and the insertion site in advance. Moreover, the recombination reaction is reversible, which makes it impossible to achieve continuous gene insertion efficiently and conveniently (Barrangou R et al., "A decade of discovery: CRISPR functions and applications." Nat Microbiol, 2017, 2: 17092.). Therefore, developing a simple, efficient and highly specific new gene site-specific insertion tool is a hot topic in the field of gene editing and a difficulty that needs to be overcome urgently.

[0004] The CRISPR-Cas system is an acquired immune system that has gradually evolved in bacteria and archaea in the face of continuous attacks by phage viruses and foreign plasmids. The acquisition of short fragments of foreign genetic information is the basis of the entire CRISPR-Cas immune function, and Cas1 is the main participant in the acquisition of immune memory in bacteria (Barrangou R et al., “CRISPR provides acquired resistance against viruses in prokaryotes.” Science, 2007, 5819:1709-1712.). In recent years, a special type of cas1 genes has been newly discovered in some organisms. There are no obvious CRISPR loci around these genes, but there are some characteristic elements of transposons, such as terminal inverted repeats (TIR) and target site duplications (TSD) (Krupovic M et al., “Casposons: a new superfamily of self-synthesizing DNA transposons at the origin of prokaryotic CRISPR-Cas immunity.” Bmc Biology, 2014, 12(1): 36.). Sequences that contain both Cas1 homologous integrases and transposon-like elements are called "casposons" , and the only Cas1 homologous nuclease with integration activity is called casposase. Taxonomic studies have shown that casposase is likely to be the evolutionary ancestor of Cas1 (Koonin E. V et al., “Evolution of adaptive immunity from transposable elements combined withinnate immune systems.” Nature Reviews Genetics, 2015, 3:184-192.). During evolution, casposase gradually lost its ability to integrate long fragments of DNA, but the site specificity of its integration reaction gradually increased, and finally Cas1, which can catalyze the high-site-specific integration reaction of short fragment sequences, was formed (Wright A. V et al., “A Functional Mini-Integrase in a Two-Protein-type V-C CRISPR System.” Molecular Cell, 2019, 4: 727-+). By virtue of the above characteristics of Cas1, bacteria can obtain immune memory of phage genetic information through short sequences of only dozens of base pairs, thus efficiently completing the entire CRISPR immune mechanism.

[0005] Despite significant differences in the composition of the integrated elements and the length of the integrated sequences, the integration reaction catalyzed by casposase is quite similar to the integration mechanism of Cas1-mediated short fragment sequences (Hickman A. B et al., “Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas.” Elife Sciences, 2020, 9.). Casposase recognizes and binds to the TIR sequences at both ends of the casposon and completes the integration of the casposon through two nucleophilic attack reactions similar to Cas1. The first nucleophilic attack is the 3′-OH end of the TIR sequence attacking the proximal leader end of the TSD sequence to form a semi-integrated intermediate product. The second nucleophilic attack occurs at the distal leader end of the TSD sequence to form a double-stranded fully integrated product, and finally a complete TSD sequence is generated through DNA repair to complete the site-specific insertion of the casposon (Wang X et al., “Sequence-specific integration by the family 1 casposase from Candidatus Nitrosopumilus koreensis AR1.” Nucleic Acids Research, 2021, 49(17): 9938-9952.). The above integration process does not involve the complete cleavage of the blunt ends of double-stranded DNA, and the TSD sequence that needs to be repaired is usually only dozens of bases. Therefore, casposase-mediated gene insertion can avoid genetic information damage caused by DNA cleavage and is more efficient than HDR-mediated gene insertion. Compared with Cas1, which can only catalyze the site-specific integration reaction of short fragment DNA, casposase has both the site-specific continuous insertion activity of Cas1 and the efficient long fragment gene insertion ability of transposons. Existing studies have focused more on the homologous aspect of casposase and Cas1, that is, its site-specific integration ability of short fragment DNA. However, its near-transposase aspect, that is, the long fragment DNA site-specific integration ability of casposase, still needs further study. Compared with short fragment DNA, the long fragment DNA site-specific integration ability of casposase has more application value, such as the construction of chimeric antigens in cell therapy, the development of stable transfected cell lines in antibody drug production, and the improvement of yield and insect resistance functions in crop genetic improvement. Therefore, it is of great significance to carry out research on the long fragment DNA site-specific integration reaction of casposase and explore the development of new gene site-specific insertion tools.

[0006] Combined with the published research, casposase has the potential to be developed into a new gene site-specific insertion tool. Compared with the existing gene site-specific insertion tools, casposase has the following excellent characteristics: (1) Compared with transposases such as PB / SB, the long-fragment DNA integration reaction of casposase has high specificity, and the reaction always occurs on both sides of the TSD sequence; (2) Compared with gene insertion mediated by damage repair such as HDR, the casposase integration reaction has higher efficiency and does not involve the complete breakage of double-stranded DNA, which can avoid the genetic information loss caused by double-strand breaks; (3) The casposase protein has a small molecular weight (~40 kDa) and can perform gene insertion functions without assembling into a complex with other proteins, which is beneficial for subsequent protein delivery into cells; (4) The integration reaction mediated by casposase does not have transpositional exclusivity and can achieve continuous gene insertion at adjacent sites of the TSD sequence. Summary of the Invention

[0007] Object of the Invention: The technical problem to be solved by the present invention is to provide a gene site-specific insertion tool and application based on casposase transposase in view of the deficiencies of the prior art, which can achieve efficient, highly specific and highly selective site-specific insertion of foreign genes through a single small-molecular-weight protein. The present invention can avoid the defects of the original gene insertion tools, such as complex and large components that are difficult to deliver, relying on the complete breakage of double-stranded DNA, inability to achieve site-specific continuous insertion, and poor directional selectivity of the inserted gene. At the same time, the application of this system is also proposed.

[0008] To solve the above technical problems, the present invention discloses a gene site-specific insertion tool and application based on casposase transposase. The specific technical solutions are as follows: A gene site-specific insertion tool based on casposase transposase, wherein the gene site-specific insertion tool includes casposase transposase, 1Donor sequence and pTarget; among them, the amino acid sequence of the casposase transposase is shown in SEQ ID No.2 or SEQ ID No.4; the casposase transposase shown in SEQ ID No.2 is the wild-type casposase transposase (casposase-WT) derived from Methanosarcina mazei GÖ 1, and the casposase transposase with the amino acid sequence shown in SEQ ID No.4 is the engineered casposase transposase (Ecasposase).

[0009] The 1Donor sequence includes the target gene sequence, and the two ends of the target gene sequence are terminal inverted repeat sequences; the target gene sequence is any target sequence to be inserted. Preferably, it is the chloramphenicol resistance gene (CmR).

[0010] The pTarget described above is a plasmid containing a leader sequence and a TSD sequence.

[0011] Among them, the terminal inverted repeat sequences are TRF and TRR, which are derived from Methanosarcina mazei GÖ 1. The nucleotide sequence of TRF is 5′-TAGAATCT- 3′; the nucleotide sequence of TRR is 5′-AGATTCTA- 3′; the structure of the 1Donor sequence is 5′-TRF - target gene sequence - TRR - 3′.

[0012] Among them, the 1Donor sequence is integrated into the first plasmid vector for site - directed gene insertion editing. The first plasmid vector includes any one of pCDFDuet - 1, pRSFDuet - 1, or pACYDuet1, preferably pCDFDuet - 1. The 1Donor sequence can also be directly used in the form of a dsDNA sequence.

[0013] Among them, the nucleotide sequence of the leader sequence is 5′-ACTCCAAGAGCAGAAGAGTTT-3′ (SEQ ID No.7); the nucleotide sequence of the TSD sequence is any one of the following a1 - a3: a1, 5′-ATTGATAAAGAGT-3′ (SEQ ID No.8), named TSD - WT; a2, 5′-ATTGATATTGAGA -3′ (SEQ ID No.9), named TSD - L; a3, 5′-TTTCATAAAGAGT-3′ (SEQ ID No.10), named TSD - S; Different TSD sequences are selected according to the different directions of the inserted gene.

[0014] Both the leader sequence and the TSD sequence are derived from Methanosarcina mazei GÖ 1.

[0015] For the pTarget described above, its starting plasmid is a plasmid containing the ccdB toxic element, preferably plasmid pKIL108.

[0016] In the pTarget described above, the connection order of the leader sequence, the TSD sequence, and the ccdB toxic element is 5′-leader sequence - TSD sequence - ccdB toxic element - 3′. Preferably, when the nucleotide sequence of the TSD is as shown in SEQ ID No.8, the nucleotide sequence of the pTarget is as shown in SEQ ID No.5.

[0017] The gene site-directed insertion tool forms a casposase-TRF / TRR protein-nucleic acid complex by the binding of casposase to the TRF and TRR sequences of the Donor sequence. The complex specifically recognizes and binds to the leader-TSD of the insertion target pTarget. Subsequently, the 3′-OH of TRF / TRR performs nucleophilic attacks on the near-leader end and the far-leader end of the leader-TSD sequence respectively. By modifying and optimizing the TSD sequence, the nucleophilic attack capabilities of the Ecasposase-TRF / TRR protein-nucleic acid complex on the near-leader end and the far-leader end of pTarget can be made different, thereby controlling the insertion direction of the target gene and achieving efficient, specific, and direction-selective site-directed insertion of the target gene.

[0018] Among them, the gene site-directed insertion tool includes a gene site-directed insertion tool with a protein as a vector and a gene site-directed insertion tool with a plasmid as a vector. Among them, for the gene site-directed insertion tool with a protein as a vector, gene site-directed insertion editing is carried out using the casposase transposase obtained by protein purification, and the expression of the casposase protein is completed in vitro. For the gene site-directed insertion tool with a plasmid as a vector, the casposase transposase is integrated into the plasmid vector, and gene site-directed insertion editing is carried out in the form of a plasmid. The plasmid vector includes a first plasmid vector and / or a second plasmid vector. The second plasmid vector is any one of pET-28a, pGEX-6P-1, pUC19, or pUC57. The gene site-directed insertion tool with a plasmid as a vector completes the expression of the casposase protein in vivo.

[0019] In a second aspect, the present invention provides a gene site-directed insertion method, which uses the gene site-directed insertion tool described in the first aspect. The gene site-directed insertion method includes the following steps: transforming the casposase transposase, the 1Donor sequence, and pTarget into a host cell to achieve site-directed insertion of the gene. Among them, the gene site-directed insertion tool includes a gene site-directed insertion tool with a protein as a vector and a gene site-directed insertion tool with a plasmid as a vector. The gene site-directed insertion tool with a plasmid as a vector includes a two-plasmid system and a single-plasmid system.

[0020] When the gene site-directed insertion tool is a protein-based gene site-directed insertion tool, the gene site-directed insertion method is as follows: incubate the casposase transposase obtained by protein purification with the 1Donor sequence obtained by PCR amplification to obtain a nucleoprotein complex, and transform the nucleoprotein complex into a host cell after mixing it with pTarget to achieve site-directed insertion of the gene; preferably, the casposase transposase obtained by protein purification is prepared as follows: transform the plasmid expressing casposase into E.coli a prokaryotic expression cell, induce expression, and purify to obtain a high-purity and correctly folded casposase protein; the plasmid expressing casposase is the pET28a-sumo plasmid expressing casposase, and the E.coli prokaryotic expression cell is E.coli BL21(DE3). When purifying, first subject the expression product to nickel column affinity chromatography and high-salt elution to obtain a crude protein, then cut off the sumo-tag with SUMO Protease, and finally subject the tag-free crude protein to gel filtration chromatography to obtain a high-purity casposase transposase. This preparation method can quickly and efficiently produce high-purity and correctly folded casposase. Through E.coli the prokaryotic expression system, a large amount of target protein can be obtained within 24 hours by affinity chromatography and gel filtration chromatography purification.

[0021] When the gene site-directed insertion tool is a two-plasmid system (consisting of two plasmids, mDonor and pTarget), the gene site-directed insertion method is as follows: clone the coding gene of the casposase transposase and the 1Donor sequence into the first plasmid vector to obtain the mDonor plasmid, and co-transform the mDonor plasmid and the pTarget plasmid into a host cell to achieve site-directed insertion of the gene; when the gene site-directed insertion tool is a single-plasmid system, the gene site-directed insertion method is as follows: clone the casposase transposase plasmid into the second plasmid vector to obtain the casp plasmid, transform the casp plasmid into a host cell to obtain recombinant bacterium 1, and co-transform the 1Donor sequence and the pTarget plasmid into recombinant bacterium 1 to achieve site-directed insertion of the gene.

[0022] Among them, the nucleotide sequence of the coding gene of the casposase transposase is shown as SEQ ID No. 1 (the nucleotide sequence of the coding gene of casposase-WT) or SEQ ID No. 3 (the nucleotide sequence of the coding gene of Ecasposase); the first plasmid vector includes any one of pCDFDuet-1, pRSFDuet-1 or pACYDuet1; the second plasmid vector includes any one of pET-28a, pGEX-6P-1, pUC19 or pUC57.

[0023] The host cell is preferably Escherichia coli BL21(DE3).

[0024] Preferably, the insertion efficiency and direction of the target gene can be determined according to the number of monoclonal colonies on the plate after transformation in combination with the Sanger sequencing results. The gene site-directed insertion efficiency can also be analyzed by NGS sequencing.

[0025] In a third aspect, the present invention provides the application of the gene site-directed insertion tool described in the first aspect in gene editing of prokaryotes or eukaryotes. Among them, the prokaryotes include Escherichia coli.

[0026] Beneficial effects: The present invention conducts research on the long-fragment DNA integration activity of casposase, develops it into a novel gene site-directed insertion tool, and improves the gene length that casposase can insert and the site-directed insertion efficiency through a series of modifications and optimizations of the casposase protein, its donor DNA Donor, and the insertion target pTarget, reduces the off-target rate of gene insertion, and controls the selection of the inserted gene direction by modifying the TSD sequence. Compared with the prior art, the casposase gene site-directed insertion tool of the present invention can selectively control the insertion direction of the target gene through a single small molecule protein, achieve efficient site-directed insertion of the target gene, and make up for the defects of the existing gene insertion tools, such as complex components, large molecular weight, and difficulty in delivery. In addition, the present invention uses the casposase transposase to efficiently and specifically insert a target gene up to 8000 bp in length into the target site, and can also selectively control the insertion direction of the target gene. The preparation method of the present invention can rapidly and efficiently prepare high-purity casposase protein in vitro, and can also achieve the in vivo expression of casposase through a plasmid vector, thereby completing the site-directed insertion of the target gene. The present invention can realize the transformation of genetic information through gene site-directed insertion, endow cells with new functions, and also lay a foundation for the subsequent development and optimization of this system. Description of the Drawings

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0028] Figure 1 Two directions for Casposase-mediated site-specific gene insertion.

[0029] Figure 2 Plasmid maps of pET-28a-Ecaspoase and pET28a-sumo-Ecasposase constructed in the present invention.

[0030] Figure 3 Plasmid map of pKIL108-leader-TSD (pTarget) constructed in Example 1 of the present invention.

[0031] Figure 4 Plasmid map of pCDFDuet1-Ecasposase-Donor (mDonor) constructed in Example 1 of the present invention.

[0032] Figure 5 Gel filtration chromatography and SDS-PAGE electrophoresis identification diagrams of Ecasposase and casposase-WT in Example 2 of the present invention.

[0033] Figure 6 Fluorescence detection of the ability of Ecasposase to specifically insert genes of different lengths in Example 3 of the present invention.

[0034] Figure 7 Process flow and insertion efficiency result diagram of the site-specific gene insertion tool with protein as the carrier in Example 4 of the present invention. Figure 7 A in is the process flow diagram of the site-specific gene insertion tool, Figure 7 B in is the insertion efficiency result diagram.

[0035] Figure 8 Effect of different TSD sequences on the direction selectivity of Ecasposase site-specific gene insertion in Example 4 of the present invention. Figure 8 A in is the insertion efficiency diagram, Figure 8 B in is the representative Sanger sequencing result of the target gene inserted into the clone.

[0036] Figure 9 Process flow diagram of the site-specific gene insertion tool with plasmid as the carrier and representative target gene insertion clone diagram in Example 5 of the present invention. Figure 9 A in is the two-plasmid system, Figure 9 B in is the single-plasmid system, Figure 9 C in is the target gene insertion clone diagram of the two-plasmid system, Figure 9D in it is the insertion efficiency diagram of the dual-plasmid system and the single-plasmid system. Detailed implementation manners

[0037] The present invention will be further described in detail below in conjunction with embodiments. However, the present invention is not limited to the given examples.

[0038] Example 1 Plasmid construction In this example, plasmids were constructed, and these plasmids were used to subsequently prepare each component of the gene site-directed insertion tool of the present invention. The gene site-directed insertion tool described in the present invention includes casposase transposase, donor DNA Donor, and insertion target pTarget. This gene site-directed insertion tool forms a casposase-TRF / TRR protein-nucleic acid complex by casposase binding to the TRF and TRR sequences of the donor DNA, and the complex specifically recognizes and binds to the leader-TSD of the insertion target pTarget; subsequently, the 3′-OH of TRF / TRR performs nucleophilic attacks on the near-leader end and the far-leader end of the leader-TSD sequence respectively; by modifying and optimizing the TSD sequence, the nucleophilic attack ability of the Ecasposase-TRF / TRR protein-nucleic acid complex on the near-leader end and the far-leader end of pTarget can be made different, thereby controlling the insertion direction of the target gene and achieving efficient, specific, and direction-selective site-directed insertion of the target gene (as Figure 1 shown).

[0039] 1. Construct a plasmid expressing casposase transposase: Use primers casp-F / casp-R to amplify the coding gene sequences of Ecasposase (amino acid sequence is shown in SEQ ID No.4, and the coding gene is shown in SEQ ID No.3) and casposase-WT protein (amino acid sequence is shown in SEQ ID No.2, and the coding gene is shown in SEQ ID No.1) by PCR, and perform enzymatic digestion (BamHI and XhoI) and ligation or homologous recombination to connect them into pET28a-sumo or pET28a. Transfer the recombinant plasmid to E.coli TOP10 competent cells by chemical transformation method. Subsequently, use a plasmid extraction kit to extract monoclonal plasmids, and after confirming correct by Sanger sequencing, the target recombinant plasmids can be obtained. The recombinant plasmids pET-28a-Ecasposase and pET28a-SUMO-Ecasposase constructed in this example are as Figure 2 shown. The primer sequences described in this example are as follows: Casp-F: 5′-CGCGGATCCATGAAACTGCTGCTGCTG-3′ (SEQ ID No.11), Casp-R: 5′-CCGCTCGAGACGCTGGGAGCTAACCAG-3′ (SEQ ID No.12).

[0040] 2. Construction of the pTarget plasmid with the inserted target: In this example, the pTarget plasmid contains the inserted target sequence, and its DNA structure is 5′-leader-TSD-3′.

[0041] Among them, the leader sequence is 5′-ACTCCAAGAGCAGAAGAGTTT-3′ (SEQ ID No.7).

[0042] The TSD sequence has three different sequences: TSD-WT, TSD-L, and TSD-S. Among them, TSD-WT is 5′-ATTGATAAAGAGT-3′ (SEQ ID No.8); TSD-L is 5′- ATTGATATTGAGA -3′ (SEQ ID No.9); TSD-S is 5′-TTTCATAAAGAGT-3′ (SEQ ID No.10). The TSD-WT sequence is derived from Methanosarcina mazei the original gene cluster of GÖ 1, and TSD-L / TSD-S are modified from TSD-WT.

[0043] The 5′-leader-TSD-3′ fragment is ligated to the pKIL108 vector, and the constructed recombinant plasmid pKIL108-leader-TSD (pTarget) is as Figure 3 shown. The pKIL108-leader-TSD plasmid includes three types according to different TSDs: pKIL108-leader-TSD-WT, pKIL108-leader-TSD-L, and pKIL108-leader-TSD-S. Among them, the nucleotide sequence of pKIL108-leader-TSD-WT is as shown in SEQ ID No.5, and the nucleotide sequences of pKIL108-leader-TSD-L and pKIL108-leader-TSD-S can be obtained by replacing the nucleotide sequence at TSD-WT in the sequence shown in SEQ ID No.5 with the TSD-S and TSD-L nucleotide sequences respectively.

[0044] 3. Construction of the donor DNA: In this embodiment, the donor DNA has two forms, namely lDonor and mDonor. Among them, lDonor is linear dsDNA generated by PCR amplification, which is composed of 5′-TRF sequence - target gene sequence - TRR - 3′. Among them, the TRF sequence is 5′-tagaatct-3′, and the TRR sequence is 5′-agattcta-3′.

[0045] In this embodiment, the dsDNA containing the chloramphenicol resistance gene (CmR) is used as the target gene sequence.

[0046] Another donor DNA, mDonor, is obtained by separately constructing and ligating the Ecasposase coding gene and the 1Donor sequence into two open reading frames of pCDFDuet-1, that is, pCDFDuet1-Ecasposase-Donor. Its plasmid map is as Figure 4 shown. When the target gene is the CmR gene, the nucleotide sequence of pCDFDuet1-Ecasposase-Donor is as shown in SEQ ID No.6. In the sequence shown in SEQ ID No.6, the sequence located between the TRF sequence and the TRR sequence is the CmR gene sequence.

[0047] Example 2 Protein Purification Based on Example 1, this example prepares and purifies the protein required for site-directed gene insertion. The pET28a-SUMO-Ecasposase and pET28a-SUMO-casposase-WT constructed in Example 1 are transformed into Escherichia coli E.coli BL21(DE3). Single colonies growing on the kanamycin plate are picked into the LB liquid medium. After overnight activation, they are transferred to the LB liquid medium and cultured at 37 °C and 200 rpm until the OD of the bacterial liquid 600 is between 0.6 and 0.8. Then, the temperature is lowered to 25 °C, and IPTG with a final concentration of 0.5 mM is added for overnight induction of protein expression.

[0048] Centrifuge to collect the overnight cultured bacteria, resuspend them in Ni-NTA buffer (20 mM HEPES pH7.5, 500 mM NaCl, 10 mM imidazole). After ultrasonic lysis, centrifuge and take the supernatant of the bacterial liquid and load it onto a nickel column. First, wash with Ni-NTA buffer containing 20 mM imidazole to remove low-affinity proteins, and then elute with Ni-NTA buffer containing 500 mM imidazole at the final concentration to obtain the crude Ecasposase / casposase-WT protein. The crude protein is digested overnight with SUMO Protease at 4 °C to remove the sumo-tag at the N-terminus of the target protein. The tag-free Ecasposase / casposase-WT can be concentrated and then loaded onto a Hiload16 / 60 Surperdex 200 pg gel filtration column, and eluted with an eluent containing 20 mM HEPES pH7.5 and 150 mM NaCl to obtain high-purity Ecasposase / casposase-WT protein. The corresponding gel filtration chromatogram and SDS-PAGE electrophoresis identification results are as Figure 5 shown.

[0049] Example 3 Gene-specific insertion ability test This example is based on Example 2 to investigate the gene-specific insertion ability of Ecasposase under in vitro conditions. First, using Lenti-dCas9-KRAB-blast (Addgene Plasmid #89567) as a template, specific primers containing TRF / TRR (including Donnor-TRF and Donnor-TRR2000 / 4000 / 6000 / 8000) are used for PCR amplification to generate target genes of about 2000, 4000, 6000, 8000 bp with 8 bp TRF / TRR sequences at both ends, namely Donor gene. High-purity Donor gene can be obtained by cutting and recovering the PCR product from the gel.

[0050] dsDNA containing the leader-TSD sequence labeled with 6-FAM and cy5 at the 3′ end is used as the specific insertion target (leader-TSD target), and dsDNA without the leader-TSD sequence is used as the non-specific fluorescent substrate.

[0051] Incubate the Donor gene at a final concentration of 0.2 μM, Ecasposase at a final concentration of 0.1 μM, and the fluorescent leader-TSD target at a final concentration of 0.2 μM in Insertion Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MnCl2) at 37 °C for 30 min to catalyze the site-specific gene insertion reaction. Load the product onto a 2% agarose gel for electrophoresis, and the fluorescence scanning results are as Figure 6 shown. It can be seen from this result that Ecasposase can catalyze the site-specific gene insertion of the target gene with a length of 2000 - 8000 bp, and the gene insertion efficiency gradually decreases as the length of the target gene increases. The DNA sequences used in this example are shown in Table 1.

[0052] Table 1 DNA sequence list used in Example 3

[0053] Example 4 Construction of a site-specific gene insertion tool with a protein as a vector Based on Example 2, this example constructs an engineered casposase gene site-specific insertion tool with a protein as a vector.

[0054] Respectively incubate the Ecasposase and casposase-WT (final concentration 0.5 μM) purified in Example 2 with 1Donor (final concentration 0.2 μM) constructed in Example 1 at 4 °C for 20 minutes in 20 mM HEPES pH 7.5 and 20 mM NaCl to form a casposase-gene nucleoprotein complex. Add the pTarget (final concentration 40 ng / μL) constructed in Example 1 to the above complex and divide it into two equal parts, and electrotransform them (Bio-Rad GenePulser Xcell electrotransformation conditions: 3 kv / cm, 300 Ω, 25 μF, 7 ms) into E.coli BL21(DE3) and DB3.1 competent cells, and use the ratio of the number of BL21(DE3) monoclonal colonies X (ampicillin-resistant plate) to the number of DB3.1 competent colonies Y (ampicillin-resistant plate) to represent the target gene insertion efficiency (as shown in Figure 7 A in).

[0055] The pTarget plasmid contains the ccdB gene and can express the CcdB toxic protein, so it cannot replicate in BL21(DE3) cells. The DB3.1 cells contain a mutant of DNA gyrase (gyrA462) that can tolerate the toxic effect of CcdB, so pTarget can replicate in DB3.1. The insertion of the new gene can cause a frameshift mutation in the ccdB gene in pTarget, interfering with the reading frame of the ccdB gene and preventing the expression of the CcdB toxic protein. Therefore, the recombinant pTarget can replicate normally in BL21(DE3) cells. The recombinant plasmid inserted with the target gene can be quickly and efficiently screened by the above method. As Figure 7 shown in B of , both casposase-WT / Ecasposase can insert the target gene of about 1000 bp into the target sequence containing leader-TSD-WT. Compared with the wild-type casposase (i.e., casposase-WT), the engineered Ecasposase can significantly improve the gene insertion efficiency.

[0056] Given the direction selectivity of casposase-mediated gene insertion ( Figure 1 ), in this example, BL21(DE3) monoclonal colonies generated on the ampicillin-resistant plate were randomly picked and subjected to Sanger sequencing to determine the direction of Ecasposase gene insertion. The results are shown in A of Figure 8 : For the insertion target containing TSD-WT, there is no obvious gene insertion selectivity (L-insertion ~56% vs. S-insertion ~40%); by modifying TSD-WT, Ecasposase can show obvious gene insertion direction selectivity. Ecasposase shows a significant L-insertion tendency for the target sequence containing TSD-L (L-insertion ~87% vs. S-insertion ~5%); while for the target sequence containing TSD-S, it shows an obvious S-insertion tendency (L-insertion ~85% vs. S-insertion ~8%). The Sanger sequencing results of BL21(DE3) monoclonal colonies on the ampicillin-resistant plate show ( Figure 8 shown in B of ): The insertion sites of the target gene are all located in the leader-TSD target sequence region. For the target sequence containing the TSD-L sequence, the gene insertion direction tends to be L-insertion, while for the target sequence of the TSD-S sequence, the gene insertion tends to be S-insertion.

[0057] Example 5 Construction of a gene site-directed insertion tool using a plasmid as a vector This example is based on Example 1 and constructs an engineered casposase gene site-directed insertion tool using a plasmid as a vector.

[0058] This example involves two plasmid-based engineered casposase gene site-directed insertion tools: the two-plasmid system based on Ecasposase (TPE) and the single-plasmid system based on Ecasposase (SPE).

[0059] The mDonor and pTarget constructed in Example 1 are the two plasmids in the TPE system. As shown in A of Figure 9 , an equal amount of mDonor and pTarget with a final concentration of 50 ng / μL were co-electroporated into BL21(DE3) and DB3.1 competent cells. Subsequently, BL21(DE3) was spread on an ampicillin-resistant LB plate containing IPTG (final concentration 0.2 mM), and DB3.1 competent cells were spread on an ampicillin-resistant LB plate containing IPTG (final concentration 0.2 mM). The plasmid in the BL21(DE3) monoclonal colonies growing on the ampicillin-resistant plate is pInsert containing the target gene. The growth results of a representative TPE screening plate are shown in C of Figure 9 .

[0060] For the single-plasmid system based on Ecasposase (SPE), first, the pET-28a-Ecasposase constructed in Example 1 was transformed into BL21(DE3), and the colonies were screened on a kanamycin-resistant plate. The monoclonal colonies generated were picked to prepare calcium chloride chemically competent cells of recombinant bacterium 1. Subsequently, the PCR product of the target gene with TRF and TRR at both ends (1Donor described in Example 1, final concentration 0.2 μM) and the pTarget plasmid (final concentration 50 ng / μL) were co-electroporated into the newly prepared chemically competent cells of recombinant bacterium 1, and finally spread on an ampicillin-resistant plate containing IPTG (final concentration 0.2 mM). The monoclonal colonies that grew contain the target gene (shown in B of Figure 9 ). As shown in D of Figure 9 , both plasmid systems can achieve site-directed insertion of the target gene, and the gene insertion efficiency of SPE is slightly higher than that of TPE (SPE ~22% vs. TPE ~17%). In addition, the qPCR method can also be used. One primer is designed for each of pTarget and the target gene, and the bacteria before transformation and plating of TPE and SPE are used as templates to quantitatively analyze the internal reference gene and the target gene to characterize the gene insertion efficiency.

[0061] Based on the above embodiments, the gene site-directed insertion tool of the present invention is derived from the type II casposase family Methanosarcina mazei GÖ 1 casposase. Through engineering transformation, it can quickly and efficiently achieve site-directed insertion of target genes up to 8000 bp in various vector forms, and at the same time can selectively control the insertion direction of the target gene. The present invention will further expand the existing gene site-directed insertion tool library and also lay a foundation for the further development and application of Ecasposase.

[0062] The present invention provides an idea and method for a gene site-directed insertion tool and application based on casposase transposase. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A gene site-directed insertion tool based on casposase transposase, characterized in that, The gene site-directed insertion tool described above includes casposase transposase, 1Donor sequence and pTarget; Among them, the amino acid sequence of the casposase transposase described above is as shown in SEQ ID No.2 or SEQ ID No.4; The 1Donor sequence described above includes a target gene sequence, and the two ends of the target gene sequence are terminal inverted repeat sequences; The pTarget is a plasmid containing a leader sequence and a TSD sequence.

2. The gene site-directed insertion tool according to claim 1, wherein The terminal inverted repeat sequence is TRF and TRR; The nucleotide sequence of the TRF is 5′-TAGAATCT-3′; The nucleotide sequence of the TRR is 5′-AGATTCTA-3′; The structure of the 1Donor sequence is 5′-TRF-target gene sequence-TRR-3′.

3. The gene site-directed insertion tool according to claim 1, wherein The 1Donor sequence is integrated into the first plasmid vector for gene site-directed insertion editing, and the first plasmid vector includes any one of pCDFDuet-1, pRSFDuet-1 or pACYDuet1.

4. The gene site-directed insertion tool according to claim 1, wherein The nucleotide sequence of the leader sequence is 5′-ACTCCAAGAGCAGAAGAGTTT-3′, as shown in SEQ ID No.7; The nucleotide sequence of the TSD sequence described above is any one of the following a1~a3: a1, 5′-ATTGATAAAGAGT-3′, as shown in SEQ ID No.8, a2, 5′- ATTGATATTGAGA -3′, as shown in SEQ ID No.9, a3, 5′-TTTCATAAAGAGT-3′, as shown in SEQ ID No.10; For the pTarget described above, the starting plasmid is a plasmid containing the ccdB toxicity element; In the pTarget described above, the connection order of the leader sequence, TSD sequence and ccdB toxicity element is 5′-leader sequence-TSD sequence-ccdB toxicity element-3′.

5. The gene site-directed insertion tool according to claim 1, characterized in that The gene site-directed insertion tool described above includes a gene site-directed insertion tool with a protein as a vector and a gene site-directed insertion tool with a plasmid as a vector; Among them, for the gene site-directed insertion tool with a protein as a vector, gene site-directed insertion editing is carried out using the casposase transposase obtained by protein purification; For the gene site-directed insertion tool with a plasmid as a vector, the casposase transposase is integrated into the plasmid vector, and gene site-directed insertion editing is carried out in the form of a plasmid. The plasmid vector includes the first plasmid vector and / or the second plasmid vector. The first plasmid vector includes any one of pCDFDuet-1, pRSFDuet-1 or pACYDuet1, and the second plasmid vector includes any one of pET-28a, pGEX-6P-1, pUC19 or pUC57.

6. A gene site-directed insertion method, characterized in that, Use the gene site-directed insertion tool according to any one of claims 1-5; the gene site-directed insertion method comprises the following steps: transforming the casposase transposase, the 1Donor sequence and pTarget into a host cell to achieve site-directed insertion of a gene.

7. The gene site-directed insertion method according to claim 6, wherein The gene site-directed insertion tool includes a gene site-directed insertion tool with a protein as a vector and a gene site-directed insertion tool with a plasmid as a vector; the gene site-directed insertion tool with a plasmid as a vector includes a two-plasmid system and a single-plasmid system; When the gene site-directed insertion tool is a gene site-directed insertion tool with a protein as a vector, the gene site-directed insertion method is: incubating the casposase transposase purified from the protein and the 1Donor sequence amplified by PCR to obtain a nucleoprotein complex, mixing the nucleoprotein complex with pTarget and then transforming it into a host cell to achieve site-directed insertion of a gene; When the gene site-directed insertion tool is a two-plasmid system, the gene site-directed insertion method is: cloning the coding gene of the casposase transposase and the 1Donor sequence into a first plasmid vector to obtain an mDonor plasmid, and co-transforming the mDonor plasmid and the pTarget plasmid into a host cell to achieve site-directed insertion of a gene; When the gene site-directed insertion tool is a single-plasmid system, the gene site-directed insertion method is: cloning the casposase transposase plasmid into a second plasmid vector to obtain a casp plasmid, transforming the casp plasmid into a host cell to obtain recombinant bacterium 1, and co-transforming the 1Donor sequence and the pTarget plasmid into recombinant bacterium 1 to achieve site-directed insertion of a gene.

8. The gene site-directed insertion method according to claim 7, wherein The nucleotide sequence of the coding gene of the casposase transposase is as shown in SEQ ID No.1 or SEQ ID No.3; The first plasmid vector includes any one of pCDFDuet-1, pRSFDuet-1 or pACYDuet1; The second plasmid vector includes any one of pET-28a, pGEX-6P-1, pUC19 or pUC57.

9. Application of the gene site-directed insertion tool according to any one of claims 1-5 in gene editing of prokaryotes or eukaryotes.

10. The application according to claim 9, wherein The prokaryotes include Escherichia coli.

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