A gene site-directed insertion tool based on casposase transposase and its application

Abstract

The present invention relates to a gene site-directed insertion tool based on casposase transposase and its application. The gene site-directed insertion tool consists of casposase transposase, 1Donor sequence and insertion target pTarget. 1Donor sequence consists of any target sequence with 8 bp homology arms at both ends, and the insertion target consists of leader-TSD target sequence. The present invention utilizes casposase transposase to efficiently and specifically insert a target gene as long as 8000 bp into the target site, and can also selectively control the insertion direction of the target gene. In addition, the casposase transposase in the present invention can realize gene site-directed insertion in the form of protein and plasmid. The application of this tool is to realize the site-directed insertion of the target gene in prokaryotic or eukaryotic cells, which is suitable for genetic information modification and giving cells new functions.

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] At present, the main tools for site-directed gene insertion include nucleases, transposases, and recombinases. Most of these tools have defects such as poor insertion site specificity, low efficiency, and large number of functional components, 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.). Site-directed gene insertion mediated by nucleases such as Cas9 is to cut a specific genomic target site, induce double-strand breaks, and then complete the site-directed insertion of the target gene through repair methods such as homology-directed repair (HDR). However, this method has low insertion efficiency and DNA double-strand breaks are prone to cause mutation defects such as chromosomal translocation and rearrangement (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 Back (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 evolved in bacteria and archaea in the face of constant attacks from bacteriophage viruses and exogenous plasmids. The acquisition of short fragments of exogenous genetic information is the foundation of the entire CRISPR-Cas immune function, while Cas1 is the main player in bacteria acquiring immune memory (Barrangou R et al., “CRISPR provides acquired resistance against viruses in prokaryotes.” Science, 2007, 5819:1709-1712.). In recent years, a special class of bacteria has been discovered in certain cas1 There is no obvious CRISPR locus around the gene, but there are some transposon-like elements, such as terminal inverted repeat (TIR) ​​and target site duplication (TSD) (Krupovic M et al., “Casposons: a new superfamily of self-synthesizing DNA transposons at the origin of prokaryotic CRISPR-Casimmunity.” Bmc Biology, 2014, 12(1): 36.). This type of sequence containing both Cas1 homologous integrase and transposon-like elements is called a “casposon”. Among them, the only Cas1-homologous nuclease with integration activity is called a casposase. Taxonomic studies suggest that casposase is likely 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, casposases gradually lost their ability to integrate long DNA fragments, but the site specificity of their integration reactions gradually increased, ultimately leading to the development of Cas1, which can catalyze the highly site-specific integration of short sequences (Wright A. V et al., “A Functional Mini-Integrase in a Two-Protein-type VC CRISPR System.” Molecular Cell, 2019, 4: 727-+.). Leveraging these properties of Cas1, bacteria can acquire immune memory of phage genetic information using only a short sequence of a few dozen base pairs, effectively completing the entire CRISPR immune mechanism.

[0005] Despite significant differences in the composition of the integration elements and the length of the integration sequences, the casposase-catalyzed integration reaction closely resembles the mechanism of Cas1-mediated integration of short 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 cassone integration through two nucleophilic attack reactions similar to those of Cas1. The first nucleophilic attack occurs when the 3′-OH terminus of the TIR sequence attacks the TSD sequence near the leader, forming a semi-integrated intermediate. The second nucleophilic attack occurs at the far leader end of the TSD sequence, forming a double-stranded fully integrated product, and finally produces a complete TSD sequence through DNA repair, completing the site-specific insertion of casposon (Wang X et al., "Sequencespecific integration by the family 1 casposase from Candidatus Nitrosopumiluskoreensis AR1." Nucleic Acids Research, 2021, 49(17): 9938-9952.). The above integration process does not involve complete breakage of the blunt end of the double-stranded DNA, and the TSD sequence that needs to be repaired is usually only a few dozen bases. Therefore, casposase-mediated gene insertion can avoid genetic information damage caused by DNA cutting 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 focus more on the homology between casposase and Cas1, that is, its site-specific integration ability of short-fragment DNA. However, further research is needed on the casposase-like aspect of its transposase-like nature, namely its ability to integrate long DNA fragments at specific sites. Compared to short DNA fragments, this ability has greater application value, such as the construction of chimeric antigens in cell therapy, the development of stably transfected cell lines for antibody drug production, and the modification of crop genetics for yield and insect resistance. Therefore, research on the long DNA fragment integration reaction of casposases and the exploration of new tools for site-specific gene insertion are of great significance.

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

[0007] Purpose of the Invention: The present invention addresses the shortcomings of existing technologies by providing a casposase-based gene insertion tool and its applications. This tool enables efficient, highly specific, and highly selective site-specific insertion of exogenous genes using a single, low-molecular-weight protein. This invention overcomes the shortcomings of existing gene insertion tools, such as their complex components, bulky size, difficulty in delivery, reliance on complete double-stranded DNA breaks, inability to achieve continuous site-specific insertion, and poor directional selectivity of inserted genes. Applications of this system are also proposed.

[0008] In order to solve the above technical problems, the present invention discloses a gene site-specific insertion tool based on casposase transposase and its application. The specific technical solution is as follows:

[0009] A gene site-directed insertion tool based on casposase transposase, the gene site-directed insertion tool comprises casposase transposase, 1Donor sequence and pTarget; wherein the amino acid sequence of the casposase transposase is as shown in SEQ ID No.2 or SEQ ID No.4; the casposase transposase shown in SEQ ID No.2 is derived from Methanosarcina mazei The wild-type casposase transposase of GÖ 1 (casposase-WT) and the casposase transposase with an amino acid sequence as shown in SEQ ID No. 4 are engineered casposase transposase (Ecasposase).

[0010] The Donor sequence includes a target gene sequence, and both ends of the target gene sequence are terminal inverted repeat sequences; the target gene sequence is any target sequence to be inserted, preferably a chloramphenicol resistance gene (CmR).

[0011] The pTarget is a plasmid containing a leader sequence and a TSD sequence.

[0012] Wherein, the terminal inverted repeat sequences are TRF and TRR, which are derived from Methanosarcina mazei The nucleotide sequence of the TRF is 5′-TAGAATCT-3′; the nucleotide sequence of the TRR is 5′-AGATTCTA-3′; and the structure of the Donor sequence is 5′-TRF-target gene sequence-TRR-3′.

[0013] The 1Donor sequence is integrated into a first plasmid vector for site-directed gene insertion and editing, and the first plasmid vector includes any one of pCDFDuet-1, pRSFDuet-1, or pACYDuet1, preferably pCDFDuet-1. The 1Donor sequence can also be used directly in the form of a dsDNA sequence.

[0014] 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 to a3:

[0015] a1, 5′-ATTGATAAAGAGT-3′ (SEQ ID No. 8), named TSD-WT;

[0016] a2, 5′-ATTGATATTGAGA -3′ (SEQ ID No. 9), named TSD-L;

[0017] a3, 5′-TTTCATAAAGAGT-3′ (SEQ ID No. 10), named TSD-S;

[0018] Different TSD sequences are selected according to the direction of the inserted gene.

[0019] The leader sequence and TSD sequence are derived from Methanosarcina mazei GÖ 1.

[0020] The starting plasmid of the pTarget is a plasmid containing the ccdB toxicity element, preferably plasmid pKIL108.

[0021] In the pTarget, the connection order of the leader sequence, TSD sequence and ccdB toxic element is 5'-leader sequence-TSD sequence-ccdB toxic element-3'. Preferably, when the nucleotide sequence of the TSD is shown as SEQ ID No. 8, the nucleotide sequence of the pTarget is shown as SEQ ID No. 5.

[0022] This gene site-directed insertion tool uses casposase to bind to the TRF and TRR sequences of the Donor sequence to form a casposase-TRF / TRR protein-nucleic acid complex. The complex specifically recognizes and binds to the leader-TSD of the insertion target pTarget; then 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; through the modification and optimization of 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 differentiated, thereby controlling the insertion direction of the target gene and achieving efficient, specific, and directionally selective site-directed insertion of the target gene.

[0023] Among them, the gene site-specific insertion tool includes a gene site-specific insertion tool with a protein as a carrier and a gene site-specific insertion tool with a plasmid as a carrier; wherein, the gene site-specific insertion tool with a protein as a carrier utilizes the casposase transposase obtained by protein purification to perform gene site-specific insertion editing, and completes the expression of the casposase protein in vitro; the gene site-specific insertion tool with a plasmid as a carrier integrates the casposase transposase into a plasmid vector, and performs gene site-specific insertion editing in the form of a plasmid, wherein the plasmid vector includes a first plasmid vector and / or a second plasmid vector, wherein the second plasmid vector includes any one of pET-28a, pGEX-6P-1, pUC19 or pUC57, and the gene site-specific insertion tool with a plasmid as a carrier then completes the expression of the casposase protein in vivo.

[0024] In a second aspect, the present invention provides a method for site-directed gene insertion, using the gene site-directed insertion tool described in the first aspect; the gene site-directed gene insertion method comprises the following steps: transforming the casposase transposase, 1Donor sequence, and pTarget into a host cell to achieve site-directed gene insertion. The gene site-directed gene insertion tool includes protein-based gene site-directed insertion tools and plasmid-based gene site-directed insertion tools; the plasmid-based gene site-directed insertion tools include dual-plasmid systems and single-plasmid systems.

[0025] When the gene site-directed insertion tool is a gene site-directed insertion tool with a protein as a carrier, the gene site-directed insertion method is: incubating the casposase transposase obtained by protein purification with the 1Donor sequence obtained by PCR amplification to obtain a nucleic acid-protein complex, mixing the nucleic acid-protein complex with pTarget and then transforming it into the host cell to achieve site-directed insertion of the gene; preferably, the casposase transposase obtained by protein purification is prepared according to the following method: transforming the plasmid expressing casposase into E. coli Induced expression is performed in prokaryotic expression cells, and high-purity, conformationally correct casposase protein is obtained through purification; the casposase-expressing plasmid is the casposase-expressing pET28a-sumo plasmid, and the E. coli Prokaryotic expression cells E. coli BL21 (DE3), during purification, the expression product is first subjected to nickel column affinity chromatography and high salt elution to obtain crude protein, and then the sumo-tag is cut off by SUMO Protease. Finally, the tag-free crude protein is subjected to gel filtration chromatography to obtain high-purity casposase transposase. This preparation method can quickly and efficiently produce high-purity and conformationally correct casposase. E. coli Prokaryotic expression system, affinity chromatography and gel filtration chromatography purification can obtain a large amount of target protein within 24 hours.

[0026] When the gene site-directed insertion tool is a dual-plasmid system (composed of two plasmids, mDonor and pTarget), the gene site-directed insertion method is: cloning the casposase transposase encoding gene and the 1Donor sequence into a first plasmid vector to obtain an mDonor plasmid, and jointly transforming 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: 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 a recombinant bacterium 1, and jointly transforming the 1Donor sequence and the pTarget plasmid into the recombinant bacterium 1 to achieve site-directed insertion of the gene.

[0027] The nucleotide sequence of the gene encoding the casposase transposase is shown as SEQ ID No. 1 (nucleotide sequence of the gene encoding casposase-WT) or SEQ ID No. 3 (nucleotide sequence of the gene encoding Ecasposase); 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.

[0028] The host cell is preferably Escherichia coli BL21 (DE3).

[0029] Preferably, the insertion efficiency and direction of the target gene can be determined based on the number of single colonies on the plate after transformation combined with Sanger sequencing results. The efficiency of gene insertion can also be analyzed by NGS sequencing.

[0030] In a third aspect, the present invention provides the use of the gene site-directed insertion tool described in the first aspect in gene editing of prokaryotes or eukaryotes, wherein the prokaryotes include Escherichia coli.

[0031] Beneficial effects:

[0032] The present invention focuses on the long-fragment DNA integration activity of casposase and develops it into a new gene site-directed insertion tool. By performing a series of modifications and optimizations on the casposase protein, its donor DNA Donor, and the insertion target pTarget, the gene length and site-directed insertion efficiency of casposase can be increased, the off-target rate of gene insertion is reduced, and the direction of the inserted gene is controlled 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, achieving efficient site-directed insertion of the target gene, and compensating for the defects of the existing gene insertion tools such as complex components and large molecular weight that are difficult to deliver. In addition, the present invention utilizes casposase transposase to efficiently and specifically insert target genes up to 8000 bp into the target site, while also selectively controlling the insertion direction of the target gene. The preparation method of the present invention can quickly and efficiently prepare high-purity casposase protein in vitro, and can also achieve in vivo expression of casposase through a plasmid vector, thereby completing the site-directed insertion of the target gene. The present invention can achieve genetic information modification through gene site-specific insertion, endow cells with new functions, and also lay the foundation for the subsequent development and optimization of the system. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described 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.

[0034] Figure 1 Two directions for casposase-mediated gene site-directed insertion.

[0035] Figure 2 The plasmid maps of pET-28a-Ecaspoase and pET28a-sumo-Ecasposase constructed in the present invention.

[0036] Figure 3 This is the plasmid map of pKIL108-leader-TSD (pTarget) constructed in Example 1 of the present invention.

[0037] Figure 4 This is the plasmid map of pCDFDuet1-Ecasposase-Donor (mDonor) constructed in Example 1 of the present invention.

[0038] Figure 5 The gel filtration chromatogram and SDS-PAGE electrophoresis identification diagram of Ecasposase and casposase-WT in Example 2 of the present invention are shown.

[0039] Figure 6 In Example 3 of the present invention, the specific insertion ability of Ecasposase into genes of different lengths was detected by fluorescence.

[0040] Figure 7 Flow chart of the gene site-directed insertion tool using protein as a carrier and the insertion efficiency results in Example 4 of the present invention. Figure 7 A in the figure is the flow chart of the gene site-directed insertion tool. Figure 7 Figure B is the insertion efficiency result diagram.

[0041] Figure 8 The influence of different TSD sequences on the directional selectivity of the Ecasposase site-directed insertion gene in Example 4 of the present invention. Figure 8 A in is the insertion efficiency graph, Figure 8 B in the figure is the representative Sanger sequencing result of the target gene insertion clone.

[0042] Figure 9 Flowchart of the gene site-directed insertion tool using a plasmid as a vector and a representative target gene insertion clone diagram in Example 5 of the present invention. Figure 9 A in is a double plasmid system, Figure 9 B in the equation is a single plasmid system. Figure 9 C in the figure is the target gene insertion clone diagram of the double plasmid system. Figure 9D in the figure is the insertion efficiency diagram of the dual-plasmid system and the single-plasmid system. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below with reference to the examples, but the present invention is not limited to the examples given.

[0044] Example 1 Plasmid construction

[0045] This embodiment is to construct plasmids, which are used for the subsequent preparation of various components of the gene site-specific insertion tool of the present invention. The gene site-specific insertion tool described in the present invention comprises a casposase transposase, a donor DNA Donor and an insertion target pTarget. The gene site-specific insertion tool is formed by casposase binding to the TRF and TRR sequences of the donor DNA to form a casposase-TRF / TRR protein nucleic acid complex, which specifically recognizes and binds to the leader-TSD of the insertion target pTarget; then 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 casposase-TRF / TRR protein nucleic acid complex on the near leader end and the far leader end of pTarget can be different, thereby controlling the insertion direction of the target gene and achieving efficient, specific and directionally selective site-specific insertion of the target gene (such as Figure 1 shown).

[0046] 1. Construction of plasmid expressing casposase transposase:

[0047] The coding gene sequences of Ecasposase (amino acid sequence shown in SEQ ID No. 4, coding gene shown in SEQ ID No. 3) and casposase-WT protein (amino acid sequence shown in SEQ ID No. 2, coding gene shown in SEQ ID No. 1) were amplified by PCR using primers casp-F / casp-R. The recombinant plasmids were then ligated into pET28a-sumo or pET28a using enzyme digestion (BamHI and XhoI) or homologous recombination. The recombinant plasmids were then transferred to pET28a-sumo or pET28a by chemical transformation. E. coli TOP10 competent cells were then extracted using a plasmid extraction kit to extract the monoclonal plasmid and the target recombinant plasmid was obtained after confirmation of the correctness by Sanger sequencing. The recombinant plasmids pET-28a-Ecasposase and pET28a-SUMO-Ecasposase constructed in this example are as follows: Figure 2 The primer sequences described in this embodiment are as follows:

[0048] Casp-F: 5′-CGCGGATCCATGAAACTGCTGCTGCTG-3′ (SEQ ID No. 11),

[0049] Casp-R: 5′-CCGCTCGAGACGCTGGGAGCTAACCAG-3′ (SEQ ID No. 12).

[0050] 2. Construction of target insertion pTarget plasmid:

[0051] In this example, the pTarget plasmid contains an inserted target sequence, and its DNA structure is 5′-leader-TSD-3′.

[0052] Among them, the leader sequence is 5′-ACTCCAAGAGCAGAAGAGTTT-3′ (SEQ ID No. 7).

[0053] There are three different TSD sequences: TSD-WT, TSD-L, and TSD-S. TSD-WT is 5′-ATTGATAAAGAGT-3′ (SEQ ID No. 8); TSD-L is 5′-ATTGATTTGAGA -3′ (SEQ ID No. 9); and 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, TSD-L / TSD-S was transformed from TSD-WT.

[0054] The 5′-leader-TSD-3′ fragment was connected to the pKIL108 vector to construct the recombinant plasmid pKIL108-leader-TSD (pTarget). Figure 3 As shown, the pKIL108-leader-TSD plasmid includes three types according to the different TSDs: pKIL108-leader-TSD-WT, pKIL108-leader-TSD-L and pKIL108-leader-TSD-S, wherein the nucleotide sequence of pKIL108-leader-TSD-WT is shown in SEQ ID No. 5, and the nucleotide sequences of pKIL108-leader-TSD-L and pKIL108-leader-TSD-S are obtained by replacing the nucleotide sequence at TSD-WT in the sequence shown in SEQ ID No. 5 with the nucleotide sequences of TSD-S and TSD-L, respectively.

[0055] 3. Construction of donor DNA:

[0056] In this example, the donor DNA has two compositions: lDonor and mDonor. lDonor is a linear dsDNA produced by PCR amplification and consists of 5′-TRF sequence-target gene sequence-TRR-3′. The TRF sequence is 5′-tagaatct-3′, and the TRR sequence is 5′-agattcta-3′.

[0057] In this example, dsDNA containing the chloramphenicol resistance gene (CmR) was used as the target gene sequence.

[0058] Another donor DNA mDonor was constructed by connecting the Ecasposase coding gene and 1Donor sequence into the two open reading frames of pCDFDuet-1, namely pCDFDuet1-Ecasposase-Donor. The plasmid map is shown in Figure 4 As shown, when the target gene is the CmR gene, the nucleotide sequence of pCDFDuet1-Ecasposase-Donor is shown as 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.

[0059] Example 2 Protein Purification

[0060] This example is based on Example 1, and the protein required for site-directed gene insertion was prepared and purified. The pET28a-SUMO-Ecasposase and pET28a-SUMO-casposase-WT constructed in Example 1 were transformed into Escherichia coli. E. coli In BL21 (DE3), pick the monoclonal colony growing on the kanamycin plate and transfer it to LB liquid medium. After overnight activation, transfer it to LB liquid medium and culture at 37℃ and 200 rpm until the bacterial liquid OD reaches 600 After the pH value was between 0.6 and 0.8, the temperature was lowered to 25°C and IPTG at a final concentration of 0.5 mM was added overnight to induce protein expression.

[0061] Overnight bacterial cultures were harvested by centrifugation and resuspended in Ni-NTA buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 10 mM imidazole). After sonication, the supernatant was centrifuged and loaded onto a nickel column. Low-affinity proteins were removed by washing with Ni-NTA buffer containing 20 mM imidazole. The crude protein was then eluted with Ni-NTA buffer containing a final concentration of 500 mM imidazole to obtain the crude E-casposase / casposase-WT protein. The crude protein was digested overnight with SUMO Protease at 4°C to remove the N-terminal SUMO tag of the target protein. The untagged E-casposase / casposase-WT was concentrated and loaded onto a Hiload 16 / 60 Surperdex 200 pg gel chromatography column. The purified protein was eluted with 20 mM HEPES pH 7.5, 150 mM NaCl. The corresponding gel chromatography chromatogram and SDS-PAGE electrophoresis identification results are as follows Figure 5 shown.

[0062] Example 3 Gene-specific insertion ability test

[0063] This example, building on Example 2, examined the specific insertion ability of E-casposase into genes of varying lengths in vitro. First, using Lenti-dCas9-KRAB-blast (Addgene Plasmid #89567) as a template, PCR amplification was performed using specific TRF / TRR primers (including Donnor-TRF and Donnor-TRR2000 / 4000 / 6000 / 8000) to generate target genes (i.e., donor genes) of approximately 2000, 4000, 6000, and 8000 bp, respectively, with 8 bp TRF / TRR sequences at either end. Highly purified donor genes were obtained by gel excision and recovery of the PCR products.

[0064] The dsDNA containing the leader-TSD sequence and labeled with 6-FAM and cy5 fluorescence at the 3′ end was used as the specific insertion target (leader-TSD target), and the dsDNA without the leader-TSD sequence was used as the nonspecific fluorescent substrate.

[0065] The Donor gene was added to a final concentration of 0.2 μM, the Ecasposase to a final concentration of 0.1 μM, and the fluorescent leader-TSD target to a final concentration of 0.2 μM in Insertion Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MnCl2) and incubated at 37°C for 30 min to catalyze the site-directed insertion reaction. The product was loaded onto a 2% agarose gel for electrophoresis, and the fluorescence scanning results were as follows: Figure 6 As shown in Table 1. The results show that Ecasposase can catalyze site-specific gene insertion of target genes of 2000-8000 bp, and the efficiency of gene insertion gradually decreases with the increase of target gene length. The DNA sequences used in this example are shown in Table 1.

[0066] Table 1 DNA sequence list used in Example 3

[0067]

[0068] Example 4 Construction of a gene-targeted insertion tool using protein as a carrier

[0069] This example is based on Example 2, and constructs an engineered casposase gene site-specific insertion tool using protein as a carrier.

[0070] Ecasposase and casposase-WT (final concentration 0.5 μM) purified in Example 2 were incubated with 1Donor (final concentration 0.2 μM) constructed in Example 1 at 4°C for 20 minutes in 20 mM HEPES pH 7.5, 20 mM NaCl to form a casposase-gene nucleic acid protein complex. pTarget (final concentration 40 ng / μL) constructed in Example 1 was added to the above complex and divided into two equal parts. Electroporation was performed on a Bio-Rad GenePulser Xcell (3 kV / cm, 300 Ω, 25 μF, 7 ms) to E. coli In BL21 (DE3) and DB3.1 competent cells, the ratio of the number of BL21 (DE3) single colonies X (ampicillin resistance plate) to the number of DB3.1 competent colonies Y (ampicillin resistance plate) is used to express the insertion efficiency of the target gene (e.g. Figure 7 (as shown in A in the figure).

[0071] Since the pTarget plasmid contains the ccdB gene, it can express the CcdB toxic protein and therefore cannot be propagated in BL21 (DE3) cells. DB3.1 cells contain a mutant of DNA gyrase (gyrA462) that can tolerate the toxic effects of CcdB, so pTarget can be propagated in DB3.1. The insertion of a new gene can cause a frameshift mutation in the ccdB gene in pTarget, disrupting the ccdB gene reading frame and making it impossible to express the CcdB toxic protein. Therefore, the recombinant pTarget can be propagated normally in BL21 (DE3) cells. The above method can be used to quickly and efficiently screen out recombinant plasmids with inserted target genes. Figure 7 As shown in Figure 1B, both casposase-WT and Ecasposase can insert a target gene of approximately 1000 bp in length 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.

[0072] Given that casposase-mediated gene insertion has a directional selection tendency ( Figure 1 ), in this example, BL21 (DE3) monoclones generated on the ampicillin-resistant plate were randomly selected and subjected to Sanger sequencing to determine the insertion direction of the Ecasposase gene. The results are shown in FIG. Figure 8 As shown in A, the target sequence containing TSD-WT showed no obvious gene insertion tendency (L-insertion ~56% vs. S-insertion ~40%). By modifying TSD-WT, Ecasposase showed a clear gene insertion direction tendency. Ecasposase showed a significant L-insertion tendency for the target sequence containing TSD-L (L-insertion ~87% vs. S-insertion ~5%), while it showed a significant S-insertion tendency for the target sequence containing TSD-S (L-insertion ~85% vs. S-insertion ~8%). The Sanger sequencing results of the BL21 (DE3) monoclonal on the ampicillin resistance plate showed ( Figure 8 B): The target gene insertion sites are all located in the leader-TSD target sequence region. The gene insertion direction of the target sequence containing the TSD-L sequence tends to be L-insertion, while the gene insertion direction of the target sequence containing the TSD-S sequence tends to be S-insertion.

[0073] Example 5 Construction of a gene site-specific insertion tool using a plasmid as a vector

[0074] This example is based on Example 1, and constructs an engineered casposase gene site-directed insertion tool using a plasmid as a vector.

[0075] This example relates to two plasmid-based engineered casposase gene site-directed insertion tools: a two-plasmid system based on Ecasposase (TPE) and a single-plasmid system based on Ecasposase (SPE).

[0076] The mDonor and pTarget constructed in Example 1 are the double plasmids in the TPE system. Figure 9 As shown in Figure A, equal amounts of mDonor and pTarget at a final concentration of 50 ng / μL were co-electroporated into BL21(DE3) and DB3.1 competent cells. BL21(DE3) cells were then plated onto ampicillin-resistant LB plates containing IPTG (final concentration 0.2 mM), and DB3.1 competent cells were plated onto ampicillin-resistant LB plates containing IPTG (final concentration 0.2 mM). The plasmid in the BL21(DE3) single colony growing on the ampicillin-resistant plate is the pInsert containing the target gene. Representative TPE screening plate growth results are shown in Figure 2. Figure 9 As shown in C.

[0077] The single plasmid system (SPE) based on Ecasposase was first transformed into BL21 (DE3) with the pET-28a-Ecasposase constructed in Example 1. Screening was performed on kanamycin-resistant plates, and the resulting single clones were picked to prepare calcium chloride chemically competent cells of recombinant bacteria 1. Subsequently, the target gene PCR product 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 bacteria 1. Finally, the cells were plated on ampicillin-resistant plates containing IPTG (final concentration 0.2 mM). The resulting single clones were the cells containing the target gene ( Figure 9 B in ). Figure 9 As shown in Figure D, both plasmid systems achieve site-specific insertion of the target gene, with SPE achieving slightly higher insertion efficiency than TPE (SPE ~22% vs. TPE ~17%). Alternatively, qPCR can be used to quantitatively analyze the internal reference gene and the target gene, using TPE and SPE transformed cells before plating as templates, to characterize gene insertion efficiency.

[0078] Based on the above embodiments, the gene site-directed insertion tool of the present invention is derived from the type 2 casposase family. Methanosarcina mazei Based on the GÖ1 casposase, through its engineering modification, rapid and efficient site-specific insertion of target genes up to 8000 bp in length can be achieved in a variety of vector formats, while also allowing for selective control of the insertion direction. This invention will further expand the existing library of tools for site-specific gene insertion and lay the foundation for the further development and application of Ecasposase.

[0079] The present invention provides a casposase-based gene site-directed insertion tool and its application concept and method. There are many methods and approaches to implement this technical solution. The above is only a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also considered to be within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A gene site-directed insertion tool based on casposase transposase, characterized in that: The gene site-directed insertion tool includes casposase transposase, 1Donor sequence and pTarget; Wherein, the amino acid sequence of the casposase transposase is shown as SEQ ID No.4; The Donor sequence includes a target gene sequence, and both ends of the target gene sequence are terminal inverted repeat sequences; The pTarget is a plasmid containing a leader sequence and a TSD sequence; The terminal inverted repeat sequences are 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 Donor sequence is 5′-TRF-target gene sequence-TRR-3′; 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 is any one of the following a1 to a3: a1, 5′-ATTGATAAAGAGT-3′, as shown in SEQ ID No. 8, a2, 5′-ATTGATATTTGAGA-3′, as shown in SEQ ID No. 9, a3, 5′-TTTCATAAAGAGT-3′, as shown in SEQ ID No.

10.

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

3. The gene-targeted insertion tool according to claim 1, characterized in that The pTarget, whose starting plasmid is a plasmid containing the ccdB toxicity element; In the pTarget, the connection order of the leader sequence, TSD sequence and ccdB toxic element is 5′-leader sequence-TSD sequence-ccdB toxic element-3′.

4. The gene-targeted insertion tool according to claim 1, characterized in that The gene site-specific insertion tool includes a gene site-specific insertion tool using protein as a carrier and a gene site-specific insertion tool using plasmid as a carrier; Among them, the gene site-specific insertion tool using protein as a carrier uses the casposase transposase obtained by protein purification to perform gene site-specific insertion editing; The gene site-directed insertion tool using a plasmid as a vector integrates the casposase transposase into the plasmid vector and performs gene site-directed insertion editing in the form of a plasmid. The plasmid vector includes a first plasmid vector and / or a 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.

5. A method for site-directed gene insertion, characterized in that: The gene site-directed insertion tool according to any one of claims 1 to 4 is used; the gene site-directed insertion method comprises the following steps: transforming the casposase transposase, 1Donor sequence and pTarget into a host cell to achieve site-directed insertion of the gene.

6. The method for site-directed gene insertion according to claim 5, wherein: The gene site-specific insertion tools include those with proteins as carriers and those with plasmids as carriers; the gene site-specific insertion tools with plasmids as carriers include dual-plasmid systems and single-plasmid systems; When the gene site-directed insertion tool is a gene site-directed insertion tool using a protein as a carrier, the gene site-directed insertion method is: incubating the casposase transposase obtained by protein purification with the Donor sequence obtained by PCR amplification to obtain a nucleic acid-protein complex, mixing the nucleic acid-protein complex with pTarget and then transforming it into a host cell to achieve site-directed insertion of the gene; When the gene site-directed insertion tool is a dual-plasmid system, the gene site-directed insertion method is as follows: cloning the casposase transposase encoding gene 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 gene insertion; 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 a recombinant bacterium 1, and co-transforming the 1Donor sequence and the pTarget plasmid into the recombinant bacterium 1 to achieve site-directed insertion of the gene.

7. The method for site-directed gene insertion according to claim 6, wherein: The nucleotide sequence of the gene encoding the casposase transposase is shown in 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.

8. Use of the gene site-directed insertion tool according to any one of claims 1 to 4 in gene editing of prokaryotes; the prokaryotic organism is Escherichia coli.

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