Retrotransposon systems for genome editing and their applications

Abstract

This application provides a retroposophyte system for editing the genome and its use therein, wherein the retroposophyte in the retroposophyte system comprises an amino acid sequence as shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530; or the retroposophyte is a truncated version of an amino acid sequence as shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530. This application also relates to engineered sequences of retrosockase coupled with other amino acid sequences; and engineered sequences of truncated retrosockase coupled with other amino acid sequences.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and in particular to a retrotransposon system for editing genomes and its uses.

[0002] Cross-referencing This application claims the benefits of Chinese patent application CN2024119768030, filed on December 30, 2024, and Chinese patent application CN2025114152934, filed on September 29, 2025, the contents of which are incorporated herein by reference in their entirety.

[0003] The sequence listing file submitted at the same time The entire contents of the following XML file are incorporated herein by reference in their entirety: Sequence listing PI05199 in Computer-readable Format (CRF) (created on December 25, 2025, file size 798KB). Background Technology

[0004] Large DNA fragment genomic integration is a key technology in genetic engineering research. However, integrating large DNA fragments into the mammalian cell genome is generally inefficient and lacks specificity. Existing genome editing methods, such as the CRISPR-Cas system, perform well in editing small DNA fragments but are not suitable for inserting long DNA fragments. Other methods for site-specific insertion of large DNA fragments also have their limitations. For example, achieving large fragment insertion through homologous recombination is inefficient, and this method involves double-strand DNA breaks, posing certain safety risks. Furthermore, recombinase systems like Cre / loxP usually require pre-insertion of loxP sites for subsequent integration. At the same time, these technologies largely rely on donor DNA, facing challenges such as high immunogenicity, difficulties in in vivo delivery, and the risk of random integration into the genome.

[0005] In contrast, retroposylases offer a more advantageous option for site-specific integration of large DNA fragments into the genome, filling a gap in this technological field. A retroposylase system typically consists of two parts: first, a retroposylase protein peptide; and second, donor RNA carrying the new gene information. Retroposylase recognizes and binds to the donor RNA, forming a protein-RNA complex, and then uses its reverse transcriptase activity to reverse transcribe the RNA into DNA, which is subsequently integrated into a specific site in the genome. This technology not only avoids the risks associated with double-strand DNA breaks but also reduces dependence on donor DNA, achieving efficient integration of large DNA fragments through a single reverse transcription reaction, and has broad application potential.

[0006] The R2 retrotransposon system is an important member of the retrotransposase family, exhibiting unique advantages, especially in the site-specific integration of large DNA fragments. R2 retrotransposons are a class of non-LTR retrotransposons widely found in eukaryotic genomes, renowned for their efficient and precise DNA insertion mechanism. This system primarily targets 28S rDNA genes, synthesizing cDNA through reverse transcription to specifically integrate the R2 gene sequence into the host genome.

[0007] The integration process of the R2 retrotransposon relies on the activity of reverse transcriptase. It first recognizes a specific RNA donor sequence via retrotransposase, which then reverse transcribes the donor RNA into cDNA, which is then integrated into the target DNA. Compared to traditional genome editing techniques, the R2 retrotransposon does not rely on homologous recombination or double-strand DNA breaks, significantly reducing genomic instability and potential safety risks. Furthermore, its precision and efficiency in integrating large fragments have immense application value in gene insertion. Summary of the Invention

[0008] In view of the technical problems existing in the prior art, there is an urgent need in the field to develop a more effective retropozid system. The R2 system described in this application exhibits highly efficient gene integration activity in mammalian cells, which greatly enriches the retropozid chassis tool library that can be applied to biotechnology fields such as gene editing.

[0009] The specific technical solution of this application is as follows: 1. A retroposomyase, said retroposomyase comprising an amino acid sequence as shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530, or comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530; or The reverse transposase is a truncated version of the amino acid sequence shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530.

[0010] 2. A system for modifying DNA, the system comprising: A retroposomyase comprising an amino acid sequence as shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530, or comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530; or The retrosockase is a truncated version of the amino acid sequence shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530, for example, truncating up to 250 amino acids from the N-terminus; or The retrososozyme is an engineered retrososozyme obtained by further coupling the amino acid sequence or a truncated version thereof, as described in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530, with other amino acid sequences. The other amino acid sequences are selected from any one, two, three, four, five, six, or seven of the following: exonucleases, single-stranded DNA-binding proteins, chromatin regulatory peptides, high-migration peptides, RNaseH domains, nucleolar localization signals (NLS), nucleolar localization sequences, and nucleoside localization signals; and The donor RNA or nucleic acid encoding the donor RNA, the donor RNA comprising: a sequence that binds to the retrososozyme and a heterologous sequence; Preferably, the heterologous sequence is at least 1-50,000 bases, for example, 1 nt or more, 10 nt or more, 50 nt or more, 60 nt or more, 70 nt or more, 80 nt or more, 90 nt or more, 100 nt or more, 150 nt or more, 200 nt or more, 250 nt or more, 300 nt or more, 350 nt or more, 400 nt or more, 450 nt or more, 500 nt or more, 550 nt or more, 600 nt or more, 650 nt or more, 700 nt or more, 750 nt or more, 800 nt or more, 850 nt or more, 900 nt or more, 950 nt or more, 1000 nt or more, 1100 nt or more, 1200 nt or more, 1300 nt or more, 1400 nt or more, 1500 nt or more, 1600 nt or more, 1700 nt or more, 1800 nt or more, 1900 nt or more. NT or higher, 2000 NT or higher, 2100 NT or higher, 2200 NT or higher, 2300 NT or higher, 2400 NT or higher, 2500 NT or higher, 2600 NT or higher, 2700 NT or higher, 2800 NT or higher, 2900 NT or higher, 3000 NT or higher, 3500 NT or higher, 4000 NT or higher, 4500 NT or higher, 5000 NT or higher, 5500 NT or higher, 6000 NT or higher, 6500 NT or higher, 7000 NT or higher, 7500 NT or higher, 8000 NT or higher, 8500 NT or higher, 9000 NT or higher, 9500 NT or higher, 10000 NT or higher, 15000 NT or higher, 20000 NT or higher, 25000 NT or higher, 30000 NT or higher, 35000 NT or higher, 40000 NT or higher, 45000 NT or higher; The system is further preferably comprised of nucleic acid encoding VPX protein or VPX protein itself; Preferably, the other amino acid sequence is an exonuclease, and the exonuclease is inserted at the N-terminus, interior, or C-terminus of the retroposal enzyme; Preferably, the exonuclease is an exonuclease derived from T5 bacteriophage; Further preferred exonuclease sequences are as shown in SEQ ID NO. 495 or have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 495; or The other amino acid sequences are single-stranded DNA-binding proteins, and the single-stranded DNA-binding proteins are inserted at the N-terminus, interior, or C-terminus of the retrospinase; Preferably, the single-stranded DNA binding protein is a single-stranded DNA binding protein from the bacteria *Sulfolobus tokodaii*. Further preferred single-stranded DNA-binding proteins have a sequence such as SEQ ID NO. 496 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence described in SEQ ID NO. 496; or The other amino acid sequences are chromatin-regulating peptides or high-mobility peptides, and the chromatin-regulating peptides or high-mobility peptides are inserted at the N-terminus, interior, or C-terminus of the retrospinase. Preferably, the chromatin-regulating peptide or high-mobility peptide is a human chromatin-regulating peptide or high-mobility peptide. The preferred chromatin-regulating peptide or high-mobility peptide sequence is as shown in SEQ ID NO. 497 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 497; or The other amino acid sequence is an RNaseH domain, and the RNaseH domain is inserted at the N-terminus, interior or C-terminus of the retrospinase; Preferably, the RNaseH domain is an RNaseH domain derived from MLV reverse transcriptase; Further preferred sequences of the RNase H domain of MLV reverse transcriptase are as shown in SEQ ID NO. 498 or have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 498; or The other amino acid sequences are nuclear localization signals (NLS), and the nuclear localization signals (NLS) are inserted at the N-terminus, interior, or C-terminus of the retrososidase; Preferably, the nuclear localization signal (NLS) sequence is as shown in SEQ ID NO. 499 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 499; or The other amino acid sequences are nucleolar positioning signals, and the nucleolar positioning signals are inserted at the N-terminus, interior, or C-terminus of the retrososidase; Preferably, the nucleolar localization signal sequence is as shown in SEQ ID NO. 521 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 521; or The other amino acid sequences are nucleoside localization signals, and the nucleoside localization signals are inserted at the N-terminus, interior, or C-terminus of the retrososozyme. Preferably, the nucleus localization signal sequence is as shown in SEQ ID NO. 500 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 500.

[0011] In this application, the N-terminus of the retrososomal enzyme refers to the amino terminus of the amino acid sequence of the retrososomal enzyme, and the C-terminus of the retrososomal enzyme refers to the carboxyl terminus of the amino acid sequence of the retrososomal enzyme. Insertion of any of the aforementioned other amino acid sequences at the N-terminus of the retrososomal enzyme is performed at the amino terminus of the amino acid sequence, and insertion of any of the aforementioned other amino acid sequences at the C-terminus of the retrososomal enzyme is performed at any position within the region from the first amino acid at the N-terminus to the last amino acid at the C-terminus.

[0012] 3. The system according to item 2, wherein, The heterologous sequence comprises one or more of the following: sequences encoding polypeptides or non-coding RNA sequences, sequences containing promoters or enhancers, sequences encoding one or more introns, and transcription termination sequences; Preferably, the polypeptide is a therapeutic polypeptide or a mammalian polypeptide; more preferably, the polypeptide is a therapeutic protein, membrane protein, intracellular protein, extracellular protein, structural protein, signal transduction protein, regulatory protein, transport protein, organelle protein, sensory protein, motor protein, defense protein, storage protein, reporter protein, antibody, enzyme, or coagulation factor; and even more preferably, the polypeptide has 20 to 10,000 amino acids, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 210 amino acids. 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 59 00, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900; Preferably, the intracellular proteins are selected from cytoplasmic proteins, nucleoproteins, organelle proteins, mitochondrial proteins, or lysosomal proteins. Further preferred is that the sequence encoding the polypeptide contains one or more introns.

[0013] 4. The system according to item 2 or 3, wherein the donor RNA further comprises a homology domain, preferably the homology domain comprising a first homology domain and a second homology domain; Further preferably, the first homologous domain consists of three or more bases located at the 5' end of the donor RNA that have 100% identity with the target DNA strand, and the second homologous domain consists of three or more bases located at the 3' end of the donor RNA that have 100% identity with the target DNA strand. Preferably, the target DNA is a genomic safe harbor GSH site or a genomic natural harbor site. TM Site.

[0014] 5. The system according to any one of items 2-4, wherein the nucleic acid encoding the retrosockase and the donor RNA or the nucleic acid encoding the donor RNA are separate nucleic acids, preferably the donor RNA does not encode the retrosockase, and more preferably the donor RNA contains one or more chemical modifications; or The nucleic acid encoding the retrosockase and the donor RNA or the nucleic acid encoding the donor RNA are covalently linked. Preferably, the nucleic acid encoding the retrosockase and the donor RNA or the nucleic acid encoding the donor RNA form a fusion nucleic acid. More preferably, the fusion nucleic acid comprises RNA or DNA. Further preferably, the nucleic acid encoding the retrososase, the donor RNA or the nucleic acid encoding the donor RNA, and the nucleic acid encoding the VPX protein are separate nucleic acids; or The nucleic acid encoding the retrososozyme and the nucleic acid encoding the VPX protein are covalently linked or fused nucleic acids, and are separate nucleic acids from the donor RNA or the nucleic acid encoding the donor RNA; The nucleic acid encoding the retrososozyme is covalently linked or fused with the donor RNA or the nucleic acid encoding the donor RNA, and is a separate nucleic acid from the nucleic acid encoding the VPX protein; The donor RNA or the nucleic acid encoding the donor RNA is covalently linked or fused with the nucleic acid encoding the VPX protein, and is a separate nucleic acid from the nucleic acid encoding the retrososomalase; or The nucleic acid encoding the retrososase, the donor RNA or the nucleic acid encoding the donor RNA, and the nucleic acid encoding the VPX protein are covalently linked or fused nucleic acids. The fused nucleic acid is further preferably composed of RNA or DNA.

[0015] 6. The system according to any one of claims 2-5, wherein the donor RNA comprises: Optional 5' untranslated sequence (5'UTR) that binds to the said retrospinase, The 3' untranslated sequence (3'UTR) that binds to the retrospinase, Heterogeneous sequences, and The promoter operatively connected to the heterogeneous sequence, Preferably, the promoter is located between the 5' untranslated sequence (5'UTR) bound by the retrosockase and the heterologous sequence, or preferably, the promoter is located between the 3' untranslated sequence (3'UTR) bound by the retrosockase and the heterologous sequence.

[0016] 7. The system according to claim 6, wherein the heterologous sequence is contained in an open reading frame or its reverse complementary sequence oriented at 5' to 3' on the donor RNA; or the heterologous sequence is contained in an open reading frame or its reverse complementary sequence oriented at 3' to 5' on the donor RNA.

[0017] 8. The system according to any one of claims 2-7, wherein the donor RNA further comprises a nuclear localization signal or the nucleic acid encoding the retrospinase comprises a nuclear localization signal and / or a nucleolar localization signal and / or an exit nuclear signal.

[0018] 9. The system according to any one of claims 2-8, wherein the nucleic acid encoding the retrosockase and the nucleic acid encoding the donor RNA are present in a ratio of 10:1 to 1:10, for example in a ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0019] 10. The system according to any one of claims 2-9, wherein the donor RNA comprises a stem-loop sequence or helix at the 5' end of a pseudoknot sequence, preferably comprising one or more (e.g., 2, 3 or more) stem-loop sequences or helices at the 3' end of a pseudoknot sequence, such as the 3' end of a pseudoknot sequence and the 5' end of a heterologous sequence, further preferably the donor RNA of the pseudoknot has catalytic activity, such as RNA cleavage activity, for example, cis-RNA cleavage activity, or The donor RNA contains, for example, at least one stem-loop sequence or helix at the 3' of the heterologous sequence, such as 1, 2, 3, 4, 5 or more stem-loop sequences, hairpin or helical sequences.

[0020] 11. The system according to any one of items 2-10, wherein The 5' untranslated sequence (5'UTR) in the donor RNA that binds to the retrosockase comprises a nucleotide sequence as shown in any one of SEQ ID NO:159-316 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence shown in any one of SEQ ID NO:159-316. The 3' untranslated sequence (3'UTR) in the donor RNA that binds to the retrosockase comprises a nucleotide sequence as shown in any one of SEQ ID NO:317-474 and SEQ ID NO:501-507, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence shown in any one of SEQ ID NO:317-474 and SEQ ID NO:501-507.

[0021] 12. The system according to any one of items 2-11, wherein, The donor RNA comprises the following structures from its 5' end to its 3' end: First homologous domain, The 5' untranslated sequence (5'UTR) that binds to the said retrospinase, Heterogeneous sequences, The 3' untranslated sequence (3'UTR) that binds to the said retrospinase, and Second homologous domain; Preferably, the first homologous domain is one or more, two or more, five or more, ten or more, twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, eighty or more, ninety or more, one hundred or one hundred ... Further preferably, the 5' untranslated sequence (5'UTR) in the donor RNA that binds to the retrosockase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence shown in any one of SEQ ID NO:159-316. The 3' untranslated sequence (3'UTR) in the donor RNA that binds to the retrosockase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequences shown in any one of SEQ ID NO:317-474 and SEQ ID NO:501-507.

[0022] 13. The system according to any one of items 2-12, wherein The heterologous sequence is inserted into the target site in the cell genome at an average copy number of at least 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, or 5 copies / genome, preferably at only one target site in the genome, and preferably at the target site of the 28S rDNA gene.

[0023] 14. The system according to any one of claims 2-13, wherein the heterologous sequence insertion results in the insertion at a target site (e.g., with one or more copies of the insertion) in approximately 1%-100% of the cells (e.g., approximately 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% of the cells) in the cell population that has come into contact with the system, for example, as measured using single-cell ddPCR, or This results in the heterologous sequence being inserted at a copy number of 1 into the target site in approximately 1%–100% of the cells in the cell population in contact with the system (e.g., approximately 1%–10%, 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100% of the cells), for example, as measured using colony isolation and ddPCR.

[0024] 15. The system according to any one of claims 2-14, wherein the heterologous sequence is inserted into the target site (intermediate-target insertion) in the cell population at a higher rate than that of insertion into a non-target site (off-target insertion), wherein the ratio of intermediate-target insertion to off-target insertion is greater than 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 500:1 or 1,000:1.

[0025] 16. A 5' untranslated sequence (5'UTR) comprising a nucleotide sequence as shown in any one of SEQ ID NO:159-316 or comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with a nucleotide sequence shown in any one of SEQ ID NO:159-316.

[0026] 17. A 3' untranslated sequence (3'UTR) comprising a nucleotide sequence as shown in any one of SEQ ID NO:317-474 and SEQ ID NO:501-507 or comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in any one of SEQ ID NO:317-474 and SEQ ID NO:501-507.

[0027] 18. An engineered transpose element, comprising, from 5' to 3': 5' untranslated sequence (5'UTR), heterologous sequence, and 3' untranslated sequence (3'UTR), The 5' untranslated sequence comprises a nucleotide sequence as shown in any one of SEQ ID NO:159-316 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with a nucleotide sequence shown in any one of SEQ ID NO:159-316; or The 3' untranslated sequence comprises a nucleotide sequence as shown in any one of SEQ ID NO:317-474 and SEQ ID NO:501-507, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in any one of SEQ ID NO:317-474 and SEQ ID NO:501-507. Preferably, it comprises, from 5' to 3': a first homology domain, a 5' untranslated sequence (5'UTR) that binds to the retrososidase, a heterologous sequence, a 3' untranslated sequence (3'UTR) that binds to the retrososidase, and a second homology domain.

[0028] 19. The engineered transposable element according to claim 18, wherein the engineered transposable element is the donor RNA or nucleic acid encoding the donor RNA mentioned in claims 2-15.

[0029] 20. A host cell comprising the retrososozyme of claim 1, the system of any one of claims 2-15, or the engineered transposable element of claim 18 or 19, wherein the host cell is preferably a mammalian cell or a plant cell, more preferably a mammalian cell, and even more preferably a human cell.

[0030] 21. A method for modifying a target DNA strand in a cell, tissue, or subject, the method comprising using the retrospoenzyme of item 1, the system of any one of items 2-15, or the engineered transposon element of item 18 or 19 on the cell, tissue, or subject, wherein the system reverse transcribes the donor RNA sequence into the target DNA strand, thereby modifying the target DNA strand in the cell, tissue, or subject.

[0031] 22. The method according to item 21, wherein the cells or tissues are mammalian cells or tissues, preferably human cells or tissues, and the subject is a mammal, preferably a human.

[0032] 23. The method according to item 22, wherein the cell is a fibroblast, a primary cell, or a cell that has not been immortalized.

[0033] 24. The method according to any one of items 21-23, wherein the method is performed in vivo or in vitro.

[0034] 25. A method for modifying the genome of a mammalian cell or inserting DNA into the genome of a mammalian cell, the method comprising using the cell with the retrospinase described in item 1, the system of any one of items 2-15, or the engineered transposon element described in item 18 or 19, preferably the mammal being human.

[0035] 26. The method of claim 25, wherein the method results in the addition of at least 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 base pairs of exogenous DNA sequence to the genome of the mammal.

[0036] 27. The method according to any one of items 21-26, wherein the cell is part of a tissue; or the mammalian cell is euploid, not immortalized, part of an organism, a primary cell, non-dividing, a hepatocyte, or derived from a subject suffering from a hereditary disease.

[0037] 28. The method according to any one of claims 21-27, wherein the method comprises contacting a cell, tissue, or subject with the retrosockase of claim 1 or the nucleic acid encoding the retrosockase of claim 1 and a donor RNA or the nucleic acid encoding the donor RNA. Preferably, the contact includes contacting the cell, tissue, or subject with a plasmid, virus, virus-like particle, virion, liposome, vesicle, exogenous body, or lipid nanoparticle; Further preferred methods of contact include the use of non-viral delivery, such as electroporation.

[0038] 29. The method according to claim 28, wherein the contact comprises intravenous administration to the subject, preferably at least twice, of the retrosockase of claim 1 or the nucleic acid encoding the retrosockase of claim 1 and the donor RNA or the nucleic acid encoding the donor RNA.

[0039] 30. The method according to any one of items 21-29, wherein, The retrosockase described in item 1 or the nucleic acid encoding the retrosockase described in item 1 and the donor RNA or the nucleic acid encoding the donor RNA are administered separately; or The retrososozyme described in item 1 or the nucleic acid encoding the retrososozyme described in item 1 and the donor RNA or the nucleic acid encoding the donor RNA are administered together.

[0040] 31. A nucleic acid encoding the retrositase described in item 1.

[0041] 32. A vector comprising the nucleic acid described in item 31.

[0042] 33. A host cell comprising the vector described in item 32.

[0043] 34. A pharmaceutical composition comprising the system described in items 2-15, the nucleic acid described in item 31, the carrier described in item 32, or the host cell described in item 20 or 33, preferably the system being placed in a pharmaceutically acceptable carrier, more preferably the carrier being a vesicle (including liposomes, natural or synthetic lipid bilayers, exogenous bodies), lipid nanoparticles, viral or plasmid carriers.

[0044] The effects of the invention The inventors of this application have discovered a novel R2 system with higher efficiency in integrating large genomic fragments. The novel R2 system can specifically integrate target genes into the 28S rDNA of the genome. This system and method have broad application prospects and can meet needs including but not limited to the following: Treatment Needs: This technology can be applied to gene therapy to restore gene function by expressing therapeutic transgenes in individuals with loss-of-function mutations or by replacing genes with gain-of-function mutations. Furthermore, therapeutic goals can be achieved by introducing regulatory sequences to suppress the expression of gain-of-function mutations or by regulating the expression of specific genes, transgenes, and their related systems. In some embodiments, the RNA sequence template can encode a host cell-specific promoter region, such as a tissue-specific promoter or enhancer, thereby enabling precise gene regulation. In other embodiments, the promoter can be operatively linked to the coding sequence to ensure the effective expression of specific genes in specific cells or tissues.

[0045] Functional cell preparation requirements: This system can provide strong technical support for the preparation of functional cells such as immune cells. For example, by integrating biomolecules with specific functions (such as chimeric antigen receptors (CARs)) into immune cells, they can be endowed with new functions, enhancing their ability to recognize and kill tumor cells, thus showing broad application prospects in cell therapy.

[0046] New Demands in Crop Breeding: In the agricultural field, this method can be used to edit plant genomes, conferring new economic traits such as stress resistance or insect resistance by integrating specific genes into plant callus tissue, thereby improving crop breeding efficiency and economic value. This gene integration method holds promise for providing more efficient and precise solutions for modern agricultural breeding technology. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the excavation process for the new R2 reversible subsystem.

[0048] Figure 2 The study involves phylogenetic analysis of the newly discovered R2 retrotransposons with known R2 retrotransposons.

[0049] Figure 3 This is a schematic diagram of obtaining mRNA expressing R2 protein and another donor RNA through in vitro transcription, where LHA represents the first homologous domain of the donor RNA and RHA represents the second homologous domain of the donor RNA.

[0050] Figure 4This method utilizes liposome transfection to introduce mRNA expressing the newly discovered R2 protein and its corresponding donor RNA into human cell lines. The diagram illustrates the gene integration efficiency of the newly discovered R2 retrotransposon system in mammalian cells, showing that the GFP gene is integrated. Finally, the proportion of GFP-positive cells is analyzed by flow cytometry.

[0051] Figure 5 This method utilizes liposome transfection to introduce mRNA expressing the R2 ancestor protein and its corresponding donor RNA into human cell lines. It is a schematic diagram of the gene integration efficiency of the newly discovered R2 retrotransposon system in mammalian cells, which integrates the GFP gene. Finally, the proportion of GFP-positive cells is analyzed by flow cytometry.

[0052] Figure 6 This is a schematic diagram illustrating the gene integration efficiency of ancestral proteins.

[0053] Figure 7 This is the result of multiple sequence alignment of the 3'UTR sequence of the novel R2 retrotransposon.

[0054] Figure 8 This is a schematic diagram illustrating the gene integration efficiency of donor RNA, in which... Figure 8 A in the diagram is a schematic diagram showing the effect of the 3'UTR sequence alteration of the donor RNA on the gene integration efficiency of the novel R2 retrososozyme in human cells. Figure 8 B in the diagram illustrates the effect of 5'UTR sequence alterations in donor RNA on the gene integration efficiency of the novel R2 retrososozyme in human cells.

[0055] Figure 9 This diagram illustrates the gene integration efficiency after the newly discovered R2 retrosockase has been engineered.

[0056] Figure 10 This demonstrates the efficient integration of engineered R2 retrotransposonin with the donor in mammalian cells.

[0057] Figure 11 This demonstrates the efficient integration of engineered R2 retrotransposonin with the donor in human T cells.

[0058] Figure 12 The results showed that the accessory protein could improve the integration efficiency of R2 retrotransposons in T cells. Detailed Implementation

[0059] The present application will now be described in detail with reference to the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0060] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0061] definition The terms “nucleic acid” and “polynucleotide” are used interchangeably to refer to a polymer of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogues. “Oligonucleotide” and “oligonucleotide” are used interchangeably to refer to short polynucleotides having no more than about 50 nucleotides. As used herein, “complementarity” refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid via conventional Watson-Crick base pairing. The complementarity percentage indicates the percentage of residues in the nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., 5, 6, 7, 8, 9, 10, approximately 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). “Complete complementarity” means that all consecutive residues in the nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. As used herein, “substantially complementary” means that in regions of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, the degree of complementarity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100, or refers to two nucleic acids that hybridize under strict conditions.

[0062] For nucleic acid sequences, the "sequence identity percentage (%)" is defined as the percentage of nucleotides in a candidate sequence that are identical to nucleotides in a specific nucleic acid sequence after sequence alignment (if necessary) to achieve the maximum sequence identity percentage, allowing gaps (gaps). For peptide, polypeptide, or protein sequences, the "sequence identity percentage (%)" is the percentage of amino acid residues in a candidate sequence that are identically substituted to amino acid residues in a specific peptide or amino acid sequence after sequence alignment (if necessary) to achieve the maximum sequence homology percentage. For the purpose of determining the amino acid sequence identity percentage, alignment can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0063] The terms “polypeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. Proteins may have one or more polypeptides. The term also covers polymers of amino acids that have been modified; for example, through disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as conjugation with a labeled component).

[0064] As used herein, “truncated variant” is defined as a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains the necessary characteristics. A typical truncated variant of a polynucleotide differs from the nucleic acid sequence of another reference polynucleotide. Changes in the nucleic acid sequence of the truncated variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as described below. A typical truncated variant of a polypeptide differs from another reference polypeptide in its amino acid sequence. Typically, the differences are limited, making the sequences of the reference polypeptide and the truncated variant very similar overall and identical in many regions. The amino acid sequences of the truncated variant and the reference polypeptide can differ by any combination of one or more substitutions, additions, or deletions. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. Truncated variants of polynucleotides or polypeptides may be naturally occurring (such as allelic variants) or may be truncated variants of unknown natural origin. Non-naturally occurring truncated variants of polynucleotides and polypeptides can be prepared by mutagenesis, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0065] As used herein, the term "wildtype" has the meaning commonly understood by those skilled in the art as referring to the typical form of an organism, strain, gene, or trait that distinguishes it from mutants or variants when it exists in nature. It can be isolated from resources in nature and is not deliberately modified.

[0066] The term "cell" as used herein should be understood not only to a specific single cell, but also to its offspring or potential offspring. Because certain modifications may occur in offspring due to mutations or environmental influences, such offspring may indeed differ from the parent cell, but are still included within the scope of this terminology.

[0067] As used herein, the terms “transduction” and “transfection” include methods known in the art for introducing DNA into cells to express a target protein or molecule using infectious agents (such as viruses) or other means. In addition to viral or virus-like reagents, there are chemical-based transfection methods, such as those using calcium phosphate, dendritic polymers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine); non-chemical methods, such as electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, plasmid delivery, or transposons; particle-based methods, such as those using gene guns, magnetic transfection or magnet-assisted transfection, particle bombardment; and hybridization methods (such as nuclear transfection).

[0068] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids.

[0069] The term "in vivo" refers to the organism from which cells are obtained. "Ex vivo" or "in vitro" refers to the organism from which cells are obtained outside of the organism.

[0070] As used herein, “treatment / treating” is a method for obtaining a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, reducing the recurrence rate of the disease, delaying or slowing the progression of the disease, improving the disease state, providing (partial or complete) remission of the disease, reducing the dosage of one or more other medications required to treat the disease, delaying the progression of the disease, improving quality of life, and / or prolonging survival. “Treatment” also includes reducing symptoms, conditions, or pathological consequences of the disease. The methods of this invention consider any or more of these aspects of treatment.

[0071] As used herein, the term "effective amount" means an amount of compound or composition sufficient to treat a particular condition, ailment, or disease (e.g., to improve, alleviate, reduce, and / or delay one or more of its symptoms). As understood in the art, an "effective amount" can be administered once or multiple times, i.e., a single or multiple administration may be required to achieve the desired therapeutic endpoint.

[0072] The terms “subject,” “individual,” or “patient” are used interchangeably herein. For therapeutic purposes, “individual” refers to any animal classified as a mammal, including humans, livestock, and farm animals, as well as zoo, farm, or pet animals such as dogs, horses, cats, and cattle. In some embodiments, “individual” refers to a human individual.

[0073] It should be understood that embodiments of the invention described herein include those "consisting of" and / or "substantially consisting of". References to "about" values ​​or parameters herein include (and describe) variations of that value or parameter itself. For example, a reference to "about X" includes a description of "X".

[0074] As used herein, references to “not” values ​​or parameters generally refer to and describe “except” values ​​or parameters. For example, “The method is not used to treat type X cancer” means that the method is used to treat cancers other than type X.

[0075] As used in this article, the term “approximately XY” has the same meaning as “approximately X to approximately Y”.

[0076] As used herein and in the appended claims, the singular forms “a / an” and “the” include the plural objects unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a preliminary basis for the use of exclusive terms such as “only” or “merely” in conjunction with the description of the elements of the claim, or for the use of the limitation of “no”.

[0077] As used herein, the term "and / or" in words such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" in words such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0078] The term "vector" is a composition of substances containing isolated nucleic acids and capable of being used to deliver said isolated nucleic acids into the cell. Many vectors are known in the art, including but not limited to: linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Typically, a suitable vector contains at least one origin of replication functioning in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selective markers. The term "vector" should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc.

[0079] In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. In some embodiments, the vector is a bacteriophage vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York), and other virology and molecular biology manuals.

[0080] In some embodiments, the rAAV construct can be administered to a subject via the enterum. In some embodiments, the rAAV construct can be administered to a subject parenterally. In some embodiments, rAAV particles can be administered to one or more cells, tissues, or organs via subcutaneous, intraocular, intravitreal, subretinal, intravenous (IV), intraventricular, intramuscular, intrathecal (IT), intracisional, intraperitoneal, inhalation, local, or direct injection. In some embodiments, rAAV particles can be administered to a subject by injection into the hepatic artery or portal vein.

[0081] Methods for introducing vectors into mammalian cells are known in the art. Vectors can be transferred into host cells by physical, chemical, or biological methods.

[0082] Physical methods for introducing a vector into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing the vector and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York. In some embodiments, the vector is introduced into the cells via electroporation.

[0083] The methods described herein are applicable to any suitable cell type. In some embodiments, the cells are bacterial, yeast, fungal, algal, plant, or animal cells (e.g., mammalian cells, such as human cells). In some embodiments, the cells are of natural origin, such as cells isolated from tissue biopsies. In some embodiments, the cells are isolated from cell lines cultured in vitro. In some embodiments, the cells are derived from primary cell lines. In some embodiments, the cells are derived from immortalized cell lines. In some embodiments, the cells are genetically engineered cells.

[0084] The nuclear localization signal, as used in this article, is a domain of a protein, typically a short amino acid sequence, that interacts with nuclear vectors to allow the protein to be transported into the nucleus. Alternatively, the nuclear localization signal may be an RNA sequence, and in some embodiments, it is located on the donor RNA. In some embodiments, the retroposylase polypeptide is encoded on a first RNA, and the donor RNA is a second separate RNA, with the nuclear localization signal located on the donor RNA rather than on the RNA encoding the retroposylase polypeptide. While not wishing to be bound by theory, in some embodiments, the RNA encoding the retroposylase primarily targets the cytoplasm to facilitate its translation, while the donor RNA primarily targets the nucleus to facilitate its retroposal entry into the genome. In some embodiments, the nuclear localization signal is located at the 3' end, 5' end, or within the donor RNA. In some embodiments, the nuclear localization signal is located at the 3' end of a heterologous sequence (e.g., directly at the 3' end of the heterologous sequence) or at the 5' end of a heterologous sequence (e.g., directly at the 5' end of the heterologous sequence). In some embodiments, the nuclear localization signal is placed outside the 5' UTR or 3' UTR of the donor RNA. In some embodiments, the nuclear localization signal is placed between the 5' UTR and the 3' UTR, wherein optionally, the nuclear localization signal is not transcribed with the transgene (e.g., the nuclear localization signal is antisense-oriented or downstream of a transcription termination signal or a polyadenylation signal). In some embodiments, the nuclear localization sequence is located inside an intron. In some embodiments, multiple identical or different nuclear localization signals are present in RNA, for example, in donor RNA. In some embodiments, the length of the nuclear localization signal is less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 bp. Various RNA nuclear localization sequences can be used.

[0085] As used herein, the term "domain" refers to the structure of a biomolecule that contributes to a specific function of that biomolecule. A domain can contain a continuous region (e.g., a continuous sequence) or distinct non-continuous regions (e.g., non-continuous sequences) of a biomolecule. Examples of protein domains include, but are not limited to, endonuclease domains, target DNA-binding domains, and reverse transcription domains; examples of nucleic acid domains are regulatory domains, such as transcription factor-binding domains.

[0086] As used herein, the term "exogenous" when used in relation to a biomolecule (e.g., a nucleic acid sequence or polypeptide) refers to the artificial introduction of the biomolecule into a host genome, cell, or organism. For example, nucleic acids added to an existing genome, cell, tissue, or subject using recombinant DNA technology or other methods are exogenous to the existing nucleic acid sequence, cell, tissue, or subject.

[0087] As used herein, the term "heterogeneous" means, when used to describe a first element with reference to a second element, that the first and second elements do not exist in nature in the arrangement described. For example, a heterologous polypeptide, nucleic acid molecule, construct, or sequence refers to (a) a polypeptide, nucleic acid molecule, or part of a polypeptide or nucleic acid molecule sequence that is not native to the cell expressing it, (b) a polypeptide or nucleic acid molecule, or part of a polypeptide or nucleic acid molecule, that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule having altered expression compared to its native expression level under similar conditions. For example, heterologous regulatory sequences (e.g., promoters, enhancers) can be used to regulate the expression of a gene or nucleic acid molecule in a manner different from how the gene or nucleic acid molecule is normally expressed in nature. In another instance, a heterologous domain of a polypeptide or nucleic acid sequence (e.g., the DNA-binding domain of the polypeptide or the nucleic acid encoding the DNA-binding domain of the polypeptide) may be arranged relative to other domains, or may be a different sequence or originate from a different source relative to other domains or portions of the polypeptide or its encoding nucleic acid. In some embodiments, the heterologous nucleic acid molecule may be present in the natural host cell genome, but may have altered expression levels or different sequences, or both. In other embodiments, the heterologous nucleic acid molecule may not be endogenous to the host cell or host genome, but may have been introduced into the host cell through transformation (e.g., transfection, electroporation), wherein the added molecule may be integrated into the host genome or may exist transiently (e.g., mRNA) or semi-stable for more than one generation as extrachromosomal genetic material (e.g., cell-free viral vectors, plasmids, or other self-replicating vectors).

[0088] As used herein, the term "host genome or host cell" refers to a cell and / or its genome in which proteins and / or genetic material have been introduced. It should be understood that these terms are intended not only to refer to a specific subject cell and / or genome, but also to the genomes of the offspring of such cells and / or the offspring of such cells. Because certain modifications may occur in offspring due to mutations or environmental influences, such offspring may actually differ from the parent cell, but are still included within the scope of the term "host cell" as used herein. A host genome or host cell can be an isolated cell or cell line grown in a culture, or genomic material isolated from such a cell or cell line, or it can be a host cell or host genome that constitutes a living tissue or organism. In some cases, the host cell can be an animal cell or a plant cell, for example, as described herein. In some cases, the host cell can be a bovine cell, a horse cell, a pig cell, a goat cell, a sheep cell, a chicken cell, or a turkey cell. In some cases, the host cell can be a corn cell, a soybean cell, a wheat cell, or a rice cell.

[0089] The genomic safe harbor sites (GSH sites) used in this article are sites in the host genome that can accommodate the integration of new genetic material, for example, ensuring that the inserted genetic element does not cause significant changes to the host genome that would pose a risk to the host cell or organism. GSH sites typically meet one, two, three, four, five, six, seven, eight, or nine of the following criteria: (i) >300 kb from cancer-related genes; (ii) >300 kb from miRNAs / other functional small RNAs; (iii) >50 kb from the 5' gene terminus; (iv) >50 kb from the origin of replication; (v) >50 kb from any highly conserved element; (vi) low transcriptional activity (i.e., no mRNA + / - 25 kb); (vii) not located in a copy number variable region; (viii) located in open chromatin; and / or (ix) unique, with one copy in the human genome. Examples of GSH loci in the human genome that meet some or all of these criteria include: (i) adeno-associated virus locus 1 (AAVS1), the natural site of AAV virus integration on chromosome 19; (ii) the chemokine (CC motif) receptor 5 (CCR5) gene, a chemokine receptor gene known as the HIV-1 co-receptor; (iii) the human ortholog of the mouse Rosa26 locus; and (iv) rDNA loci. Other GSH loci are known and described, for example, in Pellenz et al., electronically published August 20, 2018 (https: / / doi.org / 10.1101 / 396390).

[0090] In some embodiments, the genomic safe harbor site is the Natural Harbor™ site. In some embodiments, the Natural Harbor™ site is ribosomal DNA (rDNA). In some embodiments, the Natural Harbor™ site is 5S rDNA, 18S rDNA, 5.8S rDNA, or 28S rDNA. In some embodiments, the Natural Harbor™ site is the Mutsu site in 5S rDNA. In some embodiments, the Natural Harbor™ site is the R2 site in 28S rDNA.

[0091] As used in this article, "pseudoknot sequence" refers to a nucleic acid (e.g., RNA) that has a sequence with suitable self-complementarity to form a pseudoknot structure.

[0092] As used herein, a “stem-loop sequence” refers to a nucleic acid sequence (e.g., an RNA sequence) that has sufficient self-complementarity to form a stem-loop, for example, having a stem containing at least two (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs and a loop having at least three (e.g., four) base pairs. The stem may contain mismatches or protrusions.

[0093] In some embodiments, the cells are animal cells selected from the group consisting of: cattle, sheep, goats, horses, pigs, deer, chickens, ducks, geese, rabbits, and fish.

[0094] In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the human cells are human embryonic kidney 293T (HEK293T or 293T) cells or HeLa cells. In some embodiments, the cells are human embryonic kidney (HEK293T) cells. In some embodiments, the cells are mouse Hepa1-6 cells. In some embodiments, the mammalian cells are selected from the group consisting of: immune cells, hepatocytes, tumor cells, stem cells, blood cells, nerve cells, zygotes, muscle cells (such as cardiomyocytes), and skin cells.

[0095] In some embodiments, the cells are immune cells selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) T cells, iNK-T cells, NK-T-like cells, gdT cells, tumor-infiltrating T cells, and dendritic cell (DC) activated T cells. In some embodiments, the method generates modified immune cells, such as CAR-T cells or TCR-T cells.

[0096] In some embodiments, the cell is an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a progenitor cell of a gamete, a gamete, a zygote, or a cell in an embryo.

[0097] As described in this article, the first homology domain refers to the Left Homology Arm (LHA), which is interchangeable with the left homology arm. Similarly, the second homology domain refers to the Right Homology Arm (RHA), which is interchangeable with the right homology arm.

[0098] As described herein, accessory proteins are a class of proteins that play important auxiliary functions in organisms. They do not directly participate in catalytic reactions or structural support, but can regulate, stabilize, or promote specific biological processes through interactions with other proteins or molecules. In this application, the accessory protein is used to improve the integration efficiency of retrotransposons in cells. In this application, the accessory protein can be an accessory protein from any source in the art, such as a viral accessory protein. For example, the sequence of the accessory protein may contain the amino acid sequence shown in any of SEQ ID NO: 533-538 or contain an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any of SEQ ID NO: 533-538.

[0099] This application provides a retroposomyase comprising an amino acid sequence as shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530, or comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530; or The reverse transposase is a truncated version of the amino acid sequence shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530.

[0100] For example, the sequence can be obtained by removing amino acids 1-80, 1-190, 1-220, 390-500, and 1100-1120 from the amino acid sequences shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530. In some embodiments, the accessory protein is used to improve the integration efficiency of the retrotransposon in the cell. In some embodiments, the accessory protein is an accessory protein from any source in the art, such as a viral accessory protein, for example, the sequence of the accessory protein comprising the amino acid sequence shown in any one of SEQ ID NO:533-538 or comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO:533-538.

[0101] This application provides a system for modifying DNA, the system comprising: A retroposomyase comprising an amino acid sequence as shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530, or comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530; or The reverse isotope is a truncated version of the amino acid sequence shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530, for example, truncating up to 250 amino acids from the N-terminus, such as truncating amino acids 1-80, 1-190, or 1-220 from the N-terminus; or The retropososome is an engineered retropososome obtained by further conjugating the amino acid sequence or a truncated version thereof, as described in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530, with other amino acid sequences; and The donor RNA or nucleic acid encoding the donor RNA, the donor RNA comprising: a sequence that binds to the retrososozyme and a heterologous sequence; Preferably, the heterologous sequence is at least 1-50,000 bases, for example, 1 nt or more, 10 nt or more, 50 nt or more, 60 nt or more, 70 nt or more, 80 nt or more, 90 nt or more, 100 nt or more, 150 nt or more, 200 nt or more, 250 nt or more, 300 nt or more, 350 nt or more, 400 nt or more, 450 nt or more, 500 nt or more, 550 nt or more, 600 nt or more, 650 nt or more, 700 nt or more, 750 nt or more, 800 nt or more, 850 nt or more, 900 nt or more, 950 nt or more, 1000 nt or more, 1100 nt or more, 1200 nt or more, 1300 nt or more, 1400 nt or more, 1500 nt or more, 1600 nt or more, 1700 nt or more, 1800 nt or more, 1900 nt or more. NT or higher, 2000 NT or higher, 2100 NT or higher, 2200 NT or higher, 2300 NT or higher, 2400 NT or higher, 2500 NT or higher, 2600 NT or higher, 2700 NT or higher, 2800 NT or higher, 2900 NT or higher, 3000 NT or higher, 3500 NT or higher, 4000 NT or higher, 4500 NT or higher, 5000 NT or higher, 5500 NT or higher, 6000 NT or higher, 6500 NT or higher, 7000 NT or higher, 7500 NT or higher, 8000 NT or higher, 8500 NT or higher, 9000 NT or higher, 9500 NT or higher, 10000 NT or higher, 15000 NT or higher, 20000 NT or higher, 25000 NT or higher, 30000 NT or higher, 35000 NT or higher, 40000 NT or higher, 45000 NT or higher; The system is further preferably comprised of nucleic acid encoding VPX protein or VPX protein itself; Preferably, the other amino acid sequence is an exonuclease, and the exonuclease is inserted at the N-terminus, interior, or C-terminus of the retrospinase; Preferably, the exonuclease is an exonuclease derived from T5 bacteriophage; Further preferred exonuclease sequences are as shown in SEQ ID NO. 495 or have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 495; or The other amino acid sequences are single-stranded DNA-binding proteins, and single-stranded DNA-binding proteins are inserted at the N-terminus, interior, or C-terminus of the retrospinase; Preferably, the single-stranded DNA binding protein is a single-stranded DNA binding protein from the bacteria *Sulfolobus tokodaii*. Further preferred single-stranded DNA-binding proteins have a sequence such as SEQ ID NO. 496 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence described in SEQ ID NO. 496; or The other amino acid sequences are chromatin-regulating peptides or high-migration peptides, and chromatin-regulating peptides or high-migration peptides are inserted at the N-terminus, interior or C-terminus of the retrospinase. Preferably, the chromatin-regulating peptide or high-mobility peptide is a human chromatin-regulating peptide or high-mobility peptide. The preferred chromatin-regulating peptide or high-mobility peptide sequence is as shown in SEQ ID NO. 497 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 497; or The other amino acid sequences are RNaseH domains, and RNaseH domains are inserted at the N-terminus, interior, or C-terminus of the retrospinase. Preferably, the RNaseH domain is an RNaseH domain derived from MLV reverse transcriptase; Further preferred sequences of the RNase H domain of MLV reverse transcriptase are as shown in SEQ ID NO. 498 or have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 498; or The other amino acid sequences are nuclear localization signals (NLS), and nuclear localization signals (NLS) are inserted at the N-terminus, interior, or C-terminus of the retrososoylase. Preferably, the nuclear localization signal (NLS) sequence is as shown in SEQ ID NO. 499 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 499; or The other amino acid sequences are nucleoside localization signals, and nucleoside localization signals are inserted at the N-terminus, interior, or C-terminus of the retrososomalase; The other amino acid sequences are nucleolar positioning signals, and the nucleolar positioning signals are inserted at the N-terminus, interior, or C-terminus of the retrososidase; Preferably, the nucleolar localization signal sequence is as shown in SEQ ID NO. 521 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 521; or Preferably, the nucleus localization signal sequence is as shown in SEQ ID NO. 500 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 500.

[0102] For example, insert any two, three, or four of the exonuclease and / or single-stranded DNA-binding protein and / or chromatin regulatory peptide (CMP) and / or RNase H domains into the N-terminus, interior, or C-terminus of the reverse sequestrant.

[0103] An exonuclease is inserted at the N-terminus of the retroposomase, and an exonuclease, single-stranded DNA-binding protein, chromatin regulatory peptide (CMP), or RNase H domain is inserted after amino acid 383 of the retroposomase; or A single-stranded DNA-binding protein is inserted at the N-terminus of the retroposase, and an exonuclease, single-stranded DNA-binding protein, chromatin regulatory peptide (CMP), or RNase H domain is inserted after amino acid 383 of the retroposase; or A chromatin regulatory peptide (CMP) is inserted at the N-terminus of the retroposase, and an exonuclease, single-stranded DNA-binding protein, chromatin regulatory peptide (CMP), or RNase H domain is inserted after amino acid 383 of the retroposase; or A single-stranded DNA-binding protein is inserted at the N-terminus of the retroposase, and a chromatin regulatory peptide (CMP) is inserted after amino acid 383 of the retroposase, and a chromatin regulatory peptide (CMP) or RNase H domain is inserted after amino acid 1112 of the retroposase; or A single-stranded DNA-binding protein is inserted at the N-terminus of the retroposase, and an RNase H domain is inserted after amino acid 383 of the retroposase, and a chromatin regulatory peptide (CMP) or RNase H domain is inserted after amino acid 1112 of the retroposase; or A chromatin regulatory peptide (CMP) is inserted at the N-terminus of the retroposomase, and a chromatin regulatory peptide (CMP) is inserted after amino acid 383 of the retroposomase, and a chromatin regulatory peptide (CMP) or RNase H domain is inserted after amino acid 1112 of the retroposomase; or A chromatin regulatory peptide (CMP) is inserted at the N-terminus of the retrosockase, and an RNaseH domain is inserted after amino acid 383 of the retrosockase. Alternatively, a chromatin regulatory peptide (CMP) or an RNaseH domain is inserted after amino acid 1112 of the retrosockase.

[0104] In some embodiments, the DNA modification system of this application includes: the retrososidase of this application or a truncated version of the retrososidase or a nucleic acid encoding the retrososidase of this application or a truncated version of the retrososidase or an engineered sequence of the retrososidase coupled with other amino acid sequences or a nucleic acid encoding therethere; an engineered sequence of the truncated version of the retrososidase coupled with other amino acid sequences or a nucleic acid encoding therethere; and a donor RNA or a nucleic acid encoding the donor RNA, the donor RNA comprising: a sequence that binds to the retrososidase and a heterologous sequence, wherein the heterologous sequence is at least 1-50,000 bases.

[0105] In some embodiments, the amino acid sequence of the VPX protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of any one of SEQ ID No. 492, 493, or 494.

[0106] The heterologous sequence in this application is selected from one or more of the following: sequences encoding polypeptides or non-coding RNA sequences, sequences containing promoters or enhancers, and sequences encoding one or more introns.

[0107] In some embodiments, the polypeptide is a therapeutic polypeptide or a mammalian polypeptide; more preferably, the polypeptide is a therapeutic protein, membrane protein, intracellular protein, extracellular protein, structural protein, signal transduction protein, regulatory protein, transport protein, organelle protein, sensory protein, motor protein, defense protein, storage protein, reporter protein, antibody, enzyme, or coagulation factor.

[0108] In some embodiments, the polypeptide has 20 to 10,000 amino acids, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, etc. 0, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 280 0, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6 400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900.

[0109] In some embodiments, the intracellular protein is selected from cytoplasmic proteins, nucleoproteins, organelle proteins, mitochondrial proteins, or lysosomal proteins. In some embodiments, the sequence encoding the polypeptide includes one or more introns.

[0110] The system described herein can be used in vitro or in vivo. In some embodiments, the system or system components are delivered to cells (e.g., mammalian cells, such as human cells) in vitro or in vivo. In some embodiments, the cells are eukaryotic cells, such as cells of multicellular organisms, such as animals, such as mammals (e.g., humans, pigs, cattle), birds (e.g., poultry, such as chickens, turkeys, or ducks), or fish. In some embodiments, the cells are non-human animal cells (e.g., laboratory animals, livestock, or companion animals). In some embodiments, the cells are stem cells (e.g., hematopoietic stem cells), fibroblasts, or T cells. In some embodiments, the cells are non-dividing cells, such as non-dividing fibroblasts or non-dividing T cells. In some embodiments, the cells are plant cells. Those skilled in the art will understand that components of the GeneWriter system can be delivered in the form of peptides, nucleic acids (e.g., DNA, RNA), and combinations thereof.

[0111] In this application, delivery can be performed using any of the following combinations to deliver the retroposal enzyme (e.g., as DNA encoding the retroposal enzyme protein, as RNA encoding the retroposal enzyme protein, or as the protein itself) and the donor RNA (e.g., as DNA encoding the RNA, or as RNA): 1. Retroposal DNA + Donor DNA 2. Reverse spouse RNA + donor DNA 3. Retrosockase DNA + Donor RNA 4. Reverse spouse RNA + donor RNA 5. Retrosock protein + donor DNA 6. Reverse spouse protein + donor RNA 7. Retroposomal virus + virus containing donor RNA or DNA 8. Retro-associated virus + donor DNA 9. Retrosock virus + donor RNA 10. Retroposal DNA + Virus containing donor RNA or DNA 11. Retrosockase RNA + Virus containing donor RNA or DNA 12. Retroposase protein + virus containing donor RNA or DNA The aforementioned retroposomycin includes the original full-length retroposomycin mentioned in this application (e.g., the amino acid sequence shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530) or a truncated version of the retroposomycin or an engineered sequence of the retroposomycin coupled with other amino acid sequences; an engineered sequence of the truncated version of the retroposomycin coupled with other amino acid sequences.

[0112] As described above, in some embodiments, a virus is used to deliver DNA or RNA encoding a retrostase protein, and in some embodiments, a virus is used to deliver donor RNA (or DNA encoding donor RNA).

[0113] In some embodiments, the systems and / or components of the systems described herein are delivered in the form of nucleic acids. For example, the reverse stosome polypeptide may be delivered in the form of DNA or RNA encoding the polypeptide, and the donor RNA may be delivered in the form of RNA or its complementary DNA to be transcribed into RNA. In some embodiments, the systems or components of the systems described herein are delivered on 1, 2, 3, 4 or more different nucleic acid molecules. In some embodiments, the systems or components of the systems described herein are delivered as a combination of DNA and RNA. In some embodiments, the systems or components of the systems described herein are delivered as a combination of DNA and protein. In some embodiments, the systems or components of the systems described herein are delivered as a combination of RNA and protein. In some embodiments, the reverse stosome polypeptide is delivered as a protein.

[0114] In some embodiments, a vector is used to deliver the system or components thereof described herein into cells, such as mammalian or human cells. The vector may be, for example, a plasmid or a virus. In some embodiments, delivery is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the virus is adeno-associated virus (AAV), lentivirus, or adenovirus. In some embodiments, the system or components thereof described herein are delivered to cells together with virus-like particles or virions. In some embodiments, delivery uses more than one virus, virus-like particles, or virions.

[0115] In some embodiments, the pharmaceutical compositions and systems described herein can be formulated in liposomes or other similar vesicles. Liposomes are spherical vesicle structures consisting of a single or multiple lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes are biocompatible, non-toxic, capable of delivering both hydrophilic and lipophilic drug molecules, protecting their cargo from degradation by plasma enzymes, and transporting their load across biological membranes and the blood-brain barrier (BBB).

[0116] Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparing multilayered vesicular lipids are known in the art (see, for example, U.S. Patent No. 6,693,086, whose teachings concerning the preparation of multilayered vesicular lipids are incorporated herein by reference). Although vesicle formation is spontaneous when the lipid membrane is mixed with an aqueous solution, it can be accelerated by applying force in an oscillatory manner using a homogenizer, sonicator, or extrusion device. Extruded lipids can be prepared by extruding through a filter with a reduced size.

[0117] Lipid nanoparticles are another example of carriers providing biocompatible and biodegradable delivery systems for the pharmaceutical compositions described herein. Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the properties of SLNs while improving drug stability and loading capacity, and preventing drug leakage. Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively guide drug delivery to specific targets and improve drug stability and controlled drug release. Lipid polymer nanoparticles (PLNs), a novel carrier combining liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNPs and liposomes. PLNs consist of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. Thus, these two components improve drug encapsulation efficiency, facilitate surface modification, and prevent leakage of water-soluble drugs.

[0118] In some embodiments, the donor RNA further comprises a homology domain, preferably including a first homology domain and a second homology domain, that is, the donor RNA comprises the following structure from its 5' end to its 3' end: a first homology domain, a 5' untranslated sequence (5'UTR) that binds to the retrososozyme, a heterologous sequence, a 3' untranslated sequence (3'UTR) that binds to the retrososozyme, and a second homology domain.

[0119] In some embodiments, the first homologous domain is three or more bases located at the 5' end of the donor RNA that have 100% identity with the target DNA strand, and the second homologous domain is three or more bases located at the 3' end of the donor RNA that have 100% identity with the target DNA strand. Preferably, the target DNA is a genomic safe harbor GSH site or a genomic natural harbor site. TM Site.

[0120] In some embodiments, the first homologous domain is LHA, the second homologous domain is RHA, the sequence of LHA is shown in SEQ ID NO:490, and the sequence of RHA is shown in SEQ ID NO:491.

[0121] The donor RNA in this application can also be used as an engineered transposon element.

[0122] All sequences in this application are represented in the form of deoxynucleotides. If they represent RNA sequences, those skilled in the art can replace T with U. When describing sequences in this application, they can represent RNA sequences and their corresponding DNA sequences.

[0123] This application provides a method for treating a disease, comprising contacting the system, nucleic acid, vector, or cell described above with the cells or tissues of a subject in need, thereby treating the disease by expressing a therapeutic transgene in a subject with a loss-of-function mutation or by replacing a gene with a gain-of-function mutation.

[0124] This application provides a method for crop breeding, which includes contacting the above-described system, the above-described nucleic acid, the above-described vector, or the above-described cell with crop tissue to obtain a crop with specific properties.

[0125] Example This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0126] Example 1 R2 Reverse Cord Subsystem according to Figure 1 The flowchart yields the novel R2 retroreflex subsystem: First, a large amount of genomic data was obtained from public databases such as NCBI, and potential R2 systems were identified using RepeatMasker software. Then, BLAST software was used to align sequences with conserved 28S rDNA insertion sites, screening for potential R2 systems specifically inserted into 28S rDNA sites. Next, nucleic acid sequences were clustered using the cd-hit-est software with the -c 0.95 -n11 parameters. Multiple sequence alignment was performed on the nucleic acid sequences in each cluster using the L-INS-i algorithm of MAFFT software. The alignment results were used to generate a consensus sequence for each cluster using the majority rule. ORFfinder software was used to predict the open reading frames (ORFs) of the consensus sequences, screening for proteins larger than 300 amino acids. Finally, Interproscan software was used to analyze the protein domain composition of candidate proteins, screening for proteins possessing reverse transcriptase (RT) domains. Proteins containing the RT domain were sequence-aligned with R2Bm and R2Tg proteins to screen for proteins possessing a target DNA-binding zinc finger motif (ZF) and an endonuclease domain as complete R2 proteins. Simultaneously, R2 systems with a UTR length greater than 10 bp were considered complete R2 elements. Based on species information, redundant sequences of complete R2 elements were eliminated using the cd-hit software with the -c0.95 -n 5 parameter, ultimately yielding novel R2 retropox systems (as shown in Table 1).

[0127] Novel R2 retrotransposons were aligned with known R2 protein sequences using the L-INS-i algorithm in MAFFT software. The alignment results were then used to construct a phylogenetic tree with LIN1_SM as the outer branch in iqtree2 software using the -m MFP parameter. Phylogenetic analysis showed that these novel R2 retrotransposases and known R2 retrotransposons occupy different branches on the phylogenetic tree (see [link to phylogenetic tree]). Figure 2 This suggests that they may belong to different R2 systems. The nucleic acid sequences of all novel R2 class retroposomal systems, such as the 5' and 3' UTR sequences, are shown in SEQ ID NO:159-316, SEQ ID NO:317-474, and SEQ ID NO:501-507, respectively, and the amino acid sequence of the retroposomyase is shown in SEQ ID NO:1-158.

[0128] Table 1:

[0129]

[0130]

[0131] Example 2: Constructing a mammalian cell reporter system to systematically screen the activity of newly discovered R2 retrotransposons. Two mRNAs were obtained through in vitro transcription, one of which can be translated into the R2 protein, while the other can be used directly as a donor RNA in the cell. The donor RNA contains an inverted expression frame initiated by the CMV promoter. Within this frame, CMV expresses GFP. After the two RNAs are transfected into the cell, the donor RNA does not have the ability to translate into normal GFP protein because the GFP sequence is an antisense RNA strand. Only when a novel retrospinase permanently integrates the intronless donor RNA into the cell's DNA through reverse transcriptase activity can the inverted expression frame initiated by the CMV promoter express normal, intronless GFP mRNA, thereby expressing the green fluorescent GFP protein. Therefore, by detecting the presence and proportion of GFP cells using fluorescence microscopy and flow cytometry, it is possible to determine whether the novel retroposalase can exert its effect in mammalian cells and how high its activity is. It is important to note that the donor RNA of the retroposalase system generally contains five parts: the first homologous domain (LHA), the 5' UTR sequence, the 3' UTR sequence, the sequence carrying new genetic information between the 5' UTR and 3' UTR sequences, and the second homologous domain (RHA) (see...). Figure 3 ).

[0132] plasmid construction The protein-coding sequence of the newly discovered R2 retrosockase was codon-optimized (in humans) and synthesized. The DNA-coding fragment was then loaded into the IVT-NLS-3×flag vector between the XmaI and NheI restriction sites using Gibson cloning. The plasmid expressing the donor RNA was constructed by amplifying multiple sequences containing GFP, 5'-UTR, 3'-UTR, and the CMV promoter using PCR. Finally, the DNA-coding fragment was loaded into the backbone vector using Gibson cloning, thus constructing the plasmid expressing the donor RNA.

[0133] In vitro transcription Using DNA containing the sequence encoding the R2 protein and the donor RNA sequence as a template, in vitro transcription was performed using an RNA in vitro transcription kit (E2080 or E2040, NEB) to obtain mRNA encoding the retroposal protein and donor RNA (e.g., Figure 3 (As shown).

[0134] Cell culture, transfection, and fluorescence-activated cell sorting (FACS) HEK293T cell lines were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well plates (Corning) for 16 hours until the cell density reached 70%-90%. 500 ng of mRNA encoding the retrotransposase protein and 1000 ng of mRNA expressing the donor RNA were transfected into each 24-well plate using Lipofectamine 3000 (Invitrogen). Four days after transfection, cells were digested with trypsin-EDTA (0.05%) (Gibco). The proportion of cells with GFP-positive signals was then analyzed using a BD FACSAria™ Fusion Cell Sorter (BD) instrument. The analysis of the proportion of cells with GFP-positive signals, combined with observations under a fluorescence microscope, confirmed whether the newly discovered R2 retrotransposon possesses gene integration activity in mammalian cells.

[0135] The reporting system of this embodiment was used to detect the activity of newly discovered novel R2 retrotransposons. Results of the R2 retrotransposon names and their corresponding gene writing efficiencies are shown below. Figure 4 See Table 2. Among them, the R2 retrotransposons labeled #18, #19, #117, #134, #139, #146, #147, #148, #149, #150, #151, #154, and #155 have a gene integration efficiency of over 20% in mammalian cells, and the integration efficiency of #149 and #150 exceeds 40%.

[0136] Table 2:

[0137]

[0138] Example 3: Construction and Testing of Ancestor Sequences Generate ancestor sequence The novel R2 retrotransposon protein sequence was compared with known R2 protein sequences using the L-INS-i algorithm in MAFFT software. The alignment results were then used in iqtree2 software with the -m MFP parameter to construct a phylogenetic tree with LIN1_SM as the outer branch. Based on the phylogenetic tree results, the sequence was divided into 15 branches (see [link to phylogenetic tree]). Figure 5 The protein multiple sequence alignment results within each branch were extracted, and the consensus sequence of each branch was generated using the majority rule as the ancestral sequence of that branch. The amino acid sequences of these sequences are shown in SEQ ID NO:475-489.

[0139] Carrier construction The ancestral protein coding sequence of the generated R2 retrosockase was codon-optimized (human) and synthesized. The DNA coding fragment was loaded into the IVT-NLS-3×flag vector between the XmaI and NheI restriction sites using Gibson cloning. The plasmid expressing the donor RNA was constructed by amplifying multiple sequences containing GFP, 5'-UTR, 3'-UTR, and the CMV promoter using PCR. Finally, the DNA coding fragment was loaded into the backbone vector using Gibson cloning to construct the plasmid expressing the donor RNA.

[0140] In vitro transcription Using DNA containing the sequence encoding the R2 protein and the donor RNA sequence as a template, in vitro transcription was performed using an RNA in vitro transcription kit (E2080 or E2040, NEB) to obtain mRNA encoding the retrososomal protein and donor RNA (e.g., ...). Figure 3 (As shown).

[0141] Cell culture, transfection, and fluorescence-activated cell sorting (FACS) HEK293T cell lines were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well plates (Corning) for 16 hours until the cell density reached 70%-90%. 500 ng of mRNA encoding the retrotransposase protein and 1000 ng of mRNA expressing the donor RNA were transfected into each 24-well plate using Lipofectamine 3000 (Invitrogen). Four days after transfection, cells were digested with trypsin-EDTA (0.05%) (Gibco). The proportion of cells with GFP-positive signals was then analyzed using a BD FACSAria™ Fusion Cell Sorter (BD) instrument. The analysis of the proportion of cells with GFP-positive signals, combined with observations under a fluorescence microscope, confirmed whether the newly discovered R2 retrotransposon possesses gene integration activity in mammalian cells. The activity of the generated R2 retrotransposon ancestor protein was detected using the reporting system of this embodiment. The results of the ancestor protein name and its corresponding gene writing efficiency are shown in [link to report]. Figure 6 See Table 3. Among them, the gene integration efficiency of ancestral proteins 3, 7, 9, 10, 11, 12, 13, and 14 in mammalian cells exceeds 20%, with the integration efficiency of ancestral proteins 7, 9, 10, 11, 12, and 14 exceeding 40%, and the integration efficiency of ancestral proteins 9 and 12 reaching as high as 50%.

[0142] Table 3

[0143] Example 4: Enhancing gene integration efficiency using engineered donor RNA Engineered donor RNA design like Figure 3 As shown, the donor RNA includes the first homologous domain (LHA), 5'-UTR, target gene, 3'-UTR, and the first homologous domain. The engineered donor RNA primarily modifies the 5'-UTR and 3'-UTR sequences. Modification of the 5'-UTR sequence mainly involves replacing it with the 5'-UTR of another species for testing. Modification of the 3'-UTR sequence mainly involves truncating the 3'-UTR.

[0144] Multiple alignment analysis of the 3'-UTR sequences matched by 158 newly discovered novel R2 retropososases revealed a highly conserved sequence within them (starting from GGTGGACG) (see...). Figure 7 Therefore, the engineered 3'-UTR that matches the novel R2 retrospinase mainly refers to truncating the rest and retaining only this conserved sequence.

[0145] plasmid construction The protein-coding sequence of the newly discovered R2 retrosockase was codon-optimized (human) and synthesized. The DNA-coding fragment was then loaded into the IVT-NLS-3×flag vector between the XmaI and NheI restriction sites using Gibson cloning. The plasmid expressing the donor RNA was constructed by amplifying multiple sequences containing GFP, the 5'-UTR sequence, the 3'-UTR sequence (engineered 3'-UTR), and the CMV promoter using PCR. Finally, the DNA-coding fragment was loaded into the backbone vector using Gibson cloning, thus constructing the plasmid expressing the donor RNA.

[0146] In vitro transcription Using DNA containing the sequence encoding the R2 protein and the donor RNA sequence as a template, in vitro transcription was performed using an RNA in vitro transcription kit (E2080 or E2040, NEB) to obtain mRNA encoding the retroposal protein and donor RNA (e.g., Figure 3 (As shown).

[0147] Cell culture, transfection, and fluorescence-activated cell sorting (FACS) HEK293T cell lines were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well plates (Corning) for 16 hours until cell density reached 70%-90%. 500 ng of mRNA encoding the retroposophore protein and 1000 ng of mRNA expressing the donor RNA were transfected into each 24-well plate using the CALNP™ mRNA in vitro transfection reagent (D-Nano Therapeutics). Four days after transfection, cells were digested with trypsin-EDTA (0.05%) (Gibco). The proportion of cells with GFP-positive signals was then analyzed using a BD FACSAria™ Fusion Cell Sorter (BD) instrument. The analysis of the proportion of cells with GFP-positive signals was used to assess whether the newly discovered R2 retroposophore, combined with engineered donor RNA, has enhanced gene integration activity in mammalian cells.

[0148] Experimental results show that using the engineered 3'-UTR sequences (SEQ ID NO 501-507) corresponding to retroposses #18, #147, #148, #149, #150, #151, and #154 can significantly improve the gene integration efficiency of R2 retropossesses. Figure 8 The corresponding full-length donor RNA sequence is (SEQ ID NO 508-514). Replacing the 5'-UTR that matches the R2 retrospinase with the 5'-UTR sequence of another species has little impact on the efficiency of gene integration. Figure 8 (B in the middle).

[0149] Example 5: Enhancing gene integration efficiency using engineered novel R2 retrosockase Engineered novel R2 retrospinase RNA design Engineered methods include: protein truncating and / or coupling with other amino acid sequences. For the coupled amino acid sequences, exonucleases, single-stranded DNA-binding proteins, chromatin regulatory peptides or high-mobility peptides, or RNaseH domains are preferred.

[0150] plasmid construction Based on the plasmid expressing the newly discovered R2 retrosockase, an engineered R2 retrosockase was constructed using PCR and Gibson cloning. The plasmid expressing the donor RNA was constructed by amplifying multiple sequences containing GFP, 5'-UTR, 3'-UTR, and the CMV promoter using PCR. Finally, the DNA coding fragment was loaded into a backbone vector using Gibson cloning, thus constructing the plasmid expressing the donor RNA.

[0151] In vitro transcription Using DNA containing the sequence encoding the R2 protein and the donor RNA sequence as a template, in vitro transcription was performed using an RNA in vitro transcription kit (E2080 or E2040, NEB) to obtain mRNA encoding the retroposal protein and donor RNA (e.g., Figure 3 (As shown).

[0152] Cell culture, transfection, and fluorescence-activated cell sorting (FACS) HEK293T, Huh7, and HepG2 cell lines were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well plates (Corning) for 16 hours until the cell density reached 70%-90%. 500 ng of mRNA encoding the retroposophore protein and 1000 ng of mRNA expressing the donor RNA were transfected into each 24-well plate using the CALNP™ mRNA in vitro transfection reagent (D-NanoTherapeutics). Four days after transfection, cells were digested with trypsin-EDTA (0.05%) (Gibco). The proportion of cells with GFP-positive signals was then analyzed using a BD FACSAria™ Fusion Cell Sorter (BD) instrument. The analysis of the proportion of cells with GFP-positive signals was used to assess whether the newly discovered R2 retroposophore, combined with engineered donor RNA, has enhanced gene integration activity in mammalian cells.

[0153] Experimental results show that engineered retroposomal sequences (SEQ ID NO 515-520) corresponding to retroposomals #18, #147, #148, #149, #150, and #154, respectively, can further improve the gene integration efficiency of R2 retroposomals. Figure 9 The donor RNA sequences used here correspond to SEQ ID NO 508, 509, 510, 511, 512, and 514, respectively.

[0154] Example 6: Achieving efficient integration of engineered R2 retrotransposonin with donor in mammalian cells In this embodiment, the gene integration activity of wild-type, v1.1, and v1.2 engineered variants of #18, #148, #154, and R2Tg, along with their corresponding wild-type and engineered donor RNAs, in HEK293T cells was tested using the reporting system of Example 3. The gene integration activity of the R2 retrotransposon system in mammalian cells was evaluated by analyzing the proportion of cells with GFP-positive signals after 4 days. The wild-type sequence for #18 is Seq ID No. 18; the wild-type sequence for #148 is Seq ID No. 148; the wild-type sequence for #154 is Seq ID No. 154; the wild-type sequence for R2Tg is Seq ID No. 528; the v1.1 engineered variant sequence for #18 is Seq ID No. 515; the v1.2 engineered variant sequence for #18 is Seq ID No. 522; the v1.1 engineered variant sequence for #148 is Seq ID No. 517; the v1.2 engineered variant sequence for #148 is Seq ID No. 524; the v1.1 engineered variant sequence for #154 is Seq ID No. 520; the v1.2 engineered variant sequence for #154 is Seq ID No. 527; the v1.1 engineered variant sequence for R2Tg is Seq ID No. 18. No. 529; the v1.2 engineered variant of R2Tg has a Seq ID No. 530.

[0155] plasmid construction The protein-coding sequence of the newly discovered R2 retrosockase was codon-optimized (human) and synthesized. The DNA-coding fragment was then loaded into the IVT-NLS-3×flag vector between the XmaI and NheI restriction sites using Gibson cloning. The plasmid expressing the donor RNA was constructed by amplifying multiple sequences containing GFP, the 5'-UTR sequence, the 3'-UTR sequence (engineered 3'-UTR), and the CMV promoter using PCR. Finally, the DNA-coding fragment was loaded into the backbone vector using Gibson cloning, thus constructing the plasmid expressing the donor RNA.

[0156] In vitro transcription Using DNA containing the sequence encoding the R2 protein and the donor RNA sequence as a template, in vitro transcription was performed using an RNA in vitro transcription kit (E2080 or E2040, NEB) to obtain mRNA encoding the retroposal protein and donor RNA (e.g., Figure 3 (As shown).

[0157] Cell culture, transfection, and fluorescence-activated cell sorting (FACS) HEK293T cell lines were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well plates (Corning) for 16 hours until cell density reached 70%-90%. 500 ng of mRNA encoding retroposomalase protein and 1000 ng of donor RNA mRNA were transfected into each 24-well plate using the CALNP™ mRNA in vitro transfection reagent (D-Nano Therapeutics). Four days after transfection, cells were digested with trypsin-EDTA (0.05%) (Gibco). The proportion of cells with GFP-positive signals was then analyzed using a BD FACSAria™ Fusion Cell Sorter (BD) instrument. The analysis of the proportion of cells with GFP-positive signals was used to assess whether the newly discovered R2 retroposomalase, combined with engineered donor RNA, has enhanced gene integration activity in mammalian cells.

[0158] Experimental results show that using engineered #18, #148, and #154 with R2Tg v1.1 and v1.2 variants combined with their corresponding engineered donors can significantly improve gene integration in mammalian cells. The results for R2 retrotransposon names and their corresponding gene writing efficiencies are shown in [link to relevant documentation]. Figure 10 The v1.1 and v1.2 variants of #148 and #154, as well as the v1.1 variant of R2Tg, combined with engineered donor RNA, can achieve integration efficiencies of over 80%.

[0159] Example 7: Achieving efficient integration of engineered R2 retrotransposonin with donor in human T cells This embodiment uses highly efficient integrated R2 proteins #148 and R2Tg, and their variants, validated on HEK293T, combined with engineered donors. Related RNA is electroporated on T cells, and the integration efficiency of these R2 retropolloid systems on T cells is evaluated by analyzing the proportion of cells with GFP-positive signals. The wild-type sequence of #148 is Seq ID No. 148; the wild-type sequence of R2Tg is Seq ID No. 528; the v1.1 engineered variant of #148 is Seq ID No. 517; the v1.2 engineered variant of #148 is Seq ID No. 524; the v1.1 engineered variant of R2Tg is Seq ID No. 529; and the v1.2 engineered variant of R2Tg is Seq ID No. 530. The engineered donor sequence for #148 is Seq ID No. 510; the engineered donor sequence for R2Tg is Seq ID No. 532.

[0160] plasmid construction The protein-coding sequence of the R2 retrosockase was codon-optimized (human) and synthesized. The DNA-coding fragment was then loaded into the IVT-NLS-3×flag vector between the XmaI and NheI restriction sites using Gibson cloning. The plasmid expressing the donor RNA was constructed by amplifying multiple sequences containing GFP, the 5'-UTR sequence, the 3'-UTR sequence (engineered 3'-UTR), and the CMV promoter using PCR. Finally, the DNA-coding fragment was loaded into the backbone vector using Gibson cloning, thus constructing the plasmid expressing the donor RNA.

[0161] In vitro transcription Using DNA containing the sequence encoding the R2 protein and the donor RNA sequence as a template, in vitro transcription was performed using an RNA in vitro transcription kit (E2080 or E2040, NEB) to obtain mRNA encoding the retroposal protein and donor RNA (e.g., Figure 3 (As shown).

[0162] T cell culture, isolation and activation, electroporation, and fluorescence-activated cell sorting (FACS) method. Peripheral Blood Mononuclear Cells (PBMCs) were isolated using the EasySep™ Human T Cell Isolation Kit (Stemcell Technologies). The obtained T cells were activated using the human T CellTransAct (Miltenyi Biotec) reagent and cultured for 72 hours in X-VIVO15 medium (hereinafter referred to as T cell medium) containing 5% fetal bovine serum (Gibco) and 400 IU / mL IL2. Two hours before electroporation, cells were collected, centrifuged, and the supernatant was discarded. After removing the activation beads, cells were counted and cultured in T-cell medium for 2 hours. Cells were then resuspended in B1 mix buffer at a rate of 50 mg / well. The cell suspension was added to pre-allocated 500 ng mRNA encoding retroposophore protein and 1000 ng mRNA expressing donor RNA in EP tubes. After mixing, the cell suspension was added to electroporation strips of the P3 Primary Cell 4D-Nucleofector X Kit (Lonza) and electroporated using the EO-138 program. After electroporation, cells were incubated in Opti-MEM medium at 37°C for 20 minutes for retrieval. T-cell medium was then added, and the cells were transferred to 96-well plates for further culture. On day 2, the proportion of cells with GFP-positive signals was analyzed using a BD FACSAria™ Fusion Cell Sorter (BD) instrument. This was used to determine the gene integration efficiency of the R2 retroposophore in T cells.

[0163] Experimental results show that the newly discovered R2 retrotransposon #148, whether wild-type or engineered variant, exhibits significantly higher gene integration efficiency in T cells than its corresponding R2Tg retrotransposon. Results on R2 retrotransposon names and their corresponding gene writing efficiencies are shown below. Figure 11 The v1.2 variant of #148 achieved an integration efficiency of 15% within T cells.

[0164] Example 8: Using accessory proteins can improve the integration efficiency of R2 retrotransposons in T cells. In this embodiment, various accessory proteins (derived from viruses) were added to test their ability to improve the integration efficiency of R2 retrotransposon #148 and engineered donor RNA on T cells. The impact of these accessory proteins on the optimal gene integration efficiency of R2 retrotransposon #148 and engineered donor RNA on T cells was evaluated by analyzing the proportion of cells with GFP-positive signals. The v1.2 engineered variant sequence of #148 is Seq ID No. 524; the engineered donor sequence of #148 is Seq ID No. 510; and the sequences of the accessory proteins are Seq ID Nos. 533-538, respectively.

[0165] plasmid construction The protein sequence of R2 retrosockase and its accessory proteins were codon-optimized (human) and synthesized. The DNA-coding fragment was then loaded into the vector between the XmaI and NheI restriction sites using Gibson cloning. The accessory protein sequence was also codon-optimized (human) and synthesized. The DNA-coding fragment was then loaded into the backbone vector using Gibson cloning. The plasmid expressing the donor RNA was constructed by amplifying multiple sequences containing GFP, 5'-UTR, 3'-UTR (engineered 3'-UTR), and the CMV promoter using PCR. Finally, the DNA-coding fragment was loaded into the backbone vector using Gibson cloning, thus constructing the plasmid expressing the donor RNA.

[0166] In vitro transcription Using DNA containing the sequence encoding the R2 protein and the donor RNA sequence as a template, in vitro transcription was performed using an RNA in vitro transcription kit (E2080 or E2040, NEB) to obtain mRNA encoding the retroposal protein and donor RNA (e.g., Figure 3 (As shown).

[0167] T cell culture, isolation and activation, electroporation, and fluorescence-activated cell sorting (FACS) method. Peripheral Blood Mononuclear Cells (PBMCs) were isolated using the EasySep™ Human T Cell Isolation Kit (Stemcell Technologies). The obtained T cells were activated using the human T CellTransAct (Miltenyi Biotec) reagent and cultured for 72 hours in X-VIVO15 medium (hereinafter referred to as T cell medium) containing 5% fetal bovine serum (Gibco) and 400 IU / mL IL2. Two hours before electroporation, cells were collected, centrifuged, and the supernatant was discarded. After removing the activation beads, cells were counted and cultured in T-cell medium for 2 hours. Cells were then resuspended in B1 mix buffer at a rate of 50 mg / well. The cell suspension was added to pre-allocated EP tubes containing 500 ng of mRNA encoding accessory proteins, 500 ng of mRNA encoding retropososome protein, and 1000 ng of mRNA expressing donor RNA. After mixing, the cell suspension was added to electroporation strips of a P3 Primary Cell 4D-Nucleofector X Kit (Lonza) and electroporated using the EO-138 program. After electroporation, cells were incubated in Opti-MEM medium at 37°C for 20 minutes for retrieval. T-cell medium was then added, and the cells were transferred to 96-well plates for further culture. On day 2, the proportion of cells with GFP-positive signals was analyzed using a BD FACSAria™ Fusion Cell Sorter (BD) instrument. This was used to determine the gene integration efficiency of the R2 retropososome in T cells.

[0168] Experimental results showed that the accessory protein Vp4 significantly improved the gene integration efficiency of R2 retrotransposon #148 and engineered donor RNA on T cells. The names of the accessory proteins and their corresponding gene writing efficiencies are shown in [link to results]. Figure 12 Among them, the accessory protein Vp4 (sequence Seq ID No. 536) can increase the integration efficiency of R2 retrotransposon #148 with engineered donor RNA to more than 35% on T cells.

[0169] The sequences involved in this application are as follows:

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[0262] Note: For N appearing in the sequences SEQ ID NO:336 and SEQ ID NO:374, it represents any one of A, T, C or G.

[0263] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A retroposomyase, said retroposomyase comprising an amino acid sequence as shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530, or comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530; or The reverse transposase is a truncated version of the amino acid sequence shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530.

2. A system for modifying DNA, the system comprising: A retroposomyase comprising an amino acid sequence as shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530, or comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530; or The retroposal enzyme is a truncated version of the amino acid sequence shown in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520, and SEQ ID NO:522-530, for example, truncating up to 250 amino acids from the N-terminus; or The retrososozyme is an engineered retrososozyme obtained by further coupling the amino acid sequence or a truncated version thereof, as described in any one of SEQ ID NO:1-158, SEQ ID NO:475-489, SEQ ID NO:515-520 and SEQ ID NO:522-530, with other amino acid sequences. wherein The other amino acid sequences are selected from any one, two, three, four, five, six, or seven of the following: exonucleases, single-stranded DNA-binding proteins, chromatin regulatory peptides, high-mobility peptides, RNaseH domains, nuclear localization signals (NLS) for entering the nucleus, nucleolar localization sequences, and nuclear localization signals for exiting the nucleus. and The donor RNA or nucleic acid encoding the donor RNA, the donor RNA comprising: a sequence that binds to the retrososozyme and a heterologous sequence; Preferably, the heterologous sequence is at least 1-50,000 bases, for example, 1 nt or more, 10 nt or more, 50 nt or more, 60 nt or more, 70 nt or more, 80 nt or more, 90 nt or more, 100 nt or more, 150 nt or more, 200 nt or more, 250 nt or more, 300 nt or more, 350 nt or more, 400 nt or more, 450 nt or more, 500 nt or more, 550 nt or more, 600 nt or more, 650 nt or more, 700 nt or more, 750 nt or more, 800 nt or more, 850 nt or more, 900 nt or more, 950 nt or more, 1000 nt or more, 1100 nt or more, 1200 nt or more, 1300 nt or more, 1400 nt or more, 1500 nt or more, 1600 nt or more, 1700 nt or more, 1800 nt or more, 1900 nt or more. NT or higher, 2000 NT or higher, 2100 NT or higher, 2200 NT or higher, 2300 NT or higher, 2400 NT or higher, 2500 NT or higher, 2600 NT or higher, 2700 NT or higher, 2800 NT or higher, 2900 NT or higher, 3000 NT or higher, 3500 NT or higher, 4000 NT or higher, 4500 NT or higher, 5000 NT or higher, 5500 NT or higher, 6000 NT or higher, 6500 NT or higher, 7000 NT or higher, 7500 NT or higher, 8000 NT or higher, 8500 NT or higher, 9000 NT or higher, 9500 NT or higher, 10000 NT or higher, 15000 NT or higher, 20000 NT or higher, 25000 NT or higher, 30000 NT or higher, 35000 NT or higher, 40000 NT or higher, 45000 NT or higher; The system is further preferably comprised of nucleic acid encoding VPX protein or VPX protein itself; Preferably, the other amino acid sequence is an exonuclease, and the exonuclease is inserted at the N-terminus, interior, or C-terminus of the retroposal enzyme; Preferably, the exonuclease is an exonuclease derived from T5 bacteriophage; Further preferred exonuclease sequences are as shown in SEQ ID NO. 495 or have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 495; or The other amino acid sequences are single-stranded DNA-binding proteins, and the single-stranded DNA-binding proteins are inserted at the N-terminus, interior, or C-terminus of the retrospinase; Preferably, the single-stranded DNA binding protein is a single-stranded DNA binding protein from the bacteria *Sulfolobus tokodaii*. Further preferred single-stranded DNA-binding proteins have a sequence such as SEQ ID NO. 496 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence described in SEQ ID NO. 496; or The other amino acid sequences are chromatin-regulating peptides or high-mobility peptides, and the chromatin-regulating peptides or high-mobility peptides are inserted at the N-terminus, interior, or C-terminus of the retrospinase. Preferably, the chromatin-regulating peptide or high-mobility peptide is a human chromatin-regulating peptide or high-mobility peptide. The preferred chromatin-regulating peptide or high-migration peptide sequence is as shown in SEQ ID NO. 497 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 497; or The other amino acid sequence is an RNaseH domain, and the RNaseH domain is inserted at the N-terminus, interior or C-terminus of the retrospinase; Preferably, the RNaseH domain is an RNaseH domain derived from MLV reverse transcriptase; Further preferred sequences of the RNase H domain of MLV reverse transcriptase are as shown in SEQ ID NO. 498 or have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 498; or The other amino acid sequences are nuclear localization signals (NLS), and the nuclear localization signals (NLS) are inserted at the N-terminus, interior, or C-terminus of the retrososidase; Preferably, the nuclear localization signal (NLS) sequence is as shown in SEQ ID NO. 499 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 499; or The other amino acid sequences are nucleolar positioning signals, and the nucleolar positioning signals are inserted at the N-terminus, interior, or C-terminus of the retrososidase; Preferably, the nucleolar localization signal sequence is as shown in SEQ ID NO. 521 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO. 521; or The other amino acid sequences are nucleoside localization signals, and the nucleoside localization signals are inserted at the N-terminus, interior, or C-terminus of the retrososozyme. Preferably, the nucleus localization signal sequence is as shown in SEQ ID NO. 500 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No.

500.

3. A 5' untranslated sequence (5'UTR) comprising a nucleotide sequence as shown in any one of SEQ ID NO:159-316 or comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with a nucleotide sequence shown in any one of SEQ ID NO:159-316.

4. A 3' untranslated sequence (3'UTR) comprising a nucleotide sequence as shown in any one of SEQ ID NO:317-474 and SEQ ID NO:501-507 or comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in any one of SEQ ID NO:317-474 and SEQ ID NO:501-507.

5. An engineered transpose element, comprising, from 5' to 3': 5' untranslated sequence (5'UTR), heterologous sequence, and 3' untranslated sequence (3'UTR), The 5' untranslated sequence comprises a nucleotide sequence as shown in any one of SEQ ID NO:159-316 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with a nucleotide sequence shown in any one of SEQ ID NO:159-316; or The 3' untranslated sequence comprises a nucleotide sequence as shown in any one of SEQ ID NO:317-474 and SEQ ID NO:501-507, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in any one of SEQ ID NO:317-474 and SEQ ID NO:501-507. Preferably, it comprises, from 5' to 3': a first homology domain, a 5' untranslated sequence (5'UTR) that binds to the retrososidase, a heterologous sequence, a 3' untranslated sequence (3'UTR) that binds to the retrososidase, and a second homology domain.

6. A method for modifying a target DNA strand in a cell, tissue, or subject, the method comprising using the retrospoenzyme of claim 1, the system of claim 2, or the engineered transposon element of claim 5 on the cell, tissue, or subject, wherein the system reverse transcribes the donor RNA sequence into the target DNA strand, thereby modifying the target DNA strand in the cell, tissue, or subject.

7. A method for modifying the genome of a mammalian cell or inserting DNA into the genome of a mammalian cell, the method comprising using the cell with the retrospinase of claim 1, the system of claim 2, or the engineered transposon element of claim 5, preferably the mammal being human.

8. A nucleic acid encoding the retrositase of claim 1.

9. A vector comprising the nucleic acid of claim 8.

10. A pharmaceutical composition comprising the system of claim 2, the nucleic acid of claim 8, or the carrier of claim 9, preferably the system being placed in a pharmaceutically acceptable carrier, and more preferably the carrier being a vesicle (including liposomes, natural or synthetic lipid bilayers, exogenous bodies), lipid nanoparticles, a viral or plasmid carrier.

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