Active DNA transposon system and use method thereof

Abstract

An engineered transposable element, a gene transfer system comprising same, a method for using same and a kit. A disclosed composition, and the disclosed system and method can be used to insert heterologous nucleic acids into target nucleic acids in vitro or in cells.

Description

Active DNA transposon system and its application Technical Field

[0001] This application generally relates to the field of genetics. More specifically, this application relates to transposable elements and their uses. Background Technology

[0002] Typical methods for introducing DNA into cells include DNA condensing agents, such as calcium phosphate and polyethylene glycol; lipid-containing agents, such as liposomes and multilayer vesicles; and virus-mediated strategies. However, such methods may have limitations. For example, there are size limitations associated with DNA condensing agents and virus-mediated strategies. Furthermore, the amount of nucleic acid that can be transfected into cells is limited in viral strategies. Not all methods are conducive to inserting delivered nucleic acids into the cellular nucleic acid matrix; while DNA condensing methods and lipid-containing agents are relatively easy to prepare, inserting nucleic acids into viral vectors can be labor-intensive. Virus-mediated strategies can be cell-type or tissue-type specific, and the use of virus-mediated strategies in vivo can introduce immune problems.

[0003] One suitable tool to overcome these problems is through transposable elements. A transposable element (TE, transposon, or jumping gene) is a DNA sequence that can change its position within a nucleic acid, thereby creating or reversing mutations and altering sequences in the genome. Transposable elements represent the vast majority of many eukaryotic genomes. For example, approximately 50% of the human genome is derived from transposon sequences, while other genomes, such as those of plants, are composed of an even higher proportion of DNA derived from transposable elements. Transposable elements are generally classified into two classes, Class 1 and Class 2. Class 1 is represented by retrotransposons, including 1) long terminal repeat (LTR) retrotransposons, such as endogenous retroviruses (ERVs), and 2) non-LTR retrotransposons, such as long spread elements (LINEs) and short spread elements (SINEs). Class 2 TEs include 1) "cut-and-paste" DNA transposons, characterized by terminal repeat sequences (TRs, also known as terminal inverted repeats, TIRs) and capable of being moved by transposases, and 2) non-"cut-and-paste" DNA transposable elements, such as Helitron and Polinton. While class 2 transposable elements (TEs) are widespread and active in a variety of eukaryotes, not all of them possess transposable activity. Recent examples of active transposable elements include members of the hAT and piggyBac superfamily, which have shown signs of mobility over the past few million years. However, the selection of active transposable elements currently available for gene discovery research and gene therapy is limited.

[0004] Therefore, there is a need for novel transposable elements suitable for introducing DNA into cells, as well as methods and systems for efficiently inserting heterologous sequences of different sizes into the nucleic acids of cells or inserting DNA into the genome of cells via transposable elements. Summary of the Invention

[0005] This application provides engineered transposon elements, gene transfer systems comprising said engineered transposon elements, and methods and kits for using them. Methods for inserting heterologous nucleic acids into target nucleic acids in vitro or in cells are also provided. The compositions, systems, and methods described herein can be used in a variety of applications, including tagging, genome engineering, and gene discovery research.

[0006] In one aspect, this application provides an engineered transposon element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof, and wherein the transposon element exhibits transposon activity that allows the heterologous nucleic acid to be inserted into the DNA of a cell. In some embodiments, the 5'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from SEQ ID NO:1-6. In some embodiments, the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6. In some embodiments, the 3'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from SEQ ID NO:7-12. In some further embodiments, the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12.

[0007] In some embodiments of any of the engineered transposable elements described above, the transposable element further comprises a 5'5' target site repeat sequence (TSD) side-mounted with a 5' TR or a 3'3' TSD side-mounted with a 3' TR. In some embodiments, the nucleic acid sequences of the 5' TSD and the 3' TSD are identical. In some embodiments, the 5' TSD comprises a nucleic acid sequence selected from SEQ ID NO:19-24, a variant thereof, or a fragment thereof, while the 3' TSD comprises a nucleic acid sequence selected from SEQ ID NO:19-24, a variant thereof, or a fragment thereof.

[0008] In some embodiments of any of the engineered transposable elements described above, the 5'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 1 and 2, while the 3'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 7 and 8.

[0009] In some embodiments of any of the engineered transposable elements described above, the engineered transposable element is derived from Tc1-1_ST, Tc1-2_ST, Tc1-3_ST, Tc1-4_ST, Tc1-5_ST, or Tc1-1_PS. In some embodiments, the engineered transposable element is derived from Tc1-1_ST or Tc1-2_ST. In some embodiments, the 5'TR contains the nucleic acid sequence of SEQ ID NO:1, and the 3'TR contains the nucleic acid sequence of SEQ ID NO:7. In some embodiments, the 5'TR contains the nucleic acid sequence of SEQ ID NO:2, and the 3'TR contains the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the 5'TR contains the nucleic acid sequence of SEQ ID NO:3, and the 3'TR contains the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the 5'TR contains the nucleic acid sequence of SEQ ID NO:4, and the 3'TR contains the nucleic acid sequence of SEQ ID NO:10. In some embodiments, the 5'TR contains the nucleic acid sequence of SEQ ID NO:5, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:11. In some embodiments, the 5'TR contains the nucleic acid sequence of SEQ ID NO:6, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:12.

[0010] In some embodiments of any of the engineered transposable elements described above, the heterologous nucleic acid comprises a coding sequence. In some further embodiments, the heterologous nucleic acid also comprises a promoter operatively linked to the coding sequence.

[0011] In some embodiments of any of the engineered transposable elements described above, the transposable activity of the transposable element is higher than that of the piggyBac (PB) transposable, Sleeping Beauty (SB) transposable, and / or TcBuster transposable.

[0012] In some embodiments of any of the engineered transposable elements described above, the cell is an animal cell, plant cell, algal cell, fungal cell, yeast cell, or bacterial cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is selected from immune cells (e.g., T cells), hepatocytes, tumor cells, stem cells, fertilized eggs, muscle cells, and skin cells. In some embodiments, the cell is a human cell. In some embodiments, the transposable element exhibits higher transposable activity in human embryonic kidney 293T (293T) cells than in HeLa cells.

[0013] In some embodiments of any of the engineered transposable elements described above, the transposable element is contained within a carrier. In some further embodiments, the carrier is a plasmid or a viral vector.

[0014] Another aspect of this application provides a gene transfer system comprising: 1) an engineered transposable element according to any of the transposable elements described above; and 2) a transposase, or a nucleic acid encoding the transposase. In some embodiments, the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18 or a variant thereof.

[0015] In another aspect, this application provides a gene transfer system comprising: 1) an engineered transposable element; and 2) a transposase or a nucleic acid encoding a transposase, wherein the transposable element comprises, from 5' to 3', a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the transposable element exhibits transposition activity that allows the heterologous nucleic acid to be inserted into the DNA of a cell, and wherein the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18 or a variant thereof. In some embodiments, the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, a variant thereof, or a fragment thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, a variant thereof, or a fragment thereof.

[0016] In some embodiments of any of the gene transfer systems described above, the transposon element comprises a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from SEQ ID NO:1, 2, 3, 4, 5, and 6, while the 3' TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from SEQ ID NO:7, 8, 9, 10, 11, and 12. In some embodiments, the engineered transposon element is derived from Tc1-1_ST, Tc1-2_ST, Tc1-3_ST, Tc1-4_ST, Tc1-5_ST, or Tc1-1_PS. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST. In some embodiments, the 5' TR comprises the nucleic acid sequence of SEQ ID NO:1, the 3' TR comprises the nucleic acid sequence of SEQ ID NO:7, and the transposase comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, the 5'TR contains the nucleic acid sequence of SEQ ID NO:2, the 3'TR contains the nucleic acid sequence of SEQ ID NO:8, and the transposase contains the amino acid sequence of SEQ ID NO:14. In some embodiments, the 5'TR contains the nucleic acid sequence of SEQ ID NO:3, the 3'TR contains the nucleic acid sequence of SEQ ID NO:9, and the transposase contains the amino acid sequence of SEQ ID NO:15. In some embodiments, the 5'TR contains the nucleic acid sequence of SEQ ID NO:4, the 3'TR contains the nucleic acid sequence of SEQ ID NO:10, and the transposase contains the amino acid sequence of SEQ ID NO:16. In some embodiments, the 5'TR contains the nucleic acid sequence of SEQ ID NO:5, the 3'TR contains the nucleic acid sequence of SEQ ID NO:11, and the transposase contains the amino acid sequence of SEQ ID NO:17. In some embodiments, the 5'TR contains the nucleic acid sequence of SEQ ID NO:6, the 3'TR contains the nucleic acid sequence of SEQ ID NO:12, and the transposase contains the amino acid sequence of SEQ ID NO:18.

[0017] In some embodiments of any of the gene transfer systems described above, the gene transfer system comprises a nucleic acid encoding a transposase. In some further embodiments, the transposable element and the nucleic acid encoding the transposase are in different vectors. In some further specific embodiments, the transposable element and the nucleic acid encoding the transposase are in the same vector.

[0018] In another aspect, this application provides a method for inserting a heterologous nucleic acid into a target nucleic acid, comprising: contacting the target nucleic acid with a transposon element according to any one of the above-described engineered transposon elements or a gene transfer system according to any one of the above-described gene transfer systems. In some embodiments, the method is performed in vitro. In some embodiments, the target nucleic acid is in a cell. In some embodiments, the target nucleic acid is genomic DNA.

[0019] In some embodiments of any of the above methods, the target nucleic acid is in a cell, which is an animal cell, plant cell, algal cell, fungal cell, yeast cell, or bacterial cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is selected from immune cells (e.g., T cells), hepatocytes, tumor cells, stem cells, fertilized eggs, muscle cells, and skin cells. In some embodiments, the insertion of the heterologous nucleic acid inactivates the cell's genes.

[0020] In some embodiments according to any of the methods described above, the heterologous nucleic acid encodes a protein. In some embodiments, the protein is selected from reporter proteins, engineered receptors, cytokines, antibiotic resistance proteins, antigens, and therapeutic proteins.

[0021] In some embodiments according to any of the above methods, the heterologous nucleic acid encodes RNA. In some embodiments, the RNA is selected from therapeutic RNA, small interfering RNA (siRNA), microRNA, short hairpin RNA (shRNA), long non-coding RNA (lincRNA), and guide RNA (gRNA). In some embodiments, the heterologous nucleic acid encodes more than one molecule.

[0022] In some embodiments of any of the above methods, the length of the heterologous nucleic acid does not exceed about 300 kilobases (kb), for example, about 10 kb to about 300 kb, or about 100 base pairs (bp) to about 10 kb, or about 100 bp to about 5 kb, or about 100 bp to about 2 kb, or about 2 kb to about 300 kb.

[0023] In some embodiments of any of the methods described above, the insertion is random.

[0024] In another aspect, this application provides a kit comprising a transposon element according to any of the above-described engineered transposon elements, or a gene transfer system according to any of the above-described gene transfer systems, and instructions for inserting a heterologous nucleic acid into a target nucleic acid.

[0025] Specifically, this application relates to the following technical solutions:

[0026] 1. An engineered transpose element, comprising, from 5' to 3':

[0027] 5' terminal repeat (5'TR), heterologous nucleic acid, and 3' terminal repeat (3'TR)

[0028] The 5'TR contains a nucleic acid sequence selected from any one of SEQ ID NOs:1-6, a variant thereof, or a fragment thereof.

[0029] The 3'TR contains a nucleic acid sequence selected from any of SEQ ID NOs:7-12, its variants, or fragments thereof, and

[0030] The engineered transposable element described therein exhibits transposable activity that allows heterologous nucleic acids to be inserted into the DNA of cells.

[0031] 2. The engineered transposon element according to claim 1, wherein the engineered transposon element comprises a 5'TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising a nucleic acid sequence selected from any of SEQ ID NOs:25-30.

[0032] 3. The engineered transposon element according to claim 1 or 2, wherein the engineered transposon element comprises a 3'TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising a nucleic acid sequence selected from any of SEQ ID NOs:31-36.

[0033] 4. The engineered transposable element according to any one of items 1-3, wherein the 5'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs:1-6, and / or wherein the 3'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs:7-12.

[0034] 5. The engineered transposable element according to any one of claims 1-4, wherein the engineered transposable element comprises: 1) a 5' TR having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs: 1-6; and 2) a 3' TR having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs: 7-12.

[0035] 6. The engineered transposable element according to any one of items 1-5, wherein the 5'TR comprises a nucleic acid sequence selected from any one of SEQ ID NOs:1-6, and / or wherein the 3'TR comprises a nucleic acid sequence selected from any one of SEQ ID NOs:7-12.

[0036] 7. The engineered transposable element according to any one of claims 1-6, the engineered transposable element further comprising a 5' target site repeat sequence (TSD) side-attached to the 5' end of the 5' TR and / or a 3' TSD side-attached to the 3' end of the 3' TR, wherein the 5' TSD comprises a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any one of SEQ ID NOs:19-24, and wherein the 3' TSD comprises a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any one of SEQ ID NOs:19-24.

[0037] 8. The engineered transposable element according to item 7, wherein the nucleic acid sequences of the 5'TSD and 3'TSD are identical.

[0038] 9. The engineered transposable element according to any one of claims 1-8, wherein the 5'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5 and 6, and the 3'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs: 7, 8, 9, 10, 11 and 12.

[0039] 10. The engineered transducer element according to item 9, wherein:

[0040] (a) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:1, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:7;

[0041] (b) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:2, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:8;

[0042] (c) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:3, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:9;

[0043] (d) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:4, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:10;

[0044] (e) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:5, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:11. Or

[0045] (f) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:6, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:12.

[0046] 11. The engineered transducer element according to item 10, wherein:

[0047] (a) The 5'TR contains the nucleic acid sequence of SEQ ID NO:1, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:7;

[0048] (b) The 5'TR contains the nucleic acid sequence of SEQ ID NO:2, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:8;

[0049] (c) The 5'TR contains the nucleic acid sequence of SEQ ID NO:3, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:9;

[0050] (d) The 5'TR contains the nucleic acid sequence of SEQ ID NO:4, and the 3'TR contains the nucleic acid sequence of SEQ ID NO:10; or

[0051] (e) The 5'TR contains the nucleic acid sequence of SEQ ID NO:5, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:11. Or

[0052] (e) The 5'TR contains the nucleic acid sequence of SEQ ID NO:6, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:12.

[0053] 12. The engineered transposable element according to any one of items 1-11, wherein the engineered transposable element comprises an LTF containing a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any one of SEQ ID NOs:25-30.

[0054] 13. The engineered transposon element according to any one of claims 1-12, wherein the engineered transposon element comprises an RTF containing a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any one of SEQ ID NOs:31-36.

[0055] 14. The engineered transposable element according to any one of claims 1-13, wherein the engineered transposable element comprises an LTF and an RTF, the LTF having at least about 80% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs:25-30, and the RTF having at least about 80% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs:31-36.

[0056] 15. The engineered transposable element according to claim 14, wherein the engineered transposable element comprises an LTF containing a nucleic acid sequence selected from any of SEQ ID NOs: 25-30, and an RTF containing a nucleic acid sequence selected from any of SEQ ID NOs: 31-36.

[0057] 16. The engineered transposable element according to any one of claims 1-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:25; 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:31.

[0058] 17. The engineered transposable element according to claim 16, the engineered transposable element comprising: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:1, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:7, a variant thereof, or a fragment thereof.

[0059] 18. The engineered transposable element according to claim 17, the engineered transposable element comprising: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:1; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:7.

[0060] 19. The engineered transposable element according to claim 18, the engineered transposable element comprising: 1) a 5' TR comprising the nucleic acid sequence of SEQ ID NO:1; and 2) a 3' TR comprising the nucleic acid sequence of SEQ ID NO:7.

[0061] 20. The engineered transposable element according to any one of claims 16-19, the engineered transposable element further comprising a 5' TSD containing a nucleic acid sequence of TA (SEQ ID NO: 19) and a 3' TSD containing a nucleic acid sequence of SEQ ID NO: 19.

[0062] 21. The engineered transposable element according to any one of claims 16-20, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:25; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:31.

[0063] 22. The engineered transposable element according to any one of claims 16-21, wherein the engineered transposable element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:13 or a variant thereof.

[0064] 23. The engineered transposon element according to claim 22, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:13.

[0065] 24. The engineered transposable element according to any one of claims 10-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:26; and 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:32.

[0066] 25. The engineered transposable element according to claim 24, the engineered transposable element comprising: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:2, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:8, a variant thereof, or a fragment thereof.

[0067] 26. The engineered transposable element according to claim 25, the engineered transposable element comprising: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:2; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:8.

[0068] 27. The engineered transposable element according to claim 26, the engineered transposable element comprising: 1) a 5' TR comprising the nucleic acid sequence of SEQ ID NO:2; and 2) a 3' TR comprising the nucleic acid sequence of SEQ ID NO:8.

[0069] 28. The engineered transposable element according to any one of claims 24-27, the engineered transposable element further comprising a 5' TSD containing the nucleic acid sequence of SEQ ID NO:20, and a 3' TSD containing the nucleic acid sequence of SEQ ID NO:20.

[0070] 29. The engineered transposable element according to any one of items 24-28, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:26; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:32.

[0071] 30. The engineered transposon element according to any one of claims 24-29, wherein the engineered transposon element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:14 or a variant thereof.

[0072] 31. The engineered transposable element according to claim 30, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:114.

[0073] 32. The engineered transposable element according to any one of claims 10-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:3; and 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:9.

[0074] 33. The engineered transposable element according to claim 32, the engineered transposable element comprising: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:3, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:9, a variant thereof, or a fragment thereof.

[0075] 34. The engineered transposable element according to claim 33, the engineered transposable element comprising: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:3; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:9.

[0076] 35. The engineered transposable element according to claim 34, the engineered transposable element comprising: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:3; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:9.

[0077] 36. The engineered transposable element according to any one of claims 32-35, the engineered transposable element further comprising a 5' TSD containing the nucleic acid sequence of SEQ ID NO:21, and a 3' TSD containing the nucleic acid sequence of SEQ ID NO:21.

[0078] 37. The engineered transposable element according to any one of claims 32-36, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:27; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:33.

[0079] 38. The engineered transposable element according to any one of claims 32-37, wherein the engineered transposable element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:15 or a variant thereof.

[0080] 39. The engineered transposable element according to claim 38, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:15.

[0081] 40. The engineered transposable element according to any one of claims 10-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:28; and 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:34.

[0082] 41. The engineered transposable element according to claim 40, the engineered transposable element comprising: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:4, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:10, a variant thereof, or a fragment thereof.

[0083] 42. The engineered transposable element according to claim 41, the engineered transposable element comprising: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:4; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:10.

[0084] 43. The engineered transposable element according to claim 42, the engineered transposable element comprising: 1) a 5' TR comprising the nucleic acid sequence of SEQ ID NO:4; and 2) a 3' TR comprising the nucleic acid sequence of SEQ ID NO:10.

[0085] 44. The engineered transposable element according to any one of claims 40-43, the engineered transposable element further comprising a 5' TSD containing the nucleic acid sequence of SEQ ID NO:22, and a 3' TSD containing the nucleic acid sequence of SEQ ID NO:22.

[0086] 45. The engineered transposable element according to any one of claims 40-44, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:28; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:34.

[0087] 46. ​​The engineered transposon element according to any one of claims 40-45, wherein the engineered transposon element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:16 or a variant thereof.

[0088] 47. The engineered transposase according to claim 46, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:16.

[0089] 48. The engineered transposable element according to any one of claims 10-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:29; and 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:35.

[0090] 49. The engineered transposable element according to claim 48, the engineered transposable element comprising: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:5, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:11, a variant thereof, or a fragment thereof.

[0091] 50. The engineered transposable element according to claim 49, the engineered transposable element comprising: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:5; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:11.

[0092] 51. The engineered transposable element according to claim 50, the engineered transposable element comprising: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO: 5; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO: 11.

[0093] 52. The engineered transposable element according to any one of claims 48-51, the engineered transposable element further comprising a 5' TSD containing the nucleic acid sequence of SEQ ID NO:23, and a 3' TSD containing the nucleic acid sequence of SEQ ID NO:23.

[0094] 53. The engineered transposable element according to any one of claims 48-52, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:29; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:35.

[0095] 54. The engineered transposable element according to any one of claims 48-53, wherein the engineered transposable element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:17 or a variant thereof.

[0096] 55. The engineered transposon element according to claim 54, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:17.

[0097] 56. The engineered transposable element according to any one of claims 10-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:30; and 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:36.

[0098] 57. The engineered transposable element according to claim 56, the engineered transposable element comprising: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:6, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:12, a variant thereof, or a fragment thereof.

[0099] 58. The engineered transposable element according to claim 57, the engineered transposable element comprising: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:6; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:12.

[0100] 59. The engineered transposable element according to claim 58, the engineered transposable element comprising: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO: 6; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO: 12.

[0101] 60. The engineered transposable element according to any one of claims 56-59, the engineered transposable element further comprising a 5' TSD containing the nucleic acid sequence of SEQ ID NO:24 and a 3' TSD containing the nucleic acid sequence of SEQ ID NO:24.

[0102] 61. The engineered transposable element according to any one of claims 56-60, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:30; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:36.

[0103] 62. The engineered transposable element according to any one of claims 56-61, wherein the engineered transposable element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:18 or a variant thereof.

[0104] 63. The engineered transposon element according to claim 62, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:18.

[0105] 64. The engineered transpose element according to any one of items 1-15, wherein the engineered transpose element is derived from Tc1-1_ST, Tc1-2_ST, Tc1-3_ST, Tc1-4_ST, Tc1-5_ST or Tc1-1_PS.

[0106] 65. The engineered transpose element according to item 64, wherein the engineered transpose element is derived from Tc1-1_ST or Tc1-2_ST.

[0107] 66. The engineered transposable element according to any one of items 1-65, wherein the heterologous nucleic acid comprises a coding sequence.

[0108] 67. The engineered transposable element according to claim 66, wherein the heterologous nucleic acid further comprises one or more regulatory elements for regulating the expression of a coding sequence.

[0109] 68. The engineered transposable element according to claim 67, wherein the heterologous nucleic acid further comprises a promoter operatively linked to the coding sequence.

[0110] 69. An engineered transposon element according to any one of claims 66-68, wherein the coding sequence encodes one or more of a therapeutic protein, an engineered receptor, a selection marker, a reporter protein, a transposase, a polypeptide that can be used for genome editing, and an RNA molecule.

[0111] 70. The engineered transposon element according to claim 69, wherein the coding sequence encodes a chimeric antigen receptor (CAR), an engineered T-cell receptor (TCR), or an RNA molecule that can be used for genome editing.

[0112] 71. The engineered transposon element according to claim 69, wherein the coding sequence comprises a reporter gene.

[0113] 72. The engineered transposon element according to item 66, wherein the heterologous nucleic acid contains at least one restriction endonuclease recognition site, such as a restriction site.

[0114] 73. The engineered transposable element according to item 66, wherein the heterologous nucleic acid comprises a tag sequence.

[0115] 74. The engineered transposable element according to item 66, wherein the heterologous nucleic acid comprises a barcode sequence.

[0116] 75. The engineered transposable element according to item 66, wherein the heterologous nucleic acid comprises a unique molecular identifier (UMI).

[0117] 76. The engineered transposable element according to any one of items 1-75, wherein the transposable activity of the engineered transposable element is higher than that of the piggyBac (PB) transposable, the Sleeping Beauty (SB) transposable and / or the TcBuster (TB) transposable.

[0118] 77. The engineered transposable element according to any one of items 1-76, wherein the cell is an animal cell, plant cell, algal cell, fungal cell, yeast cell, or bacterial cell.

[0119] 78. The engineered transposable element according to any one of items 1-77, wherein the cell is a mammalian cell.

[0120] 79. The engineered transposable element according to claim 78, wherein the mammalian cell is selected from immune cells, hepatocytes, tumor cells, stem cells, fertilized eggs, muscle cells, and skin cells.

[0121] 80. The engineered transposable element according to item 78 or 79, wherein the cell is a human cell.

[0122] 81. The engineered transposable element according to any one of items 1-80, wherein the engineered transposable element has higher transposable activity in human embryonic kidney 293T (293T) cells than in HeLa cells.

[0123] 82. The engineered transducer element according to any one of items 1-81, wherein the engineered transducer element is present in a carrier.

[0124] 83. The engineered transposable element according to item 82, wherein the carrier is a plasmid or a viral vector.

[0125] 84. A gene transfer system comprising: 1) an engineered transposable element according to any one of claims 1-83; and 2) a transposase, or a nucleic acid encoding the transposase.

[0126] 85. The gene transfer system according to claim 84, wherein the transposase comprises an amino acid sequence selected from any of SEQ ID NOs:13-18, a variant thereof, or a fragment thereof.

[0127] 86. The gene transfer system according to claim 85, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with an amino acid sequence selected from any of SEQ ID NOs:13-18.

[0128] 87. The gene transfer system according to claim 86, wherein the transposase comprises an amino acid sequence selected from any one of SEQ ID NOs:13-18.

[0129] 88. A gene transfer system comprising: 1) an engineered transposon element; and 2) a transposase, or a nucleic acid encoding a transposase, wherein the engineered transposon element comprises, from 5' to 3':

[0130] 5' terminal repeat (5'TR), heterologous nucleic acid, and 3' terminal repeat (3'TR)

[0131] The engineered transposable element described herein exhibits transposable activity that allows heterologous nucleic acids to insert into the DNA of cells, and

[0132] The transposase therein comprises an amino acid sequence selected from any one of SEQ ID NOs:13-18 or a variant thereof.

[0133] 89. The gene transfer system according to claim 88, wherein the 5'TR comprises a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any of SEQ ID NOs:1-6, and wherein the 3'TR comprises a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any of SEQ ID NOs:7-12.

[0134] 90. The gene transfer system according to claim 89, wherein the 5'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any of SEQ ID NOs:1, 2, 3, 4, 5 and 6, and the 3'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any of SEQ ID NOs:7, 8, 9, 10, 11 and 12.

[0135] 91. The gene transfer system according to any one of claims 88-90, wherein the engineered transposon element is derived from Tc1-1_ST, Tc1-2_ST, Tc1-3_ST, Tc1-4_ST, Tc1-5_ST or Tc1-1_PS.

[0136] 92. The gene transfer system according to claim 91, wherein the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0137] 93. The gene transfer system according to any one of items 88-92, wherein:

[0138] (a) The 5'TR contains the nucleic acid sequence of SEQ ID NO:1, the 3'TR contains the nucleic acid sequence of SEQ ID NO:7, and the transposase contains the amino acid sequence of SEQ ID NO:13;

[0139] (b) The 5'TR contains the nucleic acid sequence of SEQ ID NO:2, the 3'TR contains the nucleic acid sequence of SEQ ID NO:8, and the transposase contains the amino acid sequence of SEQ ID NO:14;

[0140] (c) The 5'TR contains the nucleic acid sequence of SEQ ID NO:3, the 3'TR contains the nucleic acid sequence of SEQ ID NO:9, and the transposase contains the amino acid sequence of SEQ ID NO:15;

[0141] (d) The 5'TR contains the nucleic acid sequence of SEQ ID NO:4, the 3'TR contains the nucleic acid sequence of SEQ ID NO:10, and the transposase contains the amino acid sequence of SEQ ID NO:16;

[0142] (e) The 5'TR contains the nucleic acid sequence of SEQ ID NO:5, the 3'TR contains the nucleic acid sequence of SEQ ID NO:11, and the transposase contains the amino acid sequence of SEQ ID NO:17;

[0143] (f) The 5'TR contains the nucleic acid sequence of SEQ ID NO:6, the 3'TR contains the nucleic acid sequence of SEQ ID NO:12, and the transposase contains the amino acid sequence of SEQ ID NO:18;

[0144] 94. The gene transfer system according to any one of claims 88-93, wherein the gene transfer system comprises a nucleic acid encoding the transposase.

[0145] 95. The gene transfer system according to item 94, wherein the nucleic acid is DNA.

[0146] 96. The gene transfer system according to item 94, wherein the nucleic acid is RNA.

[0147] 97. The gene transfer system according to claim 94, wherein the engineered transposon element and the nucleic acid encoding the transposon are in different vectors.

[0148] 98. The gene transfer system according to claim 94, wherein the engineered transposon element and the nucleic acid encoding the transposon are in the same vector.

[0149] 99. The gene transfer system according to item 97 or 98, wherein the vector is a viral vector.

[0150] 100. A method for preparing a plurality of barcoded nucleic acids from a target nucleic acid, comprising contacting the target nucleic acids with a gene transfer system, said gene transfer system comprising: 1) an engineered transposon element; and 2) a transposase, or a nucleic acid encoding a transposase,

[0151] The engineered transposable element from 5' to 3' comprises: a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid containing a barcode sequence, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from any of SEQ ID NOs:1-6, a variant thereof, or a fragment thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from any of SEQ ID NOs:7-12, a variant thereof, or a fragment thereof;

[0152] The transposase described therein comprises an amino acid sequence selected from any one of SEQ ID NOs:13-18 or a variant thereof.

[0153] This provides multiple barcoded nucleic acids.

[0154] 101. The method according to claim 100, wherein the target nucleic acid is genomic DNA, cDNA, or amplified DNA, optionally wherein the amplified DNA comprises a nucleic acid sequencing library.

[0155] 102. The method according to item 100 or 101, further comprising sequencing the nucleic acid sequencing library.

[0156] 103. The method according to any one of claims 100-102, the method comprising contacting a target nucleic acid with a plurality of engineered transposable elements, wherein each engineered transposable element contains a unique barcode sequence.

[0157] 104. A transposon complex composition comprising: a transposase comprising an amino acid sequence selected from any of SEQ ID NOs:13-18 or a variant thereof, and one or two heterologous nucleic acids comprising a TR sequence and a tag sequence.

[0158] 105. The transposon complex composition according to claim 104, wherein the transposon complex comprises a single heterologous nucleic acid forming a hairpin.

[0159] 106. The transposable composite composition according to claim 105, wherein the hairpin comprises a cuttable site.

[0160] 107. The transposon complex composition according to claim 104, wherein the transposon complex comprises two transposases that bind to two heterologous nucleic acids.

[0161] 108. A transposon complex composition comprising: a first transposase that binds to a first heteronucleotide comprising a 5' TR sequence and a first tag sequence, and a second transposase that binds to a second heteronucleotide comprising a 3' TR sequence and a second tag sequence.

[0162] 109. The transposon complex composition according to claim 108, wherein the 5'TR comprises a nucleic acid sequence selected from any of SEQ ID NOs:1-6, a variant thereof, or a fragment thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from any of SEQ ID NOs:7-12, a variant thereof, or a fragment thereof.

[0163] 110. The transposon complex composition according to claim 108, wherein the first tag sequence is different from the second tag sequence.

[0164] 111. A method for preparing a library of nucleic acid fragments having first and second tag sequences for a target nucleic acid, comprising contacting the target nucleic acid with a plurality of transposon complexes, the transposon complexes comprising: (1) a first transposon complex comprising a first transposase and a first heteronucleic acid comprising a 5' TR sequence and a first tag sequence; and (2) a second transposon complex comprising a second transposase and a second heteronucleic acid comprising a 3' TR sequence and a second tag sequence, wherein the first tag sequence is different from the second tag sequence, and wherein the transposase comprises any amino acid sequence selected from SEQ ID NOs:13-18 or a variant thereof;

[0165] The first and second heteronucleotides are inserted into the target nucleic acid, and the target nucleic acid is fragmented into multiple nucleic acid fragments, each nucleic acid fragment containing one of the first or second nucleic acids linked to the 5' end of the nucleic acid fragment;

[0166] Optionally, the nucleic acid fragment is DNA;

[0167] This provides a library of nucleic acid fragments.

[0168] 112. The method according to item 111, wherein the transposon complex of (1) comprises two first heteronucleotides and the transposon complex of (2) comprises two second heteronucleotides.

[0169] 113. The method according to claim 111, wherein the 5'TR comprises a nucleic acid sequence, a variant thereof or a fragment thereof selected from any of SEQ ID NO:s 1-6, and wherein the 3'TR comprises a nucleic acid sequence, a variant thereof or a fragment thereof selected from any of SEQ ID NOs:s 7-12.

[0170] 114. The method according to item 111, the method further comprising amplifying nucleic acid fragments.

[0171] 115. A method for inserting a heterologous nucleic acid into a target nucleic acid, comprising:

[0172] The target nucleic acid is contacted with the transposon element of any one of items 1-83, the gene transfer system of any one of items 84-99, or the transposon complex composition of any one of items 94-100, thereby inserting the heterologous nucleic acid into the target nucleic acid.

[0173] 116. The method according to item 115, wherein the method is performed in vitro.

[0174] 117. The method according to item 115, wherein the target nucleic acid is in a cell.

[0175] 118. The method according to item 117, wherein the target nucleic acid is genomic DNA.

[0176] 119. The method according to item 117 or 118, wherein the cell is an animal cell, plant cell, algal cell, fungal cell, yeast cell, or bacterial cell.

[0177] 120. The method according to item 119, wherein the cell is a mammalian cell.

[0178] 121. The method according to claim 120, wherein the mammalian cells are selected from immune cells, hepatocytes, tumor cells, stem cells, fertilized eggs, muscle cells, and skin cells.

[0179] 122. The method according to any one of items 118-121, wherein the insertion of the heterologous nucleic acid inactivates the genes of the cell.

[0180] 123. The method according to any one of items 115-122, wherein the heterologous nucleic acid encodes a protein.

[0181] 124. The method according to claim 123, wherein the protein is selected from the group consisting of reporter proteins, engineered receptors, cytokines, antibiotic resistance proteins, antigens, and therapeutic proteins.

[0182] 125. The method according to any one of items 115-122, wherein the heterologous nucleic acid encodes RNA.

[0183] 126. The method according to claim 125, wherein the RNA is selected from the group consisting of: therapeutic RNA, small interfering RNA (siRNA), microRNA, short hairpin RNA (shRNA), long non-coding RNA (lincRNA), and guide RNA (gRNA).

[0184] 127. The method according to any one of items 115-126, wherein the heterologous nucleic acid encodes more than one molecule.

[0185] 128. The method according to any one of items 115-127, wherein the length of the heterologous nucleic acid does not exceed about 300 kilobases (kb).

[0186] 129. The method according to item 128, wherein the length of the heterologous nucleic acid is from about 10 kb to about 300 kb.

[0187] 130. The method according to claim 128, wherein the length of the heterologous nucleic acid is about 100 base pairs (bp) to about 10 kb.

[0188] 131. The method according to item 128, wherein the length of the heterologous nucleic acid is about 100 bp to about 5 kb.

[0189] 132. The method according to item 131, wherein the length of the heterologous nucleic acid is about 100 bp to about 2 kb.

[0190] 133. The method according to item 129, wherein the length of the heterologous nucleic acid is about 2 kb to about 300 kb.

[0191] 134. The method according to any one of items 115-133, wherein the insertion is random.

[0192] 135. A kit comprising an engineered transposon element according to any one of claims 1-83, or a gene transfer system according to any one of claims 84-99, or a transposon complex composition according to any one of claims 94-100, for inserting a heterologous nucleic acid into a target nucleic acid. Attached Figure Description

[0193] Figure 1 shows a diagram illustrating the process of identifying the active transposable element (TE).

[0194] Figure 2 shows a set of exemplary binary constructs for screening active transposable elements. The upper construct is an auxiliary construct for transposase expression, which contains, from 5' to 3': a cytomegalovirus (CMV) promoter, a transposase (Tn) gene, and a polyA (pA) signal. The lower construct is a donor construct, which contains, from 5' to 3': a 5' target site repeat (5'TSD) sequence, a 5' terminal repeat (5'TR) sequence, a phosphoglycerate kinase (PGK) promoter, a 5' sequence encoding puromycin and enhanced GFP fusion (Puro-eGFP), a polyA (pA) signal, a 3' terminal repeat (3'TR) sequence, and a 3' target site repeat (3'TSD) sequence.

[0195] Figure 3 shows the transposition activities of identified transposable elements in HEK293T cells compared to the control TE, which includes piggyBac, HyperpiggyBac, and SB100X. In HEK293T (293T), the transposition activities of the following identified transposable elements, Tc1-1_ST and Tc1-2_ST, were compared to the control TE and negative control (donor only), which included piggyBac, HyperpiggyBac (hPB), and SB100X.

[0196] Figure 4 shows the transposition (i.e., transfer) efficiency of the identified Tc1-1_ST in HEK293T (293T) cells, HeLa cells, HCT116 cells, and K562 cells compared with the control TE piggyBac (IFP2), hyperpiggyBac (hPB), and SB100X.

[0197] Figure 5 shows the transposition activity of the identified transposable element Tc1-1_ST in primary T cells compared to the control TE SB100X.

[0198] Figure 6 shows the transposition activities of the identified transposable elements SB100X and Tc1-1_ST in HeLa cells, based on different ratios of helper plasmid and transposable element plasmid, compared to the control TE SB100X. Figure 6A shows the transposition (transfer) efficiency of the control TE SB100X. Figure 6B shows the identified transposition (transfer) efficiency of Tc1-1_ST.

[0199] Figure 7 shows the loading capacity of the identified transposable element Tc1-1_ST in 293T cells compared to the control SB100X.

[0200] Figure 8 shows the common sequence of the genomic insertion locus within a 20 bp window around the target site in the genomes of the TE Tc1-1_ST original species and the stable transposon K562 cell line.

[0201] Figure 9 shows the enrichment folds of identified transposable elements compared to random insertions in gene upstream and downstream, upstream and downstream of transcription start sites, different chromatin states, and safe harbor regions. These transposable elements include piggyBac, SB100X, and Tc1-1_ST.

[0202] Figure 10 shows the enrichment fold of identified transposon elements compared to random insertions in the locations around the transcription start site (TSS) within a 5kb window. These transposon elements include piggyBac, SB100X, and Tc1-1_ST.

[0203] Figure 11 shows the enrichment fold of identified transposable elements compared to random insertions in different epigenetic modification protein regions. These transposable elements include piggyBac, SB100X, and Tc1-1_ST. Detailed Implementation

[0204] This application provides engineered transposon elements, gene transfer systems comprising engineered transposon elements, and methods and kits for using them. This application is based, at least in part, on the identification of novel transposon elements from a wide range of species through pan-genomic bioinformatics analysis (e.g., Table 2), and the surprising results demonstrating the efficient transposition of many identified transposon elements in human cells. The disclosed compositions, systems, and methods can be used to insert heterologous nucleic acids into target nucleic acids, including the introduction of heterologous DNA into the genome of cells. The transposon elements and gene transfer systems described herein can be used in a variety of applications, such as gene therapy and gene discovery research.

[0205] Therefore, in one aspect, this application provides an engineered transposon element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof, and wherein the transposon element exhibits transposon activity that allows the heterologous nucleic acid to be inserted into the DNA of a cell.

[0206] On the other hand, this application provides a gene transfer system comprising: 1) an engineered transposon element; and 2) a transposase or a nucleic acid encoding a transposase, wherein the transposon element comprises, from 5' to 3', a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the transposon element exhibits transposition activity that allows the heterologous nucleic acid to be inserted into the DNA of a cell. In some embodiments, the transposon element is an engineered transposon element. In some embodiments, the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18 or a variant thereof.

[0207] I. Definition

[0208] As used herein, the terms “transposon,” “transposon element,” or “TE” refer to a polynucleotide capable of being excised from a first nucleic acid (i.e., the donor nucleic acid, such as a vector) and integrated into a target site (such as a second nucleic acid in a cell or genomic or extrachromosomal DNA). A transposon comprises a nucleic acid sequence of a cis-acting nucleic acid sequence flanking the ends of the transposon element, provided that at least one cis-acting nucleic acid sequence is located at the 5' end of the nucleic acid sequence and at least one cis-acting nucleic acid sequence is located at the 3' end of the nucleic acid sequence. The cis-acting nucleic acid sequence includes at least one terminal repeat sequence (TR, also known as an inverted terminal repeat sequence (ITR) or terminal inverted repeat sequence (TIR)) at each end of the transposon element to which the transposase binds. The transposon element described herein may or may not contain an open reading frame (ORF) encoding a transposase.

[0209] As used herein, the term “transposation” refers to the alteration of the position of a transposable element from a first nucleic acid (e.g., a vector) and its integration into a target site (e.g., a second nucleic acid in a cell or genomic or extrachromosomal DNA).

[0210] As used herein, the term "terminal repeat" or "TR" refers to a nucleic acid sequence located at both ends of a transposable element and laterally attached to a transposable second nucleic acid sequence. A TR located at the 5' (upstream) of the second nucleic acid sequence is called a 5' TR, and a TR located at the 3' (downstream) of the second nucleic acid sequence is called a 3' TR. In both types of transposable elements, the TRs are complementary to each other.

[0211] As used herein, the term "target site repeat" or "TSD" refers to the nucleic acid sequence present at the insertion site of a transposon element. TSDs may arise from DNA repair at the sticky ends caused by the staggered cleavage of the target DNA duplex by transposases. TSDs are flanked by the transposable element's TR. A TSD located at the 5' of the 5' TR is a 5' TSD. A TSD located at the 3' of the 3' TR is a 3' TSD.

[0212] As used herein, the term "transposase" refers to a polypeptide that catalyzes the excision of a transposon from a first nucleic acid (e.g., a vector) and its integration into a target site (e.g., a second nucleic acid in a cell or genomic or extrachromosomal DNA). In some embodiments, transposases bind to one or both terminal repeat sequences.

[0213] As used herein, "left transposon fragment" or "LTF" refers to the segment of a naturally occurring transposon element from 5' TSD to the start codon of the transposase ORF sequence. As used herein, "right transposon fragment" or "RTF" refers to the segment of a naturally occurring transposon element from the stop codon of the transposase ORF sequence to 3' TSD.

[0214] The terms “nucleic acid,” “polynucleotide,” and “nucleic acid sequence” are used interchangeably to refer to a polymer of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogues. “Oligonucleotide” and “oligo” are used interchangeably to refer to short polynucleotides having no more than about 50 nucleotides.

[0215] As used herein, "heterologous nucleic acid" refers to a DNA or RNA sequence that originates from a source different from a reference nucleic acid sequence. For example, in the case of transposable elements, the heterologous nucleic acid originates from a source different from the terminal repeat sequence. For example, a nucleic acid sequence isolated from an organism that does not contain the terminal repeat sequence is considered a heterologous nucleic acid targeting the terminal repeat sequence.

[0216] As used herein, the term "operably ligated" refers to a nucleic acid sequence that is functionally related to another nucleic acid sequence. For example, if a coding sequence is operably ligated to a promoter sequence, this typically means that the promoter can promote transcription of the coding sequence. Operable ligation means that the ligated DNA sequences are usually contiguous, and when two protein-coding regions must be ligated, they are contiguous and within the reading frame. Because enhancers can function even when separated from promoters by thousands of bases and intron sequences can have variable lengths, some nucleic acid sequences may be operably ligated but not contiguous.

[0217] The "percentage of sequence identity (%)" for nucleic acid sequences 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, with the maximum percentage of sequence identity achieved by allowing vacancies if necessary. The "percentage of sequence homology (%)" for peptide, polypeptide, or protein sequences is the percentage of amino acid residues in a candidate sequence that have the same substituted amino acid residues as those in a specific peptide or amino acid sequence after sequence alignment, with the maximum percentage of sequence homology achieved by allowing vacancies if necessary. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, for example, using methods such as BLAST, BLAST-2, ALIGN, or MEGALIGN. TM The (DNASTAR) software is publicly available computer software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0218] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. Examples of vectors include, but are not limited to, bacteria, plasmids, bacteriophages, granules, free genes, viruses, and insertable DNA fragments, i.e., fragments capable of being inserted into the host cell genome through homologous recombination.

[0219] As used in this article, the term "plasmid" refers to a circular double-stranded DNA that can accept exogenous DNA fragments and replicate in prokaryotic or eukaryotic cells.

[0220] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to amino acid polymers of any length. Thus, for example, the terms peptide, oligopeptide, protein, antibody, and enzyme are included in the definition of a polypeptide. A polymer can be linear or branched, it can contain modified amino acids, and it can be broken down by non-amino acid components. A protein can have one or more polypeptides. The term also includes modified amino acid polymers; for example, those modified by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeled component.

[0221] As used herein, “variant” is defined as a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains its essential characteristics. A typical variant of a polynucleotide differs from the nucleic acid sequence of another reference polynucleotide. Changes in the nucleic acid sequence of a variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. As described below, nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. A typical variant of a polypeptide differs from the amino acid sequence of another reference polypeptide. Typically, the differences are limited, and thus the sequences of the reference polypeptide and the variant are generally very similar and identical in many regions. The amino acid sequences of the variant and the reference polypeptide can differ due to one or more substitutions, additions, or deletions in any combination of forms. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. Variants of polynucleotides or polypeptides can be naturally occurring, such as allelic variants, or they may be variants of unknown natural origin. Non-natural 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.

[0222] As used in this article, a “fragment” of a sequence refers to a portion of the sequence. For example, a fragment of a nucleic acid sequence refers to a portion of the nucleic acid sequence, and a fragment of an amino acid sequence refers to a portion of the amino acid sequence.

[0223] As used herein, the terms "genetic circuit," "biological circuit," or "synthetic circuit" refer to a set of biological components designed to perform logical functions. Typically, an input is required to activate a genetic circuit, which then produces an output based on the input.

[0224] As used herein, the term “engineering” refers to any operation that results in a detectable change in a polynucleotide or polypeptide, including but not limited to the insertion, deletion, and substitution of a portion of a polynucleotide or amino acid sequence.

[0225] As used herein, the term "transposon efficiency" refers to the efficiency with which a transposon element inserts a heterologous nucleic acid into a target cell population. For example, transposon efficiency can be determined by transfecting a plasmid containing a reporter gene or a gene encoding a selection marker, such as an antibiotic resistance gene (e.g., puromycin), into a target cell population, and determining the number of cells expressing the gene product encoded by the reporter gene or selection marker, for example, by measuring the number of cells that are antibiotic resistant.

[0226] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into host cells. A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acids. As used herein, the terms “transduction” and “transfection” include all methods known in the art for introducing DNA into cells to express a target protein or molecule using infectious agents (e.g., viruses) or other means. In addition to viruses or virus-like agents, there are chemical-based transfection methods, such as those using calcium phosphate, dendritic macromolecules, liposomes, or cationic polymers (e.g., DEAE-glucan or polyethyleneimine); non-chemical methods, such as electroporation, cell squeezing, acoustic perforation, phototransfection, puncture transfection, protoplast fusion, plasmid or transposon delivery; particle-based methods, such as those using gene guns, magnetic transfection or magnetically assisted transfection, particle bombardment; and hybrid methods, such as nuclear transfection.

[0227] The term "in vivo" refers to the body within which cells are obtained. "Ex vivo" or "in vitro" refers to the body outside of the organism in which cells are obtained.

[0228] It should be understood that the embodiments of the present invention described herein include embodiments that are “composed of” and / or “substantially composed of”.

[0229] This article mentions that the numerical value or parameter “about” includes (and describes) the variation with respect to that numerical value or parameter itself. For example, a description of “about X” includes a description of “X”.

[0230] As used in this article, mentioning "not" a numerical value or parameter generally indicates and describes something "different from" a numerical value or parameter. For example, "This method is not used to treat type X cancer" means that this method is used to treat cancers other than type X.

[0231] The term “about XY” used in this article has the same meaning as “about X to about Y”.

[0232] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly specifies otherwise.

[0233] II. Engineered Transistor Components

[0234] This application provides, in one aspect, an engineered transposon element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR). In some embodiments, the transposon element exhibits transposon activity that allows the heterologous nucleic acid to insert into a target nucleic acid (e.g., DNA) in vitro. In some embodiments, the engineered transposon element exhibits transposon activity that allows the heterologous nucleic acid to insert into a target nucleic acid (e.g., DNA in mammals or cellular plants) in a cell. In some embodiments, the transposon element contains a nucleic acid sequence encoding a transposase. In some embodiments, the transposon element does not contain a nucleic acid sequence encoding a transposase.

[0235] In some embodiments, the engineered transposon element comprises, from 5' to 3': a 5' target site repeat sequence (5'TSD), a 5' TR, a heterologous nucleic acid, a 3' TR, and a 3'TSD. In some embodiments, the transposon element exhibits transposon activity that allows the heterologous nucleic acid to insert into a target nucleic acid (e.g., DNA) in vitro. In some embodiments, the engineered transposon element exhibits transposon activity that allows the heterologous nucleic acid to insert into a target nucleic acid (e.g., DNA in mammals or cellular plants) within a cell. In some embodiments, the transposon element contains a nucleic acid sequence encoding a transposase. In some embodiments, the transposon element does not contain a nucleic acid sequence encoding a transposase.

[0236] In some embodiments, this application provides novel transposon elements from a wide range of species that can efficiently transposon in human cells. The transposon elements described herein provide direct experimental evidence for naturally active mammalian cut-and-paste DNA transposons.

[0237] A list of exemplary transposon elements and their corresponding left-terminal fragment (LTF), right-terminal fragment (RTF), transposase, 5' terminal repeat (TR), 3' TR, 5' target site repeat (TSD), and 3' TSD sequences can be found in Tables 1-2 and the sequence listing. Table 1 lists six TEs identified from the bioinformatics analysis disclosed in this application, including two TEs (TE IDs: 1 and 3) with a concordance length not exceeding 3000 bp, a MITE copy number greater than 300, and an average difference of less than 1%, suitable for efficient genome engineering. Table 2 lists the active TEs validated using transposon analysis experiments in the human cell line HEK293T.

[0238] In some embodiments, the transposon element is a Class 2 transposon element. Class 2 transposon elements can be classified into superfamilies based on the correlation of transposases and shared structural features, including the length of terminal repeats (TRs) and target site repeats (TSDs) of side-attached TRs generated during integration. The transposon elements of this application are envisioned to originate from various suitable TE superfamilies and / or families. In some embodiments, the transposon element is from the TcMariner superfamily. In some embodiments, the 5' TR is the reverse complement of the 3' TR. In some embodiments, the 5' TR is not the reverse complement of the 3' TR.

[0239] In some embodiments, the engineered transposon element comprises a 5' TR containing a nucleic acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with a 5' TR of a transposon element selected from the TcMariner superfamily. In some embodiments, the engineered transposon element comprises a 3' TR containing a nucleic acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with a 3' TR of a transposon element selected from the TcMariner superfamily.

[0240] In some embodiments, the engineered transposon element comprises a 5'TR and a 3'TR, the 5'TR comprising a nucleic acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with a 5'TR of a transposon element selected from the TcMariner superfamily, and the 3'TR comprising a nucleic acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with a 3'TR of a transposon element selected from the TcMariner superfamily.

[0241] In some embodiments, the engineered transposon element comprises 5'TR, 3'TR, LTF, RTF, transposase, 5'TSD, and / or 3'TSD derived from any TE in Table 1. In some embodiments, the engineered transposon element comprises 5'TR, 3'TR, LTF, RTF, transposase, 5'TSD, and / or 3'TSD derived from any TE in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST, Tc1-2_ST, Tc1-3_ST, Tc1-4_ST, Tc1-5_ST, or Tc1-1_PS. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0242] In some embodiments, the engineered transposable element comprises an LTF containing nucleic acid sequences selected from SEQ ID NO:25-30, their variants, or fragments thereof.

[0243] In some embodiments, the engineered transposable element comprises an RTF containing a nucleic acid sequence selected from SEQ ID NO:31-36, its variants, or fragments thereof.

[0244] In some embodiments, the engineered transposon element comprises an LTF and an RTF, wherein the LTF has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleic acid sequence selected from SEQ ID NO: 25-30, and the RTF has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleic acid sequence selected from SEQ ID NO: 31-36. In some embodiments, the engineered transposon element comprises an LTF containing a nucleic acid sequence selected from SEQ ID NO: 25-30 and an RTF containing a nucleic acid sequence selected from SEQ ID NO: 31-36.

[0245] In some embodiments, the engineered transposon element comprises a 5' TR in an LTF, the LTF comprising nucleic acid sequences selected from SEQ ID NO:25-30. In some embodiments, the engineered transposon element comprises a 3' TR in an RTF, the RTF comprising nucleic acid sequences selected from SEQ ID NO:31-36.

[0246] In some embodiments, the engineered transposable element comprises a 5'TR, a variant of 5'TR, or a fragment of 5'TR in an LTF containing nucleic acid sequences selected from SEQ ID NO:25-30; and a 3'TR, a variant of 3'TR, or a fragment of 3'TR in an RTF containing nucleic acid sequences selected from SEQ ID NO:31-36.

[0247] In some embodiments, the engineered transposon element comprises a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from SEQ ID NO:1-6. In some embodiments, the engineered transposon element of this application comprises a 5' TR having a sequence identity with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of a nucleic acid sequence selected from SEQ ID NO:1-6. In some embodiments, the 5' TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6. In some embodiments, the engineered transposon element comprises a 3' TR having a complementary sequence to the 5' TR.

[0248] In some embodiments, the engineered transposon element comprises a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from SEQ ID NO:7-12. In some embodiments, the engineered transposon element comprises a 3' TR having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleic acid sequence selected from SEQ ID NO:7-12. In some embodiments, the 3' TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12. In some embodiments, the engineered transposon element comprises a 5' TR having a complementary sequence to the 3' TR.

[0249] In some embodiments, the engineered transposable element comprises: 1) a 5' TR having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with a nucleic acid sequence selected from SEQ ID NO:1-6; and 2) a 3' TR having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with a nucleic acid sequence selected from SEQ ID NO:7-12.

[0250] In some embodiments, the engineered transposable element comprises: 1) a 5' TR having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with a nucleic acid sequence selected from SEQ ID NO:1-6; and 2) a 3' TR having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with a nucleic acid sequence selected from SEQ ID NO:7-12.

[0251] This document also considers engineered transposable elements comprising variants or fragments of any of the 5'TR and / or 3'TR, or LTF and / or RTF as described herein, for example, in Tables 1 and 2. In some embodiments, the variants comprise nucleotide substitutions of no more than one of about 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, the fragments comprise at least one of about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides.

[0252] In some embodiments, the engineered transpose element includes a 5' TSD. In some embodiments, the engineered transpose element includes a 3' TSD. In some embodiments, the engineered transpose element includes both a 5' TSD and a 3' TSD. In some embodiments, the engineered transpose element does not include a 5' TSD. In some embodiments, the engineered transpose element does not include a 3' TSD. In some embodiments, the engineered transpose element does not include either a 5' TSD or a 3' TSD. In some embodiments, the 5' TSD is the same as the 3' TSD. In some embodiments, the 5' TSD is different from the 3' TSD.

[0253] In some embodiments, the engineered transposon element comprises a nucleic acid sequence encoding a transposase. In some embodiments, the engineered transposon element does not comprise a nucleic acid sequence encoding a transposase. In some embodiments, the transposase is derived from the same species as the 5'TR and 3'TR sequences. In some embodiments, the transposase is a natural transposase targeting the 5'TR and 3'TR sequences. In some embodiments, the transposase is an engineered transposase based on a natural transposase targeting the 5'TR and 3'TR sequences.

[0254] Transposases catalyze the excision of transposons from donor polynucleotides (e.g., vectors) and subsequently the integration of the transposons into target nucleic acids, such as the genome or extrachromosomal DNA of a target cell. In some embodiments, transposases bind to the terminal repeats of transposable elements.

[0255] In some embodiments, the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18, its variants, or fragments thereof.

[0256] This application considers variants of the transposases listed in Tables 1-2 and the sequence listing in some embodiments. A reference to a transposase variant refers to a transposase polypeptide that differs from a reference transposase polypeptide (e.g., a naturally occurring transposase polypeptide) by the addition, deletion, truncation, and / or substitution of at least one amino acid residue, which retains transposition activity. In some embodiments, the transposase polypeptide variant differs from the reference transposase polypeptide by one or more substitutions, which may be conserved or non-conserved, as is known in the art. In some embodiments, the variant transposase comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the sequence identity or similarity to the corresponding sequence of the reference transposase. In some implementations, the variant transposase contains no more than about 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution.

[0257] Also considered are functional fragments of the transposases described herein with amino acid deletions or variants with amino acid additions. In some embodiments, the transposase fragment has a length of at least about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700 or more amino acid residues. In some embodiments, amino acid additions or deletions occur at the C-terminus and / or N-terminus of the reference transposase. In some embodiments, amino acid additions or deletions occur at internal locations, such as the flexible loop of the reference transposase. In some embodiments, the amino acid deletion (e.g., N-terminal and / or C-terminal truncation) comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175 or more amino acids, including all values ​​and ranges between these values. In some implementations, the variant transposase contains an N-terminal or C-terminal purification tag, a selection marker (e.g., an antibiotic resistance gene), or a reporter gene (e.g., a fluorescent reporter gene).

[0258] As described above, the transposase polypeptide of the present invention can be modified in a variety of ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the reference polypeptide can be prepared by mutation in DNA. Methods for mutagenesis and nucleic acid sequence alteration are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al. (1987, Methods in Enzymol, 154:367-382), US Pat. No. 4, 873, 192, Watson, J. De et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0259] In some embodiments, the transposase is codon-optimized compared to a reference transposase. In some embodiments, the transposase is codon-optimized for expression in mammalian cells such as human cells. In some embodiments, the transposase is codon-optimized for expression in plant cells.

[0260] In some embodiments, the transposase comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with an amino acid sequence selected from SEQ ID NO:13-18. In some embodiments, the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18.

[0261] In some embodiments, the transposable element comprises: 1) a 5' TR in an LTF containing the nucleic acid sequence of SEQ ID NO:25; and 2) a 3' TR in an RTF containing the nucleic acid sequence of SEQ ID NO:31. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO:1, a variant thereof, or a fragment thereof; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO:7, a variant thereof, or a fragment thereof. In some embodiments, the transposable element comprises: 1) a 5' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO:1; and 2) a 3' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO:7. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO:1; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO:7. In some embodiments, the transposon element comprises an LTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:25; and an RTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:31. In some embodiments, the transposon element is associated with a transposase containing the amino acid sequence of SEQ ID NO:13 or a variant thereof. In some embodiments, the transposase comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO:13. In some embodiments, the transposable element comprises a nucleic acid sequence encoding a transposase. In some embodiments, the transposable element does not comprise a nucleic acid sequence encoding a transposase.

[0262] SEQ ID NO:1(5'TR of Tc1-1_ST)

[0263] SEQ ID NO:7(3'TR of Tc1-1_ST)

[0264] SEQ ID NO:13 (Transposase of Tc1-1_ST)

[0265] In some embodiments, the transposable element comprises: 1) a 5' TR in an LTF containing the nucleic acid sequence of SEQ ID NO:26; and 2) a 3' TR in an RTF containing the nucleic acid sequence of SEQ ID NO:32. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO:2, a variant thereof, or a fragment thereof; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO:8, a variant thereof, or a fragment thereof. In some embodiments, the transposable element comprises: 1) a 5' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO:2; and 2) a 3' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO:2; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the transposable element further comprises a 5' TSD containing the nucleic acid sequence of TA (SEQ ID NO: 20) and a 3' TSD containing the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the transposable element does not contain a 5' TSD and / or a 3' TSD. In some embodiments, the transposable element comprises an LTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO: 26; and 2) an RTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO: 32. In some embodiments, the transposable element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:14 or a variant thereof. In some embodiments, the transposase comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO:14. In some embodiments, the transposable element comprises a nucleic acid sequence encoding a transposase.In some implementations, the transposable element does not contain a nucleic acid sequence encoding a transposase.

[0266] SEQ ID NO:2(Tc1-2_ST's 5'TR)

[0267] SEQ ID NO:8(3'TR of Tc1-2_ST)

[0268] SEQ ID NO:14 (Transposase of Tc1-2_ST)

[0269] In some embodiments, the transposable element comprises: 1) a 5' TR in an LTF containing the nucleic acid sequence of SEQ ID NO:27; and 2) a 3' TR in an RTF containing the nucleic acid sequence of SEQ ID NO:33. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO:3, a variant thereof, or a fragment thereof; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO:9, a variant thereof, or a fragment thereof. In some embodiments, the transposable element comprises: 1) a 5' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO:3; and 2) a 3' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO:3; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the transposable element further comprises a 5' TSD containing the nucleic acid sequence of TA (SEQ ID NO: 21) and a 3' TSD containing the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the transposable element does not contain a 5' TSD and / or a 3' TSD. In some embodiments, the transposable element comprises an LTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO: 27; and an RTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the transposable element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:15 or a variant thereof. In some embodiments, the transposase comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO:15. In some embodiments, the transposable element comprises a nucleic acid sequence encoding a transposase.In some implementations, the transposable element does not contain a nucleic acid sequence encoding a transposase.

[0270] SEQ ID NO:3(Tc1-3_ST's 5'TR)

[0271] SEQ ID NO:9(3'TR of Tc1-3_ST)

[0272] SEQ ID NO:15 (Transposase of Tc1-3_ST)

[0273] In some embodiments, the transposable element comprises: 1) a 5' TR in an LTF containing the nucleic acid sequence of SEQ ID NO:28; and 2) a 3' TR in an RTF containing the nucleic acid sequence of SEQ ID NO:34. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO:4, a variant thereof, or a fragment thereof; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO:10, a variant thereof, or a fragment thereof. In some embodiments, the transposable element comprises: 1) a 5' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO:4; and 2) a 3' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO:10. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO:4; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO:10. In some embodiments, the transposable element further includes a 5' TSD containing the nucleic acid sequence of TA (SEQ ID NO: 22) and a 3' TSD containing the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the transposable element does not contain a 5' TSD and / or a 3' TSD. In some embodiments, the transposable element comprises an LTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO: 28; and an RTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the transposable element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:16 or a variant thereof. In some embodiments, the transposase comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO:16. In some embodiments, the transposable element comprises a nucleic acid sequence encoding a transposase.In some implementations, the transposable element does not contain a nucleic acid sequence encoding a transposase.

[0274] SEQ ID NO:4(Tc1-4_ST's 5'TR)

[0275] SEQ ID NO:10(3'TR of Tc1-4_ST)

[0276] SEQ ID NO:16 (Transposase of Tc1-4_ST)

[0277] In some embodiments, the transposable element comprises: 1) a 5' TR in an LTF containing the nucleic acid sequence of SEQ ID NO:29; and 2) a 3' TR in an RTF containing the nucleic acid sequence of SEQ ID NO:35. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO:5, a variant thereof, or a fragment thereof; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO:11, a variant thereof, or a fragment thereof. In some embodiments, the transposable element comprises: 1) a 5' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO: 5; and 2) a 3' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO: 5; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the transposable element further comprises a 5' TSD containing the nucleic acid sequence of SEQ ID NO: 23 and a 3' TSD containing the nucleic acid sequence of SEQ ID NO: 23. The transposon element does not contain a 5' TSD and / or a 3' TSD. In some embodiments, the transposon element comprises an LTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:29; and an RTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:35. In some embodiments, the transposon element is associated with a transposase containing the amino acid sequence of SEQ ID NO:17 or a variant thereof. In some embodiments, the transposase comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO:17. In some embodiments, the transposable element comprises a nucleic acid sequence encoding a transposase. In some embodiments, the transposable element does not comprise a nucleic acid sequence encoding a transposase.

[0278] SEQ ID NO:5(Tc1-5_ST's 5'TR)

[0279] SEQ ID NO:11(3'TR of Tc1-5_ST)

[0280] SEQ ID NO:17 (Transposase of Tc1-5_ST)

[0281] In some embodiments, the transposable element comprises: 1) a 5' TR in an LTF containing the nucleic acid sequence of SEQ ID NO:30; and 2) a 3' TR in an RTF containing the nucleic acid sequence of SEQ ID NO:31. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO:6, a variant thereof, or a fragment thereof; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO:12, a variant thereof, or a fragment thereof. In some embodiments, the transposable element comprises: 1) a 5' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO:6; and 2) a 3' TR having a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the nucleic acid sequence of SEQ ID NO:12. In some embodiments, the transposable element comprises: 1) a 5' TR containing the nucleic acid sequence of SEQ ID NO:6; and 2) a 3' TR containing the nucleic acid sequence of SEQ ID NO:12. In some embodiments, the transposable element further comprises a 5' TSD containing the nucleic acid sequence of SEQ ID NO:24 and a 3' TSD containing the nucleic acid sequence of SEQ ID NO:24. In some embodiments, the transposable element does not contain a 5' TSD and / or a 3' TSD. In some embodiments, the transposable element comprises an LTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:30; and 2) an RTF containing a nucleic acid sequence having at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO:36. In some embodiments, the transposable element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:18 or a variant thereof. In some embodiments, the transposase comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO:18. In some embodiments, the transposable element comprises a nucleic acid sequence encoding the transposase.In some implementations, the transposable element does not contain a nucleic acid sequence encoding a transposase.

[0282] SEQ ID NO:6(5'TR of Tc1-1_PS)

[0283] SEQ ID NO:12(3'TR of Tc1-1_PS)

[0284] SEQ ID NO:18 (Transposase of Tc1-1_PS)

[0285] Heterologous nucleic acids

[0286] The engineered transposable elements described herein are applicable to transposing various heterologous nucleic acids. In some embodiments, the heterologous nucleic acid is DNA. In some embodiments, the heterologous nucleic acid is double-stranded. In some embodiments, the heterologous nucleic acid comprises one or more modified nucleotides. In some embodiments, the heterologous nucleic acid is unmodified.

[0287] The heterologous nucleic acid in the transposable element can have various suitable lengths. In some embodiments, the length of the heterologous nucleic acid is at least about 1kb, 2kb, 10kb, 20kb, 30kb, 40kb, 50kb, 60kb, 70kb, 80kb, 90kb, 100kb, 150kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb, 500kb, 600kb, 700kb, 800kb, 900kb, 1000kb or more. In some implementations, the length of the heterologous nucleic acid does not exceed any one of about 1000kb, 900kb, 800kb, 700kb, 600kb, 500kb, 450kb, 400kb, 350kb, 300kb, 250kb, 200kb, 150kb, 100kb, 90kb, 80kb, 70kb, 60kb, 50kb, 40kb, 30kb, 20kb, 10kb, 5kb, 2kb, or 1kb. In some embodiments, the length of the heterologous nucleic acid is approximately 100 bp to approximately 1 kb, approximately 1 kb to approximately 2 kb, approximately 2 kb to approximately 5 kb, approximately 5 kb to approximately 10 kb, approximately 100 bp to approximately 5 kb, approximately 100 bp to approximately 2 kb, approximately 2 kb to approximately 10 kb, approximately 1 kb to approximately 10 kb, approximately 10 kb to approximately 20 kb, approximately 20 kb to approximately 50 kb, approximately 50 kb to approximately 100 kb, approximately 1 kb to approximately 100 kb, approximately 150 kb to approximately 200 kb. b. Approximately 200kb to approximately 300kb, approximately 300kb to approximately 400kb, approximately 400kb to approximately 500kb, approximately 500kb to approximately 600kb, approximately 600kb to approximately 700kb, approximately 700kb to approximately 80kb, approximately 800kb to approximately 900kb, approximately 900kb to approximately 1000kb, approximately 10kb to approximately 100kb, approximately 100kb to approximately 500kb, approximately 500kb to approximately 1000kb, or approximately 10kb to approximately 500kb. In some embodiments, the heteronucleotide is approximately 10kb to approximately 300kb nucleotides in length. In some embodiments, the heteronucleotide is approximately 100bp to approximately 300kb nucleotides in length.

[0288] The heteronucleotide may contain one or more coding sequences, including coding sequences of any 1, 2, 3, 4, 5, 6, 10 or more. Any suitable coding sequence may be used in this application, and the coding sequence may encode any suitable target biological product. In some embodiments, the coding sequence encodes an RNA molecule. In some embodiments, the coding sequence encodes a polypeptide, such as a protein. In some embodiments, the heteronucleotide contains a first coding sequence encoding a first protein and a second coding sequence encoding a second protein. In some embodiments, the heteronucleotide contains a first coding sequence encoding a first RNA and a second coding sequence encoding a second RNA. In some embodiments, the heteronucleotide contains a first coding sequence encoding a protein and a second coding sequence encoding RNA.

[0289] In some embodiments, the coding sequence encodes a therapeutic protein. In some embodiments, the coding sequence encodes a therapeutic antibody, including monoclonal antibodies, multispecific antibodies, and antibody fragments. In some embodiments, the coding sequence encodes a cytokine. In some embodiments, the coding sequence encodes an antigen. In some embodiments, the coding sequence encodes a therapeutic agent that can be used for gene therapy. Exemplary therapeutic proteins that can be used for gene therapy include, but are not limited to, adenosine deaminase, enzymes affected in lysosomal storage diseases, apolipoprotein E, brain-derived neurotrophic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), cardiotrophic factor 1 (CT-1), CD22, CD40, ciliary neurotrophic factor (CNTF), CCL1-CCL28, CXCL1-CXCL17, CXCL1, CXCL2, CX3CL1, vascular endothelial growth factor (VEGF), dopamine, Erythropoietin, Factor IX, Factor VIII, Epidermal Growth Factor (EGF), Estrogen, FAS-ligand, Fibroblast Growth Factor 1 (FGF-1), Fibroblast Growth Factor 2 (FGF-2), Fibroblast Growth Factor 4 (FGF-4), Fibroblast Growth Factor 5 (FGF-5), Fibroblast Growth Factor 6 (FGF-6), Fibroblast Growth Factor 1 (FGF-7), Fibroblast Growth Factor 1 (FGF-10), Flt-3, Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-Macrophage Stimulating Factor (GM-CSF), Growth Hormones, hepatocyte growth factor (HGF), interferon α (IFN-a), interferon β (IFN-b), interferon γ (IFNg), insulin, glucagon, insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), leukocytes Interleukin-10 (IL-10), Interleukin-11 (IL-11), Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-15 (IL-15), Interleukin-17 (IL-17), Interleukin-19 (IL-19), Macrophage Colony-Stimulating Factor (M-CSF), Monocyte Chemoattractant Protein 1 (MCP-1), Macrophage Inflammatory Protein 3a (MIP-3a), Macrophage Inflammatory Protein 3b (MIP-3b), Nerve Growth Factor (NGF), Neurotrophic Factor 3 (NT-3), Neurotrophic Factor 4 (NT-4)Parathyroid hormone, platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB (PDGF-AB), platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor CC (PDGF-CC), platelet-derived growth factor DD (PDGF-DD), RANTES, stem cell factor (SCF), stromal cell-derived factor 1 (SDF-1), transforming growth factor α (TGF-a), transforming growth factor β (TGF-b), tumor necrosis factor α (TNF-a), Wnt1, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15 or Wnt16, Hedgehog factor, Desert hedgehog factor, and Indian hedgehog factor. ,

[0290] In some implementations, the coding sequence encodes engineered receptors, such as chimeric antigen receptors (CARs) or engineered T-cell receptors (TCRs).

[0291] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that specifically transplants one or more antigens onto cells such as T cells. CARs are also known as "artificial T-cell receptors," "chimeric T-cell receptors," or "chimeric immune receptors." In some implementations, a CAR comprises an extracellular variable domain of an antibody specific to a tumor antigen, and an intracellular signal transduction domain of a T cell or other receptor, such as one or more co-stimulatory domains. "CAR-T" refers to T cells expressing a CAR.

[0292] In some specific embodiments, the coding sequence encodes a chimeric antigen receptor (CAR). Many chimeric antigen receptors are known in the art and are applicable to this application. CARs with specificity for any cell surface marker can also be constructed by utilizing, for example, antigen-binding fragments of antibody molecules or variable domains of antibodies. Any method for generating a CAR can be used herein. See, for example, US6,410,319, US7,446,191, US7,514,537, US9765342B2, WO 2002 / 077029, WO2015 / 142675, US2010 / 065818, US2010 / 025177, US2007 / 059298, and Berger C. et al., J. Clinical Investigation 118:1 294-308 (2008), which are incorporated herein by reference.

[0293] As used herein, “T cell receptor” or “TCR” refers to an endogenous or recombinant T cell receptor comprising an extracellular antigen-binding domain that binds to a specific antigenic peptide bound to an MHC molecule. In some embodiments, the TCR comprises a TCRα polypeptide chain and a TCRβ polypeptide chain. In some embodiments, the TCR specifically binds to a tumor antigen. “TCR-T” refers to a T cell expressing a recombinant TCR. The term “recombinant” refers to a biomolecule, such as a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is wholly or partially unrelated to the polynucleotide found in nature of the gene, (3) is operatively linked to the unrelated polynucleotide, or (4) is not present in nature. The term “recombinant” can be used to refer to cloned DNA isolates, chemically synthesized polynucleotide analogs or polynucleotide analogs biosynthesized from a heterologous system, and proteins and / or mRNA encoded by such nucleic acids.

[0294] In some embodiments, the coding sequence encodes an engineered T-cell receptor (TCR). In some embodiments, the engineered TCR is specific to tumor antigens. In some embodiments, the tumor antigen is derived from an intracellular protein of tumor cells. Many TCRs specific to tumor antigens (including tumor-associated antigens) have been described, including, for example, NY-ESO-1 cancer-testis antigen, p53 tumor suppressor antigen, TCRs of tumor antigens in melanoma (e.g., MARTI, gp100), leukemia (e.g., WT1, minor histocompatibility antigen), and breast cancer (e.g., HER2, NY-BR1). Any TCR known in the art may be used in this application. In some embodiments, the TCR has enhanced affinity for tumor antigens. Exemplary TCRs and methods for producing TCRs have been described, for example, in US5830755 and Kessels et al. Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001).

[0295] In some embodiments, the coding sequence encodes a selection marker. A "selection marker" is a gene whose expression produces a detectable phenotype and facilitates the detection of host cells with heterologous nucleic acids encoding a selection marker inserted into a target nucleic acid (e.g., genomic DNA). In some embodiments, the selection marker confers resistance to antibiotics such as puromycin. Other non-limiting examples of selection markers include antibiotic resistance genes and nutrient markers. For example, a selection marker may be a gene confers resistance to antibiotics selected from: ampicillin, kanamycin, erythromycin, chloramphenicol, gentamicin, kasugamycin, rifampin, spectinomycin, D-cyclic serine, nalidixic acid, streptomycin, or tetracycline. Other non-limiting examples of selection markers include adenosine deaminase, aminoglycoside phosphotransferase, dihydrofolate reductase, hygromycin-β-phosphotransferase, thymidine kinase, and xanthine-guanine phosphoribosyltransferase. Examples of selection markers suitable for mammalian cells include DHFR, thymidine kinase, metallothionein I and II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc. In some specific embodiments, the heteronucleotide contains the coding sequence of a puromycin resistance gene.

[0296] In some embodiments, the coding sequence is a reporter gene. A "reporter gene" is a gene encoding a detectable product, and therefore the detection of the reporter gene product can be used to assess the function of the target nucleic acid. The reporter gene can be fused to any suitable target nucleic acid (e.g., a promoter, target gene, selection marker, and / or terminal repeat of a transposon element) to allow detection of whether the target nucleic acid is expressed or altered (e.g., cleaved by a transposase) under a given set of conditions. Non-limiting examples of reporter genes include: 3-galactosidase, 3-glucuronidase, glutathione S-transferase (GST), horseradish peroxidase (HRP), luciferase, chloramphenicol acetyltransferase (CAT), secretory alkaline phosphatase (SEAP), green fluorescent protein (GFP, e.g., eGFP), red fluorescent protein (RFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), catechol 2,3-oxygenase (xylE), and autofluorescein, including blue fluorescent protein (BFP). In some embodiments, the heteronucleic acid comprises a coding sequence encoding enhanced green fluorescent protein (eGFP). In some embodiments, the coding sequence encodes more than one biological product, or the coding sequence may encode a fusion protein. In some embodiments, the heteronucleic acid of this application comprises a coding sequence encoding a puromycin resistance-enhanced green fluorescent protein (eGFP) fusion protein.

[0297] In some implementations, the coding sequence encodes a transposase.

[0298] In some embodiments, the coding sequence encodes a polypeptide that can be used for genome editing. Genome editing can be accomplished using a nuclease that generates a specific double-strand break (DSB) at a desired location in the genome and utilizes the cell's endogenous mechanisms to repair the induced break via homologous directed repair (HDR) (e.g., homologous recombination) or via non-homologous end joining (NHEJ). Any suitable nuclease can be introduced into the cell to induce genome editing of the target DNA sequence, including but not limited to CRISPR-associated protein (Cas, e.g., Cas9) nucleases, zinc finger nucleases (ZFN, e.g., FokI), transcription activator effector nucleases (TALEN, e.g., TALE), homing endonucleases and their variants (Shukla et al. (2009) Nature 459:437-441; Townsend et al. (2009) Nature 459:442-445). In some embodiments, the coding sequence encodes a Cas9 polypeptide.

[0299] In some embodiments, the coding sequence encodes an RNA molecule. The RNA molecule can be protein-coding RNA such as messenger RNA (mRNA), or non-protein-coding RNA, including but not limited to transfer RNA (tRNA) and ribosomal RNA (rRNA), small RNAs such as microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), or piwi-interacting RNA (piRNA), and long non-coding RNA (lincRNA). Certain types of small RNAs, such as microRNA and siRNA, are important in RNA interference (RNAi). RNAi is a gene regulation process in which the expression of normally expressed target genes is suppressed by interfering with small RNAs through post-transcriptional degradation or translational repression. For a detailed description of RNAi technology, see, for example, US Pat. No. 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588. In some embodiments, the coding sequence encodes a regulatory RNA. In some embodiments, the coding sequence encodes an RNAi molecule. In some embodiments, the coding sequence encodes shRNA. In some embodiments, the coding sequence encodes miRNA.

[0300] In some implementations, the coding sequence encodes an RNA molecule that can be used for genome editing. Examples of such RNA molecules include, but are not limited to, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA (gRNA), and single guide RNA (sgRNA).

[0301] In some embodiments, the heteronucleotide further comprises one or more regulatory elements that regulate the expression of the coding sequence. These regulatory elements are contemplated for use with the methods and constructs described herein. The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as poly-A signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0302] Promoters are crucial regulatory elements that guide the expression pattern of coding sequences. In some embodiments, the heteronucleotide contains a promoter operatively linked to a coding sequence. Any suitable promoter can be used in this application. In some embodiments, the promoter is an endogenous promoter. In some embodiments, the promoter is a heteropromoter. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in this application. Promoters can be broadly classified into constitutive promoters or regulatory promoters, such as inducible promoters. In some embodiments, the heteronucleotide contains a coding sequence (e.g., a transposase-coding sequence) operatively linked to a constitutive promoter. In some embodiments, the heteronucleotide contains a coding sequence (e.g., a transposase-coding sequence) operatively linked to an inducible promoter.

[0303] Constitutive promoters allow heterologous nucleic acids to be constitutively expressed in host cells. Exemplary constitutive promoters considered herein include, but are not limited to, cytomegalovirus (CMV) promoters, human elongation factor-1α (hEF1α), ubiquitin C promoters (UbiC), glycerol phosphokinase promoters (PGK), simian virus 40 early promoters (SV40), and chicken β-actin promoters coupled with CMV early enhancers (CAGG). The efficiency of such constitutive promoters in driving transgene expression has been extensively compared in numerous studies. For example, Michael C. Milone et al. compared the efficiency of CMV, hEF1α, UbiC, and PGK in driving chimeric antigen receptor expression in primary human T cells and concluded that the hEF1α promoter not only induces the highest levels of transgene expression but also maintains it ideally in CD4 and CD8 human T cells (Molecular Therapy, 17(8):1453-1464(2009)). In some embodiments, the promoter in the heterologous nucleic acid is a CAG promoter. An exemplary engineered transposon element containing a heterologous nucleic acid sequence encoding a transposase or selectable marker / reporter gene driven by a constitutive promoter is shown in Figure 3. The promoter is either a CMV promoter or a PGK promoter.

[0304] For certain applications, such as some gene therapies, it may be necessary to use promoters with moderate or weak expression patterns of the coding sequence, rather than strong expression promoters (e.g., CMV promoters), to avoid or reduce transposase-based autoregulatory events, collectively known as overproduction inhibition (OPI).

[0305] Regulatory promoters, such as inducible promoters, allow heterologous nucleic acids to be expressed under certain conditions, such as at specific developmental stages, or in specific tissue types or subcellular locations. Various types of regulatory promoters are known in the art, including inducible, tissue-specific, cell-type-specific, or cell cycle-specific promoters; see, for example, Sambrook and Russell, 2001. Inducible promoters belong to the category of regulatory promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment or physiological state of engineered mammalian cells, inducers (i.e., inducers), or combinations thereof.

[0306] In some implementations, it may be desirable to use promoter expression coding sequences only in a subset of cell types, cell lineages, or tissues, or at a specific developmental stage. Examples include, but are not limited to: B29 promoter (B cell expression), dwarfing transcription factor (CBFa2) promoter (stem cell expression), CD14 promoter (monocyte expression), CD43 promoter (leukocyte and platelet expression), CD45 promoter (hematopoietic cell expression), CD68 promoter (macrophage expression), endothelial glycoprotein promoter (endothelial cell expression), fms-associated tyrosine kinase 1 (FLT1) promoter (endothelial cell expression), integrin, α2b (ITGA2B) promoter (megakaryocyte expression), intracellular adhesion molecule 2 (ICAM-2) promoter (endothelial cell expression), interferon β (IFN-β) promoter (hematopoietic cell expression), β-globin LCR (erythrocyte expression), globin promoter (erythrocyte expression), β-globin promoter (erythrocyte expression), α-globin HS40 enhancer (erythrocyte expression), ankyrin-1 promoter (erythrocyte expression), and Wescott-Aldridge syndrome protein (WASP) promoter (hemoglobin topocell expression).

[0307] Aspects of the methods described herein may utilize terminator sequences. A terminator sequence comprises a nucleic acid sequence that marks the end of a gene or operon during transcription. This sequence mediates transcriptional termination by providing signals in the newly synthesized mRNA that trigger the release of mRNA from the transcription complex. These processes include direct interactions between the mRNA secondary structure and the complex and / or indirect activity of recruited termination factors. The release of the transcription complex frees up RNA polymerase and associated transcription mechanisms to initiate the transcription of new mRNA. Terminator sequences include those known in the art. In some embodiments of this application, the terminator sequence is a polyadenylation (poly-A) signal.

[0308] In some embodiments, the heteronucleotide contains at least one restriction endonuclease recognition site, such as a restriction site, as a site for inserting the foreign nucleic acid. Various restriction sites are known in the art, including but not limited to: HindIII, PstI, SalI, AccI, HincII, XbaI, BamHI, SmaI, XmaI, KpnI, SacI, EcoRI, etc. In some embodiments, the restriction site is a multiple cloning site (MCS, also known as a multiple adapter), i.e., a closely packed series or array of sites recognized by a variety of different restriction endonucleases (such as those listed above). In other embodiments, the heteronucleotide of this application contains a recombinase recognition site, such as LoxP, FRT, or AttB / AttP sites recognized by Cre, Flp, and PhiC31 recombinases, respectively.

[0309] In some embodiments, the heterologous nucleic acid contains a tag sequence. The tag sequence can be used to identify molecules, or, for example, to provide a site for capturing molecules via hybridization.

[0310] In some embodiments, the heterologous nucleic acid comprises a barcode sequence. A "barcode sequence" refers to a nucleic acid having a sequence that can be used to identify and / or distinguish one or more first molecules conjugated to that nucleic acid barcode from one or more second molecules. Nucleic acid barcode sequences are typically short, for example, about 5 to 20 bases in length, and can be conjugated to one or more target molecules or their amplification products. Nucleic acid barcode sequences can be single-stranded or double-stranded.

[0311] In some embodiments, the heterologous nucleic acid includes a unique molecular identifier (UMI). As used herein, the term "unique molecular identifier" or "UMI" refers to a nucleic acid sequence that can be used to identify and / or distinguish one or more first molecules conjugated to a UMI from one or more second molecules. UMIs are typically short, for example, about 5 to 20 bases in length, and can be conjugated to one or more target molecules or their amplification products. UMIs can be single-stranded or double-stranded. In some embodiments, both the nucleic acid barcode sequence and the UMI are incorporated into the nucleic acid target molecule or its amplification product. Typically, the UMI is used to distinguish similar types of molecules within a population or group, while the nucleic acid barcode sequence is used to distinguish populations or groups of molecules. In some embodiments where both UMI and nucleic acid barcode sequences are used, the UMI sequence is shorter than the nucleic acid barcode sequence. In some embodiments where both UMI and nucleic acid barcode sequences are used, the UMI is incorporated into the target nucleic acid or its amplification product before the nucleic acid barcode sequence is incorporated. In some implementations, when using both UMI and nucleic acid barcode sequences, the nucleic acid barcode sequence is incorporated into the UMI or its amplification product after the UMI is incorporated into the target nucleic acid or its amplification product.

[0312] Transposable activity

[0313] In some embodiments, the transposable element of this application exhibits transposable activity in vitro or in cells.

[0314] Transposon activity can be detected using a variety of techniques known to those skilled in the art. Examples of assays for measuring the ability of transposases to excise transposable elements from vectors, integrate transposable elements into the genomic or extrachromosomal DNA of cells, and bind to inverted repeat sequences can be found, for example, in Ivies et al. Cell, 91, 501-510 (1997), WO 98 / 40510 (Hackett et al.), WO 99 / 25817 (Hackett et al.), and WO00 / 68399 (Mclvor et al.).

[0315] In some implementations, transposition assays are based on the trans complementarity of two components in a transposition element system, one component containing a selection marker / reporter gene flanked by a terminal repeat (donor), and the other component expressing a transposase that recognizes and binds to the terminal repeat to perform transposition (helper). For example, Figure 3 shows a set of exemplary binary constructs for screening active transposition elements. The upper construct is a helper construct for transposase expression, comprising from 5' to 3': a cytomegalovirus (CMV) promoter, a transposase (Tn) gene, and a poly(A) signal. The lower construct is a donor construct, comprising from 5' to 3': a phosphoglycerate kinase (PGK) promoter, a 5' target site repeat (TSD) sequence, a 5' terminal repeat (5'TR) sequence, a sequence encoding puromycin and enhanced GFP fusion (Puro-eGFP), a 3' terminal repeat (3'TR) sequence, a 3' target site repeat (TSD) sequence, and a poly(A) signal. In transposon assays, donor plasmids are co-transfected with helper or control plasmids into cultured mammalian cells (e.g., human 293T, HeLa, or Hct116 cells). The number of cell clones resistant to puromycin due to chromosomal integration and expression of puromycin resistance genes is used as an indicator of gene transfer efficiency, expressed by methylene blue staining. For suspension cells, such as K562 and primary T cells, transposon activity can be assessed based on GFP reporter-positive cells after electroporation. Figure 3-7 shows colony counts based on methylene blue staining results from transposon assays, representing the transposon efficiency of TE compared to controls. The transposon efficiency in this type of assay is also referred to as "transfer efficiency."

[0316] In some embodiments, the transfer efficiency of the engineered transposable element is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or higher. In some embodiments, the transfer efficiency of the engineered transposable element is determined in human cells, such as human 293T, HeLa, Hct116, K562, or primary T cells.

[0317] In some embodiments, the engineered transposon element exhibits higher transposon activity than the piggyBac (PB) transposon, Sleeping Beauty (SB) transposon, and / or TcBuster (TB) transposon. In some embodiments, the engineered transposon element exhibits higher transposon activity than the piggyBac (PB) transposon, for example, by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2x, 3x, 5x, 10x, or more, as determined in mammalian cells by reporter gene-based transposon assays (e.g., as described in Example 2). In some embodiments, the engineered transposon element exhibits higher transposon activity than the Sleeping Beauty (SB) transposon, for example, by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2x, 3x, 5x, 10x, or more, as determined in mammalian cells by reporter gene-based transposon assays (e.g., as described in Example 2). In some embodiments, the engineered transposon element exhibits higher transposon activity than the TcBuster (TB) transposon, for example, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2x, 3x, 5x, 10x, or more, as determined in mammalian cells by reporter gene-based transposon assays (e.g., as described in Example 2). In some embodiments, the engineered transposable element exhibits higher transposable activity than the PB and SB transposables. In some embodiments, the engineered transposable element exhibits higher transposable activity than the PB and TB transposables. In some embodiments, the engineered transposable element exhibits higher transposable activity than the TB and SB transposables. In some embodiments, the engineered transposable element exhibits higher transposable activity than the PB, SB, and TB transposables.

[0318] In some embodiments, the transposition activity of the engineered transposable element is evaluated in mammalian cells. In some embodiments, the mammalian cells are HeLa cells. In some embodiments, the mammalian cells are human embryonic kidney 293T (293T). In some embodiments, the mammalian cells are K562 cells. In some embodiments, the mammalian cells are Hct116 cells. In some embodiments, the mammalian cells are human T cells, such as primary T cells derived from a donor. In some embodiments, the engineered transposable element has higher transposition activity in 293T cells than in HeLa cells, such that the transposition activity in 293T cells is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2x, 3x, 5x, 10x or more higher than the transposition activity in HeLa cells.

[0319] cell

[0320] The engineered transposable elements described herein exhibit transposable activity in a variety of cell types and can be used to insert heterologous nucleic acids into target nucleic acids in any suitable cell.

[0321] In some embodiments, the cells are isolated cells. In some embodiments, the cells are in a cell culture. In some embodiments, the cells are ex vivo. In some embodiments, the cells are obtained from a living organism and maintained in a cell culture. In some embodiments, the cells are single-celled organisms. Cells can be classified into different types based on cell origin, tissue of origin, morphology, function, histological markers, expression profile, etc.

[0322] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell or derived from bacterial cells. In some embodiments, the cell is an archaea cell or derived from archaea cells. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a plant cell or derived from plant cells. In some embodiments, the cell is a fungal cell or derived from fungal cells. In some embodiments, the cell is an animal cell or derived from animal cells. In some embodiments, the cell is an invertebrate cell or derived from invertebrate cells. In some embodiments, the cell is a vertebrate cell or derived from vertebrate cells. In some embodiments, the cell is a mammalian cell or derived from mammalian cells. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebrafish (D. rerio) cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is synthetically manufactured, sometimes referred to as an artificial cell. In some embodiments, the cell is associated with an animal species from which 5'TR, 3'TR, and / or transposases are derived. In some embodiments, the cells are not related to the animal species from which the 5'TR, 3'TR, and / or transposases originate.

[0323] In some embodiments, the cells are bacterial, yeast, fungal, algal, plant, or animal cells. In some embodiments, the cells are cells isolated from a natural source, such as a tissue biopsy. In some embodiments, the cells are cells isolated from a cell line cultured in vitro. In some embodiments, the cells are genetically engineered cells. In some embodiments, the cells are seed cells that have undergone proliferation, differentiation, or both in the nucleus.

[0324] In some embodiments, the cells are animal cells derived from organisms selected from cattle, sheep, goats, horses, pigs, deer, chickens, ducks, geese, rabbits, and fish.

[0325] In some embodiments, the cells are plant cells derived from organisms selected from corn, wheat, barley, oats, rice, soybeans, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, pearl millet, foxtail millet, sorghum, rapeseed, hemp, vegetable crops, forage crops, cash crops, woody crops, and biomass crops.

[0326] In some embodiments, the cells are mammalian cells, including cells derived from humans, domestic animals and farm animals, zoos, sports fields, or pets such as dogs (C. familiaris), horses, cats, cattle, etc. 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.

[0327] In some embodiments, the cells are derived from primary cells. For example, cultures of primary cells may be passaged 0, 1, 2, 4, 5, 10, 15, or more times. In some embodiments, primary cells are harvested from an individual using any known method. For example, leukocytes may be harvested by blood component replacement, leukocyte separation, density gradient separation, etc. Cells may be harvested from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc., by biopsy. The harvested cells may be dispersed or suspended using a suitable solution. Such a solution may typically be a balanced salt solution (e.g., physiological saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, etc.), conveniently supplemented with fetal bovine serum or other naturally occurring factors, along with an acceptable low-concentration buffer. The buffer may include HEPES, phosphate buffer, lactate buffer, etc. Cells may be used immediately or stored (e.g., by freezing). Frozen cells can be thawed and reused. Cells can be frozen in DMSO, serum, culture medium buffers (e.g., 10% DMSO, 50% serum, 40% buffered medium) and / or some other such commonly used solutions for preserving cells at freezing temperatures.

[0328] In some embodiments, the cells are derived from cell lines. Various cell lines are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, and their transgenic varieties. Cell lines can be obtained from a variety of sources known to those skilled in the art (see, for example, the American Center for Type Culture Collection (ATCC) (Manassus, Va.)).

[0329] In some embodiments, the cells comprise adherent cells. In some embodiments, the cells comprise differentiated adherent cells. In some embodiments, the cells comprise undifferentiated adherent cells. In some embodiments, the cells comprise pluripotent stem cells. In some embodiments, the cells comprise non-adherent cells.

[0330] In some embodiments, the cells are derived from epithelial tissue, muscle tissue, nerve tissue, or connective tissue, or any combination thereof. In some embodiments, the cells are derived from tissues selected from: liver, gastrointestinal tract, pancreas, kidney, lung, trachea, blood vessels, skeletal muscle, heart, skin, smooth muscle, connective tissue, cornea, genitourinary system, mammary gland, reproductive system, endothelium, epithelium, fibroblasts, nerves, Schwann cells, fat, bone, bone marrow, cartilage, pericytes, mesothelial cells, endocrine system, matrix, lymph, blood, endoderm, ectoderm, mesoderm, and combinations thereof. In some embodiments, the cells are derived from tissues selected from: connective tissue (e.g., loose connective tissue, dense connective tissue, elastic tissue, reticular connective tissue, and adipose tissue), muscle tissue (e.g., skeletal muscle, smooth muscle, and cardiac muscle), urogenital tissue, gastrointestinal tissue, lung tissue, bone tissue, nervous tissue, and epithelial tissue (e.g., simple epithelium and stratified epithelium), tissues derived from the endoderm, tissues derived from the mesoderm, and tissues derived from the ectoderm, or any combination thereof. In some embodiments, the cells are derived from a tumor.

[0331] In some embodiments, the cells are selected from hepatocytes, gastrointestinal cells, pancreatic cells, kidney cells, lung cells, tracheal cells, vascular cells, skeletal muscle cells, cardiomyocytes, skin cells, smooth muscle cells, connective tissue cells, corneal cells, urogenital cells, mammary cells, germ cells, endothelial cells, epithelial cells, fibroblasts, nerve cells, Schwann cells, adipocytes, osteocytes, bone marrow cells, chondrocytes, pericytes, mesothelial cells, cells derived from endocrine tissues, stromal cells, stem cells, progenitor cells, lymphocytes, blood cells, cells derived from the endoderm, cells derived from the ectoderm, cells derived from the mesoderm, undifferentiated cells (such as stem cells or progenitor cells), tumor cells, iPS cells, and combinations thereof.

[0332] In some embodiments, the cells are immune cells, such as T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), and macrophages. In some embodiments, the cells are human T cells obtained from a patient or donor. In some embodiments, the cells are immune cells selected from cytotoxic T cells, helper T cells, natural killer (NK) T cells, iNK-T cells, NK-T-like cells, αβ T cells, αγδ T cells, tumor-infiltrating T cells, and dendritic cell (DC) activated T cells. In some embodiments, the cells are immune cells modified using the engineered transposon element or gene transfer system of this application. In some embodiments, the modified immune cells are CAR-T cells. In some embodiments, the modified immune cells are TCR-T cells.

[0333] In some embodiments, the cells used in this application are mammalian cells. In some embodiments, the mammalian cells are human HKT293 cells or HeLa cells. In some further embodiments, the transposable element exhibits higher transposable activity in 293T cells than in HeLa cells. In some embodiments, the mammalian cells are selected from immune cells, hepatocytes, tumor cells, stem cells, fertilized eggs, muscle cells, and skin cells.

[0334] In some embodiments, the cell is a stem cell or a progenitor cell. The cell may include stem cells (e.g., adult stem cells, embryonic stem cells, iPS cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc.). The cell may include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc.

[0335] In some embodiments, the cell is a diseased cell. Diseased cells may have altered metabolic, gene expression, and / or morphological characteristics. Diseased cells can be cancer cells, diabetic cells, and apoptotic cells. Diseased cells can be derived from a diseased object.

[0336] In some embodiments, the cells of this application belong to target cell types that can be used for gene therapy. Exemplary target cell types include hematopoietic stem cells, hematopoietic progenitor cells, bone marrow progenitor cells, lymphoprogenitor cells, platelet-producing progenitor cells, erythroid progenitor cells, granulocyte-producing progenitor cells, monocyte progenitor cells, megakaryocytes, promegakaryocytes, megakaryocytes, coagulation cells / platelets, proerythrocytes, basophils, polychromatic erythrocytes, orthochromatic erythrocytes, polychromatic erythrocytes, erythrocytes (erythrocytes or RBCs), basophilic promyelocytes, basophilic myelocytes, basophilic postmyelocytes, basophilic granulocytes, neutrophils, neutrophils, neutrophils, eosinophilic promyelocytes, eosinophilic myelocytes, macrophages, dendritic cells, lymphoblasts, prolymphocytes, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, and lymphodendrocytes. In a preferred embodiment, the target cell type is one or more red blood cells, such as proerythrocytes, basophils, polychromatic erythrocytes, orthochromatic erythrocytes, polychromatic erythrocytes, and red blood cells (RBCs).

[0337] carrier

[0338] In some implementations, the engineered transposable element and / or the nucleic acid sequence encoding the transposase are present in one or more vectors.

[0339] Various suitable vectors may be used in this application. In some embodiments, the vector is a plasmid vector, a granular vector, an artificial chromosome (e.g., a bacterial artificial chromosome, a yeast artificial chromosome, or a mammalian artificial chromosome), a viral vector such as a bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, Semliki Forest virus, or adeno-associated virus (AAV) vector, all of which are well known and available from commercial sources. Typically, a suitable vector contains a replication origin that is functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers.

[0340] In some embodiments, the vector is a plasmid. In some embodiments, the plasmid can be transformed into bacteria for storage or amplification, and can be transfected into mammalian cells.

[0341] Methods for introducing vectors into mammalian cells are known in the art. Vectors can be transferred into host cells by physical, chemical, and / or biological methods. It is anticipated that, for the purposes of this application, various vector types and vector delivery methods can be used alone or in combination.

[0342] Physical methods for introducing vectors into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. For example, see Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector is introduced into the cell via electroporation.

[0343] Biological methods for introducing heterologous nucleic acids into host cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian cells, such as human cells.

[0344] Chemical methods for introducing a carrier into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery carrier is a liposome (e.g., an artificial membrane vesicle).

[0345] In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. In some embodiments, the use of viral vectors to deliver transposon elements can be used for gene therapy, where the high efficiency of gene delivery by the viral vector is combined with the stability of gene expression achieved by the transposon element. For example, see Yant, Stephen R., et al. “Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo.” Nature biotechnology 20.10 (2002):999-1005.

[0346] III. Gene Transfer System

[0347] Another aspect of the application provides a gene transfer system comprising: 1) an engineered transposon element (such as any of the transposon elements described herein); and 2) a transposase, or a nucleic acid encoding the transposase.

[0348] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposon element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof; and 2) a transposase. In some embodiments, the transposon element exhibits transposition activity that allows the heterologous nucleic acid to be inserted into the DNA of a cell (e.g., mammalian or plant cells). In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0349] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposon element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof; and 2) a nucleic acid (e.g., DNA or RNA) encoding a transposase. In some embodiments, the transposon element exhibits transposition activity that allows the heterologous nucleic acid to insert into the DNA of a cell (e.g., mammalian or plant cells). In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0350] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposon element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR); and 2) a transposase comprising an amino acid sequence selected from SEQ ID NO:13-18 or a variant thereof. In some embodiments, the transposon element exhibits transposition activity that allows the heterologous nucleic acid to be inserted into the DNA of a cell (e.g., mammalian or plant cells). In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0351] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposon element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR); and 2) a nucleic acid (e.g., DNA or RNA) encoding a transposase comprising an amino acid sequence selected from SEQ ID NO:13-18 or a variant thereof. In some embodiments, the transposon element exhibits transposition activity that allows the heterologous nucleic acid to insert into the DNA of a cell (e.g., mammalian or plant cells). In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0352] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposon element; and 2) a transposase, wherein the transposon element comprises, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof; wherein the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18, or its variants thereof. In some embodiments, the transposon element exhibits transposition activity that allows the heterologous nucleic acid to be inserted into the DNA of a cell (e.g., mammalian or plant cells). In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0353] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposon element; and 2) a nucleic acid (e.g., DNA or RNA) encoding a transposase, wherein the transposon element comprises, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof; wherein the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18, or a variant thereof. In some embodiments, the transposon element exhibits transposition activity that allows the heterologous nucleic acid to insert into the DNA of a cell (e.g., mammalian or plant cells). In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0354] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposable element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises the nucleic acid sequence of SEQ ID NO:1, a variant thereof, or a fragment thereof, wherein the 3'TR comprises the nucleic acid sequence of SEQ ID NO:7, a variant thereof, or a fragment thereof; and 2) a transposase comprising the amino acid sequence of SEQ ID NO:13, or a nucleic acid encoding the transposase.

[0355] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposable element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises the nucleic acid sequence of SEQ ID NO:2, a variant thereof, or a fragment thereof, wherein the 3'TR comprises the nucleic acid sequence of SEQ ID NO:8, a variant thereof, or a fragment thereof; and 2) a transposase comprising the amino acid sequence of SEQ ID NO:14, or a nucleic acid encoding the transposase.

[0356] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposable element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises the nucleic acid sequence of SEQ ID NO:3, a variant thereof, or a fragment thereof, wherein the 3'TR comprises the nucleic acid sequence of SEQ ID NO:9, a variant thereof, or a fragment thereof; and 2) a transposase comprising the amino acid sequence of SEQ ID NO:15, or a nucleic acid encoding the transposase.

[0357] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposable element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises the nucleic acid sequence of SEQ ID NO:4, a variant thereof, or a fragment thereof, wherein the 3'TR comprises the nucleic acid sequence of SEQ ID NO:10, a variant thereof, or a fragment thereof; and 2) a transposase comprising the amino acid sequence of SEQ ID NO:16, or a nucleic acid encoding the transposase.

[0358] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposable element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises the nucleic acid sequence of SEQ ID NO:5, a variant thereof, or a fragment thereof, wherein the 3'TR comprises the nucleic acid sequence of SEQ ID NO:11, a variant thereof, or a fragment thereof; and 2) a transposase comprising the amino acid sequence of SEQ ID NO:17, or a nucleic acid encoding the transposase.

[0359] In some embodiments, a gene transfer system is provided comprising: 1) an engineered transposable element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises the nucleic acid sequence of SEQ ID NO:6, a variant thereof, or a fragment thereof, wherein the 3'TR comprises the nucleic acid sequence of SEQ ID NO:12, a variant thereof, or a fragment thereof; and 2) a transposase comprising the amino acid sequence of SEQ ID NO:18, or a nucleic acid encoding the transposase.

[0360] The gene transfer system described herein comprises a transposase. The transposase may exist as a polypeptide. Alternatively, the transposase may exist as a polynucleotide containing a coding sequence encoding the transposase. The polynucleotide may be RNA, such as mRNA encoding the transposase, or DNA, such as a coding sequence encoding the transposase. When the transposase exists as a coding sequence encoding the transposase, in some embodiments, the coding sequence may be present in the same vector comprising the transposase element, i.e., cis. In some embodiments, the gene transfer system comprises a first vector containing an engineered transposase element and a second vector containing the transposase coding sequence, i.e., trans.

[0361] In some embodiments, the gene transfer system comprises: 1) a vector containing engineered transposon elements (such as any of the engineered transposon elements described herein); and 2) a transposase.

[0362] In some embodiments, the gene transfer system comprises: 1) an engineered transposon element (any of the engineered transposon elements described herein); and 2) a nucleic acid encoding a transposase, wherein the nucleic acid is DNA. In some embodiments, the engineered transposon element and the nucleic acid are contained in a single vector. In some embodiments, the engineered transposon element and the nucleic acid are contained in separate vectors.

[0363] In some embodiments, the gene transfer system comprises: 1) an engineered transposon element (any of the engineered transposon elements described herein); and 2) a nucleic acid encoding a transposase, wherein the nucleic acid is RNA. In some embodiments, the engineered transposon element and the nucleic acid are contained in a single vector. In some embodiments, the engineered transposon element and the nucleic acid are contained in separate vectors.

[0364] In the gene transfer systems described herein, there are numerous potential and suitable combinations of intracellular delivery methods for engineered transposable elements and transposases or nucleic acids encoding transposases. Transposases can be delivered as DNA, RNA, or protein. Engineered transposable elements and transposases can be delivered together or separately. For example, transposon and transposase genes can be contained together in the same recombinant viral genome; two parts of a single infection delivery system, such that transposase expression directs the transposon to be cleaved from the recombinant viral genome for subsequent integration into the cell chromosome. In another instance, transposases and transposable elements can be delivered separately using a combination of viral and / or non-viral systems such as lipid-containing reagents. In these cases, the transposable element and / or transposase gene can be delivered via recombinant virus. In some embodiments, the transposase directs the release of the transposable element from its donor DNA for integration into the target site.

[0365] Transposases can be provided to cells as proteins or as nucleic acids encoding transposase proteins. The nucleic acids encoding transposase proteins can be in the form of DNA or RNA. Proteins can be introduced into cells alone or into vectors such as plasmids or viral vectors. Furthermore, the nucleic acids encoding transposase proteins can be stably or transiently incorporated into the cell's genome to promote transient or prolonged expression of the transposase protein in the cell. Additionally, promoters or other expression control regions can be operatively linked to the nucleic acids encoding transposase proteins to quantitatively or tissue-specifically regulate the expression of the protein. In some embodiments, the transposase protein contains a DNA-binding domain, a catalytic domain (with transposase activity), and / or a nuclear localization signal (NLS).

[0366] Therefore, a variety of methods and materials can be used to deliver the gene transfer system of this application into cells.

[0367] For example, one approach is to use plasmid vectors to deliver gene transfer systems. This system can consist of two plasmids: a helper plasmid carrying a transposase expression cassette and a donor plasmid carrying the transposase element. After transfection, both plasmids are oriented towards the nucleus, allowing the production of transposase-encoding RNA from the helper plasmid, followed by excision of the transposase element from the donor plasmid, facilitated by the transposase subunit directed to the nucleus. This approach can be further refined by placing the transposase gene and transposase element on a single plasmid, initially termed a helper-independent transposase-transposase vector. Alternatively, transfected in vitro transcribed mRNA can serve as a rich source of transposase, eliminating the risk of generating cells with prolonged transposase expression.

[0368] Therefore, in some embodiments, the transposable element is present in a first vector (donor vector), and the nucleic acid encoding the transposase is present in a second vector (helper vector). In some embodiments, the first and second vectors are used for co-transfection of cells to perform transposition.

[0369] Another approach is to use viral vectors to deliver gene transfer systems. While viral vectors may present immunogenicity or oncogenicity issues, high delivery efficiency may be required in certain applications. Components of a gene transfer system can be carried and delivered by a viral capsid, thus providing the ability to integrate genes and establish long-term transgenic expression with other augmented vectors—such as adenovirus or herpes simplex virus vectors. The viral capsid provides vector stability, tissue-specific transposon element delivery, and transmembrane transport, while the transposon elements facilitate viral vector integration based on their characteristic integration profiles. Methods and techniques for viral vector-based delivery are known in the art. For example, viral vector-based transfer of the Sleeping Beauty transposon system was first demonstrated in mouse livers with an adenovirus vector, and recent studies have demonstrated the applicability of this approach in larger animals. Adeno-associated virus vectors are also suitable as vectors for the Sleeping Beauty system. See, for example, Yant, Stephen R., et al. "Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo." Nature biotechnology 20.10(2002):999-1005, Hausl, Martin A., et al. "Hyperactive sleeping beauty transposase enables persistent phenotypic correction in mice and a canine model for hemophilia B." Molecular Therapy 18.11(2010):1896-1906, and Zhang, Wenli, et al. “Hybrid adeno-associated viral vectors utilizing transposase-mediated somatic integration for stable transgene expression in human cells.” PloS one 8.10(2013).

[0370] As described above, the gene transfer system of this application can be delivered to host cells by physical, chemical, biological methods, or combinations thereof. Various delivery vectors and methods are contemplated for use alone or in combination with this application to achieve desired results for certain applications. Therefore, those skilled in the art can best utilize various delivery methods and techniques with various modifications to suit specific intended uses, such as gene therapy. For gene therapy to be truly feasible, the stable integration of therapeutic transgenes into the genome of diseased tissue should be achieved to provide long-term and cost-effective treatment. For example, to achieve efficient and low-immunogenic gene transfer into a patient, synthetic compounds and plasmids can be used in combination to deliver DNA into cells. Liposomes and other nanoparticles may be sufficient to accomplish this task. For example, two plasmids can be delivered to the patient: one providing transposase expression (helper plasmid) and the other providing transposable elements containing therapeutic transgenes (donor plasmid). These DNAs can be compounded with liposomes and administered via parenteral injection. Upon entry into the cell, the transposase can bind to the transposable element in the donor plasmid, excise it, and then integrate it into the genome. This insertion will be stable and permanent. Helper and donor plasmids may eventually be lost due to cellular and host defense mechanisms, but any genomic integration transposable element containing therapeutic transgenes will be a stable and permanent modification. The transience of these plasmids also reduces excessive transposition, thereby minimizing the risk of oncogenesis.

[0371] Further details and exemplary transposon delivery methods and techniques can be found, for example, Skipper, Kristian Alsbjerg, et al. “DNA transposon-based gene vehicles-scenes from an evolutionary drive.” Journal of biomedical science 20.1(2013):92.

[0372] IV. Methods

[0373] This application further provides a method for inserting a heterologous nucleic acid into a target nucleic acid, comprising: contacting the target nucleic acid with an engineered transposon element or a gene transfer system, said engineered transposon element comprising a heterologous nucleic acid of any of the engineered transposon elements described herein, and said gene transfer system comprising a heterologous nucleic acid of any of the gene transfer systems described herein. The method may be performed in vitro or in cells.

[0374] In vitro methods

[0375] In some embodiments, a method is provided for inserting a heterologous nucleic acid into a target nucleic acid in vitro, comprising contacting the target nucleic acid with an engineered transposon element comprising: 1) an engineered transposon element; and 2) a transposase, wherein the transposon element comprises, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof; wherein the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18, or its variants thereof. In some embodiments, the target nucleic acid is circular DNA. In some embodiments, the target nucleic acid is linear DNA. In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0376] The methods described herein can be used for in vitro transposition, including in cell-free systems. See, for example, Goryshin, Igor Yu, and William S. Reznikoff. “Tn5 in vitro transposition.” Journal of Biological Chemistry 273.13(1998):7367-7374. Transposable elements exhibiting in vitro transposition activity can be used for next-generation sequencing (NGS) library construction, including, for example, tagging methods.

[0377] In some embodiments, a method is provided for preparing a plurality of barcoded nucleic acids from a target nucleic acid, comprising contacting the target nucleic acid with an engineered transposon element comprising: 1) an engineered transposon element; and 2) a transposase, wherein the transposon element comprises, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid containing the barcoded sequence, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR contains a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, and wherein the 3'TR contains a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof; wherein the transposase contains an amino acid sequence selected from SEQ ID NO:13-18 or its variants, thereby providing a plurality of barcoded nucleic acids. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid is cDNA. In some embodiments, the target nucleic acid is amplified DNA. In some embodiments, the heterologous nucleic acid further comprises a primer sequence. In some embodiments, the method further comprises amplifying a plurality of barcoded nucleic acids to provide a nucleic acid sequencing library. In some embodiments, the method further includes sequencing the nucleic acid sequencing library. In some embodiments, the method includes contacting the target nucleic acid with a plurality of engineered transposable elements, wherein each engineered transposable element contains a unique barcode sequence. In some embodiments, the nucleic acid sequencing library prepared using the in vitro methods described herein retains adjacency information in the target nucleic acid sequence. In some embodiments, the engineered transposable elements are derived from any of the TEs in Table 2. In some embodiments, the engineered transposable elements are derived from Tc1-1_ST or Tc1-2_ST.

[0378] In some embodiments, a tagging method is provided using any of the transposases and / or TR sequences described herein. Tagging methods using the Tn5 transposon are known in the art, for example in US9080211B2, which is incorporated herein by reference in its entirety. The tagging methods described herein use transposon complex compositions.

[0379] In some embodiments, a transposon complex composition is provided comprising: a transposase comprising an amino acid sequence selected from SEQ ID NO:13-18 or a variant thereof, and a heterologous nucleic acid comprising one or two TR sequences and a tag sequence. In some embodiments, the transposon complex comprises a single heterologous nucleic acid forming a hairpin. In some embodiments, the hairpin comprises a cleavable site.

[0380] In some embodiments, the transposon complex comprises two transposases that bind to two heteronucleotides. In some embodiments, a transposon complex composition is provided comprising: a first transposase that binds to a first heteronucleotide comprising a 5'TR sequence and a first tag sequence, and a second transposase that binds to a second heteronucleotide comprising a 3'TR sequence and a second tag sequence. In some embodiments, the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof. In some embodiments, the first tag sequence is different from the second tag sequence. In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0381] In some embodiments, a method is provided for preparing a library of nucleic acid (e.g., DNA) fragments having first and second tag sequences for a target nucleic acid, comprising contacting the target nucleic acid with a plurality of transposon complexes, the transposon complexes comprising: (1) a first transposon complex comprising a first transposase and a first heteronucleotide comprising a TR sequence and a first tag sequence; and (2) a second transposon complex comprising a second transposase and a second heteronucleotide comprising a TR sequence and a second tag sequence, wherein the first tag sequence is different from the second tag sequence, and wherein the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18 or a variant thereof; wherein the first and second heteronucleotides are inserted into the target nucleic acid, and the target nucleic acid is fragmented into a plurality of nucleic acid fragments, the nucleic acid fragments comprising one of a first or second nucleic acid linked to each 5' end of the nucleic acid fragment; thereby providing a library of nucleic acid fragments. In some embodiments, the transposon complex of (1) comprises two first heteronucleotides, and the transposon complex of (2) comprises two second heteronucleotides. In some embodiments, the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof. In some embodiments, the method further includes amplifying the nucleic acid fragment. In some embodiments, the method further includes sequencing the nucleic acid fragment or its amplicons. In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0382] In some embodiments, the engineered transposon element or transposon complex is inserted into the target nucleic acid in a random manner, i.e., without bias towards a specific sequence motif. In some embodiments, the engineered transposon element or transposon complex inserts into the target nucleic acid more randomly than PB, SB, or TB transposons.

[0383] In some embodiments, the engineered transposon element or transposon complex preferentially inserts into spatially free regions of the target nucleic acid, such as open chromatin regions or regions that do not bind to nucleosomes or other DNA-binding proteins. Therefore, the in vitro methods described herein can be used to prepare nucleic acid sequencing libraries for assays to study epigenomics (e.g., chromatin remodeling or DNA methylation), including but not limited to chromatin transposase accessibility sequencing (ATAC-seq), target and tag-based cleavage (CUT&TAG), transposase-accessible chromatin and DNA methylation analysis (ATAC-Me), and transposase-mediated chromatin looping analysis (Trac-looping). ATAC-seq, CUT&TAG, ATAC-Me and Trac-looping assays using the Tn5 transposon have been described, for example, in Buenrostro, Jason D., et al. "Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position." Nature methods (2013) 10(2):1213; Kaya-Okur HS, et al. al., "CUT&Tag for efficient epigenomic profiling of small samples and single cells." Nature Communications (2019), 10(1):1-10; Barnett KR et al. "ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions." Molecular Cell, 2020; accessibility,”Nature Methods, 2018, 15(9):741, the full text of which is incorporated herein by reference. In some implementations, sequencing library preparations are used for chromatin transposase accessibility sequencing (ATAC-seq).

[0384] In some embodiments, the engineered transposon element or transposon complex can be used to tag regions of a target nucleic acid (e.g., genomic DNA) that are spatially close to each other. Two regions that are spatially close to each other may contain the same tag sequence pair at their ends. Therefore, the in vitro methods described herein can be used to prepare fluorescently labeled probes that can be used for in situ hybridization with chromatin interaction boundaries in genomic DNA, for example in transposase-based fluorescence in situ hybridization (FISH). Tn5-based FISH methods have been described, for example, in Zhang X. et al. “Imaging chromatin interactions at sub-kilobase resolution via Tn5-FISH,” bioRxiv, 2019:601690, the full text of which is incorporated herein by reference.

[0385] Methods in cells

[0386] In some embodiments, a method is provided for inserting a heterologous nucleic acid into a target nucleic acid in a cell, comprising contacting the target nucleic acid with an engineered transposon element comprising: 1) an engineered transposon element; and 2) a transposase, wherein the transposon element comprises, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof; wherein the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18, or its variants thereof. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid is extrachromosomal DNA. In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0387] In some embodiments, a method is provided for inserting a heterologous nucleic acid into a target nucleic acid in a mammalian cell, comprising contacting the target nucleic acid with an engineered transposon element comprising: 1) an engineered transposon element; and 2) a transposase, wherein the transposon element comprises, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof; wherein the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18, or its variants thereof. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is an animal cell, such as a rodent cell. In some embodiments, the mammalian cell is an immune cell, such as a T cell. In some embodiments, the method is performed in vitro. In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some implementations, the engineered transpose element is derived from Tc1-1_ST or Tc1-2_ST.

[0388] In some embodiments, a method is provided for inserting a heterologous nucleic acid into a target nucleic acid in a plant cell, comprising contacting the target nucleic acid with an engineered transposon element comprising: 1) an engineered transposon element; and 2) a transposase, wherein the transposon element comprises, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof; wherein the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18, or its variants thereof. In some embodiments, the plant cell is a crop cell. In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0389] The transposon element or gene transfer system described herein can be introduced into one or more cells using any of a variety of techniques known in the art, such as, but not limited to, microinjection, binding of nucleic acid fragments to lipid vesicles (e.g., cationic lipid vesicles), particle bombardment, electroporation, DNA agglutination reagents (e.g., calcium phosphate, polylysine, or polyethyleneimine), or incorporating nucleic acid fragments into a viral vector and contacting the viral vector with the cell. When using a viral vector, the viral vector may include any of a variety of viral vectors known in the art, including viral vectors selected from retroviral vectors, adenoviral vectors, or adeno-associated virus (AAV) vectors.

[0390] It is envisioned that heteronucleotides may contain multiple operons, or that they may encode more than one biological product. In some embodiments, the heteronucleotides encode gene circuits. An exemplary gene circuit is a collection of components (the “output” of each component) that undergo transcription and / or translation to produce mRNA or protein, respectively. Component outputs may interact with other parts (e.g., regulate transcription or translation) or with other molecules in the cell (e.g., small molecules, DNA, RNA, or proteins present in the cellular environment). For example, the circuit may be a metabolic pathway or gene cascade, which may be naturally occurring or non-naturally occurring and artificially engineered. Each component in the circuit may include a set of components or gene modules, such as promoters, ribosome binding sites (RBS), coding sequences (CDS), and / or terminators. These components may be interconnected or assembled in different ways to realize different components, and the final components may be combined in different ways to create different circuits or pathways. In addition to these components, the circuit may also contain other molecular species present in the cell or in the cellular environment that interacts with the components.

[0391] Therefore, in some embodiments, a method for inserting a heterologous nucleic acid into a target nucleic acid in a cell is provided, comprising: contacting the cell with an engineered transposon element comprising: 1) an engineered transposon element; and 2) a transposase, wherein the transposon element comprises, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof; wherein the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18, or its variants thereof, wherein the heterologous nucleic acid encodes a gene circuit. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid is extrachromosomal DNA. In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0392] Gene circuits can be used in gene therapy. Methods and techniques for designing and using gene circuits are known in the art. Further reference may be made, for example, to Brophy, Jennifer AN, and Christopher A. Voigt. "Principles of genetic circuit design." Nature methods 11.5 (2014):508.

[0393] This method is applicable to any suitable cell type. In some embodiments, the cells are bacterial, yeast, fungal, algal, plant, or animal cells. In some embodiments, the cells are cells isolated from a natural source, such as a tissue biopsy. In some embodiments, the cells are cells isolated from a cell line cultured in vitro. In some embodiments, the cells are genetically engineered cells. In some embodiments, the cells are seed cells that have undergone proliferation, differentiation, or both in the nucleus.

[0394] In some embodiments, the cells are animal cells derived from organisms selected from cattle, sheep, goats, horses, pigs, deer, chickens, ducks, geese, rabbits, and fish.

[0395] In some embodiments, the cells are plant cells derived from organisms selected from corn, wheat, barley, oats, rice, soybeans, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, pearl millet, foxtail millet, sorghum, rapeseed, hemp, vegetable crops, forage crops, cash crops, woody crops, and biomass crops.

[0396] 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 mammalian cells are selected from immune cells, hepatocytes, tumor cells, stem cells, fertilized eggs, muscle cells, and skin cells.

[0397] 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, αγδ T cells, tumor-infiltrating T cells, and dendritic cell (DC) activated T cells. In some embodiments, the method produces modified immune cells, such as CAR-T cells or TCR-T cells.

[0398] In some embodiments, the heterologous nucleic acid is inserted into the cell's genome. In some further embodiments, the insertion of the heterologous nucleic acid inactivates the cell's genes. In some embodiments, the heterologous nucleic acid encodes a protein or RNAi molecule.

[0399] Furthermore, the heterologous nucleic acid can encode RNA molecules useful in genome editing. Examples of such RNA molecules include, but are not limited to, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA (gRNA), and single guide RNA (sgRNA).

[0400] In some embodiments, the heteronucleotide encodes a biological product selected from: reporter proteins, antigen-specific receptors, therapeutic proteins, antibiotic resistance proteins, RNAi molecules, cytokines, kinases, antigens, antigen-specific receptors, cytokine receptors, and suicide peptides. For example, the heteronucleotide may encode a receptor specific to tumor-associated antigens. T cells engineered using this method can recognize and specifically kill tumor cells expressing tumor-associated antigens. In another example, the heteronucleotide encodes a hygromycin resistance protein, thereby enabling the establishment of hygromycin-resistant cell lines. Alternatively, the heteronucleotide may not have any biological function and may be used to interrupt the function of another gene by inserting itself into an essential gene, thereby disrupting its function.

[0401] In some embodiments, the heteronucleotide encodes a therapeutic protein that can be used for gene therapy. In some embodiments, the heteronucleotide encodes a therapeutic antibody. In some embodiments, the heteronucleotide encodes an engineered receptor, such as a chimeric antigen receptor (CAR) or an engineered TCR.

[0402] In some implementations, the heteronucleotide includes one or more multiple cloning sites (MCS) to facilitate the insertion of the target polynucleotide (“cargo gene”).

[0403] In some embodiments, the method is performed in vitro. In some embodiments, transduced or transfected cells (e.g., mammalian cells) proliferate in vitro after the introduction of heterologous nucleic acids. In some embodiments, transduced or transfected cells are cultured for at least about 1, 2, 3, 4, 5, 6, 7, 10, 12, or 14 days to proliferate. In some embodiments, transduced or transfected cells are cultured for no more than about 1, 2, 3, 4, 5, 6, 7, 10, 12, or 14 days. In some embodiments, the transduced or transfected cells are further evaluated or screened to select engineered cells.

[0404] Reporter genes or selectable markers can be used to identify potentially transfected cells and to assess the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and the expression of its encoded polypeptide is demonstrated by easily detectable properties such as enzyme activity. Reporter gene expression is determined at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al. FEBS Letters 479:79-82 (2000)). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available.

[0405] Other methods for confirming the presence of heterologous nucleic acids in cells include, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; and biochemical assays, such as detecting the presence or absence of specific peptides, for example by immunological methods (such as ELISA and Western blotting).

[0406] This application envisions methods for generating isogenetic lines of mammalian cells to study genetic variation. This application also considers genome modifications of microorganisms, cells, plants, animals, or synthetic organisms to produce biomedically, agriculturally, and industrially useful products. These methods can be used as tools in biological research for understanding the genome, such as gene knockout or knock-in studies.

[0407] Cells modified with heterologous nucleic acids using any of the methods described herein, as well as organisms (e.g., animals, plants, or fungi) containing or produced by such cells, are also provided.

[0408] target nucleic acid

[0409] The methods described herein are applicable to inserting heterologous nucleic acids into a variety of target nucleic acids. In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is single-stranded. In some embodiments, the target nucleic acid is double-stranded. In some embodiments, the target nucleic acid comprises single-stranded and double-stranded regions. In some embodiments, the target nucleic acid is linear. In some embodiments, the target nucleic acid is circular. In some embodiments, the target nucleic acid comprises one or more modified nucleotides, such as methylated nucleotides, damaged nucleotides, or nucleotide analogs. In some embodiments, the target nucleic acid is unmodified. In some embodiments, the target nucleic acid binds to one or more proteins, such as nucleosomes.

[0410] The target nucleic acid can be of any length, such as at least any one of about 100 bp, 200 bp, 500 bp, 1000 bp, 2000 bp, 5000 bp, 10 kb, 20 kb, 50 kb, 100 kb, 200 kb, 500 kb, 1 Mb, or longer. In some embodiments, the target nucleic acid does not exceed any one of about 500 kb, 200 kb, 100 kb, 50 kb, 40 kb, 30 kb, 20 kb, 10 kb, 5 kb, 2 kb, 1 kb, 500 bp, 200 bp, or less. In some embodiments, the target nucleic acid is any one of about 100bp-500bp, 500bp-1kb, 100bp-1kb, 1kb-2kb, 100bp-5kb, 100bp-10kb, 100bp-20kb, 1kb-5kb, 1kb-10kb, 1kb-20kb, 20kb-100kb, or 100kb-1Mb. The target nucleic acid may also contain any sequence. In some embodiments, the target nucleic acid is enriched with specific sequences that are hotspots for transposition of the engineered transposon elements or gene delivery systems described herein. In some embodiments, the target nucleic acid is enriched with AT, such as having an AT content of at least about 40%, 45%, 50%, 55%, 60%, 65%, or higher. In some embodiments, the target nucleic acid is not enriched with AT. In some embodiments, the target nucleic acid is not enriched with specific hotspot sequences because the engineered transposon elements or gene delivery systems described herein do not favor the insertion of heterologous nucleic acids into specific sequences or sequence motifs. In some embodiments, the target nucleic acid has one or more secondary or higher-order structures. In some embodiments, the target nucleic acid is not in a condensed state, such as in chromatin.

[0411] In some embodiments, the target nucleic acid is present in the cell. In some embodiments, the target nucleic acid is present in the cell nucleus. In some embodiments, the target nucleic acid is endogenous to the cell. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid is chromosomal DNA. In some embodiments, the target nucleic acid is a protein-coding gene or its functional region, such as a coding region, or a regulatory element, such as a promoter, enhancer, 5' or 3' untranslated region, etc. In some embodiments, the target nucleic acid is a non-coding gene, such as a transposon, miRNA, tRNA, ribosomal RNA, ribozyme, or lincRNA. In some embodiments, the target nucleic acid is a plasmid.

[0412] In some embodiments, the target nucleic acid is exogenous to the cell. In some embodiments, the target nucleic acid is viral nucleic acid, such as viral DNA. In some embodiments, the target nucleic acid is a horizontally transferred plasmid. In some embodiments, the target nucleic acid is integrated into the cell's genome. In some embodiments, the target nucleic acid is not integrated into the cell's genome. In some embodiments, the target nucleic acid is a plasmid within the cell. In some embodiments, the target nucleic acid is present in an extrachromosomal array.

[0413] In some embodiments, the target nucleic acid is an isolated nucleic acid, such as isolated DNA. In some embodiments, the target nucleic acid is present in a cell-free environment. In some embodiments, the target nucleic acid is an isolated vector, such as a plasmid. In some embodiments, the target nucleic acid is an isolated linear DNA fragment.

[0414] V. Reagent kits and products

[0415] This application also provides kits and articles comprising any of the transposon elements or gene transfer systems described herein. In some embodiments, the kit includes instructions for inserting a heterologous nucleic acid into a target nucleic acid (e.g., using any of the methods described herein). In some embodiments, the kit is used for inserting a heterologous nucleic acid into a target nucleic acid in vitro. In some embodiments, the kit is used for inserting a heterologous nucleic acid into a target nucleic acid in cells such as mammalian cells or plant cells. The kits and articles described herein can be used to modify target nucleic acids in vitro or in vitro, in genetic studies, and in gene therapy.

[0416] In some embodiments, a kit comprising an engineered transposon element comprising, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heteronucleotide, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof, and wherein the transposon element exhibits transposition activity that allows the heteronucleotide to be inserted into the DNA of a cell (e.g., mammalian or plant cells). In some embodiments, the heteronucleotide comprises one or more multiple cloning sites (MCS) to facilitate the insertion of a target polynucleotide (“cargo gene”). In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0417] In some embodiments, a kit comprising a gene transfer system is provided, the gene transfer system comprising: 1) an engineered transposon element; and 2) a transposase or a nucleic acid encoding a transposase, wherein the transposon element comprises, from 5' to 3': a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from SEQ ID NO:1-6, its variants, or fragments thereof, wherein the 3'TR comprises a nucleic acid sequence selected from SEQ ID NO:7-12, its variants, or fragments thereof; wherein the transposase comprises an amino acid sequence selected from SEQ ID NO:13-18, or a variant thereof, and wherein the transposon element exhibits transposition activity that allows the heterologous nucleic acid to be inserted into the DNA of a cell (e.g., mammalian or plant cells). In some embodiments, the heterologous nucleic acid comprises one or more multiple cloning sites (MCS) to facilitate the insertion of a target polynucleotide (“cargo gene”). In some embodiments, the engineered transposon element is derived from any of the TEs in Table 2. In some embodiments, the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

[0418] In some embodiments, the kit contains one or more reagents for use in any of the methods described herein. Reagents may be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. Reagents may be provided in a form suitable for a specific assay, or in a form requiring the addition of one or more other components prior to use (e.g., in concentrate or lyophilized form). Buffers may be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the kit contains culture media, buffers, reagents, etc., to allow the proliferation or induction of cells modified using the engineered transposon elements or gene transfer systems described herein. In some embodiments, the kit contains buffers, reagents, etc., for isolating and / or preparing target nucleic acids modified using the engineered transposon elements or gene transfer systems described herein. In some embodiments, the kit contains primers and reagents for preparing sequencing libraries using the engineered transposon elements or gene transfer systems described herein.

[0419] The kit is packaged appropriately. Appropriate packaging includes, but is not limited to, vials, bottles, wide-mouth flasks, and flexible packaging (e.g., sealed polyester film or plastic bags). The kit may optionally provide additional components, such as buffer solutions and explanatory information. This application therefore also provides articles of manufacture including vials (e.g., sealed vials), bottles, wide-mouth flasks, and flexible packaging.

[0420] Example

[0421] The following embodiments are merely illustrative of the invention and should not be construed as limiting the invention in any way. The following embodiments and detailed descriptions are provided by way of illustration rather than limitation.

[0422] Example 1: Identification of candidate active transposable elements

[0423] This embodiment describes the computer-aided identification of candidate active transposon elements (TEs, transposons) across species. A large number of transposons exist in various species. However, only a small number of transposons exhibit transpositional activity in mammalian cells. Therefore, a method is needed to systematically identify candidate active transposons that can be used as reagents for genome engineering and gene therapy. This embodiment focuses on the identification of transposons with terminal inverted repeat sequences (TIRs, also known as terminal repeats TRs), but this method can be used to identify any other type of transposon.

[0424] Materials and methods

[0425] First, considering the relatively concentrated presence of active DNA transposons in fish genomes, we selected two newly assembled fish genomes (Sinocyclocheilus tileihornes and Pseudoliparis swirei) that had not undergone transposon annotation. Next, we used RepeatModeler v1.0.11129 to search for transposons in the genomes and generate a consensus sequence for each transposon. Similar to the previous procedure, ORFfinder was used to identify coding regions longer than 300 amino acids, and PfamScan was used to search for transposase domains encoding proteins. After this screening process, only 12 transposons from the Tc1 / mariner superfamily were retained. However, by comparing the sequences flanking the transposons, we found that some transposons lacked complete TIRs and had blurred terminal boundaries. Therefore, we manually extended the TIRs based on the transposon-derived elements with the highest similarity to the consensus sequence by searching for the longest complementary flanking sequence associated with the “TA..TA” sequence. The “TA..TA” sequence is a typical TSD of the Tc1 / mariner superfamily. After removing redundancies with identical transposases and TIRs, we ultimately retained six transposons. Based on the reconstructed consistent sequences of these six transposons, we ran RepeatMasker to reannotate transposon-derived elements in the genome and confirmed that all six transposons possess potentially autonomously mobile elements. These six transposons were named according to the Repbase database rules as follows: Tc1-1_ST transposon, Tc1-2_ST transposon, Tc1-3_ST transposon, Tc1-4_ST transposon, Tc1-5_ST transposon, and Tc1-1_PS transposon.

[0426] result

[0427] Figure 1 illustrates the flowchart of the bioinformatics pipeline and the number of candidate active transposable elements at each stage of the pipeline.

[0428] A total of 1,170 shared DNA transposons were constructed from two genomes. These transposons were then examined to see if they contained transposase genes. ORFfinder was used to detect open reading frames (ORFs), with a length cutoff of 300 amino acids. The protein sequences were then used to search for important domains against the Pfam HMM library using PfamScan (Madeira, F., et al., The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res, 2019. 47(W1): p. W636-W641). After further alignment of the transposon ends, manual expansion of TIRs by searching for the longest complementary alignment associated with the “TA..TA” sequence, and removal of redundant transposons with the same transposase and TIRs, six transposons remained in the pipeline.

[0429] Next, the copy number of each transposon was determined using RepeatMasker (Smit, AFA, Hubley, R&Green, P. RepeatMasker Open-4.0.2013-2015). The mean sequence difference between the shared transposon and its family members for each species was calculated, ranging from 0 to 0.52% across different species. As shown in Table 1, the mean difference for the 12 identified TEs was less than 1%.

[0430] In summary, these data demonstrate six candidate active transposons identified through pan-genome bioinformatics analysis of two genomes. Therefore, this example showcases the successful development of a robust bioinformatics pipeline for identifying candidate active transposon elements.

[0431] Example 2: Validation of candidate active transposable elements

[0432] This embodiment describes the experimental verification of the candidate active transposable element identified in Example 1.

[0433] Materials and methods

[0434] DNA synthesis and plasmid construction

[0435] Mammalian codon-optimized transposase ORFs flanked by EcoRI and NotI were synthesized and cloned into the CMV-hyPBase vector (K. Yusa, et al., A hyperactive piggyBac transposase for mammalian applications. Proc Natl Acad Sci USA, 2011. 108(4): p. 1531-6). These vectors are helper plasmids used to facilitate transposase expression under the human CMV promoter. The transposon donor plasmids contain left and right transposon fragments flanking the antibiotic resistance gene. As used herein, the left transposon fragment (LTF) refers to the segment from the 5' TSD to the TE sequence of the transposase ORF sequence, which is the start codon. As used herein, the right transposon element fragment (RTF) refers to the segment from the stop codon of the transposase ORF sequence to the 3' TSD of the TE sequence. Typically, the TR sequence is located within the left and right transposon fragments. For example, the 5' TR sequence is located within the LTF sequence, and the 3' TR sequence is located within the RTF sequence. The LTF and RTF sequences used in this experiment were synthesized by Qinglan Biotechnology Co., Ltd. and cloned into the pMV vector. 5' and 3' multiple cloning sites (MCS) were also synthesized in the transposon fragments for cargo gene cloning. The TSD sequence was located on the outermost side. The donor plasmid used for transposon selection in mammalian cells carried a p2A-linked puromycin resistance gene and an enhanced GFP gene expressed by the PGK promoter. Figure 3 shows a set of exemplary helper and donor constructs used to verify the active transposon element.

[0436] Transposals in mammalian cells

[0437] Four mammalian cell lines—HEK293T (also known as 293T), HeLa, Hct116, and K562—were used to screen for active transposons. The 293T and HeLa cell lines were maintained in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The Hct116 and K562 cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. In addition to these cell lines, CD3+ was isolated using the EasySep Human T Cell Enrichment Kit. + T cells were then collected via histopaque-1077 (Sigma-Aldrich) gradient separation, followed by the collection of monocytes and CD3+ cells. + T cells were cultured in X-Vivo 15 medium (Lonza) supplemented with 5% (v / v) heat-inactivated fetal bovine serum, 2 mM L-glutamine and 1 mM sodium pyruvate.

[0438] For transposon assays, 1.2 x 10⁻⁶ mg / L was administered 18 hours before transfection. 5 HEK293T cells, 0.7 x 10 5 One HeLa cell or 1.0 x 10 5 Hct116 cells were seeded into each well of a 24-well plate. Using a Lipo3000, 200 ng of helper plasmid and 100 ng of donor plasmid, or 100 ng of donor plasmid alone, were delivered to each cell line. Two days after transfection, cell counts were calculated and transfection efficiency was measured using FACS. Then, 1 / 100 th Transfected HEK293T, 1 / 100 th Transfected HeLa cells or 1 / 100 th Transfected Hct116 cells were transferred to 100-mm plates for puromycin (0.5 μg / ml) for 10 days (HEK-293T cell line) or 14 days (HeLa cell line and Hct116 cell line).

[0439] After puromycin selection, cells were washed once with 5 ml of cold PBS, fixed with 4% PFA for 15 minutes, and then stained with 0.2% methylene blue (in PBS) for 1 hour (Wu et al., piggyBac is a flexible and highly active transposon as compared to Sleeping Beauty, Tol2, and Mos1 in mammalian cells. PNAS, 2006, 103: p. 15008–15013). Finally, residual nonspecific staining was washed away with PBS. Individually stained colonies were counted using ImageJ software. Transposition efficiency was calculated based on the previously calculated total number of transfected cells and transfection efficiency.

[0440] For suspension cells, such as the K562 cell line and CD3+ T cells, transposon activity was assessed based on the percentage of GFP-positive cells on day 14 after electroporation, at which point the plasmid was diluted to a very small proportion.

[0441] Construction and bioinformatics analysis of TE insert libraries

[0442] Genomic DNA was isolated from stable transposon K562 cells using the DNeasy Blood and Tissue Kit (Qiagen, Germany) and sheared to an average length of 600 bp using a Covaris M220 sonicator (Covaris, USA). DNA samples underwent end repair, adapter ligation and amplification via nested PCR, and purification and sequencing were performed on a NovaSeq 6000 sequencer. Then, the TE integration site was compared with random insertion sites upstream and downstream of the primary sequence of the side-linked vector integration site, distances to the nearest gene and TSS, and different chromatin states.

[0443] result

[0444] First, through bioinformatics analysis, we identified six potentially active DNA transposons. Next, we tested the transposition activity of these six transposons in HEK293T cells. Table 1 lists detailed information on these six transposons, including their family, source species, sequence, mean difference, and copy number. The transposition efficiency of the Tc1-1_ST transposon was 10.21%, and that of the Tc1-2_ST transposon was 2.84%. The remaining four transposons showed no detectable activity in HEK293T cells. The results are shown in Figure 3. Table 2 summarizes the empirically observed active transposons (TEs) from the above experiments, including a total of two TEs that were validated as active in the 293T cell line. The percentage of sequence similarity between these six novel transposons and known transposases of the same family is shown in Table 3.

[0445] Next, we determined the transposition activity of the Tc1-1_ST transposon in four different tissue-derived cell lines: HEK293T cells, HeLa cells, HCT116 cells, and K562 cells (Figure 4). The Tc1-1_ST transposon exhibited higher transposition activity in K562 cells than in the other three cell lines, comparable to the SB100X transposon. Figure 5 shows the transposition activity of TE in primary T cells. No phenomenon known as overproduction inhibition (OPI) occurred in the Tc1-1_ST transposon. Figure 6 shows that transposition activity increases with an optimized ratio of helper plasmid encoding the transposase to the transposon plasmid. Figure 7 shows the cargo capacity of the five most active TEs, including gene lengths of 2kb, 5kb, and 10kb, compared to the identified control TE SB100X.

[0446] Among the six candidate TEs, a comparison between active and inactive TEs revealed that active TEs exhibited lower average diversity, slightly longer predicted TIR sequences, and a significantly increased number of autonomous TEs. These differences help distinguish the characteristics that can be used to identify active TEs in a bioinformatics pipeline, including preliminary and large-scale screening settings.

[0447] Integration sites retrieved from transposon integration sites revealed highly preferred TA target dinucleotides for the most active Tc1-1_ST transposon (Figure 8). The target dinucleotides for TE belong to the Tc / Mariner superfamily and are highly conserved. Figure 9 shows the frequency of integration into genomic features by comparing computer-generated random data with control TE SB100X and piggyBac, including distances to gene and transcription start sites (TSS), different chromatin states, and genomic safe harbors. The data show that Tc1-1_ST transposons exhibit very low preference for integration near gene sequences, but no preference for upstream or downstream sequences of genes and TSSs. Regarding gene expression levels and chromatin states, Tc1-1_ST transposons also show almost random integration patterns, and they tend not to insert into overactive expression regions.

[0448] In summary, these data demonstrate the successful establishment of a robust bioinformatics pipeline for identifying candidate active transposon elements in novel genomes, and the successful experimental validation of the identified TEs. The transposon efficiency and integration patterns of the Tc1-1_ST transposon suggest that the Tc1-1_ST transposon may be useful in genome engineering applications and gene therapy.

Claims

1. An engineered transpose element, comprising, from 5' to 3': 5' terminal repeat (5'TR), heterologous nucleic acid, and 3' terminal repeat (3'TR) The 5'TR contains a nucleic acid sequence selected from any one of SEQ ID NOs:1-6, a variant thereof, or a fragment thereof. The 3'TR contains a nucleic acid sequence selected from any of SEQ ID NOs:7-12, its variants, or fragments thereof, and The engineered transposable element described therein exhibits transposable activity that allows heterologous nucleic acids to be inserted into the DNA of cells.

2. The engineered transposon element of claim 1, wherein the engineered transposon element comprises a 5'TR in an LTF, a variant thereof, or a fragment thereof, wherein the LTF comprises a nucleic acid sequence selected from any of SEQ ID NOs:25-30.

3. The engineered transposon element according to claim 1 or 2, wherein the engineered transposon element comprises a 3'TR in an RTF, a variant thereof, or a fragment thereof, wherein the RTF comprises a nucleic acid sequence selected from any of SEQ ID NOs:31-36.

4. The engineered transposable element according to any one of claims 1-3, wherein the 5'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs:1-6, and / or wherein the 3'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs:7-12.

5. The engineered transposable element according to any one of claims 1-4, wherein the engineered transposable element comprises: 1) a 5' TR having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs: 1-6; and 2) a 3' TR having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs: 7-12.

6. The engineered transposable element according to any one of claims 1-5, wherein the 5'TR comprises a nucleic acid sequence selected from any one of SEQ ID NOs:1-6, and / or wherein the 3'TR comprises a nucleic acid sequence selected from any one of SEQ ID NOs:7-12.

7. The engineered transposable element according to any one of claims 1-6, wherein the engineered transposable element further comprises a 5' target site repeat sequence (TSD) side-attached to the 5' end of the 5' TR and / or a 3' TSD side-attached to the 3' end of the 3' TR, wherein the 5' TSD comprises a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any one of SEQ ID NOs:19-24, and wherein the 3' TSD comprises a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any one of SEQ ID NOs:19-24.

8. The engineered transposable element according to claim 7, wherein the nucleic acid sequences of the 5'TSD and 3'TSD are identical.

9. The engineered transposable element according to any one of claims 1-8, wherein the 5'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5 and 6, and the 3'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs: 7, 8, 9, 10, 11 and 12.

10. The engineered rotary element according to claim 9, wherein: (a) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:1, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:7; (b) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:2, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:8; (c) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:3, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:9; (d) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:4, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:10; (e) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:5, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:11; or (f) The 5'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:6, and the 3'TR contains a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

12.

11. The engineered transducer element according to claim 10, wherein: (a) The 5'TR contains the nucleic acid sequence of SEQ ID NO:1, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:7; (b) The 5'TR contains the nucleic acid sequence of SEQ ID NO:2, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:8; (c) The 5'TR contains the nucleic acid sequence of SEQ ID NO:3, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:9; (d) The 5'TR contains the nucleic acid sequence of SEQ ID NO:4, and the 3'TR contains the nucleic acid sequence of SEQ ID NO:10; or (e) The 5'TR contains the nucleic acid sequence of SEQ ID NO:5, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:

11. Or (e) The 5'TR contains the nucleic acid sequence of SEQ ID NO:6, while the 3'TR contains the nucleic acid sequence of SEQ ID NO:

12.

12. The engineered transposable element according to any one of claims 1-11, wherein the engineered transposable element comprises an LTF comprising a nucleic acid sequence selected from any one of SEQ ID NOs:25-30, a variant thereof, or a fragment thereof.

13. The engineered transposon element according to any one of claims 1-12, wherein the engineered transposon element comprises an RTF comprising a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any one of SEQ ID NOs:31-36.

14. The engineered transposable element according to any one of claims 1-13, wherein the engineered transposable element comprises an LTF and an RTF, wherein the LTF has at least about 80% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs:25-30, and the RTF has at least about 80% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs:31-36.

15. The engineered transposable element of claim 14, wherein the engineered transposable element comprises an LTF containing a nucleic acid sequence selected from any of SEQ ID NOs: 25-30, and an RTF containing a nucleic acid sequence selected from any of SEQ ID NOs: 31-36.

16. The engineered transposable element according to any one of claims 1-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:25; 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:

31.

17. The engineered transposable element of claim 16, wherein the engineered transposable element comprises: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:1, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:7, a variant thereof, or a fragment thereof.

18. The engineered transposable element of claim 17, wherein the engineered transposable element comprises: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:1; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

7.

19. The engineered transposable element of claim 18, wherein the engineered transposable element comprises: 1) a 5' TR comprising the nucleic acid sequence of SEQ ID NO:1; and 2) a 3' TR comprising the nucleic acid sequence of SEQ ID NO:

7.

20. The engineered transposable element according to any one of claims 16-19, the engineered transposable element further comprising a 5' TSD containing a nucleic acid sequence of TA (SEQ ID NO: 19) and a 3' TSD containing a nucleic acid sequence of SEQ ID NO:

19.

21. The engineered transposable element according to any one of claims 16-20, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:25; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

31.

22. The engineered transposon element according to any one of claims 16-21, wherein the engineered transposon element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:13 or a variant thereof.

23. The engineered transposable element according to claim 22, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:

13.

24. The engineered transposable element according to any one of claims 10-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:26; and 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:

32.

25. The engineered transposable element of claim 24, wherein the engineered transposable element comprises: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:2, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:8, a variant thereof, or a fragment thereof.

26. The engineered transposable element of claim 25, wherein the engineered transposable element comprises: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:2; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

8.

27. The engineered transposable element of claim 26, wherein the engineered transposable element comprises: 1) a 5' TR comprising the nucleic acid sequence of SEQ ID NO:2; and 2) a 3' TR comprising the nucleic acid sequence of SEQ ID NO:

8.

28. The engineered transposable element according to any one of claims 24-27, the engineered transposable element further comprising a 5' TSD containing the nucleic acid sequence of SEQ ID NO:20, and a 3' TSD containing the nucleic acid sequence of SEQ ID NO:

20.

29. The engineered transposable element according to any one of claims 24-28, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:26; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

32.

30. The engineered transposon element according to any one of claims 24-29, wherein the engineered transposon element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:14 or a variant thereof.

31. The engineered transposon element of claim 30, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:

114.

32. The engineered transposable element according to any one of claims 10-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:3; and 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:

9.

33. The engineered transposable element according to claim 32, wherein the engineered transposable element comprises: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:3, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:9, a variant thereof, or a fragment thereof.

34. The engineered transposable element of claim 33, wherein the engineered transposable element comprises: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:3; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

9.

35. The engineered transposable element according to claim 34, the engineered transposable element comprising: 1) a 5' TR comprising the nucleic acid sequence of SEQ ID NO:3; and 2) a 3' TR comprising the nucleic acid sequence of SEQ ID NO:

9.

36. The engineered transposable element according to any one of claims 32-35, the engineered transposable element further comprising a 5' TSD containing the nucleic acid sequence of SEQ ID NO:21, and a 3' TSD containing the nucleic acid sequence of SEQ ID NO:

21.

37. The engineered transposable element according to any one of claims 32-36, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:27; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

33.

38. The engineered transposable element according to any one of claims 32-37, wherein the engineered transposable element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:15 or a variant thereof.

39. The engineered transposable element according to claim 38, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:

15.

40. The engineered transposable element according to any one of claims 10-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:28; and 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:

34.

41. The engineered transposable element of claim 40, wherein the engineered transposable element comprises: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:4, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:10, a variant thereof, or a fragment thereof.

42. The engineered transposable element according to claim 41, the engineered transposable element comprising: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:4; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

10.

43. The engineered transposable element according to claim 42, wherein the engineered transposable element comprises: 1) a 5' TR comprising the nucleic acid sequence of SEQ ID NO:4; and 2) a 3' TR comprising the nucleic acid sequence of SEQ ID NO:

10.

44. The engineered transposable element according to any one of claims 40-43, the engineered transposable element further comprising a 5' TSD containing the nucleic acid sequence of SEQ ID NO:22, and a 3' TSD containing the nucleic acid sequence of SEQ ID NO:

22.

45. The engineered transposable element according to any one of claims 40-44, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:28; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

34.

46. ​​The engineered transposon element according to any one of claims 40-45, wherein the engineered transposon element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:16 or a variant thereof.

47. The engineered transposon element of claim 46, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:

16.

48. The engineered transposable element according to any one of claims 10-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:29; and 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:

35.

49. The engineered transposable element of claim 48, wherein the engineered transposable element comprises: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:5, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:11, a variant thereof, or a fragment thereof.

50. The engineered transposable element of claim 49, wherein the engineered transposable element comprises: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 5; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

11.

51. The engineered transposable element according to claim 50, wherein the engineered transposable element comprises: 1) a 5' TR comprising the nucleic acid sequence of SEQ ID NO: 5; and 2) a 3' TR comprising the nucleic acid sequence of SEQ ID NO:

11.

52. The engineered transposable element according to any one of claims 48-51, the engineered transposable element further comprising a 5' TSD containing the nucleic acid sequence of SEQ ID NO:23, and a 3' TSD containing the nucleic acid sequence of SEQ ID NO:

23.

53. The engineered transposable element according to any one of claims 48-52, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:29; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

35.

54. The engineered transposon element according to any one of claims 48-53, wherein the engineered transposon element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:17 or a variant thereof.

55. The engineered transposable element according to claim 54, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:

17.

56. The engineered transposable element according to any one of claims 10-15, the engineered transposable element comprising: 1) a 5' TR in an LTF, a variant thereof, or a fragment thereof, the LTF comprising the nucleic acid sequence of SEQ ID NO:30; and 2) a 3' TR in an RTF, a variant thereof, or a fragment thereof, the RTF comprising the nucleic acid sequence of SEQ ID NO:

36.

57. The engineered transposable element of claim 56, wherein the engineered transposable element comprises: 1) a 5' TR comprising a nucleic acid sequence of SEQ ID NO:6, a variant thereof, or a fragment thereof; and 2) a 3' TR comprising a nucleic acid sequence of SEQ ID NO:12, a variant thereof, or a fragment thereof.

58. The engineered transposable element of claim 57, the engineered transposable element comprising: 1) a 5' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:6; and 2) a 3' TR having a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

12.

59. The engineered transposable element according to claim 58, the engineered transposable element comprising: 1) a 5' TR comprising the nucleic acid sequence of SEQ ID NO:6; and 2) a 3' TR comprising the nucleic acid sequence of SEQ ID NO:

12.

60. The engineered transposable element according to any one of claims 56-59, wherein the engineered transposable element further comprises a 5' TSD containing the nucleic acid sequence of SEQ ID NO:24 and a 3' TSD containing the nucleic acid sequence of SEQ ID NO:

24.

61. The engineered transposable element according to any one of claims 56-60, the engineered transposable element comprising 1) an LTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:30; and 2) an RTF comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

36.

62. The engineered transposable element according to any one of claims 56-61, wherein the engineered transposable element is associated with a transposase comprising the amino acid sequence of SEQ ID NO:18 or a variant thereof.

63. The engineered transposon element according to claim 62, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:

18.

64. The engineered transpose element according to any one of claims 1-15, wherein the engineered transpose element is derived from Tc1-1_ST, Tc1-2_ST, Tc1-3_ST, Tc1-4_ST, Tc1-5_ST or Tc1-1_PS.

65. The engineered transpose element according to claim 64, wherein the engineered transpose element is derived from Tc1-1_ST or Tc1-2_ST.

66. The engineered transposable element according to any one of claims 1-65, wherein the heterologous nucleic acid comprises a coding sequence.

67. The engineered transposable element according to claim 66, wherein the heterologous nucleic acid further comprises one or more regulatory elements for regulating the expression of coding sequences.

68. The engineered transposable element of claim 67, wherein the heterologous nucleic acid further comprises a promoter operatively linked to the coding sequence.

69. The engineered transposon element according to any one of claims 66-68, wherein the coding sequence encodes one or more of a therapeutic protein, an engineered receptor, a selection marker, a reporter protein, a transposase, a polypeptide that can be used for genome editing, and an RNA molecule.

70. The engineered transposon element of claim 69, wherein the coding sequence encodes a chimeric antigen receptor (CAR), an engineered T-cell receptor (TCR), or an RNA molecule that can be used for genome editing.

71. The engineered transposon element of claim 69, wherein the coding sequence comprises a reporter gene.

72. The engineered transposon element of claim 66, wherein the heteronucleotide comprises at least one restriction endonuclease recognition site, such as a restriction site.

73. The engineered transposable element according to claim 66, wherein the heterologous nucleic acid comprises a tag sequence.

74. The engineered transposable element according to claim 66, wherein the heterologous nucleic acid comprises a barcode sequence.

75. The engineered transposable element of claim 66, wherein the heterologous nucleic acid comprises a unique molecular identifier (UMI).

76. The engineered transposable element according to any one of claims 1-75, wherein the transposable activity of the engineered transposable element is higher than that of the piggyBac (PB) transposable, the Sleeping Beauty (SB) transposable and / or the TcBuster (TB) transposable.

77. The engineered transposable element according to any one of claims 1-76, wherein the cell is an animal cell, plant cell, algal cell, fungal cell, yeast cell, or bacterial cell.

78. The engineered transposable element according to any one of claims 1-77, wherein the cell is a mammalian cell.

79. The engineered transposable element according to claim 78, wherein the mammalian cells are selected from immune cells, hepatocytes, tumor cells, stem cells, fertilized eggs, muscle cells, and skin cells.

80. The engineered transposable element according to claim 78 or 79, wherein the cell is a human cell.

81. The engineered transposable element according to any one of claims 1-80, wherein the engineered transposable element has higher transposable activity in human embryonic kidney 293T (293T) cells than in HeLa cells.

82. The engineered transducer element according to any one of claims 1-81, wherein the engineered transducer element is present in a carrier.

83. The engineered transposable element according to claim 82, wherein the carrier is a plasmid or a viral vector.

84. A gene transfer system comprising: 1) an engineered transposable element according to any one of claims 1-83; and 2) a transposase, or a nucleic acid encoding a transposase.

85. The gene transfer system of claim 84, wherein the transposase comprises an amino acid sequence selected from any one of SEQ ID NOs:13-18, a variant thereof, or a fragment thereof.

86. The gene transfer system of claim 85, wherein the transposase comprises an amino acid sequence having at least about 80% sequence identity with an amino acid sequence selected from any of SEQ ID NOs:13-18.

87. The gene transfer system of claim 86, wherein the transposase comprises an amino acid sequence selected from any one of SEQ ID NOs:13-18.

88. A gene transfer system comprising: 1) an engineered transposon element; and 2) a transposase, or a nucleic acid encoding a transposase, wherein the engineered transposon element comprises, from 5' to 3': 5' terminal repeat (5'TR), heterologous nucleic acid, and 3' terminal repeat (3'TR) The engineered transposable element described herein exhibits transposable activity that allows heterologous nucleic acids to insert into the DNA of cells, and The transposase therein comprises an amino acid sequence selected from any one of SEQ ID NOs:13-18 or a variant thereof.

89. The gene transfer system of claim 88, wherein the 5'TR comprises a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any of SEQ ID NOs:1-6, and wherein the 3'TR comprises a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any of SEQ ID NOs:7-12.

90. The gene transfer system of claim 89, wherein the 5'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5 and 6, and the 3'TR comprises a nucleic acid sequence having at least about 90% sequence identity with a nucleic acid sequence selected from any one of SEQ ID NOs: 7, 8, 9, 10, 11 and 12.

91. The gene transfer system according to any one of claims 88-90, wherein the engineered transposon element is derived from Tc1-1_ST, Tc1-2_ST, Tc1-3_ST, Tc1-4_ST, Tc1-5_ST or Tc1-1_PS.

92. The gene transfer system of claim 91, wherein the engineered transposon element is derived from Tc1-1_ST or Tc1-2_ST.

93. The gene transfer system according to any one of claims 88-92, wherein: (a) The 5'TR contains the nucleic acid sequence of SEQ ID NO:1, the 3'TR contains the nucleic acid sequence of SEQ ID NO:7, and the transposase contains the amino acid sequence of SEQ ID NO:13; (b) The 5'TR contains the nucleic acid sequence of SEQ ID NO:2, the 3'TR contains the nucleic acid sequence of SEQ ID NO:8, and the transposase contains the amino acid sequence of SEQ ID NO:14; (c) The 5'TR contains the nucleic acid sequence of SEQ ID NO:3, the 3'TR contains the nucleic acid sequence of SEQ ID NO:9, and the transposase contains the amino acid sequence of SEQ ID NO:15; (d) The 5'TR contains the nucleic acid sequence of SEQ ID NO:4, the 3'TR contains the nucleic acid sequence of SEQ ID NO:10, and the transposase contains the amino acid sequence of SEQ ID NO:16; (e) The 5'TR contains the nucleic acid sequence of SEQ ID NO:5, the 3'TR contains the nucleic acid sequence of SEQ ID NO:11, and the transposase contains the amino acid sequence of SEQ ID NO:17; (f) The 5'TR contains the nucleic acid sequence of SEQ ID NO:6, the 3'TR contains the nucleic acid sequence of SEQ ID NO:12, and the transposase contains the amino acid sequence of SEQ ID NO:18; 94. The gene transfer system according to any one of claims 88-93, wherein the gene transfer system comprises a nucleic acid encoding the transposase.

95. The gene transfer system according to claim 94, wherein the nucleic acid is DNA.

96. The gene transfer system of claim 94, wherein the nucleic acid is RNA.

97. The gene transfer system of claim 94, wherein the engineered transposon element and the nucleic acid encoding the transposase are in different vectors.

98. The gene transfer system of claim 94, wherein the engineered transposon element and the nucleic acid encoding the transposon are in the same vector.

99. The gene transfer system according to claim 97 or 98, wherein the vector is a viral vector.

100. A method for preparing a plurality of barcoded nucleic acids from a target nucleic acid, comprising contacting the target nucleic acid with a gene transfer system, said gene transfer system comprising: 1) Engineered rotary table components; and 2) transposases, or nucleic acids encoding transposases, The engineered transposable element from 5' to 3' comprises: a 5' terminal repeat sequence (5'TR), a heterologous nucleic acid containing a barcode sequence, and a 3' terminal repeat sequence (3'TR), wherein the 5'TR comprises a nucleic acid sequence selected from any of SEQ ID NOs:1-6, a variant thereof, or a fragment thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from any of SEQ ID NOs:7-12, a variant thereof, or a fragment thereof; The transposase described therein comprises an amino acid sequence selected from any one of SEQ ID NOs:13-18 or a variant thereof. This provides multiple barcoded nucleic acids.

101. The method of claim 100, wherein the target nucleic acid is genomic DNA, cDNA, or amplified DNA, optionally wherein the amplified DNA comprises a nucleic acid sequencing library.

102. The method according to claim 100 or 101, further comprising sequencing the nucleic acid sequencing library.

103. The method according to any one of claims 100-102, the method comprising contacting a target nucleic acid with a plurality of engineered transposable elements, wherein each engineered transposable element comprises a unique barcode sequence.

104. A transposon complex composition comprising: a transposase comprising an amino acid sequence selected from any of SEQ ID NOs:13-18 or a variant thereof, and one or two heterologous nucleic acids comprising a TR sequence and a tag sequence.

105. The transposon complex composition of claim 104, wherein the transposon complex comprises a single heterologous nucleic acid forming a hairpin.

106. The transposable composite composition of claim 105, wherein the hairpin comprises a cuttable site.

107. The transposon complex composition of claim 104, wherein the transposon complex comprises two transposases that bind to two heterologous nucleic acids.

108. A transposon complex composition comprising: a first transposase that binds to a first heteronucleotide comprising a 5' TR sequence and a first tag sequence, and a second transposase that binds to a second heteronucleotide comprising a 3' TR sequence and a second tag sequence.

109. The transposon complex composition of claim 108, wherein the 5'TR comprises a nucleic acid sequence selected from any of SEQ ID NOs:1-6, a variant thereof, or a fragment thereof, and wherein the 3'TR comprises a nucleic acid sequence selected from any of SEQ ID NOs:7-12, a variant thereof, or a fragment thereof.

110. The transposon complex composition of claim 108, wherein the first tag sequence is different from the second tag sequence.

111. A method for preparing a library of nucleic acid fragments having first and second tag sequences for a target nucleic acid, comprising contacting the target nucleic acid with a plurality of transposon complexes, the transposon complexes comprising: (1) a first transposon complex comprising a first transposase and a first heteronucleic acid comprising a 5' TR sequence and a first tag sequence; and (2) a second transposon complex comprising a second transposase and a second heteronucleic acid comprising a 3' TR sequence and a second tag sequence, wherein the first tag sequence is different from the second tag sequence, and wherein the transposase comprises any amino acid sequence selected from SEQ ID NOs:13-18 or a variant thereof; The first and second heteronucleotides are inserted into the target nucleic acid, and the target nucleic acid is fragmented into multiple nucleic acid fragments, each nucleic acid fragment containing one of the first or second nucleic acids linked to the 5' end of the nucleic acid fragment; Optionally, the nucleic acid fragment is DNA; This provides a library of nucleic acid fragments.

112. The method according to claim 111, wherein the transposon complex of (1) comprises two first heteronucleotides, and the transposon complex of (2) comprises two second heteronucleotides.

113. The method of claim 111, wherein the 5'TR comprises a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any of SEQ ID NO:s1-6, and wherein the 3'TR comprises a nucleic acid sequence, a variant thereof, or a fragment thereof selected from any of SEQ ID NOs:7-12.

114. The method according to claim 111, further comprising amplifying nucleic acid fragments.

115. A method for inserting a heterologous nucleic acid into a target nucleic acid, comprising: The target nucleic acid is contacted with the transposon element of any one of claims 1-83, the gene transfer system of any one of claims 84-99, or the transposon complex composition of any one of claims 94-100, thereby inserting the heterologous nucleic acid into the target nucleic acid.

116. The method of claim 115, wherein the method is performed in vitro.

117. The method of claim 115, wherein the target nucleic acid is in a cell.

118. The method of claim 117, wherein the target nucleic acid is genomic DNA.

119. The method according to claim 117 or 118, wherein the cell is an animal cell, plant cell, algal cell, fungal cell, yeast cell, or bacterial cell.

120. The method of claim 119, wherein the cell is a mammalian cell.

121. The method of claim 120, wherein the mammalian cells are selected from immune cells, hepatocytes, tumor cells, stem cells, fertilized eggs, muscle cells, and skin cells.

122. The method according to any one of claims 118-121, wherein the insertion of the heterologous nucleic acid inactivates the genes of the cell.

123. The method according to any one of claims 115-122, wherein the heterologous nucleic acid encodes a protein.

124. The method of claim 123, wherein the protein is selected from the group consisting of reporter proteins, engineered receptors, cytokines, antibiotic resistance proteins, antigens, and therapeutic proteins.

125. The method according to any one of claims 115-122, wherein the heterologous nucleic acid encodes RNA.

126. The method of claim 125, wherein the RNA is selected from the group consisting of: therapeutic RNA, small interfering RNA (siRNA), microRNA, short hairpin RNA (shRNA), long non-coding RNA (lincRNA), and guide RNA (gRNA).

127. The method according to any one of claims 115-126, wherein the heterologous nucleic acid encodes more than one molecule.

128. The method according to any one of claims 115-127, wherein the length of the heterologous nucleic acid does not exceed about 300 kilobases (kb).

129. The method of claim 128, wherein the length of the heterologous nucleic acid is from about 10 kb to about 300 kb.

130. The method of claim 128, wherein the length of the heterologous nucleic acid is about 100 base pairs (bp) to about 10 kb.

131. The method of claim 128, wherein the length of the heterologous nucleic acid is about 100 bp to about 5 kb.

132. The method of claim 131, wherein the length of the heterologous nucleic acid is about 100 bp to about 2 kb.

133. The method of claim 129, wherein the length of the heterologous nucleic acid is about 2 kb to about 300 kb.

134. The method according to any one of claims 115-133, wherein the insertion is random.

135. A kit comprising an engineered transposon element according to any one of claims 1-83, or a gene transfer system according to any one of claims 84-99, or a transposon complex composition according to any one of claims 94-100, for inserting a heterologous nucleic acid into a target nucleic acid.

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