Engineered mammalian gene writing system

Abstract

Provided is a retrotransposase which comprises a target DNA binding domain comprising a zinc finger binding motif, a reverse transcriptase domain, and an endonuclease domain, and which is capable of reverse transcription of RNA to DNA. The invention also relates to a system for modifying DNA. The system comprises the retrotransposase or a nucleic acid encoding the retrotransposase; and a donor RNA or a nucleic acid encoding the donor RNA, the donor RNA comprising: a sequence that binds to the retrotransposase and a heterologous sequence.

Description

Engineered mammalian gene writing system Technical Field

[0001] The present application belongs to the field of biotechnology. More specifically, the present application relates to a retrotransposase capable of inserting a large DNA fragment into the human genome at a specific site, its use, and a system utilizing the enzyme.

[0002] Cross-references

[0003] This application claims the benefit of Chinese Patent Application No. CN2023108423427 filed on July 10, 2023, and Chinese Patent Application No. CN2024101783089 filed on February 8, 2024, the contents of which are incorporated herein in their entirety.

[0004] Sequence listing file submitted at the same time

[0005] The entire contents of the following XML file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Format (CRF) (Name: FH01065PCT-sequencelisting.xml, Date: 20240705, Size: 154KB). Background Art

[0006] Targeted insertion of large DNA fragments into the genome is an important and useful technique in genomic research.

[0007] The integration of large DNA fragments into the genome of mammalian cells is generally inefficient and non-specific. Several existing genome editing methods, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, are not suitable for the integration of long DNA fragments. For example, the CRISPR / Cas9 system is limited to the editing of small systems and is less efficient when used to integrate longer sequences. Some other methods for the site-directed insertion of large DNA fragments have their own shortcomings. For example, the efficiency of large fragment insertion achieved through homologous recombination is low, and the double-stranded DNA breaks introduced pose a safety risk; recombinase systems such as Cre / loxP often require the insertion of loxP sites before the second step of integration can be performed; in addition, most current technologies rely on DNA donors, making it difficult to solve problems such as in vivo delivery.

[0008] Retrotransposases, on the other hand, have effectively filled the gap in large-fragment DNA site-specific integration technology, providing a more versatile and convenient tool for biological research. The new retrotransposase system is mainly divided into two components. First, the retrotransposase protein polypeptide itself. Second, the donor RNA carrying the new genetic information. Retrotransposases recognize and bind to the RNA donor sequence to form a protein-RNA complex. Through reverse transcriptase activity, they can convert RNA into DNA and integrate it into the genomic site. The DNA integration method based on retrotransposase avoids the generation of double-stranded DNA breaks and the risks they bring. At the same time, it avoids the preparation and dependence on DNA donors in practical applications. Only one reverse transcription reaction is required to reverse transcribe RNA into DNA, completing the integration of large DNA fragments. This is very beneficial for widespread application in various scenarios.

[0009] Summary of the Invention

[0010] Based on the above problems in the art, there is an urgent need to develop a more effective retrotransposase system in the art. This application relates to the following:

[0011] 1. An engineered retrotransposase comprising one, two or three wild-type retrotransposase TgR2 mutations or a truncation of the wild-type retrotransposase TgR2, or a truncation of the wild-type retrotransposase TgR2 comprising one, two or three wild-type retrotransposase TgR2 mutations, wherein the mutations are selected from:

[0012] (i) the amino acid of wild-type retrotransposase TgR2 is mutated to other amino acids corresponding to the corresponding sites in the multiple sequence alignment;

[0013] (ii) amino acids in the tertiary structure of wild-type retrotransposase TgR2 that have potential to interact with DNA or RNA are mutated into positively charged amino acids; and

[0014] (iii) The cysteine ​​in the wild-type retrotransposase TgR2 was mutated to serine.

[0015] 2. The engineered retrotransposase according to item 1, wherein the amino acid sequence of the wild-type retrotransposase TgR2 is as shown in SEQ ID NO.1 or the amino acid position numbering of the engineered retrotransposase is as defined in SEQ ID NO.1; or

[0016] The truncated form of the wild-type retrotransposase TgR2 is an N-terminal truncated form, a C-terminal truncated form and / or an intermediate sequence truncated form of the wild-type retrotransposase TgR2.

[0017] Preferably, the N-terminal truncation is a truncation of amino acids 1-500 of the wild-type retrotransposase TgR2, for example, truncation of amino acids 1-80, amino acids 1-190 or amino acids 1-220;

[0018] Preferably, the intermediate sequence truncation is truncated at the 300th to 1200th amino acids of the wild-type retrotransposase TgR2, for example, truncated at the 390th to 500th amino acids, or the 1100th to 1120th amino acids.

[0019] 3. The engineered retrotransposase according to item 1, wherein

[0020] The amino acids at the corresponding sites of the wild-type retrotransposase TgR2 in the multiple sequence alignment are confirmed using multiple sequence alignment software (such as MAFFT, etc.), and the amino acid position numbers are as defined in SEQ ID NO. 1;

[0021] Preferably, the corresponding sites of the wild-type retrotransposase TgR2 in the multiple sequence alignment are selected from any one, two, three, four, five, six, seven, eight, nine, or ten or more of the following:

[0022] K700, L704, V727, S732, L737, A747, A748, T759, I760, W762, S763, H792, A793, Q795, E798, P801, V807, S808, E812, S815, T819, T820, H825, K828, S860, Y864, H865, R866, Q868, S869, N889, N891, T892, S895, Q911, or D957,

[0023] It is further preferred that the amino acid mutation at the corresponding site of the wild-type retrotransposase TgR2 in the multiple sequence alignment is such that the other amino acids corresponding to the corresponding sites are selected from any one, two, three, four, five, six, seven, eight, nine, or ten or more of the following:

[0024] K700D, L704Q, V727L, S732T, L737I, A747S, A748S, A748P, T759L, I760L, W762R, W762K, W762Q, S763N, S763R, H792T, H792W, A793V, Q795K, Q795R, E798G, P801Q, V807I, S808R, E812K, E812R, S815N, T819D, T820L, H825Q, K828P, S860E, Y864F, H865Q, H865R, R866V, Q868S, S869K, N889G, N891Q, T892K, S895K, Q911E, or D957G;

[0025] It is further preferred that the amino acid mutation at the corresponding site of the wild-type retrotransposase TgR2 in the multiple sequence alignment is to be any amino acid selected from the following:

[0026] K700D, W762Q, H792W, A793V, R866V, or D957G.

[0027] 4. The engineered retrotransposase according to item 1, wherein

[0028] The amino acid in the tertiary structure of the wild-type retrotransposase TgR2 that has potential to interact with DNA or RNA is mutated into arginine; the amino acid position numbering is as defined in SEQ ID NO.1;

[0029] Preferably, the amino acids in the tertiary structure of the wild-type retrotransposase TgR2 that have potential interactions with DNA or RNA are selected from any one, two, three, four, five, six, seven, eight, nine, or ten or more of the following:

[0030] E153, G154, E160, E163, Q164, Q168, D227, K228, N229, N240, K267, E268, N269, P291, E294, H341, K34 2. C344, E350, E361, N363, N365, N367, A379, Q381, S383, D384, S390, D573, G575, K576, K579, I585, L5 88. K635, E636, E638, K639, N640, Q642, E643, S645, K646, G647, S648, V661, V693, I695, P696, S699, K700, I713, I715, T728, S732, K733, S751, E752, N753, L754, T819, N847, K950, Q955, D957, D990, K993 , T994, Y995, P998, I1001, D1005, H1006, T1044, D1046, T1052, Q1062, Q1071, S1072, S1073, D1074, D 1075, G1098, G1099, D1100, E1102, N1103, P1105, S1106, E1109, S1113, S1114, E1115, N1118, N1123, E 1125, E1127, Q1131, D1133, K1134, K1137, K1210, D1216, D1218, E1220, A1223, H1224, G1227, N1228, Q1233, D1234, I1237, K1238, H1240, N1241, E1245, E1249, N1268, E1270, E1302, E1306, K1307, or K1310;

[0031] It is further preferred that the amino acids in the tertiary structure of the wild-type retrotransposase TgR2 that have potential interactions with DNA or RNA are selected from any one, two, three, four, five, six, seven, eight, nine, or ten or more of the following:

[0032] D227, K228, N229, N240, K267, N269, P291, E294, H341, K342, C344, E361, N36 5. N367, A379, Q381, S383, D384, S390, K576, K579, K639, K646, K733, T819, K 950, Q955, D990, K993, T1044, T1052, Q1062, Q1071, D1074, D1075, G1098, G1099, P1105, E1109, E1125, E1127, Q1131, A1223, I1237, E1245, E1249, or E1306.

[0033] 5. The engineered retrotransposase according to item 1, wherein

[0034] The cysteine ​​residues in the wild-type retrotransposase TgR2 are selected from any one, two, three, four, five, six, seven, eight, nine, or ten or more of the following: the amino acid positions are numbered as defined in SEQ ID NO. 1;

[0035] C4, C145, C170, C282, C344, C404, C518, C521, C690, C735, C750, C855, C901, C913, C930, C1034, C1036, C1079, C1139, or C1244;

[0036] Preferably, the cysteine ​​residues in the wild-type retrotransposase TgR2 are selected from any one, two, three or four of the following:

[0037] C344, C1034, C1036, or C1244.

[0038] 6. The engineered retrotransposase according to any one of items 1 to 5, wherein the amino acid position numbers are as defined in SEQ ID NO. 1; and the engineered retrotransposase comprises any one or more of the following mutations:

[0039] (1)G1098R; (2)G1098R+H341R; (3)G1098R+K700D; (4)G1098R+W762Q; (5)G1098R+H792W; (6)G 1098R+A793V; (7) G1098R+R866V; (8) G1098R+Q955R; (9) G1098R+T1052R; (10) G1098R+Q1062R; (11)G1098R+P1105R; (12)G1098R+C1034S; (13)G1098R+I1237R; (14)G1098R+K1310R; (15)G1 098R+D957G; (16)H341R+C1036S; (17)H341R+Q1062R; (18)H341R+I1237R; (19)H341R+K1310R; (20)I1237R+K1310R; (21)C1034S+C1036S; (22)K700D+R866V; (23)W762Q+R866V; (24)H792W+ R866V; (25)A793V+R866V; (26)G1098R+A793V+K700D; (27)G1098R+A793V+W762Q; (28)G1098R+ A793V+H792W; (29)G1098R+A793V+Q955R; (30)G1098R+A793V+W762Q+Q955R; (31)G1098R+A79 3V+W762Q+H341R; (32)G1098R+A793V+Q955R+H341R; (33)G1098R+A793V+W762Q+Q955R+H341R;

[0040] Preferably, the engineered retrotransposase comprises any one or more of the following mutations:

[0041] G1098R+H341R; G1098R+K700D; G1098R+W762Q; G1098R+H792W, G1098R+A793V; G1098R+R866V; G1098R+Q955R; G 1098R+A793V+W762Q; G1098R+A793V+Q955R; G1098R+A793V+Q955R+H341R, G1098R+A793V+W762Q+Q955R+H341R.

[0042] 7. An engineered retrotransposase comprising any one or more of the following mutations:

[0043] (1)G1098R; (2)G1098R+H341R; (3)G1098R+K700D; (4)G1098R+W762Q; (5)G1098R+H792W; (6)G 1098R+A793V; (7) G1098R+R866V; (8) G1098R+Q955R; (9) G1098R+T1052R; (10) G1098R+Q1062R; (11)G1098R+P1105R; (12)G1098R+C1034S; (13)G1098R+I1237R; (14)G1098R+K1310R; (15)G1 098R+D957G; (16)H341R+C1036S; (17)H341R+Q1062R; (18)H341R+I1237R; (19)H341R+K1310R; (20)I1237R+K1310R; (21)C1034S+C1036S; (22)K700D+R866V; (23)W762Q+R866V; (24)H792W+ R866V; (25)A793V+R866V; (26)G1098R+A793V+K700D; (27)G1098R+A793V+W762Q; (28)G1098R+ A793V+H792W; (29)G1098R+A793V+Q955R; (30)G1098R+A793V+W762Q+Q955R; (31)G1098R+A79 3V+W762Q+H341R; (32)G1098R+A793V+Q955R+H341R; (33)G1098R+A793V+W762Q+Q955R+H341R;

[0044] Preferably, the engineered retrotransposase comprises any one or more of the following mutations:

[0045] G1098R+H341R; G1098R+K700D; G1098R+W762Q; G1098R+H792W, G1098R+A793V; G1098R+R866V; G1098R+Q955R; G 1098R+A793V+W762Q; G1098R+A793V+Q955R; G1098R+A793V+Q955R+H341R, G1098R+A793V+W762Q+Q955R+H341R;

[0046] The amino acid position numbers are as defined in SEQ ID NO.1.

[0047] 8. An engineered retrotransposase, which is an engineered retrotransposase obtained by further coupling the engineered retrotransposase or wild-type retrotransposase TgR2 shown in any one of items 1 to 7 with other amino acid sequences.

[0048] 9. The engineered retrotransposase according to item 8, wherein the other amino acid sequences are selected from any one, two, three, four, five, six, or seven of a nuclease exonuclease, a single-stranded DNA binding protein, a chromatin regulatory peptide, a high-mobility peptide, an RNaseH domain, a nuclear import localization signal (NLS), and a nuclear export localization signal.

[0049] 10. The engineered retrotransposase according to item 9, wherein the other amino acid sequence is an exonuclease, and the exonuclease is inserted at the N-terminus, after amino acid 191, after amino acid 432, after amino acid 1112, or at the C-terminus of the retrotransposase according to any one of items 1 to 7;

[0050] Preferably, the exonuclease is an exonuclease from T5 bacteriophage;

[0051] It is further preferred that the sequence of the exonuclease is as shown in SEQ ID NO.8 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No.8.

[0052] 11. The engineered retrotransposase according to item 9, wherein the other amino acid sequence is a single-stranded DNA binding protein, and the single-stranded DNA binding protein is inserted at the N-terminus, after amino acid 191, after amino acid 432, after amino acid 1112, or at the C-terminus of the retrotransposase according to any one of items 1 to 7;

[0053] Preferably, the single-stranded DNA binding protein is a single-stranded DNA binding protein from Sulfolobus tokodaii bacteria;

[0054] It is further preferred that the sequence of the single-stranded DNA binding protein is as shown in SEQ ID NO.9 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No.9.

[0055] 12. The engineered retrotransposase according to item 9, wherein the other amino acid sequence is a chromatin regulatory peptide or a high-mobility peptide, and the chromatin regulatory peptide or high-mobility peptide is inserted at the N-terminus, after amino acid 191, after amino acid 432, after amino acid 1112, or at the C-terminus of the retrotransposase according to any one of items 1 to 7;

[0056] Preferably, the chromatin regulatory peptide or high-mobility peptide is a chromatin regulatory peptide or high-mobility peptide from human;

[0057] Preferably, the sequence of the chromatin regulatory peptide or high-mobility peptide is as shown in SEQ ID NO.10 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No.10.

[0058] 13. The engineered retrotransposase according to item 9, wherein the other amino acid sequence is an RNaseH domain, and the RNaseH domain is inserted at the N-terminus, after amino acid 191, after amino acid 432, after amino acid 1112, or at the C-terminus of the retrotransposase according to any one of items 1 to 7 or the wild-type retrotransposase TgR2;

[0059] Preferably, the RNaseH domain is the RNaseH domain from MLV reverse transcriptase;

[0060] It is further preferred that the sequence of the RNaseH domain of MLV reverse transcriptase is as shown in SEQ ID NO.11 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID No.11.

[0061] 14. The engineered retrotransposase according to item 9, wherein

[0062] Inserting any two, three, or four of the exonuclease and / or single-stranded DNA binding protein and / or chromatin regulatory peptide (CMP) and / or RNaseH domains into the N-terminus, after amino acid 191, after amino acid 432, after amino acid 1112, or the C-terminus of the retrotransposase shown in any one of items 1 to 7 or the wild-type retrotransposase TgR2;

[0063] It is further preferred that a nuclease is inserted into the N-terminus of the retrotransposase or the wild-type retrotransposase TgR2 as shown in any one of items 1 to 7, and an exonuclease or a single-stranded DNA binding protein or a chromatin regulatory peptide (CMP) or an RNaseH domain is inserted after amino acid 432 of the retrotransposase or the wild-type retrotransposase TgR2 as shown in any one of items 1 to 7; or

[0064] It is further preferred that a single-stranded DNA binding protein is inserted into the N-terminus of the retrotransposase or the wild-type retrotransposase TgR2 as shown in any one of items 1 to 7, and an exonuclease or a single-stranded DNA binding protein or a chromatin regulatory peptide (CMP) or an RNaseH domain is inserted after amino acid 432 of the retrotransposase or the wild-type retrotransposase TgR2 as shown in any one of items 1 to 7; or

[0065] It is further preferred that a chromatin regulatory peptide (CMP) is inserted into the N-terminus of the retrotransposase or the wild-type retrotransposase TgR2 as shown in any one of items 1 to 7, and an exonuclease or a single-stranded DNA binding protein or a chromatin regulatory peptide (CMP) or an RNaseH domain is inserted after amino acid 432 of the retrotransposase or the wild-type retrotransposase TgR2 as shown in any one of items 1 to 7; or

[0066] It is further preferred that a single-stranded DNA binding protein is inserted into the N-terminus of the retrotransposase shown in any one of items 1 to 7 or the wild-type retrotransposase TgR2, and a chromatin regulatory peptide (CMP) is inserted after amino acid 432 in the retrotransposase shown in any one of items 1 to 7 or the wild-type retrotransposase TgR2, and a chromatin regulatory peptide (CMP) or RNaseH domain is inserted after amino acid 1112 in the retrotransposase shown in any one of items 1 to 7; or

[0067] It is further preferred that a single-stranded DNA binding protein is inserted into the N-terminus of the retrotransposase or wild-type retrotransposase TgR2 as shown in any one of items 1 to 7, an RNaseH domain is inserted after amino acid 432 of the retrotransposase or wild-type retrotransposase TgR2 as shown in any one of items 1 to 7, and a chromatin regulatory peptide (CMP) or RNaseH domain is inserted after amino acid 1112 of the retrotransposase or wild-type retrotransposase TgR2 as shown in any one of items 1 to 7; or

[0068] It is further preferred that a chromatin regulatory peptide (CMP) is inserted into the N-terminus of the retrotransposase shown in any one of items 1 to 7 or the wild-type retrotransposase TgR2, and a chromatin regulatory peptide (CMP) is inserted after amino acid 432 of the retrotransposase shown in any one of items 1 to 7 or the wild-type retrotransposase TgR2, and a chromatin regulatory peptide (CMP) or an RNaseH domain is inserted after amino acid 1112 of the retrotransposase shown in any one of items 1 to 7 or the wild-type retrotransposase TgR2; or

[0069] It is further preferred that a chromatin regulatory peptide (CMP) is inserted at the N-terminus of the retrotransposase shown in any one of items 1 to 7 or the wild-type retrotransposase TgR2, and an RNaseH domain is inserted after amino acid 432 in the retrotransposase shown in any one of items 1 to 7 or the wild-type retrotransposase TgR2, and a chromatin regulatory peptide (CMP) or RNaseH domain is inserted after amino acid 1112 in the retrotransposase shown in any one of items 1 to 7 or the wild-type retrotransposase TgR2.

[0070] 15. The engineered retrotransposase according to item 14, wherein

[0071] The exonuclease sequence is as shown in SEQ ID NO.8 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID No.8; or

[0072] The sequence of the single-stranded DNA binding protein is as shown in SEQ ID NO.9 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID No.9; or

[0073] The sequence of the chromatin regulatory peptide or high mobility peptide is as shown in SEQ ID NO.10 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID No.10; or

[0074] The sequence of the RNaseH domain is shown in SEQ ID NO.11 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID No.11.

[0075] 16. An engineered retrotransposase according to any one of items 1 to 15, wherein the engineered retrotransposase comprises an amino acid sequence as shown in any one of SEQ ID NOs. 2 to 7, 12, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55 to 84, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs. 2 to 7, 12, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55 to 84; or

[0076] The engineered retrotransposase is a truncated version of the amino acid sequence shown in any one of SEQ ID NOs. 1-7, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55 to 84, preferably a sequence obtained by truncating the wild-type sequence SEQ ID NO. 1 corresponding to amino acids 1-80, or 1-190, or 1-220, 390-500, or 1100-1120 from the amino acid sequence shown in any one of SEQ ID NOs. 1-7, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55 to 84.

[0077] 17. A system for modifying DNA, the system comprising:

[0078] A retrotransposase, wherein the retrotransposase is the wild-type retrotransposase TgR2 shown in SEQ ID NO. 1 or the retrotransposase shown in any one of items 1 to 16, or a nucleic acid encoding the wild-type retrotransposase TgR2 shown in SEQ ID NO. 1 or the retrotransposase shown in any one of items 1 to 16; and

[0079] A donor RNA or a nucleic acid encoding the donor RNA, wherein the donor RNA comprises: a sequence that binds to the retrotransposase and a heterologous sequence,

[0080] Preferably, the heterologous sequence is at least 1-50,000 bases, for example, 1 nt or more, 10 nt or more, 50 nt or more, 60 nt or more, 70 nt or more, 80 nt or more, 90 nt or more, 100 nt or more, 150 nt or more, 200 nt or more, 250 nt or more, 300 nt or more, 350 nt or more, 400 nt or more, 450 nt or more, 500 nt or more, 550 nt or more, 600 nt or more, 650 nt or more, 700 nt or more, 750 nt or more, 800 nt or more, 850 nt or more, 900 nt or more, 950 nt or more, 1000 nt or more, 1100 nt or more, 1200 nt or more, 1300 nt or more, 1400 nt or more, 1500 nt or more, 1600 nt or more, 1700 nt or more, 1800 nt or more, 1900 nt or more. nt or more, 2000nt or more, 2100nt or more, 2200nt or more, 2300nt or more, 2400nt or more, 2500nt or more, 2600nt or more, 2700nt or more, 2800nt or more, 2900nt or more, 3000nt or more, 3500nt or more, 4000nt or more, 4500nt or more, 5000nt or more, 5500nt or more, 6000nt or more, 6500nt or more, 7000nt or more, 7500nt or more, 8000nt or more, 8500nt or more, 9000nt or more, 9500nt or more, 10000nt or more, 15000nt or more, 20000nt or more, 25000nt or more, 30000nt or more, 35000nt or more, 40000nt or more, 45000nt or more;

[0081] It is further preferred that the system comprises a nucleic acid encoding a VPX protein or the VPX protein itself.

[0082] 18. The system according to item 17, wherein the heterologous sequence comprises one or more of the following: a sequence encoding a polypeptide or a non-coding RNA sequence, a sequence comprising a promoter or enhancer, a sequence encoding one or more introns, and a transcription termination sequence;

[0083] Preferably, the polypeptide is a therapeutic polypeptide or a mammalian polypeptide; further preferably, the polypeptide is a therapeutic protein, a membrane protein, an intracellular protein, an extracellular protein, a structural protein, a signal transduction protein, a regulatory protein, a transport protein, an organelle protein, a sensory protein, a motor protein, a defense protein, a storage protein, a reporter protein, an antibody, an enzyme, or a coagulation factor; further preferably, the number of amino acids in the polypeptide is 20 to 10,000, for example, the number of amino acids is 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800 , 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900 00, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900;

[0084] Preferably, the intracellular protein is selected from cytoplasmic proteins, nuclear proteins, organelle proteins, mitochondrial proteins or lysosomal proteins,

[0085] It is further preferred that the sequence encoding the polypeptide contains one or more introns.

[0086] 19. The system according to item 17 or 18, wherein the donor RNA further comprises a homology domain, preferably the homology domain comprises a first homology domain and a second homology domain,

[0087] More preferably, the first homology domain is 3 or more bases located at the 5' end of the donor RNA and having 100% identity with the target DNA chain, and the second homology domain is 3 or more bases located at the 3' end of the donor RNA and having 100% identity with the target DNA chain, and the target DNA is preferably a genomic safe harbor GSH site or a genomic Natural Harbor TM Location.

[0088] 20. The system according to any one of items 17 to 19, wherein the nucleic acid encoding the retrotransposase according to any one of items 1 to 16 and the donor RNA or the nucleic acid encoding the donor RNA are separate nucleic acids, preferably the donor RNA does not encode the retrotransposase, further preferably the donor RNA comprises one or more chemical modifications; or

[0089] The nucleic acid encoding the retrotransposase according to any one of items 1 to 16 and the donor RNA or the nucleic acid encoding the donor RNA are covalently linked. Preferably, the nucleic acid encoding the retrotransposase according to any one of items 1 to 16 and the donor RNA or the nucleic acid encoding the donor RNA form a fusion nucleic acid. More preferably, the fusion nucleic acid comprises RNA or DNA.

[0090] It is further preferred that the nucleic acid encoding the retrotransposase according to any one of items 1 to 16, the donor RNA or the nucleic acid encoding the donor RNA, and the nucleic acid encoding the VPX protein are separate nucleic acids; or

[0091] The nucleic acid encoding the retrotransposase according to any one of items 1 to 16 and the nucleic acid encoding the VPX protein are covalently linked or fused nucleic acids, and are separate nucleic acids from the donor RNA or the nucleic acid encoding the donor RNA;

[0092] The nucleic acid encoding the retrotransposase according to any one of items 1 to 16 is covalently linked to or fused to the donor RNA or the nucleic acid encoding the donor RNA, and is a separate nucleic acid from the nucleic acid encoding the VPX protein;

[0093] The donor RNA or the nucleic acid encoding the donor RNA is covalently linked to or fused with the nucleic acid encoding the VPX protein, and is a separate nucleic acid from the nucleic acid encoding the retrotransposase according to any one of items 1 to 16; or

[0094] The nucleic acid encoding the retrotransposase according to any one of items 1 to 16, the donor RNA or the nucleic acid encoding the donor RNA, and the nucleic acid encoding the VPX protein are covalently linked or fused nucleic acids,

[0095] It is further preferred that the fusion nucleic acid comprises RNA or DNA.

[0096] 21. The system according to any one of items 17 to 20, wherein the donor RNA comprises:

[0097] optionally a 5' untranslated sequence (5'UTR) to which the retrotransposase binds,

[0098] a 3' untranslated sequence (3'UTR) to which the retrotransposase binds,

[0099] Heterologous sequences, and

[0100] a promoter operably linked to the heterologous sequence,

[0101] Preferably, the promoter is located between the 5' untranslated sequence (5'UTR) to which the retroposase binds and the heterologous sequence or preferably, the promoter is located between the 3' untranslated sequence (3'UTR) to which the retroposase binds and the heterologous sequence.

[0102] 22. A system according to claim 21, wherein the heterologous sequence comprises an open reading frame or its reverse complement sequence in a 5' to 3' orientation on the donor RNA; or the heterologous sequence comprises an open reading frame or its reverse complement sequence in a 3' to 5' orientation on the donor RNA.

[0103] 23. The system according to any one of items 17 to 14, wherein the donor RNA further comprises a nuclear localization signal or the nucleic acid encoding the retrotransposase according to any one of items 1 to 8 comprises a nuclear localization signal and / or a nucleolar localization signal and / or a nuclear export signal.

[0104] 24. A system according to any one of items 9 to 23, wherein the nucleic acid encoding the retrotransposase according to any one of items 1 to 16 and the nucleic acid encoding the donor RNA are present in a ratio of 10:1 to 1:10, for example, in a ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0105] 25. The system according to any one of items 17 to 24, wherein the donor RNA comprises a stem-loop sequence or helix 5' to the pseudoknot sequence, preferably comprises one or more (e.g., 2, 3 or more) stem-loop sequences or helices 3' to the pseudoknot sequence, e.g., 3' to the pseudoknot sequence and 5' to the heterologous sequence, further preferably the pseudoknot donor RNA has catalytic activity, e.g., RNA cleavage activity, e.g., cis-RNA cleavage activity, or

[0106] The donor RNA comprises, for example, at least one stem-loop sequence or helix, such as 1, 2, 3, 4, 5 or more stem-loop sequences, hairpin or helix sequences, 3' to the heterologous sequence.

[0107] 26. The system according to any one of items 17 to 25, wherein:

[0108] The 5' untranslated sequence (5'UTR) in the donor RNA that binds to the retrotransposase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence of any one of SEQ ID Nos. 16, 27, 29, 31, 33, 35, 37, 39, and 41;

[0109] The 3' untranslated sequence (3'UTR) in the donor RNA that binds to the retrotransposase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence of any one of SEQ ID No. 17, 32, 34, 44, 46, 48, 50, 52, or 54; or

[0110] The amino acid sequence of the VPX protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID No. 85, 86, or 87.

[0111] 27. The system according to any one of items 17 to 26, wherein the donor RNA comprises the following structures in order from its 5' end to its 3' end:

[0112] The first homology domain,

[0113] a 5' untranslated sequence (5'UTR) to which the retrotransposase binds,

[0114] Heterologous sequences,

[0115] a 3' untranslated sequence (3'UTR) to which the retrotransposase binds, and

[0116] second homeodomain;

[0117] Preferably, the first homology domain is one or more, or two or more, or five or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more, or 50 or more, or 60 or more, or 70 or more, or 80 or more, or 90 or more, or 100 or 110 or 120 or 130 or 140 or 150 or more bases located at the 5' end of the donor RNA and having 100% identity with the target DNA chain, and the second homology domain is one or more, or two or more, or five or more, or 10 or more, or 20 or more, or 30 or more, or 40 or more, or 50 or more, or 60 or more, or 70 or more, or 80 or more, or 90 or more, or 100 or 110 or 120 or 130 or 140 or 150 or more bases located at the 3' end of the donor RNA and having 100% identity with the target DNA chain;

[0118] It is further preferred that the 5' untranslated sequence (5'UTR) in the donor RNA to which the retrotransposase binds has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence of any one of SEQ ID Nos. 16, 27, 29, 31, 33, 35, 37, 39, and 41;

[0119] The 3' untranslated sequence (3'UTR) in the donor RNA to which the retrotransposase binds has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of any one of SEQ ID Nos. 17, 32, 34, 44, 46, 48, 50, 52, and 54.

[0120] 28. The system according to any one of items 17 to 27, wherein:

[0121] The 5' untranslated sequence (5'UTR) to which the retrotransposase binds is a non-natural 5' untranslated sequence (5'UTR); or

[0122] The 3' untranslated sequence (5'UTR) to which the retrotransposase binds is a non-natural 3' untranslated sequence (3'UTR);

[0123] Further preferred are non-natural 5' untranslated sequences (5'UTRs) having additions, deletions and / or substitutions of nucleotides relative to the natural 5'UTR sequence;

[0124] Further preferred are non-natural 3' untranslated sequences (3'UTRs) having additions, deletions and / or substitutions of nucleotides relative to the natural 3'UTR sequence;

[0125] Further preferred is a non-natural 5' untranslated sequence (5'UTR) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide sequence shown in one of SEQ ID No. 16, 27, 29, 31, 33, 35, 37, 39, 41;

[0126] Further preferred is a non-natural 3' untranslated sequence (3'UTR) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence of any one of SEQ ID Nos. 17, 32, 34, 44, 46, 48, 50, 52, or 54.

[0127] 29. A system according to any one of items 17 to 27, wherein the heterologous sequence is inserted into a target site in the cell genome at an average copy number of at least 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4 or 5 copies per genome, preferably only at one target site in the genome.

[0128] 30. The system of any one of items 17 to 29, wherein the heterologous sequence results in insertion into the target site (e.g., at a copy number of 1 insertion or more than 1 insertion) in about 1%-100% of the cells (e.g., about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90% or 90%-100% of the cells) in a cell population contacted with the system, e.g., as measured using single-cell ddPCR, or

[0129] The heterologous sequence is inserted into the target site at a copy number of 1 insertion in about 1%-100% of the cells (e.g., about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% of the cells) in a population of cells contacted with the system, e.g., as measured using colony isolation and ddPCR.

[0130] 31. A system according to any one of items 17 to 30, wherein the heterologous sequence is caused to be inserted into a target site (on-target insertion) in a cell population at a higher rate than insertion into a non-target site (off-target insertion), wherein the ratio of on-target insertion to off-target insertion is greater than 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 500:1 or 1,000:1.

[0131] 32. A system according to any one of items 17 to 31, wherein

[0132] The sequence of the first homology domain is as shown in SEQ ID NO. 15, 19, 21, 23, 25 or 28, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the nucleotide sequence shown in SEQ ID No. 15, 19, 21, 23, 25 or 28; or

[0133] The sequence of the second homology domain is as shown in SEQ ID NO. 18, 20, 22, 24, 26 or 36, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID NO. 18, 20, 22, 24, 26 or 36.

[0134] 33. A non-natural 5' untranslated sequence (5'UTR) having nucleotide additions, deletions and / or substitutions relative to a natural 5'UTR sequence, preferably having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence shown in SEQ ID No. 16, 27, 29, 31, 33, 35, 37, 39, or 41.

[0135] 34. A non-natural 3' untranslated sequence (3'UTR) having nucleotide additions, deletions and / or substitutions relative to a natural 3'UTR sequence, preferably having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide sequence of SEQ ID No. 17, 32, 34, 44, 46, 48, 50, 52, 54.

[0136] 35. An engineered transposable element comprising, from 5' to 3':

[0137] 5' untranslated sequence (5'UTR), heterologous sequence and 3' untranslated sequence (3'UTR),

[0138] wherein the 5' untranslated sequence comprises a nucleotide sequence selected from SEQ ID No. 16, 27, 29, 31, 33, 35, 37, 39, 41 having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity; or

[0139] wherein the 3' non-translated sequence comprises a nucleotide sequence selected from SEQ ID No. 17, 32, 34, 44, 46, 48, 50, 52, 54 having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity,

[0140] Preferably, it comprises, from 5' to 3', a first homology domain, a 5' untranslated sequence (5'UTR) that binds to the retrotransposase, a heterologous sequence, a 3' untranslated sequence (3'UTR) that binds to the retrotransposase, and a second homology domain.

[0141] It is further preferred that the sequence of the first homology domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence shown in SEQ ID NO. 15, 19, 21, 23, 25 or 28; or

[0142] The sequence of the second homology domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO. 18, 20, 22, 24, 26, or 36.

[0143] 36. The element according to item 35, wherein the element is the donor RNA mentioned in items 17 to 32 or a nucleic acid encoding the donor RNA.

[0144] 37. A host cell comprising the engineered retrotransposase described in any one of items 1 to 16, or the element described in item 35 or 36, or the system described in any one of items 17 to 32, wherein the host cell is preferably a mammalian cell or a plant cell, and more preferably a human cell.

[0145] 38. A method for modifying a target DNA chain in a cell, tissue or subject, the method comprising using the engineered retrotransposase described in any one of items 1 to 16, or the element described in item 35 or 36, or the system described in any one of items 17 to 32 on the cell, tissue or subject, wherein the system reverse transcribes the donor RNA sequence into the target DNA chain, thereby modifying the target DNA chain in the cell, tissue or subject.

[0146] 39. The method according to item 38, wherein the cells and tissues are mammalian cells and tissues, preferably human cells and tissues, and the subject is a mammal, preferably a human.

[0147] 40. The method of claim 39, wherein the cells are fibroblasts or primary cells or cells that have not been immortalized.

[0148] 41. The method according to any one of items 38 to 40, wherein the method is performed in vivo or in vitro.

[0149] 42. A method for modifying the genome of a mammalian cell or inserting DNA into the genome of a mammal, the method comprising applying to the cell an engineered retrotransposase as described in any one of items 1 to 16, or an element as described in item 35 or 36, or a system as described in any one of items 17 to 32, preferably wherein the mammal is a human.

[0150] 43. The method of claim 42, wherein the method results in the addition of at least 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 base pairs of exogenous DNA sequence to the genome of the mammal.

[0151] 44. A method according to any one of items 38 to 43, wherein the cell is part of a tissue; or the mammalian cell is euploid, is not immortalized, is part of an organism, is a primary cell, is non-dividing, is a hepatocyte or is from a subject with a genetic disease.

[0152] 45. The method according to any one of items 38 to 44, wherein

[0153] The method comprises contacting a cell, a tissue or a subject with the retrotransposase as set forth in any one of items 1 to 16 or a nucleic acid encoding the retrotransposase as set forth in any one of items 1 to 16 and a donor RNA or a nucleic acid encoding the donor RNA,

[0154] Preferably, said contacting comprises contacting said cell, tissue or subject with a plasmid, virus, virus-like particle, virosome, liposome, vesicle, exosome or lipid nanoparticle;

[0155] It is further preferred that said contacting comprises the use of non-viral delivery, such as electroporation.

[0156] 46. ​​The method of claim 45, wherein:

[0157] The contacting comprises intravenously administering to the subject, preferably at least twice, the retrotransposase or the nucleic acid encoding the retrotransposase as described in any one of Items 1 to 16 and the donor RNA or the nucleic acid encoding the donor RNA.

[0158] 47. The method according to any one of items 38 to 46, wherein

[0159] The retrotransposase according to any one of items 1 to 16 or the nucleic acid encoding the retrotransposase according to any one of items 1 to 16 and the donor RNA or the nucleic acid encoding the donor RNA are administered separately; or

[0160] The retrotransposase described in any one of Items 1 to 16 or a nucleic acid encoding the retrotransposase described in any one of Items 1 to 16 is administered together with a donor RNA or a nucleic acid encoding the donor RNA.

[0161] 48. A nucleic acid encoding the retrotransposase according to any one of items 1 to 16.

[0162] 49. A vector comprising the nucleic acid of item 48.

[0163] 50. A host cell comprising the vector of item 49.

[0164] 51. A pharmaceutical composition comprising the system described in any one of items 17 to 32, or the nucleic acid described in item 48, or the vector described in item 49, or the host cell described in item 37 or 50. Preferably, the system is placed in a pharmaceutically acceptable carrier, and further preferably, the carrier is a vesicle (including liposomes, natural or synthetic lipid bilayers, exosomes), lipid nanoparticles, viruses or plasmid vectors.

[0165] Effects of the Invention

[0166] The system and method constructed in this application can achieve gene writing at the DNA level using only RNA donors, which is a technical innovation. By integrating the coding gene into the RNA sequence template, the system and method of this application can meet but are not limited to:

[0167] Therapeutic needs can be addressed, for example, by providing expression of a therapeutic transgene in an individual with a loss-of-function mutation, by replacing a gain-of-function mutation with a normal transgene, by providing regulatory sequences to eliminate expression of a gain-of-function mutation, and / or by controlling the expression of operably linked genes, transgenes, and systems thereof. In certain embodiments, the RNA sequence template encodes a promoter region specific for the host cell in need of treatment, such as a tissue-specific promoter or enhancer. In other embodiments, the promoter can be operably linked to the coding sequence.

[0168] The need to prepare functional cells, such as integrating biological macromolecules with specific functions (such as chimeric antigen receptors (CARs)) into immune cells to give them new tumor-killing functions.

[0169] New demands for crop breeding. For example, by integrating specific genes into plant callus tissue, plants can be given new economic traits (such as stress resistance, insect resistance, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0170] Figure 1a shows the integration principle of the R2 retrotransposase system. The R2 retrotransposase system consists of two components: the retrotransposase and the donor RNA. The R2 retrotransposase has DNA binding, endonuclease, and reverse transcriptase domains, while the donor RNA contains a left homology arm, a 5' untranslated region (UTR) sequence, the target gene, a 3' untranslated region (UTR), and a right homology arm. When the donor RNA is carried by the R2 retrotransposase to the target DNA sequence, the endonuclease domain of the R2 retrotransposase cleaves the target DNA, generating a nicked strand. This nicked strand then initiates reverse transcription of the donor RNA from the exposed 3' end of the donor RNA, integrating the newly synthesized DNA strand into the genomic DNA.

[0171] Figure 1b shows the construction of the GFP reporter system. The GFP reporter system consists of an R2 retrotransposase expression plasmid and a donor RNA expression plasmid, wherein the donor RNA contains a homology arm-left, a 5'UTR sequence, a 3'UTR sequence, a reverse expression frame between the 5'UTR sequence and the 3'UTR sequence, which is expressed by a CMV promoter (and the GFP sequence expressed in the reverse expression frame is separated by an intron sequence inserted in the opposite direction), and a homology arm-right. After transfection of the reporter system, the R2 retrotransposase and the donor RNA are expressed separately. After the donor RNA is expressed by the CAG promoter, the intron sequence contained in the donor RNA is cleaved from the expressed RNA. At this time, the GFP sequence in the expression frame expressed by the CMV promoter returns to normal at the RNA level. However, the GFP sequence at this time is an antisense RNA chain and does not have the ability to translate into GFP protein. Only when the retrotransposase permanently integrates the intron-less donor RNA into the cell's DNA through reverse transcriptase activity can the reverse expression cassette, expressed from the CMV promoter, express normal intron-less GFP mRNA, thereby expressing the green fluorescent GFP protein. Therefore, this GFP reporter system was transfected into 293T cells. 24 hours after transfection, 50,000 mCherry-positive cells were sorted using flow cytometry. After another 6 days of culture, the presence and proportion of GFP cells were detected using fluorescence microscopy and flow cytometry.

[0172] Figure 2 shows how introducing amino acid point mutations based on homologous sequence alignment improves the integration efficiency of wild-type TgR2. Multiple sequence alignments within the same branch of the TgR2 protein were extracted based on its position in the phylogenetic tree. The amino acids in TgR2 were mutated to alternative amino acid sequences corresponding to the corresponding sites in the multiple sequence alignment. GFP reporter system testing showed that, after mutation of 36 selected amino acid sites, six of the mutants with amino acid substitutions (K700D, W762Q, H792W, A793V, R866V, and D957G) significantly improved integration efficiency compared to wild-type TgR2. The remaining mutants showed little or no significant improvement, or even decreased efficiency.

[0173] Figure 3: Shows that the introduction of arginine point mutations based on the predicted 3D structure improves the gene integration efficiency of wild-type TgR2. The test results of the GFP reporter system show that among the 132 selected amino acids: E153, G154, E160, E163, Q164, Q168, D227R, K228R, N229R, N240R, K267R, E268R, N269R, P291R, E294R, H341R, K342R, C344R, E350R, E361R, N363R, N365R, N367R, A379R, Q381R, S383R, D384R, S390R, D573, G575, K576, K579, I585, L588, K 635, E636, E638, K639, N640, Q642, E643, S645, K646, G647, S648, V661, V693, I695, P696, S699, K700, I713, I715, T728, S732, K733, S 751, E752, N753, L754, T819, N847, K950, Q955, D957, D990, K993, T994, Y995, P998, I1001, D1005, H1006, T1044, D1046, T1052, Q1062 , Q1071, S1072, S1073, D1074, D1075, G1098, G1099, D1100, E1102, N1103, P1105, S1106, E1109, S1113, S1114, E1115, N1118, N1123, E 1125, E1127, Q1131, D1133, K1134, K1137, K1210R, D1216R, D1218R, E1220R, A1223R, H1224R, G1227R, N1228R, Q1233R, D1234R, I1237 R, K1238R, H1240R, N1241R, E1245R, E1249R, N1268R, E1270R, E1302R, E1306R, K1307R, and K1310R, after arginine substitution, compared with the wild-type TgR2, 10 arginine-substituted mutants: Q168R, T819R, Q955R, Q1062R, D1074R, G1098R, P1105R, E1125R, E1127R, and Q1131R can significantly improve the efficiency of gene integration, while the remaining mutants have little improvement or no significant improvement or even a decrease in efficiency.

[0174] Figure 4: Replacing cysteine ​​(C) in the protein sequence with serine (S) improves the integration efficiency of TgR2. The 20 cysteines (C) in the R2Tg protein were replaced with serine (S) to obtain the corresponding mutant proteins: C4, C145, C170, C282, C344, C404, C518, C521, C690, C735, C750, C855, C901, C913, C930, C1034, C1036, C1079, C1139, C1244. Based on the GFP reporter system designed in Example 1, compared with the wild-type TgR2 protein, four mutant proteins: C344S, C1034S, C1036S, and C1244S can significantly improve the efficiency of gene integration, while the remaining mutants have little or no improvement or even a decrease in efficiency.

[0175] Figure 5: Combining the selected point mutations with improved efficiency yielded a composite protein with significantly improved integration. The most efficient mutant, G1098R, from Examples 2, 3, and 4 was selected as a benchmark and combined with other high-efficiency mutant proteins. Compared to proteins containing the single point mutation G1098R, several mutant combinations—G1098R+H341R, G1098R+K700D, G1098R+W762Q, G1098R+H792W, G1098R+A793V, G1098R+R866V, and G1098R+Q955R—significantly improved gene integration efficiency, demonstrating a significant additive effect. The G1098R+A793V mutant with the highest integration efficiency was further combined with additional point mutations. Compared with the G1098R+A793V mutant, the mutants G1098R+A793V+W762Q, G1098R+A793V+Q955R, G1098R+A793V+Q955R+H341R, and G1098R+A793V+W762Q+Q955R+H341R can significantly improve the efficiency of gene integration.

[0176] Figure 6: Coupling of T5 exonuclease can improve the gene integration efficiency of wild-type TgR2. T5 exonuclease was coupled or inserted at the N-terminus, after amino acid 191 (the position was marked as IN-1), after amino acid 432 (the position was marked as IN-2), after amino acid 1112 (the position was marked as IN-3), and at the C-terminus of wild-type TgR2. It should be noted that two additional glycine (-GG-) were added at both ends of the T5 exonuclease inserted after amino acids 191, 432, and 1112. With reference to wild-type TgR2, the results of the GFP reporter system designed based on Example 1 showed that, with reference to wild-type TgR2, coupling or inserting T5 exonuclease at the N-terminus, IN-2, and IN-3 of wild-type TgR2 can significantly improve the efficiency of gene integration. Inserting T5 exonuclease at the IN-1 position of wild-type TgR2 slightly improved the efficiency of gene integration, while coupling T5 exonuclease at the C-terminus of wild-type TgR2 decreased the efficiency of gene integration.

[0177] Figure 7: Coupling of Sto single-stranded DNA binding protein can improve the gene integration efficiency of wild-type TgR2. Sto single-stranded DNA binding protein was coupled or inserted at the N-terminus, after amino acid 191 (the position was marked as IN-1), after amino acid 432 (the position was marked as IN-2), after amino acid 1112 (the position was marked as IN-3) and C-terminus of wild-type TgR2. It should be noted that two additional glycine (-GG-) were added at both ends of the Sto single-stranded DNA binding protein inserted after amino acids 191, 432 and 1112. The results of the GFP reporter system designed based on Example 1 showed that, compared with the wild-type TgR2, coupling or inserting Sto single-stranded DNA binding protein at the N-terminus, IN-1 and IN-2 of the wild-type TgR2 can significantly improve the efficiency of gene integration. Inserting Sto single-stranded DNA binding protein at the IN-3 position of wild-type TgR2 slightly improved the efficiency of gene integration, while coupling Sto single-stranded DNA binding protein at the C-terminus of wild-type TgR2 decreased the efficiency of gene integration.

[0178] Figure 8: Coupling of CMP epitopes can improve the gene integration efficiency of wild-type TgR2. CMP epitopes were coupled or inserted at the N-terminus, after amino acid 191 (labeled IN-1), after amino acid 432 (labeled IN-2), after amino acid 1112 (labeled IN-3), and at the C-terminus of wild-type TgR2. It should be noted that two additional glycines (-GG-) were added to both ends of the CMP chromatin regulatory peptides inserted after amino acids 191, 432, and 1112, respectively. Results based on the GFP reporter system designed in Example 1 showed that, compared with wild-type TgR2, coupling or inserting CMP epitopes at the N-terminus, IN-2, and IN-3 of wild-type TgR2 significantly improved the gene integration efficiency. Coupling or inserting CMP epitopes at the IN-1 and C-terminus of wild-type TgR2 slightly improved the gene integration efficiency.

[0179] Figure 9: Coupling the RNaseH domain of MLV reverse transcriptase can improve the gene integration efficiency of wild-type TgR2. The RNaseH domain of MLV reverse transcriptase was coupled or inserted at the N-terminus, after amino acid 191 (the position is marked as IN-1), after amino acid 432 (the position is marked as IN-2), after amino acid 1112 (the position is marked as IN-3) and the C-terminus of wild-type TgR2. It should be noted that two additional glycine (-GG-) were added at both ends of the RNaseH domain inserted after amino acids 191, 432 and 1112. The results of the GFP reporter system designed based on Example 1 showed that, compared with wild-type TgR2, inserting the RNaseH domain of MLV reverse transcriptase at the IN-2 and IN-3 positions of wild-type TgR2 can significantly improve the efficiency of gene integration. Coupling or inserting the RNaseH domain of MLV reverse transcriptase at the N-terminus and IN-1 position of wild-type TgR2 slightly improved the efficiency of gene integration, while coupling the RNaseH domain of MLV reverse transcriptase at the C-terminus of wild-type TgR2 decreased the efficiency of gene integration.

[0180] Figure 10: Coupling multiple domains further improves the gene integration efficiency of wild-type TgR2. Any two of the following: T5 exonuclease, the single-stranded DNA binding protein sto7d (Sto) from Sulfolobus tokodaii, a chromatin regulatory peptide (CMP), or the RNaseH domain of MLV reverse transcriptase (RH) (SEQ) were coupled or inserted into the N-terminus of wild-type TgR2 after amino acid 432 (position labeled IN-2). Alternatively, CMP or RH were inserted into any of three positions: the N-terminus, after amino acid 432 (position labeled IN-2), and after amino acid 1112 (position labeled IN-3). The combinations of T5(N)-CMP(IN-2), T5(N)-RH(IN-2), STO(N)-CMP(IN-2), STO(N)-RH(IN-2), and CMP(N)-CMP(IN-2) significantly improved gene integration efficiency. The further combinations of STO(N)+CMP(IN-2)+CMP(IN-3), CMP(N)+CMP(IN-2)+RH(IN-3), and CMP(N)+CMP(IN-2)+CMP(IN-3) showed significant improvements in gene integration efficiency compared to the original combinations. The other combinations showed little or no improvement.

[0181] Figure 11: Effect of changing the order of the 5' homology arm, 5' UTR, 3' homology arm, and 3' UTR on the gene integration efficiency of wild-type TgR2. In the figure, WT represents the donor used for wild-type R2Tg, A represents the donor with the order of the 5' homology arm and 5' UTR changed, B represents the donor with the 5' UTR deleted, C represents the donor with the order of the 3' homology arm and 3' UTR changed, D represents the donor with the 3' UTR deleted, E represents the donor with both the order of the 5' homology arm, 5' UTR changed and the order of the 3' homology arm and 3' UTR changed, and F represents the donor with both the 5' UTR and 3' UTR deleted. Based on the GFP reporter system designed in Example 1, the results showed that the gene integration efficiency of the six donors A, B, C, D, E, and F that changed the element order or deleted the UTR element was significantly reduced compared to the wild-type donor.

[0182] FIG12 : Effects of changing the length of the 5′ homology arm and the 3′ homology arm on the gene integration efficiency of wild-type TgR2. Among them, A1 represents a donor with a 5' homology arm (LHA) length of 150 bp and a 3' homology arm (RHA) length of 150 bp, A2 represents a donor with a 5' homology arm (LHA) length of 100 bp and a 3' homology arm (RHA) length of 100 bp, A3 represents a donor with a 5' homology arm (LHA) length of 50 bp and a 3' homology arm (RHA) length of 50 bp, A4 represents a donor with a 5' homology arm (LHA) length of 20 bp and a 3' homology arm (RHA) length of 20 bp, A5 represents a donor with a 5' homology arm (LHA) length of 10 bp and a 3' homology arm (RHA) length of 10 bp, and A6 represents a 5' homology arm (LHA) length of 100 bp and a 3' homology arm (RHA) length of 50 bp. Length of donor, A7 represents a donor with a 5' homology arm (LHA) length of 100 bp and a 3' homology arm (RHA) length of 20 bp, A8 represents a donor with a 5' homology arm (LHA) length of 100 bp and a 3' homology arm (RHA) length of 10 bp, A9 represents a donor with a 5' homology arm (LHA) length of 50 bp and a 3' homology arm (RHA) length of 100 bp, A10 represents a donor with a 5' homology arm (LHA) length of 20 bp and a 3' homology arm (RHA) length of 100 bp, A11 represents a donor with a 5' homology arm (LHA) length of 10 bp and a 3' homology arm (RHA) length of 100 bp, and A12 represents a donor without a 5' homology arm (LHA) and a 3' homology arm (RHA). Based on the GFP reporter system designed in Example 1, the results showed that the experimental group using homology arm lengths of A1 and A2 donors had the highest gene integration efficiency.

[0183] Figure 13: Effect of 5'UTR and 3'UTR modification on the gene integration efficiency of wild-type TgR2. By modifying the 5'UTR or 3'UTR sequence of the wild-type TgR2 retrotransposase donor RNA, respectively denoted as UTR modification sequences 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, based on the GFP reporter system designed in Example 1, in combination with the wild-type TgR2 retrotransposase, the gene integration efficiency of the wild-type donor RNA and the donor RNA using the UTR modification sequence was tested. Among them, the donors using UTR modification sequences 9, 10, and 11 can significantly improve the gene integration efficiency, while the gene integration efficiency of the donors with the other modification sequences is not significantly improved or decreases.

[0184] Figure 14: Engineered proteins and engineered RNA donors synergistically improve gene editing efficiency. Based on the optimal donor element sequence determined in Example 12, the optimal donor homology arm length determined in Example 13, and the optimal engineered RNA donors with modified 5'UTR and 3'UTR determined in Example 14, the GFP reporter system designed in Example 1 was used to test the G1098R+A793V+Q955R, G1098R+A793V+W762Q+Q955R, G1098R+A793V+W762Q+Q955R+H341R, and G1098R+A793V+Q955R obtained in Example 5. The TgR2 retrotransposase system is a combination of R+H341R (simplified and named PM1, PM2, PM3, and PM4 in sequence) and T5(N)-CMP(IN-2), T5(N)-RH(IN-2), STO(N)-CMP(IN-2), STO(N)-RH(IN-2), CMP(N)-CMP(IN-2), and CMP(N)+CMP(IN-2)+CMP(IN-3) (simplified and named TD1, TD2, TD3, TD4, TD5, and TD6 in sequence) screened in Example 10. Compared with the wild-type TgR2 retrotransposase system and the uncombined preferred proteins (TD1, TD2, TD3, TD4, TD5, TD6, PM1, PM2, PM3, PM4), the combinations of TD1+PM1, TD1+PM2, TD1+PM4, TD2+PM1, TD2+PM2, TD2+PM4, TD3+PM1, TD3+PM2, TD3+PM4, TD4+PM1, TD4+PM2, TD5+PM1, TD5+PM2, and TD5+PM4 significantly improved the gene integration efficiency, while the gene integration efficiency of the other combinations was not significantly improved or the efficiency decreased.

[0185] FIG15 a : Schematic diagram of the in vitro transcription vector for expressing TgR2 protein mRNA and donor RNA.

[0186] Figure 15b: Use of Lipo3000 liposomes to deliver mRNA and RNA donors expressing the R2 retrotransposase protein. The mRNA obtained by in vitro transcription and purification in Example 16 was transfected into HEK 293T cells using Lipo3000 liposomes. 96 hours after transfection, the presence and proportion of GFP cells were detected by fluorescence microscopy and flow cytometry. The engineered RNA donor containing a 5'UTR (SEQ ID NO.16) and a non-natural 3'UTR (SEQ ID NO.46) was named en-Donor, and the engineered R2 retrotransposase protein was named en-TgR2 (SEQ ID NO.77). The results show that the use of engineered retrotransposase protein and engineered RNA donor can significantly improve gene integration efficiency.

[0187] Figure 16: Using LNP to package mRNA expressing the engineered protein (SEQ ID NO.77) and an engineered RNA donor (5'UTR, SEQ ID NO.16; 3'UTR, SEQ ID NO.46), and then transfecting HEK 293T cell lines, Huh7 cell lines, and N2A cell lines, efficient gene integration can be achieved.

[0188] Figure 17: Co-transfection of a plasmid expressing VPX protein can improve the efficiency of retrotransposase gene integration. Based on the GFP reporter system designed in Example 1, the effect of additional co-transfection of plasmids expressing VPX protein from different viral sources (SIV-VPX (SEQ ID No. 85), SIV2-VPX (SEQ ID No. 86), and HIV2-VPX (SEQ ID No. 87)) on gene insertion efficiency was tested. Co-transfection of a VPX protein plasmid derived from HIV2 significantly improved gene integration efficiency.

[0189] Figure 18: The engineered TgR2 system can integrate multiple genes. Based on the GFP reporter system in Example 1, the GFP gene sequence of the donor RNA plasmid was replaced with the corresponding GDNF (SEQ ID No. 88), EPO (SEQ ID No. 89), and Factor VI (SEQ ID No. 53) gene sequences. Combined with the reporter system, the GDNF, EPO, and Factor VI genes were tested on cells. Combined with Elisa and Biophen detection methods, the results showed that the engineered TgR2 system can effectively integrate the GDNF, EPO, and Factor VI genes.

[0190] Figure 19: Truncations of the en-TgR2 protein can improve gene integration efficiency. Based on the GFP reporter system designed in Example 1, the gene insertion efficiency of different truncations of the en-TgR2 protein was tested. The truncations of the en-TgR2 protein were compared with the wild-type TgR2 protein sequence, and the corresponding amino acids at positions 1-80, 1-190, and 1-220 were deleted. The corresponding truncated proteins were named en-TgR2-v1 (SEQ ID No. 91), en-TgR2-v2 (SEQ ID No. 92), and en-TgR2-v3 (SEQ ID No. 93). The results showed that compared with the en-TgR2 protein, the truncations en-TgR2-v1 and en-TgR2-v3 were able to improve the gene insertion efficiency.

[0191] FIG20 : Engineered TgR2:TD5 and TD5-v1 were used to integrate the GFP gene using RNA donors of different structures.

[0192] Figure 21: Amino acid point mutations were introduced into the engineered TgR2:TD5-v1 to test the gene integration efficiency of these mutants.

[0193] Figure 22 shows that various truncated forms of TgR2 can function in mammalian cells, indicating that there is a minimal core structure in the TgR2 protein that is required for function.

[0194] Specific implementation methods

[0195] It should be noted that certain words are used in the specification and claims to refer to specific components. It should be understood by those skilled in the art that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present invention, but the description is based on the general principles of the specification and is not intended to limit the scope of the invention. The scope of protection of the present invention shall be as defined in the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which this disclosure belongs.

[0196] The terms "nucleic acid," "polynucleotide," and "nucleotide sequence" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof. "Oligonucleotide" and "oligonucleotide" are used interchangeably to refer to short polynucleotides having no more than about 50 nucleotides. As used herein, "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. The complementarity percentage represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., 5, 6, 7, 8, 9, 10 out of 10, which are about 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Fully complementary" means that all contiguous residues of a nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to the degree of complementarity of at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

[0197] "Percentage (%) sequence identity" for nucleic acid sequences is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in a particular nucleic acid sequence, after alignment of the sequences (if necessary) by allowing gaps to achieve maximum sequence identity. "Percentage (%) sequence identity" for peptide, polypeptide or protein sequences is the percentage of amino acid residues in a candidate sequence that are identically replaced with the amino acid residues in a particular peptide or amino acid sequence, after alignment of the sequences (if necessary) by allowing gaps to achieve maximum sequence homology. For the purpose of determining amino acid sequence identity percentage, alignment can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0198] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. A protein may have one or more polypeptides. The term also encompasses amino acid polymers that have been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0199] As used herein, "variant" is interpreted as a polynucleotide or polypeptide that is different from a reference polynucleotide or polypeptide but retains the necessary characteristics. A typical variant of a polynucleotide is different from the nucleic acid sequence of another reference polynucleotide. Changes in the variant nucleic acid sequence may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide is different from another reference polypeptide in amino acid sequence. Generally, the differences are limited so that the sequences of the reference polypeptide and the variant are very similar overall and are identical in many regions. The amino acid sequences of the variant and the reference polypeptide may differ by any combination of one or more substitutions, additions, or deletions. The amino acid residues replaced or inserted may or may not be amino acid residues encoded by the genetic code. The variant of a polynucleotide or polypeptide may be naturally occurring (such as an allelic variant), or may be an unknown naturally occurring variant. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0200] As used herein, the term "wild type" has the meaning commonly understood by those skilled in the art to refer to an organism, strain, gene, or characteristic in its typical form as it exists in nature, distinguishing it from mutants or variants. It can be isolated from a source in nature and has not been intentionally modified.

[0201] As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably to refer to the intervention of human effort. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it is naturally associated or naturally occurring.

[0202] As used herein, "cell" should be understood to refer not only to a particular individual cell, but also to the progeny or potential progeny of that cell. Because certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term herein.

[0203] As used herein, the terms "transduction" and "transfection" include methods known in the art for introducing DNA into cells using infectious agents (e.g., viruses) or other means to express a protein or molecule of interest. In addition to viruses or virus-like agents, there are chemical-based transfection methods, such as transfection methods using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine); non-chemical methods, such as electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, plasmid delivery, or transposons; particle-based methods, such as using a gene gun, magnetofection or magnet-assisted transfection, particle bombardment; and hybrid methods (such as nuclear transfection).

[0204] As used herein, the terms "transfected," "transformed," or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid.

[0205] The term "in vivo" refers to inside the organism from which the cell was obtained. "Ex vivo" or "in vitro" refers to outside the organism from which the cell was obtained.

[0206] As used herein, "treatment (treatment / treating)" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, reducing the recurrence rate of the disease, delaying or slowing the progression of the disease, improving the disease state, providing (partial or complete) relief of the disease, reducing the dosage of one or more other drugs required for treating the disease, delaying the progression of the disease, improving the quality of life, and / or prolonging life. "Treatment" also includes reducing the pathological consequences of a disease, condition, or disease. The method of the present invention contemplates any one or more of these therapeutic aspects.

[0207] As used herein, the term "effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease (e.g., to improve, alleviate, mitigate, and / or delay one or more symptoms thereof). As understood in the art, an "effective amount" can be administered in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired therapeutic endpoint.

[0208] "Subject," "individual," or "patient" are used interchangeably herein for purposes of treatment and refer to any animal classified as a mammal, including humans, livestock and farm animals, and zoo, farm, or pet animals such as dogs, horses, cats, cows, etc. In some embodiments, the individual is a human individual.

[0209] It is understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments. Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, a description referring to "about X" includes a description of "X."

[0210] As used herein, reference to an "other than" value or parameter generally means and describes an "other than" value or parameter. For example, the method is not for treating cancer type X, meaning that the method is for treating cancers other than type X.

[0211] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0212] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that claims can be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "only," "only" and the like in connection with the recitation of claim elements, or the use of a limitation such as "no."

[0213] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" in phrases such as "A, B and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0214] Unless otherwise specified, the mutations described herein may include one or more of: insertion, deletion, substitution, and may be mutations of a single amino acid or multiple amino acids.

[0215] In this application, when describing the position of an amino acid, SEQ ID No. 1, i.e., the numbering of the wild-type TgR2 enzyme, is used as the reference sequence. Those skilled in the art will fully understand how to determine the position of an amino acid with reference to the numbering of the reference sequence. For example, when describing K700, it is intended to indicate that the 700th amino acid of the reference SEQ ID No. 1 is K. If the amino acid sequence of the enzyme is modified, for example, if there is a deletion or insertion of an amino acid, resulting in a change in the numbering of the sequence, those skilled in the art should compare the reference sequence to confirm the position of the amino acid. For example, if the first 1-10 amino acids of the reference sequence are deleted, then K700 should refer to the 690th amino acid in the deleted sequence. Those skilled in the art can confirm the specific amino acid to be modified based on the modification of the sequence and by comparing it with the reference sequence.

[0216] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Typically, a suitable vector comprises an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selective markers. The term "vector" should also be interpreted to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like.

[0217] In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. In some embodiments, the vector is a phage vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals.

[0218] In some embodiments, the rAAV construct can be administered to a subject enterally. In some embodiments, the rAAV construct can be administered to a subject parenterally. In some embodiments, the rAAV particles can be administered subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intraventricularly, intramuscularly, intrathecally (IT), intracisternal, intraperitoneally, via inhalation, topically, or by direct injection into one or more cells, tissues, or organs. In some embodiments, the rAAV particles can be administered to a subject by injection into the hepatic artery or portal vein.

[0219] Methods for introducing vectors into mammalian cells are known in the art. Vectors can be transferred into host cells by physical, chemical or biological methods.

[0220] Physical methods for introducing vectors into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector is introduced into the cells by electroporation.

[0221] The methods described herein are applicable to any suitable cell type. In some embodiments, the cell is a bacterium, a yeast cell, a fungal cell, an algae cell, a plant cell or an animal cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cell is a cell of natural origin, such as a cell isolated by a tissue biopsy. In some embodiments, the cell is a cell isolated from a cell line cultured in vitro. In some embodiments, the cell is from a primary cell line. In some embodiments, the cell is from an immortalized cell line. In some embodiments, the cell is a genetically engineered cell.

[0222] The nuclear localization signal herein is a domain of a protein, typically a short amino acid sequence that interacts with a nuclear import vector, allowing the protein to be transported into the cell nucleus. Meanwhile, the nuclear localization signal may also be an RNA sequence. In certain embodiments, the nuclear localization signal is located on a donor RNA. In certain embodiments, the retrotransposase polypeptide is encoded on a first RNA, and the donor RNA is a second, separate RNA, and the nuclear localization signal is located on the donor RNA rather than on the RNA encoding the retrotransposase polypeptide. Although not wishing to be bound by theory, in some embodiments, the RNA encoding the retrotransposase primarily targets the cytoplasm to promote its translation, while the donor RNA primarily targets the nucleus to promote its retrotransposition into the genome. In some embodiments, the nuclear localization signal is at the 3' end, 5' end, or within the donor RNA. In some embodiments, the nuclear localization signal is at the 3' end (e.g., directly at the 3' end) or at the 5' end (e.g., directly at the 5' end) of the heterologous sequence. In some embodiments, the nuclear localization signal is placed outside the 5' UTR or outside the 3' UTR of the donor RNA. In some embodiments, the nuclear localization signal is placed between the 5'UTR and the 3'UTR, wherein optionally, the nuclear localization signal is not transcribed with the transgene (e.g., the nuclear localization signal is in an antisense orientation or downstream of a transcription termination signal or polyadenylation signal). In some embodiments, the nuclear localization sequence is located within an intron. In some embodiments, multiple identical or different nuclear localization signals are in RNA, for example, in a donor RNA. In some embodiments, the length of the nuclear localization signal is less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 bp. Various RNA nuclear localization sequences can be used.

[0223] As used herein, the term "domain" refers to the structure of a biomolecule that contributes to a specific function of a biomolecule. A domain can comprise a continuous region (e.g., a continuous sequence) or different non-continuous regions (e.g., a non-continuous sequence) of a biomolecule. Examples of protein domains include, but are not limited to, endonuclease domains, target DNA binding domains, and reverse transcription domains; examples of nucleic acid domains are regulatory domains, such as transcription factor binding domains.

[0224] In some embodiments, the present application relates to a target DNA binding domain comprising a zinc finger binding motif, a reverse transcriptase domain, and an endonuclease domain.

[0225] In some embodiments, the reverse transcriptase domain refers to a domain with reverse transcription function, and those skilled in the art can use conventional tools as basic local comparison search tools (e.g., BLAST) to identify the reverse transcriptase domain based on homology with other known reverse transcriptase domains. In certain embodiments, the reverse transcriptase domain is modified, for example, by site-specific mutation. In an embodiment, the reverse transcriptase domain is engineered to bind to a heterologous sequence.

[0226] In some embodiments, the endonuclease domain refers to a domain with endonuclease function, and the endonuclease element is a heterologous endonuclease element, such as Fok1 nuclease, type II restriction endonuclease (RLE type nuclease) or another RLE type endonuclease (also referred to as REL). In certain embodiments, the heterologous endonuclease activity has nickase activity and does not form double-strand breaks. Those skilled in the art can use tools as basic local comparison search tools (e.g., BLAST) to identify endonuclease domains based on homology with other known endonuclease domains.

[0227] In some embodiments, the target DNA binding domain is a target DNA binding domain containing a zinc finger binding motif, wherein the zinc finger binding motif is an amino acid sequence responsible for binding to a target DNA of a specific sequence.

[0228] As used herein, the term "exogenous," when used with respect to a biomolecule (e.g., a nucleic acid sequence or polypeptide), refers to a biomolecule that is artificially introduced into a host genome, cell, or organism. For example, a nucleic acid that is added to an existing genome, cell, tissue, or subject using recombinant DNA technology or other methods is exogenous to the existing nucleic acid sequence, cell, tissue, or subject.

[0229] As used herein, the term "heterologous" means that when used to describe a first element with reference to a second element, the term heterologous means that the first element and the second element do not exist in the arrangement as described in nature. For example, a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or a portion of a polypeptide or nucleic acid molecule sequence that is not natural for the cell expressing it, (b) a polypeptide or nucleic acid molecule or a portion of a polypeptide or nucleic acid molecule that has been changed or mutated relative to its natural state, or (c) a polypeptide or nucleic acid molecule with expression that is changed compared to the natural expression level under similar conditions. For example, a heterologous regulatory sequence (e.g., a promoter, an enhancer) can be used to regulate the expression of a gene or nucleic acid molecule in a manner different from that in which the gene or nucleic acid molecule is typically expressed in nature. In another example, the heterologous domains of a polypeptide or nucleic acid sequence (e.g., a DNA binding domain of a polypeptide or a nucleic acid encoding a DNA binding domain of a polypeptide) can be arranged relative to other domains, or can be different sequences or come from different sources relative to other domains or portions of the polypeptide or its encoding nucleic acid. In certain embodiments, the heterologous nucleic acid molecule may be present in the native host cell genome, but may have an altered expression level or a different sequence, or both. In other embodiments, the heterologous nucleic acid molecule may not be endogenous to the host cell or host genome, but may have been introduced into the host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may be integrated into the host genome or may exist transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vectors, plasmids, or other self-replicating vectors) as extrachromosomal genetic material.

[0230] As used herein, the term "gene expression unit" is a nucleic acid sequence comprising at least one regulatory nucleic acid sequence operably linked to at least one effector sequence. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous or non-contiguous. In cases where it is desired to link two protein coding regions, the operably linked sequences can be in the same reading frame.

[0231] As used herein, the term "host genome or host cell" refers to a cell and / or its genome into which proteins and / or genetic material have been introduced. It should be understood that these terms are intended to refer not only to a specific subject cell and / or genome, but also to the offspring of such a cell and / or the genome of the offspring of such a cell. Because certain modifications may occur in offspring due to mutations or environmental influences, such offspring may actually be different from the parent cell, but are still included in the scope of the term "host cell" used herein. A host genome or host cell can be an isolated cell or cell line grown in culture, or a genomic material isolated from such a cell or cell line, or can be a host cell or host genome that constitutes a living tissue or organism. In some cases, a host cell can be an animal cell or a plant cell, for example, as described herein. In some cases, a host cell can be a bovine cell, a horse cell, a pig cell, a goat cell, a sheep cell, a chicken cell, or a turkey cell. In some cases, a host cell can be a corn cell, a soybean cell, a wheat cell, or a rice cell.

[0232] As used herein, genomic safe harbor sites (GSH sites) are sites in the host genome that can accommodate the integration of new genetic material, for example, so that the inserted genetic elements do not cause significant changes in the host genome that pose a risk to the host cell or organism. GSH sites typically meet 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the following conditions: (i) >300 kb from cancer-related genes; (ii) >300 kb from miRNA / other functional small RNAs; (iii) >50 kb from the 5' gene end; (iv) >50 kb from the replication origin; (v) >50 kb from any extremely conserved element; (vi) low transcriptional activity (i.e., no mRNA + / - 25 kb); (vii) not in a copy number variable region; (viii) in open chromatin; and / or (ix) unique, with 1 copy in the human genome. Examples of GSH sites in the human genome that meet some or all of these criteria include: (i) adeno-associated virus site 1 (AAVS1), which is the natural site of integration of the AAV virus on chromosome 19; (ii) the chemokine (CC motif) receptor 5 (CCR5) gene, a chemokine receptor gene known as the HIV-1 co-receptor; (iii) the human ortholog of the mouse Rosa26 locus; and (iv) rDNA loci. Additional GSH sites are known and described, for example, in Pellenz et al., published electronically on August 20, 2018 (https: / / doi.org / 10.1101 / 396390).

[0233] In some embodiments, the genomic safe harbor site is a Natural Harbor TMIn some embodiments, Natural Harbor TM The locus is ribosomal DNA (rDNA). In some embodiments, Natural Harbor TM The locus is 5S rDNA, 18S rDNA, 5.8S rDNA or 28S rDNA. In some embodiments, Natural Harbor TM The site is the Mutsu site in 5S rDNA. In some embodiments, the Natural Harbor T The M site is the R2 site in the 28S rDNA.

[0234] As used herein, a "pseudoknot sequence" refers to a nucleic acid (eg, RNA) having a sequence with appropriate self-complementarity to form a pseudoknot structure.

[0235] As used herein, a "stem-loop sequence" refers to a nucleic acid sequence (e.g., an RNA sequence) that has sufficient self-complementarity to form a stem-loop, e.g., having a stem comprising at least two (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs and a loop having at least three (e.g., four) base pairs. The stem may contain mismatches or bulges.

[0236] In some embodiments, the cell is an animal cell of an organism selected from the group consisting of cow, sheep, goat, horse, pig, deer, chicken, duck, goose, rabbit, and fish.

[0237] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a human embryonic kidney 293T (HEK293T or 293T) cell or a HeLa cell. In some embodiments, the cell is a human embryonic kidney (HEK293T) cell. In some embodiments, the cell is a mouse Hepa1-6 cell. In some embodiments, the mammalian cell is selected from the group consisting of an immune cell, a liver cell, a tumor cell, a stem cell, a blood cell, a neural cell, a zygote, a muscle cell (such as a cardiomyocyte) and a skin cell.

[0238] In some embodiments, the cell is an immune cell selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) T cells, iNK-T cells, NK-T-like cells, γδT 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.

[0239] In some embodiments, the cell is an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a gamete progenitor cell, a gamete, a zygote, or a cell in an embryo.

[0240] The retrotransposase of the present application comprises a target DNA binding domain containing a zinc finger binding motif, a reverse transcriptase domain, and a nuclease endonuclease domain, and is capable of reverse transcribing RNA into DNA.

[0241] The present application relates to an engineered retrotransposase, which is a mutant of the wild-type retrotransposase TgR2. The sites described herein as capable of mutation can be combined, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 sites can be selected for mutation.

[0242] For example, the engineered retrotransposase is based on a sequence change of the wild-type retrotransposase TgR2, which is equivalent to the wild-type retrotransposase TgR2, and the amino acid at position 1098 is mutated, such as a mutation with G1098R. In one embodiment, the engineered retrotransposase is based on a sequence change of the wild-type retrotransposase TgR2, which is equivalent to the wild-type retrotransposase TgR2, and the amino acid at positions 1098 and 793 is mutated, such as a mutation with G1098R and A793V. In one embodiment, the engineered retrotransposase is based on a sequence change of the wild-type retrotransposase TgR2, which is equivalent to the wild-type retrotransposase TgR2, and the amino acid at positions 1098, 793, and 955 is mutated, such as a mutation with G1098R, A793V, and Q955R (also referred to herein as PM1). In one embodiment, the engineered retrotransposase is based on a sequence variation of the wild-type retrotransposase TgR2, equivalent to the wild-type retrotransposase TgR2, wherein the amino acids at positions 1098, 793, 955, and 762 are mutated, for example, with mutations of G1098R, A793V, Q955R, and W762Q (also referred to herein as PM2). In one embodiment, the engineered retrotransposase is based on a sequence variation of the wild-type retrotransposase TgR2, equivalent to the wild-type retrotransposase TgR2, wherein the amino acids at positions 1098, 793, 955, 762, and 341 are mutated, for example, with mutations of G1098R, A793V, Q955R, W762Q, and H341R (also referred to herein as PM3). In one embodiment, the engineered retrotransposase is based on sequence changes of the wild-type retrotransposase TgR2, which is equivalent to the wild-type retrotransposase TgR2, with mutations at amino acids 1098, 793, 955 and 341, for example, with mutations G1098R, A793V, Q955R and H341R (also referred to as PM4 in this article).

[0243] In this application, in addition to the mutations described above, those skilled in the art may further perform conservative mutations on amino acids not involved in the active site based on their knowledge, that is, mutate one amino acid into another amino acid with similar physicochemical properties.

[0244] In this application, all amino acid numbering is based on the amino acid sequence of wild-type transposase TgR2 (sequence shown in SEQ ID No. 1). Those skilled in the art will fully understand that the amino acid sequence itself will change if truncations, insertions, etc. occur in the amino acid sequence, but the description is still based on the original sequence of wild-type transposase TgR2. When describing mutations in this application, the letter before the position number represents the amino acid before the mutation, and the letter after the position number represents the amino acid after the mutation. For example, G1098R indicates that the amino acid at position 1098 of wild-type transposase TgR2 has mutated from G to R. The remaining mutations are described in the same manner.

[0245] In a specific embodiment, the amino acid sequence of the retrotransposase of the present application is as shown in any one of SEQ ID No. 2-7, or has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of any one of SEQ ID No. 2-7.

[0246] The present application relates to an engineered retrotransposase, which is a truncated version of the wild-type retrotransposase TgR2.

[0247] In a specific embodiment, the retrotransposase of the present application is a truncation of the wild-type retrotransposase TgR2, for example, the 1-1200th amino acid can be truncated from the N-terminus of the wild-type retrotransposase TgR2, for example, the 1-10th, 1-20th, 1-30th, 1-40th, 1-50th, 1-60th, 1-70th, 1-80th, 1-90th, 1-100th, 1-110th, 1-120th, 1-130th, 1-140th, 1-150th, 1-160th, 1-170th, 1-180th, 1-190th, 1-200th, 1-210th, , No. 1-220, No. 1-230, No. 1-240, No. 1-250, No. 1-260, No. 1-270, No. 1-280, No. 1-290, No. 1-300, No. 1-310, No. 1-320, No. 1-330, No. 1-340, No. 1-350, No. 1-360, No. 1-370, No. 1-380, No. 1-390, No. 1-400, No. 1-410, No. 1-420, No. 1-430, No. 1-440, No. 1-450, No. 1-460, No. 1-470, No. 1-480, No. 1-490, No. 1-500. It can also be truncated, for example, at amino acids 200-500, 210-500, 220-500, 230-500, 240-500, 250-500, 260-500, 270-500, 280-500, 290-500, 300-500, 310-500, 320-500, 330-500, 340-500, 350-500, 360-500, 370-500, 380-500, 390-500, and 400-500. It can also be truncated, for example, at amino acids 1000-1200, 1010-1190, 1020-1180, 1030-1180, 1040-1170, 1050-1160, 1060-1150, 1070-1140, 1080-1150, 1090-1140, 1100-1130, 1100-1120.

[0248] When describing the number of amino acids truncated in this application, the amino acid numbering is also based on the determination of the wild-type retroposase TgR2, that is, the description is based on the sequence set forth in SEQ ID No. 1. For example, when describing the 1st to 220th amino acid of the N-terminal truncation, it corresponds to the 1st to 220th amino acid of the sequence set forth in SEQ ID No. 1, as counted from the N-terminus. For example, when describing the 390th to 550th amino acid of the N-terminal truncation, it corresponds to the 390th to 550th amino acid of the sequence set forth in SEQ ID No. 1, as counted from the N-terminus. For example, when describing the 1100th to 1120th amino acid of the N-terminal truncation, it corresponds to the 1100th to 1120th amino acid of the sequence set forth in SEQ ID No. 1, as counted from the N-terminus.

[0249] In the present application, the truncation positions described above can be combined, as long as those skilled in the art confirm that these positions will not affect the activity of the retrotransposase of the present application. For example, amino acids 1-10 and 490-500 can be truncated. For example, amino acids 1-220 and amino acids 390-550 can be truncated simultaneously from segment N.

[0250] In a specific embodiment, the amino acid sequence of the retrotransposase of the present application is as shown in any one of SEQ ID No. 13, 38, 40, and 51, or has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of any one of SEQ ID No. 13, 38, 40, and 51.

[0251] In a specific embodiment, the retroposotase of the present application can be a truncated version of the retroposotase mutant described in the present application. For example, amino acids 1-500 corresponding to the wild-type sequence SEQ ID NO. 1 can be truncated from the mutant of the wild-type retroposotase TgR2, for example, amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, 1-250, 1-260, 1-270, 1-280, 1-290, 1-300, 1-310, 1-320, 1-330, 1-340, 1-351, 1-360, 1-370, 1-380, 1-390, 1-401, 1-410, 1-420, 1-430, 1-440, 1-451, 1-460, 1-470 1-250th, 1-260th, 1-270th, 1-280th, 1-290th, 1-300th, 1-310th, 1-320th, 1-330th, 1-340th, 1-350th, 1-360th, 1-370th, 1-380th, 1-390th, 1-400th, 1-410th, 1-420th, 1-430th, 1-440th, 1-450th, 1-460th, 1-470th, 1-480th, 1-490th, 1-500th. It can also be truncated, for example, at amino acids 200-500, 210-500, 220-500, 230-500, 240-500, 250-500, 260-500, 270-500, 280-500, 290-500, 300-500, 310-500, 320-500, 330-500, 340-500, 350-500, 360-500, 370-500, 380-500, 390-500, and 400-500. It can also be truncated, for example, at amino acids 1000-1200, 1010-1190, 1020-1180, 1030-1180, 1040-1170, 1050-1160, 1060-1150, 1070-1140, 1080-1150, 1090-1140, 1100-1130, 1100-1120.

[0252] In the present application, those skilled in the art will fully understand that the wild-type retrotransposase TgR2 can be truncated first and then mutated, or of course the wild-type retrotransposase TgR2 can be mutated first and then truncated, but the described amino acid sequences are all based on the numbering of the wild-type retrotransposase TgR2.

[0253] The engineered retrotransposase of the present application may also be obtained by further coupling other amino acid sequences to the wild-type retrotransposase TgR2. The engineered retrotransposase of the present application may also be obtained by further coupling other amino acid sequences to a truncated form of the wild-type retrotransposase TgR2. The engineered retrotransposase of the present application may also be obtained by further coupling other amino acid sequences to a mutant of the wild-type retrotransposase TgR2. The engineered retrotransposase of the present application may also be obtained by further coupling other amino acid sequences to a truncated form or mutant of the wild-type retrotransposase TgR2.

[0254] The other amino acids in this application refer to any other amino acid sequence different from the retrotransposase function, which can be any peptide sequence with regulatory function, any peptide sequence with connecting function, or a non-functional peptide sequence without any function.

[0255] For example, other amino acid sequences can be selected from nuclease exonucleases, single-stranded DNA binding proteins, chromatin regulatory peptides, high-mobility peptides, RNaseH domains, nuclear import localization signals (NLS), and nuclear export localization signals.

[0256] In the present application, the above-mentioned other amino acid sequences can be inserted into the N-terminal position of the wild-type retrotransposase TgR2, or after amino acid No. 191, amino acid No. 432, or amino acid No. 1112.

[0257] In the present application, the above-mentioned other amino acid sequences can be inserted into the N-terminal position of the mutant of the wild-type retrotransposase TgR2, or after amino acid No. 191, amino acid No. 432, or amino acid No. 1112.

[0258] In the present application, the above-mentioned other amino acid sequences can be inserted into the N-terminal position of the truncated form of the wild-type retrotransposase TgR2, or after amino acid No. 191, after amino acid No. 432, or after amino acid No. 1112. In this case, the amino acid numbering described still corresponds to the numbering of the wild-type retrotransposase TgR2.

[0259] In the present application, the above-mentioned other amino acid sequences can be inserted into the N-terminal position of the truncated and mutant forms of the wild-type retrotransposase TgR2, or after amino acid No. 191, after amino acid No. 432, or after amino acid No. 1112. In this case, the amino acid numbering described still corresponds to the numbering of the wild-type retrotransposase TgR2.

[0260] In addition, those skilled in the art will also understand that when other amino acid sequences are inserted in combination, the multiple other amino acid sequences can be inserted in series at any of the above positions, such as the N-terminal position, or after amino acid No. 191, after amino acid No. 432, or after amino acid No. 1112. At this time, the amino acid numbering described still corresponds to the numbering of the wild-type retrotransposase TgR2.

[0261] In the present application, when describing insertion at the N-terminal position, it can usually be inserted before the first amino acid at the N-terminus or after the first amino acid, and those skilled in the art can make the selection based on the situation.

[0262] In one embodiment, the exonuclease in the present application is a nuclease from T5 phage, for example, as shown in SEQ ID NO. 8 or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No. 8.

[0263] In one embodiment, the single-stranded DNA binding protein in the present application is a single-stranded DNA binding protein from the bacterium Sulfolobus tokodaii, for example, as shown in SEQ ID NO.9 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No.9.

[0264] In one embodiment, the chromatin regulatory peptide or high mobility peptide in the present application is a chromatin regulatory peptide or high mobility peptide from human, for example, as shown in SEQ ID NO.10 or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No.10.

[0265] In one embodiment, the RNaseH domain in the present application is an RNaseH domain from MLV reverse transcriptase, for example, as shown in SEQ ID NO.11 or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No.11.

[0266] Those skilled in the art will fully appreciate that the other amino acids inserted above can be inserted in combination, can be inserted randomly, or can be inserted at one position, such as the N-terminal position, after the RNaseH domain and amino acid No. 191 are inserted into the chromatin regulatory peptide. In the present application, the engineered retrotransposase can be selected from the amino acid sequence shown in any one of SEQ ID NOs. 2-7, 12, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55-84, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of any one of SEQ ID NOs. 2-7, 12, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55-84.

[0267] In the present application, the engineered retrotransposase is a truncated version of the amino acid sequence shown in any one of SEQ ID NOs. 1-7, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55 to 84. For example, the sequence is obtained by truncating the amino acid sequence shown in any one of SEQ ID NOs. 1-7, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55 to 84 from the wild-type sequence SEQ ID NO. 1 corresponding to amino acids 1-80, or 1-190, or 1-220, 390-500, or 1100-1120.

[0268] The present application relates to a system for modifying DNA, comprising: the retrotransposase of the present application (including wild-type retrotransposase or engineered retrotransposase) or a nucleic acid encoding the retrotransposase of the present application (including wild-type retrotransposase or engineered retrotransposase); and a donor RNA or a nucleic acid encoding the donor RNA, wherein the donor RNA comprises: a sequence that binds to the retrotransposase and a heterologous sequence, preferably the heterologous sequence is at least 1-50,000 bases, for example, more than 1 nt, more than 10 nt, more than 50 nt, more than 60 nt, or more than 1 nt. Above t, above 70nt, above 80nt, above 90nt, above 100nt, above 150nt, above 200nt, above 250nt, above 300nt, above 350nt, above 400nt, above 450nt, above 500nt Above, above 550nt, above 600nt, above 650nt, above 700nt, above 750nt, above 800nt, above 850nt, above 900nt, above 950nt, above 1000nt, above 1100nt, 12 00nt or more, 1300nt or more, 1400nt or more, 1500nt or more, 1600nt or more, 1700nt or more, 1800nt or more, 1900nt or more, 2000nt or more, 2100nt or more, 2200nt or more, 2300nt or more, 2400nt or more, 2500nt or more, 2600nt or more, 2700nt or more, 2800nt or more, 2900nt or more, 3000nt or more, 3500nt or more, 4000nt or more, 45 00nt or more, 5000nt or more, 5500nt or more, 6000nt or more, 6500nt or more, 7000nt or more, 7500nt or more, 8000nt or more, 8500nt or more, 9000nt or more, 9500nt or more, 10000nt or more, 15000nt or more, 20000nt or more, 25000nt or more, 30000nt or more, 35000nt or more, 40000nt or more, or 45000nt or more, wherein the retrotransposase is the retrotransposase shown in SEQ ID NO. 1 or the engineered retrotransposase described in the present application.

[0269] In one embodiment, the system for modifying DNA of the present application includes: the retrotransposase of the present application (including wild-type retrotransposase or engineered retrotransposase) or a nucleic acid encoding the retrotransposase described in the present application (including wild-type retrotransposase or engineered retrotransposase); and a donor RNA or a nucleic acid encoding the donor RNA, wherein the donor RNA comprises: a sequence that binds to the retrotransposase and a heterologous sequence, wherein the heterologous sequence is at least 1-50,000 bases.

[0270] In one embodiment, the system for modifying DNA of the present application includes, in addition to the retrotransposase of the present application (including wild-type retrotransposase or engineered retrotransposase) or a nucleic acid encoding the retrotransposase of the present application (including wild-type retrotransposase or engineered retrotransposase); and a donor RNA or a nucleic acid encoding the donor RNA; a nucleic acid encoding a VPX protein or the VPX protein itself.

[0271] In one embodiment, the amino acid sequence of the VPX protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID No. 85, 86, or 87.

[0272] In a specific embodiment, as shown in Figure 1, the retrotransposase and donor RNA are separately encoded by two plasmids, the retrotransposase gene is expressed by the CAG promoter, and the donor RNA is expressed by the CAG promoter. The expression frame of the donor RNA is also embedded with a reverse expression frame that is expressed by the CMV promoter. In the expression frame that is expressed by the CMV promoter, CMV expresses GFP, but the GFP here is interrupted by an intron sequence inserted in the opposite direction. Therefore, the final mode of action of the system of the present application is: after the donor RNA is expressed by the CAG promoter, the intron sequence contained in the donor RNA is sheared off from the expressed RNA, and the GFP sequence in the expression frame that is expressed by the CMV promoter returns to normal at the RNA level. However, the GFP sequence at this time is an antisense RNA chain and does not have the ability to translate into GFP protein. When and only when the retrotransposase permanently integrates the intron-less donor RNA into the cell's DNA through reverse transcriptase activity, the reverse expression frame initiated by the CMV promoter can express normal intron-free GFP mRNA, and then express GFP protein with green fluorescence.

[0273] The heterologous sequence of the present application is selected from one or more of the following: a sequence encoding a polypeptide, a sequence containing an organizational promoter or enhancer, and a sequence encoding one or more introns.

[0274] In a specific embodiment, the polypeptide is a therapeutic polypeptide or a mammalian polypeptide; further preferably, the polypeptide is a therapeutic protein, a membrane protein, an intracellular protein, an extracellular protein, a structural protein, a signal transduction protein, a regulatory protein, a transport protein, an organelle protein, a sensory protein, a motor protein, a defense protein, a storage protein, a reporter protein, an antibody, an enzyme, or a coagulation factor.

[0275] In a specific embodiment, the number of amino acids in the polypeptide is 20 to 10,000, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670 60, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, 8 00, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100 , 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600 , 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 78 00, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900.

[0276] In a specific embodiment, the intracellular protein is selected from cytoplasmic proteins, nuclear proteins, organelle proteins, mitochondrial proteins or lysosomal proteins. In a specific embodiment, the sequence encoding the polypeptide contains one or more introns.

[0277] The systems described herein can be used in vitro or in vivo. In some embodiments, the system or system components are delivered to cells (e.g., mammalian cells, such as human cells) in vitro or in vivo. In some embodiments, the cells are eukaryotic cells, such as cells of multicellular organisms, such as animals, such as mammals (e.g., humans, pigs, cattle), birds (e.g., poultry, such as chickens, turkeys, or ducks), or fish. In some embodiments, the cells are non-human animal cells (e.g., experimental animals, livestock, or companion animals). In some embodiments, the cells are stem cells (e.g., hematopoietic stem cells), fibroblasts, or T cells. In some embodiments, the cells are non-dividing cells, such as non-dividing fibroblasts or non-dividing T cells. In some embodiments, the cells are plant cells. It will be understood by those skilled in the art that the components of the Gene Writer system can be delivered in the form of polypeptides, nucleic acids (e.g., DNA, RNA), and combinations thereof.

[0278] In the present application, delivery can use any combination of the following to deliver the retrotransposase (e.g., as DNA encoding the retrotransposase protein, as RNA encoding the retrotransposase protein, or as the protein itself) and the donor RNA (e.g., as DNA encoding the RNA, or as RNA):

[0279] 1. Retrotransposase DNA + donor DNA

[0280] 2. Retrotransposase RNA + donor DNA

[0281] 3. Retrotransposase DNA + donor RNA

[0282] 4. Retrotransposase RNA + donor RNA

[0283] 5. Retrotransposase protein + donor DNA

[0284] 6. Retrotransposase protein + donor RNA

[0285] 7. Retrotransposase virus + virus containing donor RNA or DNA

[0286] 8. Retrovirus + donor DNA

[0287] 9. Retrotransposase virus + donor RNA

[0288] 10. Retrotransposase DNA + virus containing donor RNA or DNA

[0289] 11. Retrotransposase RNA + virus containing donor RNA or DNA

[0290] 12. Retrotransposase protein + virus containing donor RNA or DNA

[0291] As described above, in some embodiments, viruses are used to deliver DNA or RNA encoding a retrotransposase protein, and in some embodiments, viruses are used to deliver the donor RNA (or DNA encoding the donor RNA).

[0292] In one embodiment, the system and / or components of the system are delivered in the form of nucleic acids. For example, the retrotransposase polypeptide can be delivered in the form of DNA or RNA encoding the polypeptide, and the donor RNA can be delivered in the form of RNA or its complementary DNA to be transcribed into RNA. In some embodiments, the system or components of the system are delivered on 1, 2, 3, 4 or more different nucleic acid molecules. In some embodiments, the system or components of the system are delivered as a combination of DNA and RNA. In some embodiments, the system or components of the system are delivered as a combination of DNA and protein. In some embodiments, the system or components of the system are delivered as a combination of RNA and protein. In some embodiments, the retrotransposase polypeptide is delivered as a protein.

[0293] In some embodiments, a system or a component of a system is delivered to a cell, such as a mammalian cell or a human cell, using a vector. The vector can be, for example, a plasmid or a virus. In some embodiments, delivery is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the virus is an adeno-associated virus (AAV), a lentivirus, or an adenovirus. In some embodiments, the system or a component of a system is delivered to a cell together with a virus-like particle or a virion. In some embodiments, delivery uses more than one virus, virus-like particle, or virion.

[0294] In one embodiment, compositions and systems as described herein can be formulated in liposomes or other similar vesicles. Liposomes are spherical vesicle structures that are composed of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer. Liposomes can be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their loads across biological membranes and the blood-brain barrier (BBB).

[0295] Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to produce liposomes as drug carriers. Methods for preparing multilamellar vesicle lipids are known in the art (see, for example, U.S. Patent No. 6,693,086, which is incorporated herein by reference for its teachings on the preparation of multilamellar vesicle lipids). Although the formation of vesicles is spontaneous when the lipid film is mixed with an aqueous solution, the formation of vesicles can also be accelerated by applying force in the form of oscillation using a homogenizer, sonicator, or extrusion device. Extruded lipids can be prepared by extruding through a filter with a reduced size.

[0296] Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for the pharmaceutical compositions described herein. Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the properties of SLNs, improve drug stability and drug loading, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively guide drug delivery to specific targets and improve drug stability and controlled drug release. Lipopolymer nanoparticles (PLNs) can also be used, which are a new type of carrier that combines liposomes and polymers. These nanoparticles have the complementary advantages of PNPs and liposomes. PLNs are composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. In this way, these two components improve drug encapsulation efficiency, promote surface modification, and prevent leakage of water-soluble drugs.

[0297] In one embodiment, the donor RNA described in the present application comprises, from the 5' end to the 3' end, a 5' untranslated sequence (5'UTR) that binds to the retrotransposase, a heterologous sequence, and a 3' untranslated sequence (3'UTR) that binds to the retrotransposase.

[0298] In one embodiment, the donor RNA described in the present application comprises, from the 5' end to the 3' end, a 5' untranslated sequence (5'UTR) to which the retrotransposase binds, a promoter operably linked to a heterologous sequence, a heterologous sequence, and a 3' untranslated sequence (3'UTR) to which the retrotransposase binds.

[0299] In one embodiment, the donor RNA described in the present application comprises, from the 5' end to the 3' end, a 5' untranslated sequence (5'UTR) to which the retrotransposase binds, a heterologous sequence, a promoter operably linked to the heterologous sequence, and a 3' untranslated sequence (3'UTR) to which the retrotransposase binds.

[0300] In one embodiment, the 5' untranslated sequence (5'UTR) is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID Nos. 16, 27, 29, 31, 33, 35, 37, 39, or 41.

[0301] In one embodiment, the 3' untranslated sequence (3'UTR) is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID Nos. 17, 32, 34, 44, 46, 48, 50, 52, or 54.

[0302] In one embodiment, the donor RNA of the present application comprises the following structures from its 5' end to its 3' end: a first homologous domain, a 5' untranslated sequence (5'UTR) that binds to the retrotransposase, a heterologous sequence, a 3' untranslated sequence (3'UTR) that binds to the retrotransposase, and a second homologous domain.

[0303] In one embodiment, the sequence of the first homology domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO. 15, 19, 21, 23, 25, or 28.

[0304] In one embodiment, the sequence of the second homology domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO. 18, 20, 22, 24, 26, or 36.

[0305] The donor RNA of the present application can also become an engineered transposable element.

[0306] The sequences of this application are all expressed in the form of deoxynucleotides. If it represents an RNA sequence, those skilled in the art can replace T with U. When this application describes a sequence, it can represent an RNA sequence and its corresponding DNA sequence. Example

[0307] Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although specific embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0308] Experimental plan

[0309] Plasmid construction

[0310] The wild-type TgR2 retrotransposase protein sequence was codon-optimized (human) and synthesized. The DNA encoding the fragment was inserted into the pCAG-SV40 poly(A) vector between the XmaI and NheI restriction sites by Gibson cloning. A point mutation in the TgR2 retrotransposase was introduced by PCR using primers harboring the mutant sequence. The DNA encoding the fragment was then inserted into the pCAG-SV40 poly(A) vector between the XmaI and NheI restriction sites by Gibson cloning. The plasmid expressing the donor RNA was constructed by separately amplifying multiple sequences containing GFP(N)-intron-GFP(C), the 5'-UTR sequence, the 3'-UTR sequence, and the CMV promoter by overlapping PCR. Finally, the DNA encoding the fragment was inserted into the pSV40-mCherry vector by Gibson cloning.

[0311] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0312] HEK293T cell lines were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-well plates-cell culture dishes (Corning) for 16 hours until the cell density reached 70%-90%. Using Lipofectamine 3000 (Invitrogen), 250 ng of a plasmid encoding a retrotransposase protein and 250 ng of a plasmid expressing a donor RNA were transfected into each 24-well plate-cell culture dish. 24 hours after transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco). Cells with mCherry signals were then sorted using a MoFlo XDP (Beckman Coulter) instrument and reseeded back into 12-well plates. After 6 days of culture, the cells were digested with trypsin-EDTA (0.05%) (Gibco) and analyzed using a BD FACSAria. TM The proportion of cells with GFP-positive signals was analyzed using a Fusion Cell Sorter (BD). Comparison with the proportion of cells with GFP-positive signals in a negative control and observation under a fluorescence microscope confirmed whether the engineered retrotransposase system had enhanced gene integration efficiency in mammalian cells.

[0313] Example 1: Construction of GFP reporter system

[0314] This example designs a reporter system that accurately reflects whether the novel retrotransposase system is functional in mammalian cells (see Figure 1). Specifically, the retrotransposase protein and donor RNA are separately encoded by two plasmids, reflecting the modularity of the novel retrotransposase system. The donor RNA is expressed from the CAG promoter. It is worth noting that the expression cassette that initiates expression of the donor RNA from the CAG promoter also incorporates a reverse expression cassette that initiates expression from the CMV promoter. In the expression cassette initiated by the CMV promoter, CMV expresses GFP, but the GFP here is interrupted by an intron sequence inserted in the reverse direction.

[0315] Therefore, the final mode of action of the reporter system constructed in Example 1 of the present application is: after the donor RNA is expressed by the CAG promoter, the intron sequence contained in the donor RNA is cut off from the expressed RNA, and the GFP sequence in the expression frame expressed by the CMV promoter returns to normal at the RNA level. However, the GFP sequence at this time is an antisense RNA chain and does not have the ability to translate GFP protein. When and only when the new retrotransposase permanently integrates the donor RNA that has lost introns into the DNA of the cell through reverse transcriptase activity, the reverse expression frame expressed by the CMV promoter can express normal GFP mRNA without introns, and then express GFP protein with green fluorescence. Therefore, by detecting whether the final GFP cells exist and the proportion they occupy by fluorescence microscopy and flow cytometry, we can judge whether the new retrotransposase can work in mammalian cells and how high its activity is.

[0316] Example 2: Truncated wild-type TgR2 is still active

[0317] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of the wild-type TgR2 retrotransposase system and its multiple mutants. It should be noted that the donor RNA of the retrotransposase system generally consists of five parts: the homology arm-left, the 5'UTR sequence, the 3'UTR sequence, the sequence between the 5'UTR sequence and the 3'UTR sequence carrying the new gene information, and the homology arm-right (see Figure 1).

[0318] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0319] The plasmids used in this example were constructed using the methods described in the experimental protocol above. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the experimental protocol above. This example constructed the following truncations of wild-type TgR2: Δ1-220 (SEQ ID No. 51); Δ390-500 (SEQ ID No. 13); Δ1100-1200 (SEQ ID No. 38); and Δ1-220+390-500 (SEQ ID No. 40).

[0320] The results showed that, compared with the wild-type TgR2 (SEQ ID No. 1), its truncated forms all maintained a certain activity. The experimental results of Example 2 are shown in Table 1 and Figure 22:

[0321] Table 1

[0322] Example 3: Introducing amino acid point mutations based on homologous sequence alignment to improve the gene integration efficiency of wild-type TgR2

[0323] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of the wild-type TgR2 retrotransposase system and its multiple mutants. It should be noted that the donor RNA for the retrotransposase system generally consists of five parts: a left homology arm, a 5'UTR sequence, a 3'UTR sequence, a sequence between the 5'UTR and 3'UTR sequences carrying the new genetic information, and a right homology arm (see Figure 1).

[0324] Sequence alignment

[0325] A multiple sequence alignment of the reverse transcriptase sequences in the R2 protein was performed using the FFT-NS-I x1000 algorithm of MAFFT. The best-fitting model was determined using the Bayesian Information Criterion (BIC) of ModelFinder software. A total of 543 protein models were tested. The most suitable model was: LG+R7+F. A maximum likelihood (ML) phylogenetic tree was then constructed using the LG+R7+F model of IQ-TREE and ultrafast bootstrapping parameters. Multiple sequence alignment results within the same branch were extracted based on the position of the TgR2 protein in the phylogenetic tree. Amino acids in TgR2 were mutated to other amino acid sequences corresponding to the corresponding positions in the multiple sequence alignment.

[0326] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0327] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0328] Based on the sequence alignment results, this example selected 36 amino acids in TgR2 (SEQ ID No. 1): K700, L704, V727, S732, L737, A747, A748, T759, I760, W762, S763, H792, A793, Q795, E798, P801, V807, S808, E812, S815, T819, T820, H825, K828, S860, Y864, H865, R866, Q868, S869, N889, N891, T892, S895, Q911, and D957, and mutated them into other amino acid sequences corresponding to the corresponding sites in the multiple sequence alignment. Using the GFP reporter system in Example 1, engineered TgR2 systems with single amino acid mutations were expressed. The preferred amino acid replacement methods and their corresponding gene insertion efficiencies are shown in Figure 2. The results showed that, compared with the wild-type TgR2, the following six amino acid replacement mutants: K700D, W762Q, H792W, A793V, R866V, and D957G were able to significantly improve the efficiency of gene integration, but the efficiency of most mutants was not significantly improved, and some were even reduced. The experimental results of Example 2 are shown in Table 2 and Figure 2:

[0329] Table 2

[0330] Example 4: Introducing amino acid point mutations based on predicted protein 3D structure alignment to improve gene integration efficiency of wild-type TgR2

[0331] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of the wild-type TgR2 retrotransposase system and its multiple mutants. It should be noted that the donor RNA for the retrotransposase system generally consists of five parts: a left homology arm, a 5'UTR sequence, a 3'UTR sequence, a sequence between the 5'UTR and 3'UTR sequences carrying the new genetic information, and a right homology arm (see Figure 1).

[0332] Protein 3D structure prediction and simulation

[0333] The tertiary structure of the wild-type TgR2 protein was predicted using the default parameters of ColabFold software (Mirdita, M., Schutze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S., and Steinegger, M. (2022). ColabFold: making protein folding accessible to all. Nat Methods 19, 679-682.). MAFFT software was used for multiple sequence alignment. The boundaries of each domain of TgR2 were determined based on the multiple sequence alignment results and the predicted tertiary structure results. The predicted TgR2 structure was fitted with a reported group-II intron reverse transcriptase structure (Stamos, JL, Lentzsch, AM, and Lambowitz, AM (2017). Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications. Mol Cell 68, 926-939e924) (PDB: 6AR3), and the DNA-RNA was docked into the predicted TgR2 structure.

[0334] Mutant design

[0335] The amino acids in the predicted TgR2 tertiary structure that have potential interactions with DNA or RNA are mutated into positively charged arginine (R), thereby increasing the affinity of the TgR2 protein for nucleic acid substrates and thus improving the efficiency of gene integration.

[0336] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0337] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0338] According to the 3D structure prediction and multiple sequence alignment results, 132 amino acids in TgR2 were selected in this example: E153, G154, E160, E163, Q164, Q168, D227, K228, N229, N240, K267, E268, N269, P291, E294, H341, K342, C344, E350, E361, N363, N365, N367, A379, Q381, S383, D384, S390, D573, G575, K576, K579, I585, L588, K635, E636, and E637. 8. K639, N640, Q642, E643, S645, K646, G647, S648, V661, V693, I695, P696, S699, K700, I713, I715, T728, S732, K733, S751, E752, N75 3. L754, T819, N847, K950, Q955, D957, D990, K993, T994, Y995, P998, I1001, D1005, H1006, T1044, D1046, T1052, Q1062, Q1071, S1072, S1073, D1074, D1075, G1098, G1099, D1100, E1102, N1103, P1105, S1106, E1109, S111 3. S1114, E1115, N1118, N1123, E1125, E1127, Q1131, D1133, K1134, K1137, K1210, D1 216, D1218, E1220, A1223, H1224, G1227, N1228, Q1233, D1234, I1237, K1238, H1240, N1241, E1245, E1249, N1268, E1270, E1302, E1306, K1307, K1310, and a point mutation test of arginine (R) was performed. The GFP reporter system in Example 1 was used to express the engineered TgR2 system with a single amino acid mutation. The preferred amino acid substitution sites and their corresponding gene editing efficiencies are shown in Figure 3. It was found that multiple reference wild-type TgR2s were able to significantly improve the efficiency of gene integration. The experimental results of Example 3 are shown in Table 3 and Figure 3.

[0339] Table 3

[0340] Example 5: Replacing Cysteine ​​(C) with Serine (S) in the protein sequence to improve the integration efficiency of TgR2

[0341] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of the wild-type TgR2 retrotransposase system and its multiple mutants. It should be noted that the donor RNA for the retrotransposase system generally consists of five parts: a left homology arm, a 5'UTR sequence, a 3'UTR sequence, a sequence between the 5'UTR and 3'UTR sequences carrying the new genetic information, and a right homology arm (see Figure 1).

[0342] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0343] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0344] In this example, 20 cysteines (C) were selected and replaced with serines (S): C4, C145, C170, C282, C344, C404, C518, C521, C690, C735, C750, C855, C901, C913, C930, C1034, C1036, C1079, C1139, and C1244. The GFP reporter system designed in Example 1 was used, and the experimental results were as follows (Figure 4, Table 4). Compared with the wild-type TgR2 protein, four mutant proteins: C344S, C1034S, C1036S, and C1244S were able to significantly improve the efficiency of gene integration, while the remaining mutants had little or no improvement or decreased efficiency.

[0345] Table 4

[0346] Example 6: Point mutations have additive effects

[0347] This example combines the protein mutations screened in Examples 2 and 3 that significantly improve gene integration efficiency. Based on the GFP reporter system designed in Example 1, this example tests the gene integration efficiency of single-point mutant TgR2 retrotransposase proteins and their combined mutant proteins. It should be noted that the donor RNA for the retrotransposase system generally consists of five parts: a left homology arm, a 5'UTR sequence, a 3'UTR sequence, a sequence between the 5'UTR and 3'UTR sequences carrying the new gene information, and a right homology arm (see Figure 1).

[0348] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0349] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0350] The experimental results are as follows (Figure 5, Table 5). Compared with the protein with the single point mutation G1098R, multiple mutant combinations: G1098R+H341R, G1098R+K700D, G1098R+W762Q, G1098R+H792W, G1098R+A793V, G1098R+R866V, and G1098R+Q955R can significantly improve the efficiency of gene integration and show a significant additive effect. The G1098R+A793V mutant with the highest integration efficiency was further combined with additional point mutations. The experimental data are shown in Figure 5 and Table 6. Compared with the G1098R+A793V mutant, the mutants G1098R+A793V+W762Q, G1098R+A793V+Q955R, G1098R+A793V+Q955R+H341R, and G1098R+A793V+W762Q+Q955R+H341R can further improve the efficiency of gene integration.

[0351] Table 5

[0352] Table 6

[0353] Example 7: Coupling exonuclease to improve integration efficiency

[0354] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of the wild-type TgR2 retrotransposase system and the wild-type TgR2 retrotransposase system coupled with T5 exonuclease at different positions on the protein. It is important to note that the donor RNA for the retrotransposase system generally consists of five parts: a left homology arm, a 5'UTR sequence, a 3'UTR sequence, a sequence between the 5'UTR and 3'UTR sequences carrying the new genetic information, and a right homology arm (see Figure 1).

[0355] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0356] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0357] T5 exonuclease (SEQ ID No.8) was coupled or inserted at the N-terminus, after amino acid No. 191 (the position was marked as IN-1), after amino acid No. 432 (the position was marked as IN-2), after amino acid No. 1112 (the position was marked as IN-3) and the C-terminus of the wild-type TgR2, respectively. It should be noted that two additional glycine (-GG-) was added to both ends of the T5 exonuclease inserted after amino acids No. 191, No. 432 and No. 1112. The results of the GFP reporter system designed based on Example 1 showed that, compared with the wild-type TgR2, coupling or inserting T5 exonuclease at the N-terminus, IN-2 and IN-3 of the wild-type TgR2 can significantly improve the efficiency of gene integration. The efficiency of gene integration by inserting T5 exonuclease at the position of IN-1 of the wild-type TgR2 was slightly improved, while coupling T5 exonuclease at the C-terminus of the wild-type TgR2 instead reduced the efficiency of gene integration. The experimental results are shown in Table 7 and Figure 6.

[0358] Table 7

[0359] Example 8: Coupling single-stranded DNA binding protein to improve integration efficiency

[0360] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of the wild-type TgR2 retrotransposase system and the wild-type TgR2 retrotransposase system coupled at different positions with the single-stranded DNA binding protein sto7d (abbreviated as Sto) from the bacterium Sulfolobus tokodaii (SEQ ID No. 9). It should be noted that the donor RNA of the retrotransposase system generally consists of five parts: a homology arm (left), a 5'UTR sequence, a 3'UTR sequence, a sequence between the 5'UTR and 3'UTR sequences carrying the new genetic information, and a homology arm (right) (see Figure 1).

[0361] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0362] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0363] The single-stranded DNA binding protein sto7d (abbreviated as Sto) from Sulfolobus tokodaii bacteria was coupled or inserted (this place was coupled or inserted) at the N-terminus, after amino acid No. 191 (this position was marked as IN-1), after amino acid No. 432 (this position was marked as IN-2), after amino acid No. 1112 (this position was marked as IN-3) and the C-terminus of the wild-type TgR2, respectively. It should be noted that two additional glycine (-GG-) were added to both ends of the Sto single-stranded DNA binding protein inserted after amino acids No. 191, No. 432 and No. 1112, respectively. The results of the GFP reporter system designed based on Example 1 showed that, compared with the wild-type TgR2, coupling or inserting the Sto single-stranded DNA binding protein at the N-terminus, IN-1 and IN-2 of the wild-type TgR2 can significantly improve the efficiency of gene integration. Inserting the Sto single-stranded DNA binding protein into the IN-3 position of wild-type TgR2 slightly improved gene integration efficiency, whereas coupling the Sto single-stranded DNA binding protein to the C-terminus of wild-type TgR2 decreased gene integration efficiency. The experimental results are shown in Figure 7 and Table 8.

[0364] Table 8

[0365] Example 9: Coupling protein peptides to improve integration efficiency

[0366] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of the wild-type TgR2 retrotransposase system and the wild-type TgR2 retrotransposase system coupled with high-mobility peptides (CMPs) at different positions. It is important to note that the donor RNA for the retrotransposase system generally consists of five parts: a left homology arm, a 5'UTR sequence, a 3'UTR sequence, a sequence between the 5'UTR and 3'UTR sequences carrying the new genetic information, and a right homology arm (see Figure 1).

[0367] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0368] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0369] A high-mobility peptide (CMP) (Ding, X., Seebeck, T., Feng, Y., Jiang, Y., Davis, GD, and Chen, F. (2019). Improving CRISPR-Cas9 Genome Editing Efficiency by Fusion with Chromatin-Modulating Peptides. The CRISPR Journal 2, 51-63.) (SEQ ID No. 10) was coupled or inserted at the N-terminus, after amino acid 191 (the position is marked as IN-1), after amino acid 432 (the position is marked as IN-2), after amino acid 1112 (the position is marked as IN-3), and at the C-terminus of wild-type TgR2. It should be noted that two additional glycine residues (-GG-) were added at both ends of the CMP chromatin regulatory peptides inserted after amino acids 191, 432, and 1112, respectively. Results from the GFP reporter system designed in Example 1 showed that, compared to wild-type TgR2, coupling or inserting a CMP epi-peptide at the N-terminus, IN-2, and IN-3 of wild-type TgR2 significantly improved gene integration efficiency. Coupling or inserting a CMP epi-peptide at the IN-1 and C-terminus of wild-type TgR2 also slightly improved gene integration efficiency. The experimental results are shown in Figure 8 and Table 9.

[0370] Table 9

[0371] Example 10: Coupling RNaseH domain to improve integration efficiency

[0372] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of the wild-type TgR2 retrotransposase system and the wild-type TgR2 retrotransposase system coupled to the RNaseH domain of MLV reverse transcriptase (abbreviated as RH) at different positions. It should be noted that the donor RNA of the retrotransposase system generally consists of five parts: the homology arm (left), the 5'UTR sequence, the 3'UTR sequence, the sequence between the 5'UTR sequence and the 3'UTR sequence carrying the new genetic information, and the homology arm (right) (see Figure 1).

[0373] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0374] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0375] The RNaseH domain of MLV reverse transcriptase (abbreviated as RH) (SEQ ID No. 11) was coupled or inserted at the N-terminus, after amino acid 191 (the position is marked as IN-1), after amino acid 432 (the position is marked as IN-2), after amino acid 1112 (the position is marked as IN-3), and at the C-terminus of wild-type TgR2, respectively. It should be noted that two additional glycine residues (-GG-) were added at both ends of the RNaseH domain inserted after amino acids 191, 432, and 1112, respectively. The results of the GFP reporter system designed in Example 1 showed that, compared with wild-type TgR2, inserting the RNaseH domain of MLV reverse transcriptase at positions IN-2 and IN-3 of wild-type TgR2 can significantly improve the efficiency of gene integration. Coupling or inserting the RNaseH domain of MLV reverse transcriptase at the N-terminus and IN-1 of wild-type TgR2 slightly improved gene integration efficiency. However, coupling the RNaseH domain of MLV reverse transcriptase at the C-terminus of wild-type TgR2 actually decreased gene integration efficiency. The experimental results are shown in Figure 9 and Table 10.

[0376] Table 10:

[0377] Example 11: Superposition transformation of coupling domains

[0378] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of any two of the following: T5 exonuclease, the single-stranded DNA binding protein sto7d from Sulfolobus tokodaii (abbreviated as Sto), the chromatin regulatory peptide (CMP), or the RNaseH domain of MLV reverse transcriptase (abbreviated as RH) coupled or inserted into the N-terminus and after amino acid 432 of wild-type TgR2 (this position is labeled IN-2). Furthermore, the gene integration efficiency of inserting the CMP or RH domain into any of three positions of TgR2: the N-terminus, after amino acid 432 (this position is labeled IN-2), and after amino acid 1112 (this position is labeled IN-3). It should be noted that the donor RNA of the retrotransposase system generally contains five parts: a homology arm-left, a 5'UTR sequence, a 3'UTR sequence, a sequence between the 5'UTR sequence and the 3'UTR sequence carrying the new genetic information, and a homology arm-right (see Figure 1).

[0379] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0380] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0381] At the N-terminus of wild-type TgR2, after amino acid 432 (labeled IN-2), any two of the following: T5 exonuclease, the single-stranded DNA binding protein Sto7d from the bacterium Sulfolobus tokodaii (abbreviated as Sto), a chromatin regulatory peptide or high-mobility peptide (CMP), or the RNaseH domain of MLV reverse transcriptase (abbreviated as RH) were coupled or inserted. Alternatively, CMPs or RHs were inserted at any of three positions: the N-terminus of TgR2, after amino acid 432 (labeled IN-2), and after amino acid 1112 (labeled IN-3). The combinations of T5(N)-CMP(IN-2), T5(N)-RH(IN-2), STO(N)-CMP(IN-2), STO(N)-RH(IN-2), and CMP(N)-CMP(IN-2) significantly improved gene integration efficiency. The further combinations of STO(N)+CMP(IN-2)+CMP(IN-3), CMP(N)+CMP(IN-2)+RH(IN-3), and CMP(N)+CMP(IN-2)+CMP(IN-3) significantly improved gene integration efficiency compared to the original combinations. The remaining combinations showed little or no improvement. The experimental results are shown in Table 11 and Figure 10.

[0382] Table 11

[0383] Example 12: Effect of Changing the Order of 5' Homology Arm, 5' UTR, 3' Homology Arm, and 3' UTR on the Gene Integration Efficiency of Wild-Type TgR2

[0384] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of a wild-type TgR2 retrotransposase system and a system with modified UTR sequences in the wild-type TgR2 retrotransposase donor RNA. It should be noted that the donor RNA for the retrotransposase system generally consists of five parts: a left homology arm, a 5'UTR sequence, a 3'UTR sequence, a sequence between the 5'UTR and 3'UTR sequences carrying the new genetic information, and a right homology arm (see Figure 1).

[0385] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0386] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0387] The effects of altering the order of the 5' homology arm, 5' UTR, 3' homology arm, and 3' UTR on the integration efficiency of wild-type TgR2 were investigated. WT represents the donor used in wild-type R2Tg. A in Table 12 and Figure 11 represents a donor with altered 5' homology arm and 5' UTR order, B represents a donor with deleted 5' UTR, C represents a donor with altered 3' homology arm and 3' UTR order, D represents a donor with deleted 3' UTR, E represents a donor with altered 5' homology arm, 5' UTR order, and both 3' homology arm and 3' UTR order, and F represents a donor with deleted 5' UTR and 3' UTR order. The results showed that donors A, B, C, D, E, and F, with altered element order or deleted UTR elements, all significantly decreased integration efficiency compared to the wild-type donor. A schematic diagram of the donor element sequence is shown in Figure 11, and the experimental data are shown in Table 12.

[0388] Table 12

[0389] Example 13: Effect of Changing the Length of the 5' Homology Arm and the 3' Homology Arm on the Gene Integration Efficiency of Wild-Type TgR2

[0390] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of a wild-type TgR2 retrotransposase system and a system in which the lengths of the 5' and 3' homology arms of the wild-type TgR2 retrotransposase donor RNA were altered. It should be noted that the donor RNA for the retrotransposase system generally consists of five parts: a wild-type or altered-length left homology arm, a 5'UTR sequence, a 3'UTR sequence, a sequence between the 5'UTR and 3'UTR sequences carrying the new genetic information, and a wild-type or altered-length right homology arm (see Figure 1).

[0391] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0392] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0393] The effect of changing the length of the 5' homology arm and the 3' homology arm on the gene integration efficiency of wild-type TgR2 was investigated. 12 ), A1 represents a donor with a 150 bp 5' homology arm (LHA) (SEQ ID No. 15) and a 150 bp 3' homology arm (RHA) (SEQ ID No. 18) length, A2 represents a donor with a 100 bp 5' homology arm (LHA) (SEQ ID No. 19) and a 100 bp 3' homology arm (RHA) (SEQ ID No. 20) length, A3 represents a donor with a 50 bp 5' homology arm (LHA) (SEQ ID No. 21) and a 50 bp 3' homology arm (RHA) (SEQ ID No. 22) length, A4 represents a donor with a 20 bp 5' homology arm (LHA) (SEQ ID No. 23) and a 20 bp 3' homology arm (RHA) (SEQ ID No. 24) length, and A5 represents a 10 bp 5' homology arm (LHA) (SEQ ID No. 26) length. A6 represents a donor with a 100 bp 5' homology arm (LHA) (SEQ ID No. 19) length and a 50 bp 3' homology arm (RHA) (SEQ ID No. 22) length, A7 represents a donor with a 100 bp 5' homology arm (LHA) (SEQ ID No. 19) length and a 20 bp 3' homology arm (RHA) (SEQ ID No. 23) length, A8 represents a donor with a 100 bp 5' homology arm (LHA) (SEQ ID No. 19) length and a 10 bp 3' homology arm (RHA) (SEQ ID No. 26) length, A9 represents a donor with a 50 bp 5' homology arm (LHA) (SEQ ID No. 21) length and a 100 bp 3' homology arm (RHA) (SEQ ID No. A10 represents a donor with a 20 bp 5' homology arm (LHA) (SEQ ID No. 23) and a 100 bp 3' homology arm (RHA) (SEQ ID No. 20), A11 represents a donor with a 10 bp 5' homology arm (LHA) (SEQ ID No. 25) and a 100 bp 3' homology arm (RHA) (SEQ ID No. 20), and A12 represents a donor without a 5' homology arm (LHA) or a 3' homology arm (RHA). Experimental data showed that the experimental groups using homology arm lengths of A1 and A2 had the highest gene integration efficiency. Specific experimental results are shown in Table 13 and Figure 12.

[0394] Table 13

[0395] Example 14: Effect of Modification of 5'UTR and 3'UTR on Gene Integration Efficiency of Wild-Type TgR2

[0396] Based on the GFP reporter system designed in Example 1, this example tested the gene integration efficiency of a wild-type TgR2 retrotransposase system and a system with modified UTR sequences in the wild-type TgR2 retrotransposase donor RNA. It should be noted that the donor RNA for the retrotransposase system generally consists of five parts: a left homology arm, a wild-type or modified 5'UTR sequence, a wild-type or modified 3'UTR sequence, a sequence between the 5'UTR and 3'UTR sequences carrying the new genetic information, and a right homology arm (see Figure 1).

[0397] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0398] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0399] The 5'UTR and 3'UTR sequences of the wild-type TgR2 retrotransposase donor RNA were modified and designated as UTR1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, respectively (Table 14 and Figure 13).

[0400] The sequence of the 5'UTR used by UTR1 is shown in SEQ ID No. 27, and the sequence of the 3'UTR used by UTR1 is shown in SEQ ID No. 17;

[0401] The sequence of the 5'UTR used by UTR2 is shown as SEQ ID No. 29, and the sequence of the 3'UTR used by UTR2 is shown as SEQ ID No. 17;

[0402] The sequence of the 5'UTR used by UTR3 is shown as SEQ ID No. 31, and the sequence of the 3'UTR used by UTR3 is shown as SEQ ID No. 17;

[0403] The sequence of the 5'UTR used by UTR4 is shown in SEQ ID No. 33, and the sequence of the 3'UTR used by UTR4 is shown in SEQ ID No. 17;

[0404] The sequence of the 5'UTR used by UTR5 is shown in SEQ ID No. 35, and the sequence of the 3'UTR used by UTR5 is shown in SEQ ID No. 17;

[0405] The sequence of the 5'UTR used by UTR6 is shown in SEQ ID No. 37, and the sequence of the 3'UTR used by UTR6 is shown in SEQ ID No. 17;

[0406] The sequence of the 5'UTR used by UTR7 is shown in SEQ ID No. 39, and the sequence of the 3'UTR used by UTR7 is shown in SEQ ID No. 17;

[0407] The sequence of the 5'UTR used by UTR8 is shown as SEQ ID No. 41, and the sequence of the 3'UTR used by UTR8 is shown as SEQ ID No. 17;

[0408] The sequence of the 5'UTR used by UTR9 is shown in SEQ ID No. 16, and the sequence of the 3'UTR used by UTR9 is shown in SEQ ID No. 44;

[0409] The sequence of the 5'UTR used by UTR10 is shown as SEQ ID No. 16, and the sequence of the 3'UTR used by UTR10 is shown as SEQ ID No. 46;

[0410] The sequence of the 5'UTR used by UTR11 is shown as SEQ ID No. 16, and the sequence of the 3'UTR used by UTR11 is shown as SEQ ID No. 48;

[0411] The sequence of the 5'UTR used by UTR12 is shown as SEQ ID No. 16, and the sequence of the 3'UTR used by UTR12 is shown as SEQ ID No. 50;

[0412] The sequence of the 5'UTR used by UTR13 is shown as SEQ ID No. 16, and the sequence of the 3'UTR used by UTR13 is shown as SEQ ID No. 52;

[0413] The sequence of the 5'UTR used by UTR14 is shown as SEQ ID No. 16, and the sequence of the 3'UTR used by UTR14 is shown as SEQ ID No. 54.

[0414] Based on the GFP reporter system designed in Example 1, the gene integration efficiency of the wild-type TgR2 retrotransposase system and the system using donor RNA with modified UTR sequences were tested. Experimental data showed that using donor RNA with modified UTR sequences 9, 10, and 11 significantly improved gene integration efficiency, while the remaining modified sequences showed little improvement or decreased integration efficiency. Specific experimental results are shown in Figure 13 and Table 14.

[0415] Table 14

[0416] Example 15: Engineered proteins and engineered RNA donors synergistically improve gene editing efficiency

[0417] Using an engineered RNA donor with the optimal donor element sequence determined in Example 12, the optimal donor homology arm length determined in Example 13, and the optimal modified 5'UTR and 3'UTR determined in Example 14, we tested the TgR2 retrotransposase system, which combined the engineering approaches screened in Examples 6 and 11, using the GFP reporter system designed in Example 1. It should be noted that the donor RNA for a retrotransposase system generally consists of five parts: a left homology arm, a wild-type or modified 5'UTR sequence, a wild-type or modified 3'UTR sequence, a sequence between the 5'UTR and 3'UTR sequences carrying the novel genetic information, and a right homology arm (see Figure 1).

[0418] Plasmid construction, cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0419] The plasmids used in this example were constructed using the methods described in the above experimental protocol. The cell culture, transfection, and FACS experiments in this example were performed using the methods described in the above experimental protocol.

[0420] Based on the optimal donor element sequence determined in Example 12, the optimal donor homology arm length determined in Example 13, and the engineered RNA donors with the optimal modified 5'UTR and 3'UTR determined in Example 14, we used the GFP reporter system designed in Example 1 to test the G1098R+A793V+Q955R, G1098R+A793V+W762Q+Q955R, G1098R+A793V+W762Q+Q955R+H341R, and G1098R+A793V+Q955R+H341R (respectively simplified and named PM1, PM2, PM3, and PM4, corresponding to the sequences: SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID The TgR2 retrotransposase system is a combination of T5(N)-RH(IN-2), CMP(N)+CMP(IN-2)+CMP(IN-3), STO(N)-RH(IN-2), T5(N)-CMP(IN-2), STO(N)-CMP(IN-2), and CMP(N)-CMP(IN-2) obtained in Example 11 (simplified and named TD1, TD2, TD3, TD4, TD5, and TD6, corresponding to the sequences: SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, SEQ ID No. 59, and SEQ ID No. 60, respectively). The combinations are:

[0421] TD1+PM1 (SEQ ID No. 61)

[0422] TD1+PM2 (SEQ ID No. 62)

[0423] TD1+PM3 (SEQ ID No. 63)

[0424] TD1+PM4 (SEQ ID No. 64)

[0425] TD2+PM1 (SEQ ID No. 65)

[0426] TD2+PM2 (SEQ ID No. 66)

[0427] TD2+PM3 (SEQ ID No. 67)

[0428] TD2+PM4 (SEQ ID No. 68)

[0429] TD3+PM1 (SEQ ID No. 69)

[0430] TD3+PM2 (SEQ ID No. 70)

[0431] TD3+PM3 (SEQ ID No. 71)

[0432] TD3+PM4 (SEQ ID No. 72)

[0433] TD4+PM1 (SEQ ID No. 73)

[0434] TD4+PM2 (SEQ ID No. 74)

[0435] TD4+PM3 (SEQ ID No. 75)

[0436] TD4+PM4 (SEQ ID No. 76)

[0437] TD5+PM1 (SEQ ID No. 77)

[0438] TD5+PM2 (SEQ ID No. 78)

[0439] TD5+PM3 (SEQ ID No. 79)

[0440] TD1+PM4 (SEQ ID No. 80)

[0441] TD6+PM1 (SEQ ID No. 81)

[0442] TD6+PM2 (SEQ ID No. 82)

[0443] TD6+PM3 (SEQ ID No. 83)

[0444] TD6+PM4 (SEQ ID No. 84)

[0445] Compared with the wild-type TgR2 retrotransposase system and uncombined preferred proteins (TD1, TD2, TD3, TD4, TD5, TD6, PM1, PM2, PM3, PM4), the combinations of TD1+PM1, TD1+PM2, TD1+PM4, TD2+PM1, TD2+PM2, TD2+PM4, TD3+PM1, TD3+PM2, TD3+PM4, TD4+PM1, TD4+PM2, TD5+PM1, TD5+PM2, and TD5+PM4 significantly improved gene integration efficiency. The remaining combinations showed little improvement or decreased gene integration efficiency. The experimental results are shown in Table 15 and Figure 14.

[0446] Table 15:

[0447] Example 16: Using Lipo3000 liposomes to deliver mRNA expressing R2 retrotransposase protein as an RNA donor

[0448] In this example, an in vitro transcription system of mRNA expressing TgR2 protein and donor RNA was constructed and the gene integration efficiency was tested when Lipo3000 liposomes were used to deliver mRNA and donor RNA of the TgR2 retrotransposase system.

[0449] Plasmid construction and in vitro transcription

[0450] The plasmid containing the sequence of en-TgR2 and en-Donor was used as a template and HiScribe TM In vitro transcription was performed using the T7 mRNA synthesis kit.

[0451] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0452] HEK293T cell lines were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-well plates / cell culture dishes (Corning) for 16 hours until the cell density reached 70%-90%. 1 μg of mRNA encoding the retrotransposase protein and 1 μg of donor RNA were transfected into each 24-well plate / cell culture dish using Lipofectamine 3000 (Invitrogen). 96 hours after transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco). BD FACSAria TM The proportion of cells with GFP-positive signals was analyzed using a Fusion Cell Sorter (BD) instrument.

[0453] In this example, Lipo3000 liposomes were used to deliver mRNA of the wild-type or optimized TgR2 retrotransposase system (the TgR2 retrotransposase system was constructed using SEQ ID NO.77 and named en-TgR2), and the engineered RNA donor containing 5'UTR (SEQ ID NO.16) and non-natural 3'UTR (SEQ ID NO.46) was named en-Donor. The gene insertion efficiency of the two is shown in Table 16. The results show that, compared with the wild-type TgR2 system, the preferred system using en-Donor and en-TgR2 (SEQ ID NO.77) can significantly improve the gene integration efficiency. The experimental results of Example 16 are shown in Table 16 and Figure 15.

[0454] Table 16:

[0455] Example 17: Using LNPs to package mRNA expressing the TgR2 retrotransposase system and donor RNA to infect HEK293T cell lines, Huh7 cell lines, and N2A cell lines

[0456] Based on the in vitro transcription system constructed in Example 16, mRNA expressing the TgR2 protein (SEQ ID NO. 77) and donor RNA were used to obtain the corresponding mRNA through in vitro transcription and purification steps, and then the mRNA was packaged using lipid nanoparticles (LNPs). This example tested the gene integration efficiency after using LNPs to package the mRNA of the optimized TgR2 retrotransposase system and infecting HEK293T cell lines, Huh7 cell lines, and N2A cell lines.

[0457] Cell culture, LNP transfection, and fluorescence-activated cell sorting (FACS)

[0458] HEK293T cell line, Huh7 cell line and N2A cell line were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-well plate-cell culture dishes (Corning) for 16 hours until the cell density reached 70%-90%. LNPs packaged with 1 μg of mRNA encoding retrotransposase protein and 2 μg of donor RNA were transfected into each 24-well plate-cell culture dish. After 72 hours of transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco). The cells were then analyzed using a BD FACSAria TM The proportion of cells with GFP-positive signals was analyzed using a Fusion Cell Sorter (BD). Comparison with the proportion of cells with GFP-positive signals in a negative control and observation under a fluorescence microscope confirmed whether the engineered retrotransposase system had enhanced gene integration efficiency in mammalian cells.

[0459] In this example, LNPs were used to package the mRNA of the optimized TgR2 retrotransposase (SEQ ID NO. 77) system, and then HEK293T cell lines, Huh7 cell lines, and N2A cell lines were infected. The gene writing efficiency of the two on the HEK 293T cell line, Huh7 cell line, and N2A cell line is shown in Table 17. The results showed that after infection with LNPs, the optimized TgR2 retrotransposase system can achieve gene integration efficiencies of 16.9%, 25.4%, and 14% on the HEK 293T cell line, Huh7 cell line, and N2A cell line, respectively. The experimental results of Example 17 are shown in Table 17 and Figure 16.

[0460] Table 17:

[0461] Example 18: Expression of VPX protein can improve the gene integration efficiency of the TgR2 system

[0462] Based on the GFP reporter system in Example 1, this example tested the effect of co-transfection of VPX protein plasmids expressing different viral sources on the gene integration efficiency of the TgR2 system.

[0463] Plasmid construction

[0464] The plasmids used in this example were constructed using the methods described in the experimental protocol above. Furthermore, the DNA coding sequences for the VPX protein from SIV (SEQ ID NO. 85), the VPX protein from SIV2 (SEQ ID NO. 86), and the VPX protein from HIV2 (SEQ ID NO. 87) were synthesized. These DNA coding fragments were then inserted into the pCAG-SV40 poly(A) vector between the XmaI and NheI restriction sites via Gibson cloning.

[0465] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0466] The experiment was conducted using the same cell culture conditions as in Example 16. Using Lipofectamine 3000 (Invitrogen), 250 ng of a plasmid encoding the retrotransposase protein (en-TgR2) and 250 ng of a plasmid expressing the donor RNA (en-Donor) were transfected into each 24-well plate-cell culture dish, along with the VPX plasmid. 120 hours after transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco). The cells were then stained using a BD FACSAria. TM The proportion of cells with GFP-positive signals was analyzed using a Fusion Cell Sorter (BD) instrument.

[0467] Based on the GFP reporter system in Example 1, this example tested the gene integration efficiency of the TgR2 system by co-transfection of VPX protein plasmids expressing different viral sources (SIV-VPX (SEQ ID No. 85), SIV2-VPX (SEQ ID No. 86), and HIV2-VPX (SEQ ID No. 87)). The results showed that co-transfection with the SIV-VPX protein plasmid reduced the gene integration efficiency of the TgR2 system, while co-transfection with the SIV 2-VPX protein plasmid slightly increased the gene integration efficiency of the TgR2 system. Co-transfection with the HIV2-VPX protein plasmid can improve the gene integration efficiency of the TgR2 system. The experimental results of Example 18 are shown in Table 18 and Figure 17.

[0468] Table 18:

[0469] Example 19: Engineered TgR2 system can integrate genes other than GFP

[0470] This example tested the integration efficiency of the engineered TgR2 system for the GDNF (SEQ ID No. 88), EPO (SEQ ID No. 89), and Factor VI (SEQ ID No. 53) genes. The GFP gene sequence in the donor RNA plasmid of the GFP reporter system described in Example 1 was replaced with the GDNF, EPO, and Factor VI genes. This system was then used to integrate the corresponding genes into cells. The integration efficiency of the engineered TgR2 system (en-TgR2) for the GDNF, EPO, and Factor VI genes was then tested using an ELISA assay.

[0471] Plasmid construction and in vitro transcription

[0472] The plasmid used in this example was constructed using the method described in the experimental protocol of Example 16. In addition, the DNA coding sequences of the GDNF, EPO, and Factor VI genes were synthesized. The plasmid expressing the donor RNA of the corresponding gene was used to replace the GFP gene in the original plasmid by overlapping PCR. Using the constructed in vitro transcription plasmid as a template, HiScribe TM In vitro transcription was performed using the T7 mRNA synthesis kit. Human erythropoietin (EPO) ELISA kit was used to measure EPO content, Human GDNF ELISA kit was used to measure GDNF content, and the Biophen kit was used to measure Factor IX content.

[0473] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0474] The experiment was conducted using the same cell culture conditions as in Example 16. Using Lipofectamine 3000 (Invitrogen), 1 μg of mRNA encoding the retrotransposase protein (en-TgR2) and 2 μg of donor RNA (en-Donor) were transfected into each 24-well plate-based cell culture dish. 96 hours after transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco).

[0475] This example tested the integration efficiency of the engineered TgR2 system for the GDNF, EPO, and Factor VI genes. In the GFP reporter system described in Example 1, the GFP gene sequence of the donor RNA plasmid was replaced with the corresponding GDNF, EPO, and Factor VI genes. This reporter system was then used to test the integration of the corresponding genes in cells. Combined with ELISA assays, the results demonstrated that the engineered TgR2 system successfully integrated the GDNF, EPO, and Factor VI genes. The experimental results of Example 19 are shown in Figure 18.

[0476] Example 20: en-TgR2 truncated protein can improve gene integration efficiency

[0477] Based on the GFP reporter system described in Example 1, this example tested the gene integration efficiency of different en-TgR2 truncated proteins (SEQ ID No. 42, SEQ ID No. 43, and SEQ ID No. 45, respectively). By referring to the wild-type TgR2 protein sequence, corresponding truncated proteins were designed and combined with en-Donor to test the gene integration efficiency.

[0478] Plasmid construction

[0479] The plasmids used in this example were constructed using the methods described in the above experimental protocol.

[0480] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0481] HEK293T cell lines were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-well plates / cell culture dishes (Corning) for 16 hours until the cell density reached 70%-90%. 250 ng of a plasmid encoding a retrotransposase protein and 250 ng of a plasmid expressing a donor RNA were transfected into each 24-well plate / cell culture dish using Lipofectamine 3000 (Invitrogen). 120 hours after transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco) and stained using a BD FACSAriaTM The proportion of cells with GFP-positive signals was analyzed using a Fusion Cell Sorter (BD). Comparison with the proportion of cells with GFP-positive signals in a negative control and observation under a fluorescence microscope confirmed whether the engineered retrotransposase system had enhanced gene integration efficiency in mammalian cells.

[0482] This example tested the effects of different en-TgR2 truncated proteins on gene integration efficiency. By referring to the wild-type TgR2 protein sequence, the corresponding amino acids at positions 1-80, 1-190, and 1-220 were deleted. The corresponding en-TgR2 truncated proteins were named en-TgR2-v1 (SEQ ID No. 42), en-TgR2-v2 (SEQ ID No. 43), and en-TgR2-v3 (SEQ ID No. 45). The results showed that compared to the reference en-TgR2 protein, the truncated en-TgR2-v1 and en-TgR2-v3 proteins were able to significantly improve gene insertion efficiency. The results of Example 20 are shown in Table 19 and Figure 19.

[0483] Table 19:

[0484] Example 21: Gene integration using engineered R2 protein and donor RNA containing modified bases

[0485] In this example, an in vitro transcription system was constructed using mRNA expressing the TgR2 protein and donor RNA. Uracil (U) in the donor RNA was replaced with N1-methyl-pseudouracil (m1Ψ). The gene integration efficiency was tested using Lipo3000 liposomes to deliver the mRNA and donor RNA of the TgR2 retrotransposase system.

[0486] Plasmid construction and in vitro transcription

[0487] The plasmid containing TgR2 sequence and donor sequence was used as template and HiScribe TM In vitro transcription was performed using the T7 mRNA synthesis kit.

[0488] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0489] HEK293T cell lines were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-well plates / cell culture dishes (Corning) for 16 hours until the cell density reached 70%-90%. 500 ng of mRNA encoding the retrotransposase protein and 1 μg of donor RNA were transfected into each 24-well plate / cell culture dish using Lipofectamine 3000 (Invitrogen). 96 hours after transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco). BD FACSAria TM The proportion of cells with GFP-positive signals was analyzed using a Fusion Cell Sorter (BD) instrument.

[0490] This example uses engineered TgR2 sequences: TD5 (SEQ ID No. 59) and TD5-v1 (SEQ ID No. 12). This example uses an engineered donor RNA sequence (optDonor), which consists of the following components.

[0491] First homology domain:

[0492] A1 (SEQ ID No. 19)

[0493] A2 (SEQ ID No. 28);

[0494] 5' untranslated sequence (5'UTR) to which the retrotransposase binds:

[0495] B (SEQ ID No. 16);

[0496] Heterologous sequence:

[0497] C (SEQ ID No. 30);

[0498] 3' untranslated sequence (3'UTR) to which the retrotransposase binds:

[0499] D1 (SEQ ID No. 32)

[0500] D2 (SEQ ID No. 34);

[0501] Second homology domain:

[0502] E1 (SEQ ID No. 20)

[0503] E2 (SEQ ID No. 36).

[0504] optDonor is composed of the above sequences arranged in sequence, wherein 22 A's are added at the ends of optDonor-2, 3, 4, 5, and 6.

[0505] optDonor-1:A1+B+C+D1+E1

[0506] optDonor-2:A1+B+C+D1+E2

[0507] optDonor-3:A1+B+C+D1+E1

[0508] optDonor-4:A2+B+C+D1+E2

[0509] optDonor-5:A1+B+C+D2+E2

[0510] optDonor-6:A2+B+C+D2+E2

[0511] The experimental results (Table 20, Figure 20) showed that T5D-v1 could achieve a GFP gene integration efficiency greater than 60% in human cells.

[0512] Table 20:

[0513] Example 22: Further design of R2 protein mutants and gene integration with donor RNA containing modified bases

[0514] In this example, an in vitro transcription system was constructed using mRNA expressing the TgR2 protein and donor RNA. Uracil (U) in the donor RNA was replaced with N1-methyl-pseudouracil (m1Ψ). The gene integration efficiency was tested using Lipo3000 liposomes to deliver the mRNA and donor RNA of the TgR2 retrotransposase system.

[0515] Plasmid construction and in vitro transcription

[0516] The plasmid containing TgR2 sequence and donor sequence was used as template and HiScribe TM In vitro transcription was performed using the T7 mRNA synthesis kit.

[0517] Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

[0518] HEK293T cell lines were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-well plates / cell culture dishes (Corning) for 16 hours until the cell density reached 70%-90%. 500 ng of mRNA encoding the retrotransposase protein and 1 μg of donor RNA were transfected into each 24-well plate / cell culture dish using Lipofectamine 3000 (Invitrogen). 96 hours after transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco). BD FACSAria TM The proportion of cells with GFP-positive signals was analyzed using a Fusion Cell Sorter (BD) instrument.

[0519] This example uses the engineered TgR2 sequence: TD5-v1 (SEQ ID No. 12), as well as mutants further designed based on TD5-v1. (If the engineered TgR2 protein contains amino acid truncations or insertions, such as SEQ ID No. 59 or SEQ ID No. 12, the amino acids where the amino acid mutation occurs are numbered according to the sequence of the wild-type protein (SEQ ID No. 1).

[0520] This example uses the engineered optDonor-6 sequence from Example 20, in which 22 A residues are added to the end of optDonor-6. The experimental results are shown in Table 21 and Figure 21.

[0521] Table 21

[0522] The sequences involved in this application are as follows:

[0523] The final combined mutation sequence:

Claims

1. An engineered retrotransposase, comprising one, two or three mutations of wild-type retrotransposase TgR2 or a truncation of wild-type retrotransposase TgR2 or a truncation of wild-type retrotransposase TgR2 comprising one, two or three mutations of wild-type retrotransposase TgR2, wherein the mutations are selected from: (i) the amino acid of the wild-type retrotransposase TgR2 is mutated to other amino acids corresponding to the corresponding sites in the multiple sequence alignment; (ii) amino acids in the tertiary structure of wild-type retrotransposase TgR2 that have potential to interact with DNA or RNA are mutated into positively charged amino acids; and (iii) The cysteine ​​in the wild-type retrotransposase TgR2 was mutated to serine.

2. The engineered retrotransposase according to claim 1, wherein The amino acid sequence of the wild-type retrotransposase TgR2 is as shown in SEQ ID NO.1 or the amino acid position numbering of the engineered retrotransposase is as defined in SEQ ID NO.1; or The truncated form of the wild-type retrotransposase TgR2 is an N-terminal truncation, a C-terminal truncation and / or an intermediate sequence truncation of the wild-type retrotransposase TgR2. Preferably, the N-terminal truncation is a truncation of the 1st to 500th amino acid of the wild-type retrotransposase TgR2, for example, a truncation of the 1st to 80th amino acid, the 1st to 190th amino acid or the 1st to 220th amino acid; Preferably, the intermediate sequence truncation is truncated at the 300th to 1200th amino acids of the wild-type retrotransposase TgR2, for example, truncated at the 390th to 500th amino acids, or the 1100th to 1120th amino acids.

3. The engineered retrotransposase according to claim 1, wherein: The amino acids at the corresponding positions of the wild-type retrotransposase TgR2 in the multiple sequence alignment are confirmed using multiple sequence alignment software (such as MAFFT, etc.), and the amino acid position numbering is as defined in SEQ ID NO.1; Preferably, the corresponding site of the wild-type retrotransposase TgR2 in the multiple sequence alignment is selected from any one or two or three or four or five or six or seven or eight or nine or ten or more of the following: K700, L704, V727, S732, L737, A747, A748, T759, I760, W762, S763, H792, A793, Q795, E798, P801, V807, S808, E812, S815, T819, T820, H825, K828, S860, Y864, H865, R866, Q868, S869, N889, N891, T892, S895, Q911, or D957, It is further preferred that the amino acid mutation of the corresponding site of the wild-type retrotransposase TgR2 in the multiple sequence alignment is other amino acids corresponding to the corresponding site selected from any one or two or three or four or five or six or seven or eight or nine or ten or more of the following: K700D, L704Q, V727L, S732T, L737I, A747S, A748S, A748P, T759L, I760L, W762R, W762K, W762Q, S763N, S763R, H792T, H792W, A793V, Q795K, Q795R, E798G, P801Q, V807I, S808R, E812K, E812R, S815N, T819D, T820L, H825Q, K828P, S860E, Y864F, H865Q, H865R, R866V, Q868S, S869K, N889G, N891Q, T892K, S895K, Q911E, or D957G; It is further preferred that the amino acid mutation at the corresponding site of the wild-type retrotransposase TgR2 in the multiple sequence alignment is other amino acids corresponding to the corresponding site selected from any of the following: K700D, W762Q, H792W, A793V, R866V, or D957G.

4. The engineered retrotransposase according to claim 1, wherein The amino acid in the tertiary structure of the wild-type retrotransposase TgR2 that has potential to interact with DNA or RNA is mutated into arginine; the amino acid position number is as defined in SEQ ID NO.1; Preferably, the amino acids in the tertiary structure of the wild-type retrotransposase TgR2 that have potential interactions with DNA or RNA are selected from any one, two, three, four, five, six, seven, eight, nine, or ten or more of the following: E153, G154, E160, E163, Q164, Q168, D227, K228, N229, N240, K267, E268, N269, P291, E294, H341, K34 2. C344, E350, E361, N363, N365, N367, A379, Q381, S383, D384, S390, D573, G575, K576, K579, I585, L5 88. K635, E636, E638, K639, N640, Q642, E643, S645, K646, G647, S648, V661, V693, I695, P696, S699, K700, I713, I715, T728, S732, K733, S751, E752, N753, L754, T819, N847, K950, Q955, D957, D990, K993 , T994, Y995, P998, I1001, D1005, H1006, T1044, D1046, T1052, Q1062, Q1071, S1072, S1073, D1074, D 1075, G1098, G1099, D1100, E1102, N1103, P1105, S1106, E1109, S1113, S1114, E1115, N1118, N1123, E 1125, E1127, Q1131, D1133, K1134, K1137, K1210, D1216, D1218, E1220, A1223, H1224, G1227, N1228, Q1233, D1234, I1237, K1238, H1240, N1241, E1245, E1249, N1268, E1270, E1302, E1306, K1307, or K1310; It is further preferred that the amino acids in the tertiary structure of the wild-type retrotransposase TgR2 that have potential interactions with DNA or RNA are selected from any one, two, three, four, five, six, seven, eight, nine, or ten or more of the following: D227, K228, N229, N240, K267, N269, P291, E294, H341, K342, C344, E361, N36 5. N367, A379, Q381, S383, D384, S390, K576, K579, K639, K646, K733, T819, K 950, Q955, D990, K993, T1044, T1052, Q1062, Q1071, D1074, D1075, G1098, G1099, P1105, E1109, E1125, E1127, Q1131, A1223, I1237, E1245, E1249, or E1306.

5. The engineered retrotransposase according to claim 1, wherein The cysteine ​​in the wild-type retrotransposase TgR2 is any one or two or three or four or five or six or seven or eight or nine or ten or more selected from the following: the amino acid position numbering is as defined in SEQ ID NO.1; C4, C145, C170, C282, C344, C404, C518, C521, C690, C735, C750, C855, C901, C913, C930, C1034, C1036, C1079, C1139, or C1244; Preferably, the cysteine ​​in the wild-type retrotransposase TgR2 is any one, two, three or four selected from the following: C344, C1034, C1036, or C1244.

6. The engineered retrotransposase according to any one of claims 1 to 5, wherein: The amino acid position numbering is as defined in SEQ ID NO.1; the engineered retrotransposase comprises any one or more of the following mutations: (1)G1098R; (2)G1098R+H341R; (3)G1098R+K700D; (4)G1098R+W762Q; (5)G1098R+H792W; (6)G 1098R+A793V; (7)G1098R+R866V; (8)G1098R+Q955R; (9)G1098R+T1052R; (10)G1098R+Q1062R; (11)G1098R+P1105R; (12)G1098R+C1034S; (13)G1098R+I1237R; (14)G1098R+K1310R; (15)G1 098R+D957G; (16)H341R+C1036S; (17)H341R+Q1062R; (18)H341R+I1237R; (19)H341R+K1310R; (20)I1237R+K1310R; (21)C1034S+C1036S; (22)K700D+R866V; (23)W762Q+R866V; (24)H792W+ R866V; (25)A793V+R866V; (26)G1098R+A793V+K700D; (27)G1098R+A793V+W762Q; (28)G1098R+ A793V+H792W; (29)G1098R+A793V+Q955R; (30)G1098R+A793V+W762Q+Q955R; (31)G1098R+A79 3V+W762Q+H341R; (32)G1098R+A793V+Q955R+H341R; (33)G1098R+A793V+W762Q+Q955R+H341R; Preferably, the engineered retrotransposase comprises any one or more of the following mutations: G1098R+H341R; G1098R+K700D; G1098R+W762Q; G1098R+H792W, G1098R+A793V; G1098R+R866V; G1098R+Q955R; G 1098R+A793V+W762Q; G1098R+A793V+Q955R; G1098R+A793V+Q955R+H341R, G1098R+A793V+W762Q+Q955R+H341R.

7. An engineered retrotransposase, the engineered retrotransposase comprising any one or more of the following mutations: (1)G1098R; (2)G1098R+H341R; (3)G1098R+K700D; (4)G1098R+W762Q; (5)G1098R+H792W; (6)G 1098R+A793V; (7)G1098R+R866V; (8)G1098R+Q955R; (9)G1098R+T1052R; (10)G1098R+Q1062R; (11)G1098R+P1105R; (12)G1098R+C1034S; (13)G1098R+I1237R; (14)G1098R+K1310R; (15)G1 098R+D957G; (16)H341R+C1036S; (17)H341R+Q1062R; (18)H341R+I1237R; (19)H341R+K1310R; (20)I1237R+K1310R; (21)C1034S+C1036S; (22)K700D+R866V; (23)W762Q+R866V; (24)H792W+ R866V; (25)A793V+R866V; (26)G1098R+A793V+K700D; (27)G1098R+A793V+W762Q; (28)G1098R+ A793V+H792W; (29)G1098R+A793V+Q955R; (30)G1098R+A793V+W762Q+Q955R; (31)G1098R+A79 3V+W762Q+H341R; (32)G1098R+A793V+Q955R+H341R; (33)G1098R+A793V+W762Q+Q955R+H341R; Preferably, the engineered retrotransposase comprises any one or more of the following mutations: G1098R+H341R; G1098R+K700D; G1098R+W762Q; G1098R+H792W, G1098R+A793V; G1098R+R866V; G1098R+Q955R; G 1098R+A793V+W762Q; G1098R+A793V+Q955R; G1098R+A793V+Q955R+H341R, G1098R+A793V+W762Q+Q955R+H341R; The amino acid position numbers are as defined in SEQ ID NO.

1.

8. An engineered retrotransposase, which is an engineered retrotransposase obtained by further coupling the engineered retrotransposase or wild-type retrotransposase TgR2 as shown in any one of claims 1 to 7 with other amino acid sequences.

9. The engineered retrotransposase according to claim 8, wherein: The other amino acid sequences are selected from any one, two, three, four, five, six, or seven of nuclease exonucleases, single-stranded DNA binding proteins, chromatin regulatory peptides, high-mobility peptides, RNaseH domains, nuclear import localization signals (NLS), and nuclear export localization signals.

10. The engineered retrotransposase according to claim 9, wherein: The other amino acid sequence is a nuclease exonuclease, and the exonuclease is inserted at the N-terminus, after amino acid No. 191, after amino acid No. 432, after amino acid No. 1112, or at the C-terminus of the retrotransposase or the wild-type retrotransposase TgR2 as shown in any one of claims 1 to 7; Preferably, the exonuclease is an exonuclease from T5 bacteriophage; It is further preferred that the sequence of the exonuclease is as shown in SEQ ID NO.8 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence described in SEQ ID No.

8.

11. The engineered retrotransposase according to claim 9, wherein: The other amino acid sequence is a single-stranded DNA binding protein, and the single-stranded DNA binding protein is inserted into the N-terminus, after amino acid 191, after amino acid 432, after amino acid 1112, or at the C-terminus of the retrotransposase or wild-type retrotransposase TgR2 as shown in any one of claims 1 to 7; Preferably, the single-stranded DNA binding protein is a single-stranded DNA binding protein from Sulfolobus tokodaii bacteria; It is further preferred that the sequence of the single-stranded DNA binding protein is as SEQ ID NO.9 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence described in SEQ ID No.

9.

12. The engineered retrotransposase according to claim 9, wherein: The other amino acid sequence is a chromatin regulatory peptide or a high-mobility peptide, and the chromatin regulatory peptide or the high-mobility peptide is inserted at the N-terminus, after amino acid 191, after amino acid 432, after amino acid 1112, or at the C-terminus of the retrotransposase or the wild-type retrotransposase TgR2 as shown in any one of claims 1 to 7; Preferably, the chromatin regulatory peptide or high-mobility peptide is a chromatin regulatory peptide or high-mobility peptide from human; Preferably, the sequence of the chromatin regulatory peptide or high-mobility peptide is as shown in SEQ ID NO.10 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No.

10.

13. The engineered retrotransposase according to claim 9, wherein: The other amino acid sequence is an RNaseH domain, and the RNaseH domain is inserted at the N-terminus, after amino acid 191, after amino acid 432, after amino acid 1112, or at the C-terminus of the retrotransposase or wild-type retrotransposase TgR2 as shown in any one of claims 1 to 7; Preferably, the RNaseH domain is the RNaseH domain from MLV reverse transcriptase; It is further preferred that the sequence of the RNaseH domain of MLV reverse transcriptase is as shown in SEQ ID NO.11 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID No.

11.

14. The engineered retrotransposase according to claim 9, wherein: Any two, three or four of the exonuclease and / or single-stranded DNA binding protein and / or chromatin regulatory peptide (CMP) and / or RNaseH domains are inserted into the N-terminus, after amino acid 191, after amino acid 432, after amino acid 1112 or the C-terminus of the retrotransposase or wild-type retrotransposase TgR2 as described in any one of claims 1 to 7; It is further preferred that an exonuclease is inserted into the N-terminus of the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7, and an exonuclease or a single-stranded DNA binding protein or a chromatin regulatory peptide (CMP) or an RNaseH domain is inserted after amino acid 432 of the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7; or It is further preferred that a single-stranded DNA binding protein is inserted into the N-terminus of the retrotransposase or the wild-type retrotransposase TgR2 as shown in any one of claims 1 to 7, and an exonuclease or a single-stranded DNA binding protein or a chromatin regulatory peptide (CMP) or an RNaseH domain is inserted after amino acid 432 of the retrotransposase or the wild-type retrotransposase TgR2 as shown in any one of claims 1 to 7; or It is further preferred that a chromatin regulatory peptide (CMP) is inserted into the N-terminus of the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7, and an exonuclease or a single-stranded DNA binding protein or a chromatin regulatory peptide (CMP) or an RNaseH domain is inserted after amino acid 432 of the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7; or It is further preferred that a single-stranded DNA binding protein is inserted into the N-terminus of the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7, and a chromatin regulatory peptide (CMP) is inserted after amino acid 432 in the reverse transposase as shown in any one of claims 1 to 7, and a chromatin regulatory peptide (CMP) or RNaseH domain is inserted after amino acid 1112 in the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7; or It is further preferred that a single-stranded DNA binding protein is inserted into the N-terminus of the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7, and an RNaseH domain is inserted after amino acid 432 of the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7, and a chromatin regulatory peptide (CMP) or an RNaseH domain is inserted after amino acid 1112 of the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7; or It is further preferred that a chromatin regulatory peptide (CMP) is inserted into the N-terminus of the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7, and a chromatin regulatory peptide (CMP) is inserted after amino acid No. 432 in the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7, and a chromatin regulatory peptide (CMP) or an RNaseH domain is inserted after amino acid No. 1112 in the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7; or It is further preferred that a chromatin regulatory peptide (CMP) is inserted at the N-terminus of the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7, and an RNaseH domain is inserted after amino acid 432 in the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7, and a chromatin regulatory peptide (CMP) or RNaseH domain is inserted after amino acid 1112 in the reverse transposase or the wild-type reverse transposase TgR2 as shown in any one of claims 1 to 7.

15. The engineered retrotransposase according to claim 14, wherein: The sequence of the exonuclease is as shown in SEQ ID NO.8 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence of SEQ ID No.8; or The sequence of the single-stranded DNA binding protein is as shown in SEQ ID NO.9 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence as shown in SEQ ID No.9; or The sequence of the chromatin regulatory peptide or high mobility peptide is as shown in SEQ ID NO.10 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence of SEQ ID No.10; or The sequence of the RNaseH domain is as shown in SEQ ID NO.11 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence described in SEQ ID No.

11.

16. An engineered retrotransposase according to any one of claims 1 to 15, wherein The engineered retrotransposase comprises an amino acid sequence as shown in any one of SEQ ID NOs. 2-7, 12, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55-84, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence of any one of SEQ ID Nos. 2-7, 12, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55-84; or The engineered retrotransposase is a truncated version of the amino acid sequence shown in any one of SEQ ID NOs. 1-7, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55 to 84, preferably a sequence obtained by truncating the wild-type sequence SEQ ID NO. 1 corresponding to the 1st to 80th, or the 1st to 190th, or the 1st to 220th, the 390th to 500th, or the 1100th to 1120th amino acids from the amino acid sequence shown in any one of SEQ ID NOs. 1-7, 13, 38, 40, 42, 43, 45, 47, 49, 51, 55 to 84.

17. A system for modifying DNA, the system comprising: A reverse transposase, wherein the reverse transposase is the wild-type reverse transposase TgR2 shown in SEQ ID NO.1 or the reverse transposase shown in any one of claims 1 to 16, or a nucleic acid encoding the wild-type reverse transposase TgR2 shown in SEQ ID NO.1 or the reverse transposase shown in any one of claims 1 to 16; and A donor RNA or a nucleic acid encoding the donor RNA, wherein the donor RNA comprises: a sequence that binds to the retrotransposase and a heterologous sequence, Preferably, the heterologous sequence is at least 1-50000 bases, for example, more than 1 nt, more than 10 nt, more than 50 nt, more than 60 nt, more than 70 nt, more than 80 nt, more than 90 nt, more than 100 nt, more than 150 nt, more than 200 nt, more than 250 nt, more than 300 nt, more than 350 nt, more than 400 nt, more than 450 nt, more than 500 nt, more than 550 nt, more than 600 nt, more than 650 nt, more than 700 nt, more than 750 nt, more than 800 nt, more than 850 nt, more than 900 nt, more than 950 nt. nt or more, 1000nt or more, 1100nt or more, 1200nt or more, 1300nt or more, 1400nt or more, 1500nt or more, 1600nt or more, 1700nt or more, 1800nt or more, 1900nt or more, 2000nt or more, 2100nt or more, 2200nt or more, 2300nt or more, 2400nt or more, 2500nt or more, 2600nt or more, 2700nt or more, 2800nt or more, 2900nt or more, 3000nt or more, 3500nt or more 00nt or more, 4000nt or more, 4500nt or more, 5000nt or more, 5500nt or more, 6000nt or more, 6500nt or more, 7000nt or more, 7500nt or more, 8000nt or more, 8500nt or more, 9000nt or more, 9500nt or more, 10000nt or more, 15000nt or more, 20000nt or more, 25000nt or more, 30000nt or more, 35000nt or more, 40000nt or more, 45000nt or more; It is further preferred that the system comprises a nucleic acid encoding a VPX protein or the VPX protein itself.

18. The system of claim 17, wherein: The heterologous sequence comprises one or more of the following: a sequence encoding a polypeptide or a non-coding RNA sequence, a sequence comprising a promoter or an enhancer, a sequence encoding one or more introns, and a transcription termination sequence; Preferably, the polypeptide is a therapeutic polypeptide or a mammalian polypeptide; further preferably, the polypeptide is a therapeutic protein, a membrane protein, an intracellular protein, an extracellular protein, a structural protein, a signal transduction protein, a regulatory protein, a transport protein, an organelle protein, a sensory protein, a motor protein, a defense protein, a storage protein, a reporter protein, an antibody, an enzyme, a coagulation factor, and further preferably, the number of amino acids in the polypeptide is 20 to 10000, for example, the number of amino acids is 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800 , 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900 00, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900; Preferably, the intracellular protein is selected from cytoplasmic protein, nuclear protein, organelle protein, mitochondrial protein or lysosomal protein, It is further preferred that the sequence encoding the polypeptide contains one or more introns.

19. The system according to claim 17 or 18, wherein: The donor RNA further comprises a homology domain, preferably the homology domain comprises a first homology domain and a second homology domain, More preferably, the first homology domain is 3 or more bases located at the 5' end of the donor RNA and having 100% identity with the target DNA chain, and the second homology domain is 3 or more bases located at the 3' end of the donor RNA and having 100% identity with the target DNA chain, and the target DNA is preferably a genomic safe harbor GSH site or a genomic Natural Harbor TM Location.

20. The system according to any one of claims 17 to 19, wherein: The nucleic acid encoding the retrotransposase according to any one of claims 1 to 16 and the donor RNA or the nucleic acid encoding the donor RNA are separate nucleic acids, preferably the donor RNA does not encode the retrotransposase, and further preferably the donor RNA comprises one or more chemical modifications; or The nucleic acid encoding the reverse transposase according to any one of claims 1 to 16 and the donor RNA or the nucleic acid encoding the donor RNA are covalently linked, preferably the nucleic acid encoding the reverse transposase according to any one of claims 1 to 16 and the donor RNA or the nucleic acid encoding the donor RNA form a fusion nucleic acid, and further preferably the fusion nucleic acid comprises RNA or DNA; It is further preferred that the nucleic acid encoding the retrotransposase according to any one of claims 1 to 16, the donor RNA or the nucleic acid encoding the donor RNA, and the nucleic acid encoding the VPX protein are separate nucleic acids; or The nucleic acid encoding the retrotransposase according to any one of claims 1 to 16 and the nucleic acid encoding the VPX protein are covalently linked or fused nucleic acids, and are separate nucleic acids from the donor RNA or the nucleic acid encoding the donor RNA; The nucleic acid encoding the retrotransposase according to any one of claims 1 to 16 is covalently linked or fused to the donor RNA or the nucleic acid encoding the donor RNA, and is a separate nucleic acid from the nucleic acid encoding the VPX protein; The donor RNA or the nucleic acid encoding the donor RNA is covalently linked or fused to the nucleic acid encoding the VPX protein, and is a separate nucleic acid from the nucleic acid encoding the retrotransposase according to any one of claims 1 to 16; or The nucleic acid encoding the retrotransposase according to any one of claims 1 to 16, the donor RNA or the nucleic acid encoding the donor RNA, and the nucleic acid encoding the VPX protein are covalently linked or fused nucleic acids, It is further preferred that the fusion nucleic acid comprises RNA or DNA.

21. The system according to any one of claims 17 to 20, wherein: The donor RNA comprises: optionally a 5' untranslated sequence (5'UTR) to which the retrotransposase binds, a 3' untranslated sequence (3'UTR) to which the retrotransposase binds, Heterologous sequences, and a promoter operably linked to the heterologous sequence, Preferably, the promoter is located between the 5' untranslated sequence (5'UTR) to which the retroposase binds and the heterologous sequence or preferably, the promoter is located between the 3' untranslated sequence (3'UTR) to which the retroposase binds and the heterologous sequence.

22. The system of claim 21, wherein: The heterologous sequence comprises an open reading frame or its reverse complement in 5' to 3' orientation on the donor RNA; or the heterologous sequence comprises an open reading frame or its reverse complement in 3' to 5' orientation on the donor RNA.

23. The system according to any one of claims 17 to 14, wherein: The donor RNA further comprises a nuclear localization signal or the nucleic acid encoding the retrotransposase according to any one of claims 1 to 8 comprises a nuclear localization signal and / or a nucleolar localization signal and / or a nuclear export signal.

24. The system according to any one of claims 9 to 23, wherein: The nucleic acid encoding the retroposase according to any one of claims 1 to 16 and the nucleic acid encoding the donor RNA are present in a ratio of 10:1 to 1:10, for example, in a ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:

10.

25. The system according to any one of claims 17 to 24, wherein: wherein the donor RNA comprises a stem-loop sequence or helix 5' of the pseudoknot sequence, preferably comprises one or more (e.g. 2, 3 or more) stem-loop sequences or helices 3' of the pseudoknot sequence, such as 3' of the pseudoknot sequence and 5' of the heterologous sequence, and further preferably the donor RNA of the pseudoknot has catalytic activity, such as RNA cleavage activity, such as cis-RNA cleavage activity, or The donor RNA comprises, for example, at least one stem-loop sequence or helix, such as 1, 2, 3, 4, 5 or more stem-loop sequences, hairpin or helix sequences, 3' to the heterologous sequence.

26. The system according to any one of claims 17 to 25, wherein: The 5' untranslated sequence (5'UTR) in the donor RNA that binds to the retrotransposase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence of any one of SEQ ID No. 16, 27, 29, 31, 33, 35, 37, 39, 41; The 3' untranslated sequence (3'UTR) in the donor RNA that binds to the retrotransposase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence of any one of SEQ ID No. 17, 32, 34, 44, 46, 48, 50, 52, 54; or The amino acid sequence of the VPX protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of any one of SEQ ID No. 85, 86 or 87.

27. The system according to any one of claims 17 to 26, wherein: The donor RNA includes the following structures from its 5' end to its 3' end: The first homology domain, a 5' untranslated sequence (5'UTR) to which the retrotransposase binds, Heterologous sequences, a 3' untranslated sequence (3'UTR) to which the retrotransposase binds, and The second homology domain; Preferably, the first homology domain is one or more, two or more, five or more, ten or more, twenty or more, thirty or more, forty or more homologous domains located at the 5' end of the donor RNA and having 100% identity with the target DNA strand. or 50 or more or 60 or more or 70 or more or 80 or more or 90 or more or 100 or 110 or 120 or 130 or 140 or 150 or more bases, the second homology domain is 1 or more or 2 or more or 5 or more or 10 or more or 20 or more or 30 or more or 40 or more or 50 or more or 60 or more or 70 or more or 80 or more or 90 or more or 100 or 110 or 120 or 130 or 140 or 150 or more bases located at the 3' end of the donor RNA and having 100% identity with the target DNA strand; It is further preferred that the 5' untranslated sequence (5'UTR) in the donor RNA that binds to the retrotransposase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence of any one of SEQ ID No. 16, 27, 29, 31, 33, 35, 37, 39, 41; The 3' untranslated sequence (3'UTR) in the donor RNA to which the retrotransposase binds has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence of any one of SEQ ID No.17, 32, 34, 44, 46, 48, 50, 52, 54.

28. The system according to any one of claims 17 to 27, wherein: The 5' untranslated sequence (5'UTR) to which the retrotransposase binds is a non-natural 5' untranslated sequence (5'UTR); or The 3' untranslated sequence (5'UTR) to which the retrotransposase binds is a non-natural 3' untranslated sequence (3'UTR); Further preferred is a non-native 5' untranslated sequence (5'UTR) having additions, deletions and / or substitutions of nucleotides relative to a native 5'UTR sequence; Further preferred are non-native 3' untranslated sequences (3'UTRs) having additions, deletions and / or substitutions of nucleotides relative to the native 3'UTR sequence; Further preferred is a non-native 5' untranslated sequence (5'UTR) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide sequence shown in one of SEQ ID No. 16, 27, 29, 31, 33, 35, 37, 39, 41; Further preferred non-native 3' untranslated sequence (3'UTR) has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence of any one of SEQ ID No.17, 32, 34, 44, 46, 48, 50, 52, 54.

29. The system according to any one of claims 17 to 27, wherein: The heterologous sequence is inserted into the target site in the genome of the cell at an average copy number of at least 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4 or 5 copies per genome, preferably only at one target site in the genome.

30. The system according to any one of claims 17 to 29, wherein: causing the heterologous sequence to be inserted into the target site (e.g., at a copy number of 1 insertion or more than one insertion) in about 1%-100% of the cells (e.g., about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% of the cells) in a population of cells contacted with the system, e.g., as measured using single cell ddPCR, or Resulting in insertion of the heterologous sequence at 1 inserted copy number into approximately 1% of the cell population contacted with the system -100% of the cells (e.g., about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% of the cells), e.g., as measured using colony isolation and ddPCR.

31. A system according to any one of claims 17 to 30, wherein: The heterologous sequence is caused to be inserted into a target site (on-target insertion) in a cell population at a higher rate than insertion into non-target sites (off-target insertion), wherein the ratio of on-target insertion to off-target insertion is greater than 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 500:1, or 1,000:

1.

32. A system according to any one of claims 17 to 31, wherein: The sequence of the first homology domain is as shown in SEQ ID NO.15, 19, 21, 23, 25 or 28 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID No.15, 19, 21, 23, 25 or 28; or The sequence of the second homology domain is as shown in SEQ ID NO.18, 20, 22, 24, 26 or 36 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID NO.18, 20, 22, 24, 26 or 36.

33. A non-natural 5' untranslated sequence (5'UTR) having additions, deletions and / or substitutions of nucleotides relative to a natural 5'UTR sequence, preferably having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence shown in SEQ ID No. 16, 27, 29, 31, 33, 35, 37, 39, 41.

34. A non-native 3' untranslated sequence (3'UTR) having additions, deletions and / or substitutions of nucleotides relative to a native 3'UTR sequence, preferably having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence of SEQ ID No. 17, 32, 34, 44, 46, 48, 50, 52, 54.

35. An engineered transposable element comprising, from 5' to 3': 5' untranslated sequence (5'UTR), heterologous sequence and 3' untranslated sequence (3'UTR), wherein the 5' untranslated sequence comprises a nucleotide sequence selected from SEQ ID No. 16, 27, 29, 31, 33, 35, 37, 39, 41 having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity; or wherein the 3' non-translated sequence comprises a nucleotide sequence selected from SEQ ID No. 17, 32, 34, 44, 46, 48, 50, 52, 54 having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity, Preferably, it comprises, from 5' to 3', a first homologous domain, a 5' untranslated sequence (5'UTR) that binds to the reverse transposase, a heterologous sequence, a 3' untranslated sequence (3'UTR) that binds to the reverse transposase, and a second homologous domain, It is further preferred that the sequence of the first homologous domain is as shown in SEQ ID NO. 15, 19, 21, 23, 25 or 28 The nucleotide sequences are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical; or The sequence of the second homology domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence shown in SEQ ID NO. 18, 20, 22, 24, 26 or 36.

36. The element according to claim 35, wherein The element is the donor RNA mentioned in claims 17 to 32 or a nucleic acid encoding the donor RNA.

37. A host cell comprising the engineered retrotransposase according to any one of claims 1 to 16, or the element according to claim 35 or 36, or the system according to any one of claims 17 to 32, wherein the host cell is preferably a mammalian cell or a plant cell, and more preferably a human cell.

38. A method for modifying a target DNA chain in a cell, tissue or subject, the method comprising using the engineered retrotransposase of any one of claims 1 to 16, the element of claim 35 or 36, or the system of any one of claims 17 to 32 on the cell, tissue or subject, wherein the system reverse transcribes the donor RNA sequence into the target DNA chain, thereby modifying the target DNA chain in the cell, tissue or subject.

39. The method of claim 38, wherein: The cells and tissues are mammalian cells and tissues, preferably human cells and tissues, and the subject is a mammal, preferably a human.

40. The method of claim 39, wherein: The cells are fibroblasts or primary cells or cells that have not been immortalized.

41. The method according to any one of claims 38 to 40, wherein: The method is performed in vivo or in vitro.

42. A method for modifying the genome of a mammalian cell or inserting DNA into the genome of a mammal, the method comprising applying the engineered retrotransposase of any one of claims 1 to 16 or the element of claim 35 or 36 or the system of any one of claims 17 to 32 to the cell, preferably the mammal is a human.

43. The method of claim 42, wherein: The method results in the addition of at least 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 base pairs of exogenous DNA sequence to the genome of the mammal.

44. The method according to any one of claims 38 to 43, wherein: The cell is part of a tissue; or the mammalian cell is euploid, is not immortalized, is part of an organism, is a primary cell, is non-dividing, is a hepatocyte or is from a subject suffering from a genetic disease.

45. The method according to any one of claims 38 to 44, wherein: The method comprises contacting a cell, a tissue or a subject with a retrotransposase as described in any one of claims 1 to 16 or a nucleic acid encoding the retrotransposase as described in any one of claims 1 to 16 and a donor RNA or a nucleic acid encoding the donor RNA, Preferably, the contacting comprises contacting the cell, tissue or subject with a plasmid, a virus, a virus-like particle, a virosome, a liposome, a vesicle, an exosome or a lipid nanoparticle; It is further preferred that said contacting comprises the use of non-viral delivery, such as electroporation.

46. ​​The method of claim 45, wherein: The contacting comprises intravenously administering to the subject, preferably administering to the subject at least twice, the retroposase as claimed in any one of claims 1 to 16 or a nucleic acid encoding the retroposase as claimed in any one of claims 1 to 16 and a donor RNA or a nucleic acid encoding the donor RNA.

47. The method according to any one of claims 38 to 46, in, The retroposase according to any one of claims 1 to 16 or a nucleic acid encoding the retroposase according to any one of claims 1 to 16 and the donor RNA or a nucleic acid encoding the donor RNA are administered separately; or The retroposase according to any one of claims 1 to 16 or a nucleic acid encoding the retroposase according to any one of claims 1 to 16 is administered together with a donor RNA or a nucleic acid encoding the donor RNA.

48. A nucleic acid encoding the retrotransposase according to any one of claims 1 to 16.

49. A vector comprising the nucleic acid of claim 48.

50. A host cell comprising the vector of claim 49.

51. A pharmaceutical composition comprising the system of any one of claims 17 to 32, or the nucleic acid of claim 48, or the vector of claim 49, or the host cell of claim 37 or 50, preferably the system is placed in a pharmaceutically acceptable carrier, and further preferably the carrier is a vesicle (including liposomes, natural or synthetic lipid bilayers, exosomes), lipid nanoparticles, viruses or plasmid vectors.

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