RETROTRANSPOSON COMPOSITIONS AND METHODS OF USE
The present disclosure provides systems and methods for transposing a cargo nucleotide sequence to a target nucleic acid sequence. These systems and methods can comprise a nucleic acid comprising the cargo nucleotide sequence, wherein the cargo nucleotide sequence is configured to interact with a retrotransposase, and the retrotransposase, wherein the retrotransposase is configured to transpose the cargo nucleotide sequence to the target nucleic acid sequence. The systems and methods can also involve use of functional fragments of retrotransposases.
1 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1-29, 393-735, 799-895, 1020-1476, 1544-1554, 1850-2160, 2165-2210, and 2258-2266.
2 . The engineered retrotransposase system of claim 1 , wherein the retrotransposase comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-29, 393-735, 799-895, 1020-1476, 1544-1554, 1850-2160, 2165-2210, and 2258-2266.
3 . The engineered retrotransposase system of claim 1 , wherein the retrotransposase comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-29, 393-735, 799-895, 1020-1476, 1544-1554, 1850-2160, 2165-2210, and 2258-2266.
4 . The engineered retrotransposase system of claim 1 , wherein the retrotransposase comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1-29, 393-735, 799-895, 1020-1476, 1544-1554, 1850-2160, 2165-2210, and 2258-2266.
5 . The engineered retrotransposase system of claim 1 , wherein the retrotransposase is encoded by a nucleic acid having at least 75% sequence identity to any one of SEQ ID NOs: 120-173, 181-187, 193-197, 203-207, 217-225, 231-235, 241-245, 251-255, 267-277, 288-297, 303-307, 324-339, 964-981, 1003-1019, 1504-1520, 1521-1536, 1539-1543, 1556-1568, and 1611-1806.
6 . The engineered retrotransposase system of claim 1 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 120-173, 181-187, 193-197, 203-207, 217-225, 231-235, 241-245, 251-255, 267-277, 288-297, 303-307, 324-339, 964-981, 1003-1019, 1504-1520, 1521-1536, 1539-1543, 1556-1568, and 1611-1806.
7 . The engineered retrotransposase system of claim 1 , wherein retrotransposase is encoded by a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 120-173, 181-187, 193-197, 203-207, 217-225, 231-235, 241-245, 251-255, 267-277, 288-297, 303-307, 324-339, 964-981, 1003-1019, 1504-1520, 1521-1536, 1539-1543, 1556-1568, and 1611-1806.
8 . The engineered retrotransposase system of claim 1 , wherein retrotransposase is encoded by a nucleic acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 120-173, 181-187, 193-197, 203-207, 217-225, 231-235, 241-245, 251-255, 267-277, 288-297, 303-307, 324-339, 964-981, 1003-1019, 1504-1520, 1521-1536, 1539-1543, 1556-1568, and 1611-1806.
9 . The engineered retrotransposase system of any one of claims 1-8 , wherein the double-stranded nucleic acid comprises a 5′ recognition sequence comprising a GG nucleotide sequence and a 3′ recognition sequence comprising a TGAC nucleotide sequence.
10 . The engineered retrotransposase system of claim 9 , wherein the 5′ recognition sequence and the 3′ recognition sequence are configured to interact with the retrotransposase.
11 . The engineered retrotransposase system of any one of claims 1-10 , wherein the double-stranded nucleic acid comprising a cargo nucleotide sequence is RNA.
12 . The engineered retrotransposase system of claim 11 , wherein the RNA is an in vitro transcribed RNA.
13 . The engineered retrotransposase system of any one of claims 11-12 , wherein the RNA comprises a sequence 5′ to the cargo sequence or a sequence 3′ to the cargo sequence that has at least 80% sequence identity to an RNA cognate of any one of SEQ ID NOs: 761-798, 2161-2164, and 2211-2257, a complement thereof, or a reverse complement thereof.
14 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1-29, 393-401, 799-894, 1476, 1850-1926, and 2165-2210.
15 . The engineered retrotransposase system of claim 14 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: SEQ ID NOs: 1535-1536, 1542-1543, 1611-1623, 1663-1691, and 1786-1806.
16 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 402 or SEQ ID NO: 895.
17 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 388.
18 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 403-426.
19 . The engineered retrotransposase system of claim 18 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 389-392 and 1504-1507.
20 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 427-439.
21 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 440-554 and 1020-1037.
22 . The engineered retrotransposase system of claim 21 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 356-373, 964-981, and 1003-1019.
23 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 555-608 and 1927-2010.
24 . The engineered retrotransposase system of claim 23 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 66-173, 740-756, 1521-1534, 1539-1541, 1624-1637, 1645-1662, and 1701-1782.
25 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 609-610 and 1555.
26 . The engineered retrotransposase system of claim 25 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 308-309 and 324-325.
27 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 611-615 and 1544-1545.
28 . The engineered retrotransposase system of claim 27 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 310-312, 326-328, 1556-1557, and 1569-1570.
29 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 616 or SEQ ID NO: 617.
30 . The engineered retrotransposase system of claim 29 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 313-314 and 329-330.
31 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 618-622 and 2258-2266.
32 . The engineered retrotransposase system of claim 31 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 315-319 and 331-335.
33 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 623.
34 . The engineered retrotransposase system of claim 33 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 320 or SEQ ID NO: 336.
35 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 624-626.
36 . The engineered retrotransposase system of claim 35 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 321-323, 337-339, and 1785.
37 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 624-626.
38 . The engineered retrotransposase system of claim 35 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 321-323, 337-339, and 1785.
39 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 627-673, 1039-1475, and 2011-2026.
40 . The engineered retrotransposase system of claim 39 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 174-187 and 1508-1520.
41 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 674-678.
42 . The engineered retrotransposase system of claim 41 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 188-197.
43 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 679-683.
44 . The engineered retrotransposase system of claim 43 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 198-207.
45 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 684-692 and 2027-2046.
46 . The engineered retrotransposase system of claim 45 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 208-225 and 757-759.
47 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 693-697 and 2047-2090.
48 . The engineered retrotransposase system of claim 47 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 226-235.
49 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 698-702 and 2091-2119.
50 . The engineered retrotransposase system of claim 49 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 236-245 and 759-760.
51 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 703-707.
52 . The engineered retrotransposase system of claim 51 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 246-255.
53 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 708-718 and 2121-2159.
54 . The engineered retrotransposase system of claim 53 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 256-277, 1638-1644, and 1693-1700.
55 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 719-728.
56 . The engineered retrotransposase system of claim 55 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 278-297.
57 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 729-733.
58 . The engineered retrotransposase system of claim 57 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 298-307.
59 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 734-735 and 1546-1553.
60 . The engineered retrotransposase system of claim 59 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1558-1567, 1571-1580, and 1783-1784.
61 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 1038 or SEQ ID NO: 2160.
62 . The engineered retrotransposase system of claim 61 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1692.
63 . An engineered retrotransposase system, comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence configured to form a complex with a retrotransposase; and
(b) a retrotransposase configured to transpose the cargo nucleotide sequence to a target nucleic acid sequence and comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 1554.
64 . The engineered retrotransposase system of claim 63 , wherein the retrotransposase is encoded by a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1568 or SEQ ID NO: 1594.
65 . The engineered retrotransposase system of any one of claims 1-64 , wherein the retrotransposase comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of the retrotransposase.
66 . The engineered retrotransposase system of claim 65 , wherein the NLS comprises a sequence at least 80% identical to a sequence from the group consisting of SEQ ID NO: 1477-1492.
67 . The engineered retrotransposase system of claim 65 , wherein the NLS comprises SEQ ID NO: 1478.
68 . The engineered retrotransposase system of claim 65 , wherein the NLS is proximal to the N-terminus of the retrotransposase.
69 . The engineered retrotransposase system of claim 65 , wherein the NLS comprises SEQ ID NO: 1477.
70 . The engineered retrotransposase system of claim 65 , wherein the NLS is proximal to the C-terminus of the retrotransposase.
71 . A polypeptide comprising a reverse transcriptase comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1-29, 393-735, 799-895, 1020-1476, 1544-1554, 1850-2160, 2165-2210, and 2258-2266 fused N- or C-terminally to a non-retrotransposase domain or an affinity tag.
72 . The polypeptide of claim 71 , wherein the non-retrotransposase domain is an RNA-binding protein domain.
73 . The polypeptide of claim 72 , wherein the RNA binding protein domain comprises a bacteriophage MS2 coat protein (MCP) domain.
74 . A nucleic acid encoding the engineered retrotransposase system of any one of claims 1-64 or the polypeptide of any one of claims 71-73 .
75 . A method for modifying a target nucleic acid sequence comprising contacting the target nucleic acid sequence using the engineered nuclease system of any one of claims 1-64 .
76 . The method of claim 75 , wherein modifying the target nucleic acid sequence comprises binding, nicking, or cleaving, the target nucleic acid sequence.
77 . The method of any one of claims 75-76 , wherein the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.
78 . The method of any one of claims 75-76 , wherein the target nucleic acid sequence comprises deoxyribonucleic acid (DNA).
79 . The method of any one of claims 75-78 , wherein the modification is in vitro.
80 . The method of any one of claims 75-78 , wherein the modification is in vivo.
81 . The method of any one of claims 75-78 , wherein the modification is ex vivo.
82 . A method of modifying a target nucleic acid sequence in a mammalian cell comprising contacting the mammalian cell using the engineered nuclease system of any one of claims 1-64 .
83 . A method for synthesizing complementary DNA (cDNA), comprising:
(a) providing an RNA molecule as a template for cDNA synthesis,
(b) providing a primer oligonucleotide to initiate cDNA synthesis from the RNA molecule; and
(c) synthesizing cDNA initiated by the primer oligonucleotide from the template using a reverse transcriptase comprising a sequence having at least 80% sequence identity to a reverse transcriptase domain of any one of SEQ ID NOs: 1-29, 393-735, 799-895, 1020-1476, 1544-1554, 1850-2160, 2165-2210, and 2258-2266.
84 . The method of claim 83 , wherein the primer oligonucleotide comprises an oligo (dT) sequence or a degenerate sequence of at least six oligonucleotides.
85 . A vector comprising the nucleic acid of claim 74 .
86 . The vector of claim 85 , wherein the vector is a plasmid, a minicircle, a CELiD, an adeno-associated virus (AAV) derived virion, or a lentivirus.
87 . A cell comprising the engineered nuclease system of any one of claims 1-64 or the polypeptide of any one of claims 71-73 .
88 . The cell of claim 87 , wherein the cell is a eukaryotic cell.
89 . The cell of claim 87 , wherein the cell is a mammalian cell.
90 . The cell of claim 87 , wherein the cell is an immortalized cell.
91 . The cell of claim 87 , wherein the cell is an insect cell.
92 . The cell of claim 87 , wherein the cell is a yeast cell.
93 . The cell of claim 87 , wherein the cell is a plant cell.
94 . The cell of claim 87 , wherein the cell is a fungal cell.
95 . The cell of claim 87 , wherein the cell is a prokaryotic cell.
96 . The cell of claim 87 , wherein the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof.
97 . The cell of claim 87 , wherein the cell is an engineered cell.
98 . The cell of claim 87 , wherein the cell is a stable cell.