Transposition of nucleic acid constructs into eukaryotic genomes with a transposase from amyelois
The present invention provides polynucleotide vectors for high expression of heterologous genes. Some vectors further comprise novel transposons and transposases that further improve expression. Further disclosed are vectors that can be used in a gene transfer system for stably introducing nucleic acids into the DNA of a cell. The gene transfer systems can be used in methods, for example, gene expression, bioprocessing, gene therapy, insertional mutagenesis, or gene discovery.
1. A polynucleotide comprising an open reading frame encoding a transposase, the amino acid sequence of which is at least 90% identical to SEQ ID NO: 18, operably linked to a heterologous promoter.
2. The polynucleotide of claim 1 , wherein the transposase comprises a mutation at a position as shown below, relative to the sequence of SEQ ID NO: 18
Position
Mutation
2
T, R, D, or N
3
S, Q, F, or I
4
S, F, R, L, Y, or E
5
G, T, D, S, R, or A
6
R, A, N, D, or Q
7
K, E, Q, or H
8
R, E, D, H, or N
9
S, A, I, or R
10
L or A
11
G, L, A, S, or R
12
N, L, T, A, or H
13
V, F, L, C, or M
14
H, F, or M
15
N, D, Q, or A
16
Q, S, N, or E
17
R, D, S or V
18
A, S, L or E
19
A, E, D, S, or T
20
K, Y, L, or D
21
E, S, V, F, or Y
22
R, I, P, G, or S
23
R, S, L, E, G, D, or Y
24
A, E, D, or S
25
V, I, S, or D
26
V, E, or S
27
D
28
L or Y
29
P, S, V, or E
30
G, E, or P
31
T, A, K, V, or P
32
R, E, A, or T
33
S
34
F, N, R, C, or E
35
G, D, S, or C
36
T, I, V, or D
37
T, D, E, or S
38
L, D, S, or H
39
S, D, or E
40
W, F, Q, or N
41
L, Q, or Y
42
F or W
43
N, S, or C
44
D or S
45
D, S, or Q
46
S, E, or A
47
S, M, I, F, T, R, or A
48
G, D, or V
49
S or D
50
E, Y, F, A, S, or P
51
V or D
52
H or D
53
D, S, E, V, or L
54
D, V, N, or E
55
T, V, N, I, M, or E
56
I, S, L, E, or T
57
P, A, D, V, or Y
58
Q, E, or D
59
S, Q, R, or E
60
E, N, D, or P
61
S, N, V, or D
62
E, L, A, P, I, or D
63
E, G, or N
64
M, L, or V
65
V or E
66
A, Q, V, G, or D
67
D, T, or Q
68
H, S, E, L, or A
69
V, S, or A
70
T, G, or R
71
E, S, D, or Q
72
E, G, H, or N
73
H, T, R, A, or M
74
S, T, or Y
75
F or I
76
Y, C, R, T, or S
77
S
79
N or G
80
R, K, E, or G
81
Y, I, H, or P
82
K, V, C, or A
84
A, S, G, or Y
85
C, T, P, or K
86
Q, S, T, P, or N
87
K or C
88
L, H, G, S, N, or Q
89
S, Q, R, T, or F
90
R, S, or T
91
A, T, or N
92
G, S, or I
94
V or L
95
P or S
96
Q, A, or E
97
H, S, I, or L
98
P
99
P
100
I or F
101
Q, K, or R
102
R, M, T, S, or E
103
N, Q, V, or R
104
N, R, or A
105
V, Q, or L
106
S or P
107
N or T
108
L, V, N, or T
109
T, F, M, D, I, or G
110
E, C, or T
111
D, V, R, or S
112
D or T
113
P, V, or I
114
K, D, V, Y, F, or S
115
D, E, or T
116
P or I
117
F, Q, I, Y, or S
118
S, L, N, E, or K
119
I, A, or F
120
W
121
N, Q, or K
122
K or I
123
L
124
M, I, or F
125
D, S, or T
126
D, Q, or S
127
E, P, A, or D
128
M
129
E or I
130
Q, R, H, or D
131
E, I, or D
132
T or M
133
V
134
K, D, L, or T
135
W, M, or Y
138
E, L, V, A, or S
139
E, Y, or S
140
G, M, or A
141
I, S, or E
142
Q, R, V, L, or H
143
Y, V, K, R, or S
144
F, L, or Q
145
S, R, T, or V
146
K, N, or Q
147
F, L, E, N, T, or S
148
S, I, K, P, or A
149
D, M, L, or A
150
K, I, T, P, or E
151
D, S, or R
152
A or S
153
P, E, A, H, F, or V
154
E, K, S, T, Y, or H
155
L, W, F, M, or K
156
R, K, H, or Q
157
N, S, D, or P
158
L or I
159
D or N
160
M, Q, T, E, or I
161
V, M, D, A, C, or S
162
D
163
M or I
164
H, N, W, Y, or K
165
R
166
F, Y, or L
167
V or F
168
A
169
I
170
T
171
L, I, T, or V
172
F, M, A, or I
173
T or M
174
A
175
V
176
Y, M, R, I, or T
177
R
178
D or A
179
N or K
180
H or G
181
E, L, M, or S
182
A, S, or L
183
V, T, or E
184
N, S, D, or K
185
Y, S, or E
186
W
187
F or D
188
N, D, T, or R
189
T, A, or R
190
E, T, or S
191
E, F, L, or V
192
N, S, or L
193
S
194
R or V
195
P, T, M, or D
196
R or V
197
Y
198
R, V, or S
199
C, A, S, or T
200
V
203
K or R
204
N, E, D, or Q
205
T
206
Y
207
L, H, E, D, Q, or Y
208
V, M, or L
209
L
210
L, S, I, or Q
211
H or R
212
V, F, N, or S
213
I or M
214
H
215
M
216
N
217
N
218
P, R, K, or T
219
T, S, or A
220
D, L, I, or V
221
P
222
V, P, or D
223
G, T, or D
224
L or Q
225
R, P, or K
226
E, S, G, Q, or K
227
S, D, N, H, or T
229
K, A, V, or N
230
I, F, or M
231
I, L, T, or H
232
A or K
233
V, L, or F
234
S
235
Y, D, K, P, or S
236
V, L, or M
237
F, W, or I
238
T or D
239
K, I, L, S, or Q
240
W or L
241
I or S
242
G, E, N, H, or Q
243
I, Q, R, or C
244
L or F
245
Q, P, R, I, or A
246
K, L, Q, A, or N
247
I, L, N, or A
248
H
249
N, V, or S
250
V
251
C, G, or S
252
P, G, S, A, or Q
253
Y, H, or F
254
A, V, or I
255
C
256
V
259
M, R, Q, or S
261
L
262
P, G, L, or S
264
K
267
T or L
268
H, P, Q, or L
269
L
270
M
271
I, M, or V
273
M or I
275
M or S
277
R
278
D or S
279
R
282
L
283
R
284
L or F
285
M, W, P, L, Y, or F
286
C, M, or K
287
A or M
288
C
289
A or E
290
S
291
N, K, A, G, or S
292
N or S
293
G, S, K, or F
295
F, A, M, T, or V
296
Y, W, I, S, or V
297
K, D, or Y
298
C, A, G, F, or L
299
Y, Q, I, M, or L
300
V or P
302
T, L, or E
304
R or K
305
G, S, or D
306
S or T
307
G, K, Q, S, or L
308
T or L
309
A, N, or D
310
G or P
311
L, E, V, P, or A
312
T, K, P, or G
313
E, N, or C
314
E, Q, or P
315
V, L, R, or T
316
G, S, or A
317
T, M, E, G, or F
318
Q, R, F, Y, K, or E
319
S, Y, or I
321
I, L, K, W, or E
322
H, E, or D
323
L or M
324
A, S, T, or I
325
K, E, or S
326
G or T
327
L or V
328
F, Q, H, L, or A
329
G or R
330
S or Q
331
N, C, or H
332
H or F
333
H
334
I or L
335
Y
336
C, V, or F
339
F
340
Y
341
S
342
G
343
I
344
E, R, or T
345
L
346
I, G, A, F, or M
347
E, K, T, or L
348
Y, E, N, or A
349
M
350
K, Q, or Y
351
Q, C, or N
352
K, R, A, E, or L
353
Y
354
G, N, D, or R
355
T
356
P
357
C, M, I, or S
358
L, C, or T
361
V or I
362
R or N
363
K or R
365
R
366
R, P, or K
367
E, G, or Q
368
L or M
370
K, S, E, R, or D
371
E, V, A, or S
372
I or L
373
L, K, I, or T
374
P, K, N, D, or R
375
S, I, K, or T
376
N
377
D, P, R, or Q
378
V, M, G, or P
379
G, H, E, or A
380
S
381
Y
382
I, M, A, or V
383
Y or L
384
A, C, or R
385
Y or K
386
A, T, D, or N
387
G, E, or K
388
Q, K, P, or L
389
N, A, L, or I
390
A
391
V or L
392
L or K
393
F
394
H or F
395
V, C, K, D, or A
398
K or P
399
S, A, or K
400
R
401
A, M, or V
403
I, L, Y, or V
404
L, V, or A
405
M
406
T
407
T
408
M, C, or I
409
D
410
T, E, or D
411
A, N, or E
412
E, A, or N
413
A, S, or V
414
V or L
415
S, N, or R
416
E, T, or Q
417
T, R, S, or Q
418
D, N, or R
419
D or V
420
G
421
K
422
M or N
425
E, Q, D, or L
426
M or C
427
V or S
428
G, L, M, or K
429
D, Y, or E
431
S
432
S or Q
433
Y
434
M
435
G or A
439
E, S, T, R, or V
440
I, L, F, or T
442
Q or E
443
K, V, or M
444
C, T, Q, or S
445
A, K, S, or H
446
I, S, V, Y, or N
447
M
448
T, S, or N
449
S, C, or A
450
Q, N, or T
452
R or K
453
T
454
A, N, or K
455
A
457
Y
458
M or K
459
V, A, or K
460
V or L
461
L or G
462
Y or I
463
R, N, G, or Y
464
M, L, or V
465
V or I
466
D or Q
467
I, V, or M
468
S
469
T, A, F, C, L, or S
470
V, Y, or R
472
S
473
H, Y, F, or C
474
L or V
475
I or L
476
Y or W
477
D, S, C, K, R, or Q
478
I, E, H, A, or T
479
H, I, or A
480
H, N, K, or V
481
Q, I, P, or N
482
E, N, S, or G
483
K, V, or A
484
L, P, or V
485
T, Y, I, Q, S, or V
486
E, R, N, S, Y, or K
487
Y
488
G, K, or T
489
M, L, E, K, or Y
490
Q
491
L or M
492
E, R, or Q
493
Q, E, N, I, or S
495
A, S, Y, or P
496
R, K, T, I, A, or L
497
T, A, D, Q, or L
498
M
499
V, T, or F
500
L, T, S, G, or Y
501
S, G, or E
502
Q, K, W, F, or V
503
M, I, E, or L
504
K, Q, R, E, or H
505
R, K, Q, or S
506
T
507
A, L, V, or N
508
L, R, K, Q, E, or P
509
P, A, E, or K
510
E, A, P, M, or K
511
R, T, P, or N
512
L, S, or P
513
K or S
514
R, A, D, T, or F
515
E, A, T, Y, N, or H
516
A, V, or I
517
A
518
L, V, D, R, or Q
519
S, I, N, or L
520
L or E
521
A, K, T, S, or E
522
R, I, N, or K
523
V, K, I, or Q
524
F
525
R, K, P, or I
526
P, T, N, K, or D
527
D, V, E, T, or P
528
M, N, V, L, or T
529
P or Q
530
V, A, T, or R
531
M, S, or H
532
D, E, G, or V
533
P, N, or D
534
Q, V, N, S, or M
535
E, D, S, T, or R
536
V, T, or E
537
F, P, or M
538
K
539
T, K, P, or R
540
R, Q, or Y
541
R, S, T, or V
542
Y or G
544
H, Q, G, T, K, or R
545
T, V, F, Y, D, or E
547
S or R
548
L, S, Y, K, or N
549
R
550
L, D, or I
551
Q or R
552
S
553
M or D
554
T or A
555
T, S, N, or R
556
H, T, A, or R
557
T, S, Q, or Y
558
F
559
Y, V, C, K, P, or N
560
T, K, or A
562
K, P, or A
563
K, N, or R
564
F, A, V, N, or P
565
V or L
567
R, G, F, or M
568
Q or K
569
C or P
570
T, N, or C
571
K, V, I, or F
572
Q, T, D, or E
573
F, M, I, or L
574
Y
575
A, P, Q, or H
576
D, S, or T
577
Q
578
V, G, I, F, L, or A.
3. The polynucleotide of claim 2 , wherein the transposase comprises a mutation at an amino acid position selected from 79, 95, 115, 116, 121, 139, 166, 179, 187, 198, 203, 211, 238, 273, 304, 329, 345, 362, 366, 408, 416, 435, 458, 475, 483, 491, 529, 540, 560 and 563, relative to the sequence of SEQ ID NO: 18.
4. The polynucleotide of claim 3 , wherein the transposase comprises a mutation selected from D79N, R95S, L115D, E116P, H121Q, K139E, V166F, G179N, W187F, P198R, L203R, N211R, E238D, L273M, L273I, D304R, D304K, Q329G, T345L, K362R, T366R, L408M, S416E, S435G, L458M, V475I, N483K, I491M, A529P, K540R, S560K and S563K, relative to the sequence of SEQ ID NO: 18.
5. The polynucleotide of claim 4 , comprising the mutated V166F.
6. The polynucleotide of claim 4 , comprising at least two mutations selected from D79N, R95S, L115D, E116P, H121Q, K139E, V166F, G179N, W187F, P198R, L203R, N211R, E238D, L273M, L273I, D304R, D304K, Q329G, T345L, K362R, T366R, L408M, S416E, S435G, L458M, V475I, N483K, I491M, A529P, K540R, S560K and S563K, relative to the sequence of SEQ ID NO: 18.
7. The polynucleotide of claim 4 , comprising at least five mutations selected from D79N, R95S, L115D, E116P, H121Q, K139E, V166F, G179N, W187F, P198R, L203R, N211R, E238D, L273M, L273I, D304R, D304K, Q329G, T345L, K362R, T366R, L408M, S416E, S435G, L458M, V475I, N483K, I491M, A529P, K540R, S560K and S563K, relative to the sequence of SEQ ID NO: 18.
8. The polynucleotide of claim 2 , wherein the amino acid sequence of the transposase is selected from SEQ ID NO: 96-170.
9. The polynucleotide of claim 1 , wherein the transposase can excise or transpose a transposon from SEQ ID NO: 192, wherein the transposase differs from SEQ ID NO:18 by one or more mutation selected from D79N, R95S, L115D, E116P, H121Q, K139E, V166F, G179N, W187F, P198R, L203R, N211R, E238D, L273M, L273I, D304R, D304K, Q329G, T345L, K362R, T366R, L408M, S416E, S435G, L458M, V475I, N483K, I491M, A529P, K540R, S560K and S563K.
10. The polynucleotide of claim 9 , wherein the excision activity or transposition activity of the transposase is at least 2-fold higher than the activity of SEQ ID NO: 18.
11. The polynucleotide of claim 1 , wherein the promoter is active in an in vitro transcription reaction.
12. The polynucleotide of claim 1 , wherein the promoter is active in a eukaryotic cell.
13. The polynucleotide of claim 12 , wherein the eukaryotic cell is a mammalian cell.
14. The polynucleotide of claim 1 , wherein the open reading frame further encodes a nuclear localization sequence fused to the transposase.
15. The polynucleotide of claim 1 , wherein the open reading frame further encodes a heterologous DNA binding domain fused to the transposase.
16. The polynucleotide of claim 15 , wherein the DNA binding domain is from a CRISPR Cas protein, a zinc finger protein, or a TALE protein.
17. A non-naturally occurring polypeptide encoded by the polynucleotide of claim 1 .
18. A non-naturally occurring polynucleotide encoding a polypeptide, the sequence of which is at least 90% identical to SEQ ID NO: 18, wherein the polynucleotide sequence comprises at least 10 synonymous codon differences relative to SEQ ID NO: 929.
19. The non-naturally occurring polynucleotide of claim 18 , which is an mRNA.
20. The polynucleotide of claim 18 , wherein codons of the polynucleotide at positions of the at least ten synonymous codon differences are selected using codon preferences of a mammalian cell.
21. A method of integrating a transposon into a eukaryotic cell, the method comprising
(a) introducing into the cell a transposon comprising SEQ ID NO: 9 and SEQ ID NO: 10 as left and right ends respectively flanking a heterologous polynucleotide; and
(b) introducing into the cell a transposase, the sequence of which comprises any of SEQ ID NOS:19-35, or 51-170 wherein the transposase transposes the transposon to produce a genome comprising SEQ ID NO: 9 and SEQ ID NO: 10 flanking the heterologous polynucleotide at left and right ends respectively.
22. The method of claim 21 , wherein the transposase is introduced as a polynucleotide encoding the transposase.
23. The method of claim 22 , wherein the polynucleotide encoding the transposase is an mRNA molecule.
24. The method of claim 22 , wherein the polynucleotide encoding the transposase is a DNA molecule.
25. The method of claim 21 , wherein the transposase is introduced as a protein.
26. The method of claim 21 , wherein the heterologous polynucleotide encodes a selectable marker, and the method further comprises
(c) selecting a cell comprising the selectable marker.
27. The method of claim 21 , wherein the cell is an animal cell.
28. The animal cell produced by the method of claim 27 , wherein the cell is a mammalian cell.
29. The animal cell of claim 28 , wherein the cell is a rodent cell.
30. The animal cell of claim 28 , wherein the cell is a human cell.