RU2016110827A - Эффективная немейотическая интрогрессия аллелей - Google Patents
Эффективная немейотическая интрогрессия аллелей Download PDFInfo
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- RU2016110827A RU2016110827A RU2016110827A RU2016110827A RU2016110827A RU 2016110827 A RU2016110827 A RU 2016110827A RU 2016110827 A RU2016110827 A RU 2016110827A RU 2016110827 A RU2016110827 A RU 2016110827A RU 2016110827 A RU2016110827 A RU 2016110827A
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- 238000000034 method Methods 0.000 claims 27
- 239000011159 matrix material Substances 0.000 claims 19
- 108700028369 Alleles Proteins 0.000 claims 17
- 101710163270 Nuclease Proteins 0.000 claims 16
- 210000004027 cell Anatomy 0.000 claims 15
- 239000013611 chromosomal DNA Substances 0.000 claims 14
- 230000004568 DNA-binding Effects 0.000 claims 9
- 241001465754 Metazoa Species 0.000 claims 7
- 108010042407 Endonucleases Proteins 0.000 claims 6
- 102000004533 Endonucleases Human genes 0.000 claims 6
- 108020004414 DNA Proteins 0.000 claims 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims 4
- 238000010459 TALEN Methods 0.000 claims 4
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 claims 4
- 238000012217 deletion Methods 0.000 claims 4
- 230000037430 deletion Effects 0.000 claims 4
- 238000003780 insertion Methods 0.000 claims 4
- 230000037431 insertion Effects 0.000 claims 4
- 239000002773 nucleotide Substances 0.000 claims 4
- 125000003729 nucleotide group Chemical group 0.000 claims 4
- 230000009870 specific binding Effects 0.000 claims 4
- 125000006850 spacer group Chemical group 0.000 claims 3
- 108010073062 Transcription Activator-Like Effectors Proteins 0.000 claims 2
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 claims 2
- 241000251468 Actinopterygii Species 0.000 claims 1
- 241000283690 Bos taurus Species 0.000 claims 1
- 241000283707 Capra Species 0.000 claims 1
- 241000283073 Equus caballus Species 0.000 claims 1
- 241000282326 Felis catus Species 0.000 claims 1
- 241000287828 Gallus gallus Species 0.000 claims 1
- 241000282414 Homo sapiens Species 0.000 claims 1
- 241000699666 Mus <mouse, genus> Species 0.000 claims 1
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 241000283973 Oryctolagus cuniculus Species 0.000 claims 1
- 241001494479 Pecora Species 0.000 claims 1
- 241000009328 Perro Species 0.000 claims 1
- 241000288906 Primates Species 0.000 claims 1
- 241000700159 Rattus Species 0.000 claims 1
- 241000282898 Sus scrofa Species 0.000 claims 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims 1
- 238000010171 animal model Methods 0.000 claims 1
- 238000003776 cleavage reaction Methods 0.000 claims 1
- 230000007423 decrease Effects 0.000 claims 1
- 238000010353 genetic engineering Methods 0.000 claims 1
- 244000144972 livestock Species 0.000 claims 1
- 210000001161 mammalian embryo Anatomy 0.000 claims 1
- 108020004999 messenger RNA Proteins 0.000 claims 1
- 210000000287 oocyte Anatomy 0.000 claims 1
- 102000054765 polymorphisms of proteins Human genes 0.000 claims 1
- 210000005132 reproductive cell Anatomy 0.000 claims 1
- 230000007017 scission Effects 0.000 claims 1
- 210000001082 somatic cell Anatomy 0.000 claims 1
- 241000894007 species Species 0.000 claims 1
- 210000000130 stem cell Anatomy 0.000 claims 1
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
- C12N15/877—Techniques for producing new mammalian cloned embryos
- C12N15/8778—Swine embryos
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- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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Claims (31)
1. Способ направляемой гомологией репарации для интрогрессии экзогенного аллеля в хромосомную ДНК клетки, включающий введение нацеленной системы эндонуклеаз и матрицы для направляемой гомологией репарации (НГР-матрицы), которая содержит экзогенный аллель, в клетку, с нацеленной системой нуклеаз, содержащей ДНК-связывающий член для специфического связывания эндогенной родственной последовательности в хромосомной ДНК, где нацеленная система нуклеаз и НГР-матрица действуют с целью внесения изменений в хромосомную ДНК для ее идентичности последовательности НГР-матрицы, чтобы провести интрогрессию экзогенного аллеля в хромосомную ДНК вместо эндогенного аллеля, с нацеленной системой эндонуклеаз и/или НГР-матрицей, содержащей признак, снижающий специфическое связывание нацеленной системы эндонуклеаз с ДНК.
2. Способ по п. 1, отличающийся тем, что признак, снижающий специфическое связывание, включает несовпадение в последовательности ДНК-связывающего члена относительно эндогенной родственной последовательности и/или несовпадение в последовательности ДНК-связывающего члена относительно последовательности НГР-матрицы.
3. Способ по п. 2, отличающийся тем, что нацеленная система эндонуклеаз содержит множество последовательностей TAL-эффекторных повторов, которые слиты с нуклеазой (TALEN-нуклеаза), с TALEN-нуклеазой, содержащей последовательность вариабельных диостатков в повторе (ВДП), и несовпадения в последовательности ВДП относительно эндогенной родственной последовательности.
4. Способ по п. 2, отличающийся тем, что нацеленная система нуклеаз содержит Cas9-нуклеазу и руководящую РНК, с несовпадением, существующим в последовательности руководящей РНК относительно эндогенной родственной последовательности.
5. Способ по п. 2, отличающийся тем, что нацеленная система эндонуклеаз содержит множество последовательностей TAL-эффекторных повторов, которые слиты с нуклеазой (TALEN-нуклеаза), с TALEN-нуклеазой, содержащей последовательность вариабельных диостатков в повторе (ВДП), и несовпадения в последовательности ВДП относительно последовательности НГР-матрицы.
6. Способ по п. 2, отличающийся тем, что нацеленная система нуклеаз содержит Cas9-нуклеазу и руководящую РНК, с несовпадением, существующим в последовательности руководящей РНК, относительно последовательности НГР-матрицы.
7. Способ по п. 2, отличающийся тем, что экзогенный аллель является природным аллелем и НГР-матрица содержит несовпадение, с несовпадением, создающим последовательность, которая не встречается в природе.
8. Способ по п. 2, отличающийся тем, что экзогенный аллель не содержит несовпадений и содержит ДНК, экспрессируемую клеткой.
9. Способ по п. 2, отличающийся тем, что экзогенный аллель содержит несовпадение и содержит ДНК, экспрессируемую клеткой.
10. Способ по п. 2, включающий выбор последовательности ДНК-связывающего члена и эндогенной родственной последовательности, так что внесение изменений в хромосомную ДНК для ее идентичности последовательности НГР-матрицы создает несовпадение в последовательности ДНК-связывающего члена относительно измененной последовательности хромосомной ДНК.
11. Способ по п. 10, отличающийся тем, что экзогенный аллель является природным аллелем и НГР-матрица состоит из природного аллеля и ДНК, которая идентична последовательности хромосомной ДНК.
12. Способ по п. 10, отличающийся тем, что выбор последовательности ДНК-связывающего члена и эндогенной родственной последовательности дополнительно включает размещение второго несовпадения в эндогенной родственной последовательности, которое не изменяется, когда вносят изменения в хромосомную ДНК для ее идентичности НГР-матрице.
13. Способ по п. 2, включающий выбор последовательности ДНК-связывающего члена и эндогенной родственной последовательности, чтобы разместить несовпадение в эндогенной родственной последовательности относительно ДНК-связывающей последовательности, и внесение изменений в хромосомную ДНК для ее идентичности последовательности НГР-матрицы не убирало данное несовпадение.
14. Способ по п. 2, отличающийся тем, что несовпадение включает вставку, делецию или замену.
15. Способ по п. 14, отличающийся тем, что вставка, делеция или замена имеет длину от 1 до 20 остатков.
16. Способ по п. 14, отличающийся тем, что вставка, делеция или замена имеет длину от 1 до 20 остатков.
17. Способ по п. 2, отличающийся тем, что несовпадение является одним однонуклеотидным полиморфизмом.
18. Способ по п. 2 включающий множество несовпадений.
19. Способ по п. 1, отличающийся тем, что нацеленная система эндонуклеаз содержит пару TALEN-нуклеаз, которые локализуют хромосомную ДНК посредством спейсерной последовательности между парой нуклеаз, отличающийся тем, что признак включает выбор НГР-матрицы для получения изменения в длине спейсерной последовательности, чтобы блокировать расщепление ДНК парой TALEN-нуклеаз.
20. Способ по п. 19, отличающийся тем, что длина спейсера уменьшается путем делеции или увеличивается путем вставки.
21. Способ по любому из пп. 1-20, отличающийся тем, что клетка выбрана из группы, состоящей из первичной клетки, первичной соматической клетки, зиготы, половой клетки, стволовой клетки, ооцита, сперматозоида и эмбриона.
22. Способ по любому из пп. 1-20, отличающийся тем, что систему нуклеаз вводят в клетку в виде мРНК.
23. Способ по любому из пп. 1-20, отличающийся тем, что клетка является клеткой домашнего скота.
24. Способ по любому из пп. 1-20, отличающийся тем, что клетка является клеткой позвоночного, отличного от человека, примата, свиньи, крупного рогатого скота, лошади, овцы, козы, курицы, кролика, рыбы, собаки, мыши, кошки, крысы или лабораторного животного.
25. Способ по любому из пп. 1-20, отличающийся тем, что клетка является гомозиготной по экзогенному аллелю.
26. Клетка, полученная способом по любому из пп. 1-25.
27. Набор, содержащий нацеленную систему нуклеаз и НГР-матрицу по любому из пп. 1-26.
28. Генетически модифицированное животное, полученное методами генной инженерии с изменениями в месте однонуклеотидного полиморфизма, животное, относящееся к породе, имеющей эндогенный аллель в хромосомной ДНК животного, животное, содержащее
экзогенный аллель, обнаруживаемый у другого вида или другой породы животного, с экзогенным аллелем, содержащим изменение в месте однонуклеотидного полиморфизма относительно эндогенного аллеля.
29. Животное по п. 28, содержащее множество однонуклеотидных полиморфизмов относительно эндогенного аллеля.
30. Способ получения «посадочной площадки» в хромосомной ДНК клетки, включающий введение нацеленной системы нуклеаз и НГР-матрицы в клетку, с нацеленной системой нуклеаз, содержащей ДНК-связывающий член для специфического связывания эндогенной родственной последовательности в хромосомной ДНК, где нацеленная система нуклеаз и НГР-матрица действуют с целью внесения изменений в хромосомную ДНК для ее идентичности последовательности НГР-матрицы, где последовательность НГР-матрицы содержит «посадочную площадку».
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201361870401P | 2013-08-27 | 2013-08-27 | |
| US61/870,401 | 2013-08-27 | ||
| PCT/US2014/035854 WO2015030881A1 (en) | 2013-08-27 | 2014-04-29 | Efficient non-meiotic allele introgression |
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| Publication Number | Publication Date |
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| RU2016110827A true RU2016110827A (ru) | 2017-10-03 |
| RU2016110827A3 RU2016110827A3 (ru) | 2018-03-19 |
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| RU2016110827A RU2016110827A (ru) | 2013-08-27 | 2014-04-29 | Эффективная немейотическая интрогрессия аллелей |
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| MX2016014066A (es) | 2014-04-28 | 2017-05-03 | Recombinetics Inc | Edicion de genes multiples en cerdos. |
| MX2017003770A (es) | 2014-09-23 | 2017-06-30 | Aggenetics Inc | Materiales y metodos para la produccion de animales con pelo corto. |
| HK1258264A1 (zh) | 2015-09-21 | 2019-11-08 | 重组股份有限公司 | 具有增加的熱耐受性的經遺傳修飾的動物 |
| US20170099813A1 (en) * | 2015-10-07 | 2017-04-13 | Recombinetics, Inc. | Method of generating sterile terminal sires in livestock and animals produced thereby |
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