RU2014149120A - Способы и композиции для опосредованной нуклеазой направленной интеграции трансгенов - Google Patents

Способы и композиции для опосредованной нуклеазой направленной интеграции трансгенов Download PDF

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RU2014149120A
RU2014149120A RU2014149120A RU2014149120A RU2014149120A RU 2014149120 A RU2014149120 A RU 2014149120A RU 2014149120 A RU2014149120 A RU 2014149120A RU 2014149120 A RU2014149120 A RU 2014149120A RU 2014149120 A RU2014149120 A RU 2014149120A
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nucleic acid
acid sequence
polynucleotide
exogenous nucleic
double
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RU2014149120A
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RU2650819C2 (ru
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Грегори Дж. Кост
Федор Урнов
Уилльям М. Эйнли
Джозеф Ф. Петолино
Джаякумар Пон СЭМЬЮЭЛ
Стивен Р. Уэбб
Лакшми САСТРИ-ДЕНТ
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Сангамо Байосайенсиз, Инк.
ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи
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Abstract

1. Двухцепочечный полинуклеотид, содержащий:экзогенную последовательность нуклеиновой кислоты;последовательность, содержащую один или более участков-мишеней для одной или более нуклеаз,где один или более участков-мишеней находятся не внутри последовательности экзогенной нуклеиновой кислоты и дополнительно, где двухцепочечный полинуклеотид не содержит плеч гомологии.2. Двухцепочечный полинуклеотид по п. 1, где экзогенная последовательность нуклеиновой кислоты составляет по меньшей мере 1 тысячу оснований в длину.3. Двухцепочечный полинуклеотид по п. 1 или 2, где полинуклеотид с экзогенной последовательностью нуклеиновой кислоты представляет собой плазмиду.4. Двухцепочечный полинуклеотид по любому из п. 1 или 2, где экзогенная последовательность нуклеиновой кислоты содержит трансген.5. Двухцепочечный полинуклеотид по любому из п. 1 или 2, где последовательность содержит два участка-мишени и спейсер по меньшей мере из 5 нуклеотидов между двумя участками-мишенями.6. Клетка, содержащая двухцепочечный полинуклеотид по любому из пп. 1-5.7. Способ интеграции полинуклеотида с экзогенной последовательностью нуклеиновой кислоты в эндогенный локус клетки, где способ включает:введение двухцепочечного полинуклеотида по любому из пп. 1-5 в клетку;введение одной или более нуклеаз в клетку, где нуклеазы расщепляют двухцепочечный полинуклеотид и расщепляют эндогенный локус, так что полинуклеотид с экзогенной последовательностью нуклеиновой кислоты интегрируется в эндогенный локус.8. Способ по п. 7, где полинуклеотид с экзогенной последовательностью нуклеиновой кислоты интегрируется в прямой ориентации.9. Способ по п. 7, где полинуклеотид с экзогенно

Claims (18)

1. Двухцепочечный полинуклеотид, содержащий:
экзогенную последовательность нуклеиновой кислоты;
последовательность, содержащую один или более участков-мишеней для одной или более нуклеаз,
где один или более участков-мишеней находятся не внутри последовательности экзогенной нуклеиновой кислоты и дополнительно, где двухцепочечный полинуклеотид не содержит плеч гомологии.
2. Двухцепочечный полинуклеотид по п. 1, где экзогенная последовательность нуклеиновой кислоты составляет по меньшей мере 1 тысячу оснований в длину.
3. Двухцепочечный полинуклеотид по п. 1 или 2, где полинуклеотид с экзогенной последовательностью нуклеиновой кислоты представляет собой плазмиду.
4. Двухцепочечный полинуклеотид по любому из п. 1 или 2, где экзогенная последовательность нуклеиновой кислоты содержит трансген.
5. Двухцепочечный полинуклеотид по любому из п. 1 или 2, где последовательность содержит два участка-мишени и спейсер по меньшей мере из 5 нуклеотидов между двумя участками-мишенями.
6. Клетка, содержащая двухцепочечный полинуклеотид по любому из пп. 1-5.
7. Способ интеграции полинуклеотида с экзогенной последовательностью нуклеиновой кислоты в эндогенный локус клетки, где способ включает:
введение двухцепочечного полинуклеотида по любому из пп. 1-5 в клетку;
введение одной или более нуклеаз в клетку, где нуклеазы расщепляют двухцепочечный полинуклеотид и расщепляют эндогенный локус, так что полинуклеотид с экзогенной последовательностью нуклеиновой кислоты интегрируется в эндогенный локус.
8. Способ по п. 7, где полинуклеотид с экзогенной последовательностью нуклеиновой кислоты интегрируется в прямой ориентации.
9. Способ по п. 7, где полинуклеотид с экзогенной последовательностью нуклеиновой кислоты интегрируется в обратной ориентации.
10. Способ по любому из пп. 7-9, где одинаковые нуклеазы расщепляют эндогенный локус и донорный полинуклеотид.
11. Способ по любому из пп. 7-9, где различные нуклеазы расщепляют эндогенный локус и донорный полинуклеотид.
12. Способ по любому из пп. 7-9, где полинуклеотид с экзогенной последовательностью нуклеиновой кислоты интегрируется в эндогенный локус посредством независимых от гомологии механизмов.
13. Способ по любому из пп.7-9, где нуклеиновая кислота, содержащая два участка-мишени и спейсер, не является природной, так что участки-мишени не воссоздаются после интеграции полинуклеотида с экзогенной последовательностью нуклеиновой кислоты.
14. Способ по любому из пп.7-9, где нуклеазы образуют делецию в эндогенном локусе, и полинуклеотид с экзогенной последовательностью нуклеиновой кислоты интегрируют в делецию.
15. Способ по любому из пп.7-9, где клетка представляет собой эукариотическую клетку.
16. Способ по п. 15, где клетка представляет собой клетку растения или млекопитающего.
17. Способ по п. 16, где клетка растения представляет собой клетку двудольного или однодольного растения.
18. Трансгенный организм, содержащий полинуклеотид с экзогенной последовательностью нуклеиновой кислоты, интегрированный способом по любому из пп. 7-17.
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