RU2019114217A - Геномная инженерия - Google Patents
Геномная инженерия Download PDFInfo
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Claims (37)
1. Способ изменения ДНК-мишени в стволовой клетке, экспрессирующий фермент Cas, который формирует комплекс колокализации с направляющей РНК, комплементарной ДНК-мишени и который расщепляет ДНК-мишень сайт-специфическим образом, включающий
введение в стволовую клетку направляющую РНК, комплементарную ДНК-мишени, где РНК и фермент Cas являются членами комплекса колокализации на ДНК-мишени,
введение в стволовую клетку донорной последовательности нуклеиновой кислоты,
где направляющая РНК и фермент Cas колокализуются с ДНК-мишенью, фермент Cas расщепляет ДНК-мишень и донорная последовательность нуклеиновой кислоты встраивается в ДНК-мишень, с получением измененной ДНК в стволовой клетке.
2. Способ по п. 1, где фермент Cas представляет собой Cas9.
3. Способ по п. 1, где длина РНК составляет от около 10 до около 500 нуклеотидов.
4. Способ по п. 1, где длина РНК составляет от около 20 до около 100 нуклеотидов.
5. Способ по п. 1, где направляющая РНК представляет собой crRNA-tracrRNA.
6. Способ по п. 1, где ДНК представляет собой геномную ДНК, митохондриальную ДНК, вирусную ДНК или экзогенную ДНК.
7. Способ по п. 1, где донорная последовательность нуклеиновой кислоты встраиваются в результате рекомбинации.
8. Способ по п. 1, где донорная последовательность нуклеиновой кислоты встраивается путем гомологичной рекомбинации.
9. Способ по п. 1, где донорная последовательность нуклеиновой кислоты встраивается негомологичным соединением концов.
10. Способ по п. 1, где фермент Cas и направляющая РНК вводятся в стволовую клетку в виде чужеродной нуклеиновой кислоты, которая экспрессируется стволовой клеткой.
11. Способ по п. 1 дополнительно включающий введение множества донорных нуклеиновых кислот и множества направляющих РНК, чтобы осуществить множество изменений в ДНК в клетке.
12. Способ по п. 1, где после введения измененной ДНК в стволовую клетку, нуклеиновая кислота, кодирующая фермент Cas, удаляется из генома стволовой клетки.
13. Способ по п. 1, где РНК и донорные нуклеотидные последовательности экспрессируются как связанная последовательность нуклеиновой кислоты.
14. Стволовая клетка, включающая первую чужеродную нуклеиновую кислоту, кодирующую фермент Cas, который образует комплекс колокализации с РНК, комплементарной ДНК-мишени и который расщепляет ДНК-мишень сайт-специфическим образом.
15. Стволовая клетка по п. 14 дополнительно включающая вторую чужеродную нуклеиновую кислоту, кодирующую направляющую РНК, комплементарную ДНК-мишени, где РНК и фермент являются членами комплекса колокализации на ДНК-мишени.
16. Стволовая клетка по п. 14, дополнительно включающая индуцируемый промотор для стимуляции экспрессии фермента.
17. Стволовая клетка по п. 14, отличающаяся тем, что первая чужеродная нуклеиновая кислота удаляется из геномной ДНК клетки с помощью транспозазы.
18. Стволовая клетка по п. 14, отличающаяся тем, что фермент является Cas9.
19. Стволовая клетка по п. 14, отличающаяся тем, что РНК представляет собой crRNA-tracrRNA.
20. Стволовая клетка по п. 14, отличающаяся тем, что ДНК-мишень представляет собой геномную ДНК, митохондриальную ДНК, вирусную ДНК или экзогенную ДНК.
21. Стволовая клетка по п. 14 отличающаяся тем, что дополнительно включает донорную последовательность нуклеиновой кислоты.
22. Способ по п.1, где направляющая РНК вводится в стволовую клетку из среды, окружающей клетку.
23. Способ по п. 22 в котором направляющая РНК включает структуру 5' Cap.
24. Способ по п. 22, где направляющая РНК утратила фосфатные группы.
25. Способ по п.1. где направляющая РНК непрерывно поступает в стволовую клетку из среды, окружающей клетку.
26. Способ по п .1, где стволовая клетка генетически модифицирована путем встраивания в геном клетки удаляемых ферментом вектора или кассеты, содержащих нуклеиновую кислоту, кодирующую фермент Cas.
27. Способ по п.26, где вектор представляет собой вектор PiggyBac.
28. Способ по п.26 где нуклеиновая кислота, кодирующая фермент Cas, удаляется с помощью фермента.
29. Способ по п.26, где удаляемые ферментом вектор или кассета, содержащие нуклеиновую кислоту, кодирующую фермент Cas, удаляются с помощью фермента.
30. Способ по п.26, где удаляемые ферментом вектор или кассета, содержащие нуклеиновую кислоту, кодирующую фермент Cas, встраиваются с помощью фермента.
31. Способ по п.26, где удаляемые ферментом вектор или кассета, содержащие нуклеиновую кислоту, кодирующую фермент Cas, удаляются с помощью транспозазы.
32. Способ по п.26, где нуклеиновая кислота, кодирующая фермент Cas, является индуцибельной.
33. Способ по п.26, где нуклеиновая кислота, кодирующая фермент Cas, находится под влиянием промотора, индуцируемого dox.
34. Способ по п.26, где нуклеиновая кислота, кодирующая фермент Cas индуцируется с помощью dox.
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US201361858866P | 2013-07-26 | 2013-07-26 | |
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US (4) | US10563225B2 (ru) |
EP (4) | EP3024964B1 (ru) |
JP (4) | JP6739335B2 (ru) |
KR (2) | KR20230096134A (ru) |
CN (3) | CN105473773B (ru) |
AU (4) | AU2014293015B2 (ru) |
BR (1) | BR112016001721B1 (ru) |
CA (1) | CA2918540A1 (ru) |
ES (1) | ES2715666T3 (ru) |
HK (1) | HK1218940A1 (ru) |
IL (2) | IL243560B (ru) |
RU (2) | RU2688462C2 (ru) |
SG (3) | SG11201600217QA (ru) |
WO (1) | WO2015013583A2 (ru) |
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Publication number | Priority date | Publication date | Assignee | Title |
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US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
SG11201406547YA (en) | 2012-04-25 | 2014-11-27 | Regeneron Pharma | Nuclease-mediated targeting with large targeting vectors |
IL308158A (en) | 2012-12-17 | 2023-12-01 | Harvard College | RNA-guided human genome engineering |
EP2981617B1 (en) | 2013-04-04 | 2023-07-05 | President and Fellows of Harvard College | Therapeutic uses of genome editing with crispr/cas systems |
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