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RU2018101666A - Новые ферменты и системы crispr - Google Patents

Новые ферменты и системы crispr Download PDF

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RU2018101666A
RU2018101666A RU2018101666A RU2018101666A RU2018101666A RU 2018101666 A RU2018101666 A RU 2018101666A RU 2018101666 A RU2018101666 A RU 2018101666A RU 2018101666 A RU2018101666 A RU 2018101666A RU 2018101666 A RU2018101666 A RU 2018101666A
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cell
interest
effector protein
cpf1
plant
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RU2018101666A
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RU2018101666A3 (ru
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Фэн ЧЖАН
Бернд ЦЕТЧЕ
Йонатан С. ГУТЕНБЕРГ
Омар О. АБУДАЙЕ
Йан СЛЕЙМЕЙКЕР
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Те Брод Инститьют Инк.
Массачусетс Инститьют Оф Текнолоджи
Президент Энд Феллоуз Оф Харвард Коллидж
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Claims (62)

1. Сконструированная не встречающаяся в природе система на основе коротких палиндромных повторов, регулярно расположенных группами (CRISPR)-CRISPR-ассоциированного фермента (Cas) (CRISPR-Cas), содержащая
a) одну или несколько полинуклеотидных последовательностей CRISPR-Cas V типа, предусматривающих направляющую РНК, которая содержит направляющую последовательность, связанную с последовательностью прямого повтора, где направляющая последовательность способна гибридизироваться с целевой последовательностью, или одну или несколько нуклеотидных последовательностей, кодирующих одну или несколько полинуклеотидных последовательностей CRISPR-Cas V типа, и
b) эффекторный белок Cpf1 или одну или несколько нуклеотидных последовательностей, кодирующих эффекторный белок Cpf1;
где одна или несколько направляющих последовательностей гибридизируются с указанной целевой последовательностью, причем указанная целевая последовательность находится в направлении 3' от мотива, смежного с протоспейсером (PAM), и при этом указанная направляющая РНК образует комплекс с эффекторным белком Cpf1.
2. Сконструированная не встречающаяся в природе векторная система на основе коротких палиндромных повторов, регулярно расположенных группами (CRISPR)-CRISPR-ассоциированного фермента (Cas) (CRISPR-Cas), содержащая один или несколько векторов, содержащих
a) первый регуляторный элемент, функционально связанный с одной или несколькими нуклеотидными последовательностями, кодирующими одну или несколько полинуклеотидных последовательностей CRISPR-Cas V типа, предусматривающих направляющую РНК, которая содержит направляющую последовательность, связанную с последовательностью прямого повтора, где направляющая последовательность способна гибридизироваться с целевой последовательностью,
b) второй регуляторный элемент, функционально связанный с нуклеотидной последовательностью, кодирующей эффекторный белок Cpf1;
где компоненты (a) и (b) находятся в одном и том же или в разных векторах системы,
где, будучи транскрибированными, одна или несколько направляющих последовательностей гибридизируются с указанной целевой последовательностью, причем указанная целевая последовательность находится в направлении 3' от мотива, смежного с протоспейсером (PAM), и при этом указанная направляющая РНК образует комплекс с эффекторным белком Cpf1.
3. Система по п. 1 или 2, где целевые последовательности находятся в клетке.
4. Система по п. 3, где клетка предусматривает эукариотическую клетку.
5. Система по п. 1 или 2, где, будучи транскрибированными, одна или несколько направляющих последовательностей гибридизируются с целевой последовательностью, и при этом направляющая РНК образует комплекс c эффекторным белком Cpf1, который вызывает расщепление отдаленно от целевой последовательности.
6. Система по п. 5, где указанное расщепление приводит к образованию ступенчатого двухнитевого разрыва с "липким" 5'-концом длиной 4 или 5 нуклеотидов.
7. Система по п. 1 или 2, где PAM предусматривает 5'-мотив с высоким содержанием T.
8. Система по п. 1 или 2, где эффекторный белок представляет собой эффекторный белок Cpf1, происходящий от одного из видов бактерий, приведенных на фигуре 64.
9. Система по п. 8, где эффекторный белок Cpf1 происходит от вида бактерий, выбранного из группы, состоящей из Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens и Porphyromonas macacae.
10. Система по п. 9, где последовательность PAM представляет собой TTN, где N представляет собой A/C/G или T, а эффекторный белок представляет собой FnCpf1, или где последовательность PAM представляет собой TTTV, где V представляет собой A/C или G, а эффекторный белок представляет собой PaCpf1p, LbCpf1 или AsCpf1.
11. Система по п. 1 или 2, где эффекторный белок Cpf1 содержит один или несколько сигналов ядерной локализации.
12. Система по п. 1 или 2, где последовательности нуклеиновой кислоты, кодирующие эффекторный белок Cpf1, являются кодон-оптимизированными для экспрессии в эукариотической клетке.
13. Система по п. 1 или 2, где компоненты (a) и (b) или нуклеотидные последовательности находятся в одном векторе.
14. Способ модифицирования представляющего интерес целевого локуса, включающий доставку системы по п. 1 или 2 в указанный локус или клетку, содержащую локус.
15. Способ модифицирования представляющего интерес целевого локуса, причем способ включает доставку в указанный локус не встречающейся в природе или сконструированной композиции, содержащей эффекторный белок Cpf1 и один или несколько компонентов на основе нуклеиновой кислоты, где эффекторный белок Cpf1 образует комплекс с одним или несколькими компонентами на основе нуклеиновой кислоты, и после связывания указанного комплекса с представляющим интерес целевым локусом, который находится в направлении 3' от мотива, смежного с протоспейсером (PAM), эффекторный белок индуцирует модификацию представляющего интерес целевого локуса.
16. Способ по п. 15, где представляющий интерес целевой локус находится в клетке.
17. Способ по п. 16, где клетка является эукариотической клеткой.
18. Способ по п. 16, где клетка является клеткой животного или человека.
19. Способ по п. 16, где клетка является растительной клеткой.
20. Способ по п. 15, где представляющий интерес целевой локус содержится в молекуле ДНК in vitro.
21. Способ по п. 15, где указанную не встречающуюся в природе или сконструированную композицию, содержащую эффекторный белок Cpf1 и один или несколько компонентов на основе нуклеиновой кислоты, доставляют в клетку в виде одной или нескольких полинуклеотидных молекул.
22. Способ по п. 15, где представляющий интерес целевой локус предусматривает ДНК.
23. Способ по п. 22, где ДНК является релаксированной или суперспирализованной.
24. Способ по п. 15, где композиция содержит один компонент на основе нуклеиновой кислоты.
25. Способ по п. 24, где один компонент на основе нуклеиновой кислоты предусматривает направляющую последовательность, связанную с последовательностью прямого повтора.
26. Способ по п. 15, где модификация представляющего интерес целевого локуса представляет собой разрыв нити.
27. Способ по п. 26, где разрыв нити предусматривает ступенчатый двухнитевой разрыв ДНК с "липким" 5'-концом длиной 4 или 5 нуклеотидов.
28. Способ по п. 26, где представляющий интерес целевой локус является модифицированным посредством интеграции ДНК-вставки в ступенчатый двухнитевой разрыв ДНК.
29. Способ по п. 15, где эффекторный белок Cpf1 содержит один или несколько сигналов ядерной локализации (NLS).
30. Способ по п. 21, где одна или несколько полинуклеотидных молекул содержатся в одном или нескольких векторах.
31. Способ по п. 21, где одна или несколько полинуклеотидных молекул содержат один или несколько регуляторных элементов, функционально сконфигурированных для обеспечения экспрессии эффекторного белка Cpf1 и/или компонента(компонентов) на основе нуклеиновой кислоты, где один или несколько регуляторных элементов необязательно предусматривают индуцируемые промоторы.
32. Способ по п. 21, где одна или несколько полинуклеотидных молекул или один или несколько векторов содержатся в системе доставки.
33. Способ по п. 21, где систему или одну или несколько полинуклеотидных молекул доставляют посредством частиц, везикул или одного или нескольких вирусных векторов.
34. Способ по п. 33, где частицы предусматривают липид, сахар, металл или белок.
35. Способ по п. 33, где везикулы предусматривают экзосомы или липосомы.
36. Способ по п. 33, где один или несколько вирусных векторов предусматривают одно или несколько из аденовируса, одного или нескольких лентивирусов или одного или нескольких аденоассоциированных вирусов.
37. Способ по п. 15, который представляет собой способ модифицирования клетки, линии клеток или организма путем манипуляции с одной или несколькими целевыми последовательностями в представляющих интерес локусах генома.
38. Клетка, полученная в результате осуществления способа по п. 37, или ее потомство, где клетка содержит модификацию, не присутствующую в клетке, в отношении которой не осуществляли способ.
39. Клетка по п. 38 или ее потомство, где клетка, в отношении которой не осуществляли способ, содержит аномалию, а клетка, полученная в результате осуществления способа, характеризуется устраненной или скорректированной аномалией.
40. Продукт клетки, полученный из клетки или ее потомства по п. 38, где продукт является модифицированным по своей природе или количеству по сравнению с продуктом клетки, полученным из клетки, в отношении которой не осуществляли способ.
41. Продукт клетки по п. 40, где клетка, в отношении которой не осуществляли способ, содержит аномалию, и при этом продукт клетки отражает аномалию, которая устраняется или корректируется с помощью способа.
42. In vitro, ex vivo или in vivo клетка-хозяин или линия клеток или их потомство, содержащие систему по п. 1 или 2.
43. Клетка-хозяин или линия клеток или их потомство по п. 42, где клетка является эукариотической клеткой.
44. Клетка-хозяин или линия клеток или их потомство по п. 43, где клетка является клеткой животного.
45. Клетка-хозяин или линия клеток или их потомство по п. 33, где клетка является клеткой человека.
46. Клетка-хозяин, линия клеток или их потомство по п. 31, предусматривающие стволовую клетку или линию стволовых клеток.
47. Клетка-хозяин или линия клеток или их потомство по п. 30, где клетка является растительной клеткой.
48. Способ получения растения с модифицированным представляющим интерес признаком, кодируемым представляющим интерес геном, причем указанный способ включает приведение растительной клетки в контакт с системой по п. 1 или п. 2 или осуществление в отношении растительной клетки способа по п. 15, за счет чего обеспечивается либо модифицирование, либо введение указанного представляющего интерес гена, и регенерацию растения из указанной растительной клетки.
49. Способ идентификации представляющего интерес признака у растения, причем указанный представляющий интерес признак кодируется представляющим интерес геном, причем указанный способ включает приведение растительной клетки в контакт с системой по п. 1 или 2 или осуществление в отношении растительной клетки способа по п. 15, за счет чего обеспечивается идентификация указанного представляющего интерес гена.
50. Способ по п. 49, дополнительно включающий введение идентифицированного представляющего интерес гена в растительную клетку, или линию растительных клеток, или растительную зародышевую плазму и получение из них растения, в результате чего растение содержит представляющий интерес ген.
51. Способ по п. 50, где у растения проявляется представляющий интерес признак.
52. Частица, содержащая систему по п. 1 или 2.
53. Частица по п. 52, где частица содержит эффекторный белок Cpf1 в комплексе с направляющей РНК.
54. Система или способ по пп. 1, 2 или 15, где комплекс, направляющая РНК или белок конъюгированы по меньшей мере с одним сахарным фрагментом, необязательно N-ацетилгалактозамином (GalNAc), в частности, с трехразветвленным GalNAc.
55. Система или способ по пп. 1, 2 или 15, где концентрация Mg2+ составляет от приблизительно 1 мМ до приблизительно 15 мМ.
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RU2745637C1 (ru) * 2020-04-15 2021-03-29 Федеральное бюджетное учреждение науки "Центральный научно-исследовательский институт эпидемиологии" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ЦНИИ Эпидемиологии Роспотребнадзора) Способ получения препарата рибонуклеопротеинового комплекса CRISPR/Cas и препарат для выявления гена антибиотикоустойчивости bla VIM-2 (металло-бета-лактамаза класс B VIM-2) Pseudomonas aeruginosa в ультранизких концентрациях
WO2021211010A1 (ru) * 2020-04-15 2021-10-21 Федеральное Бюджетное Учреждение Науки "Центральный Научно-Исследовательский Институт Эпидемиологии" Федеральной Службы По Надзору В Сфере Защиты Прав Потребителей И Благополучия Человека Crispr/cas для выявления гена антибиотикоустойчивости bla vim-2

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