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В поисках противоядия от CRISPR: можно ли обращать изменения в генах?

Когда ученые, стоящие за Манхэттенским проектом, узнали о разрушении Хиросимы и Нагасаки, восторженный энтузиазм сменился мрачным сожалением. То, что начиналось как революция в области физики, мутировало в оружие массового поражения — от которого невозможно было защититься. С точки зрения биологии, у CRISPR есть разрушительная сила подобных масштабов. И ученые не хотят, чтобы история снова повторилась.

В поисках противоядия от CRISPR: можно ли обращать изменения в генах?

CRISPR: ядерное оружие в мире биологии

Всего через пять лет после открытия CRISPR Агентство перспективных оборонных исследований DARPA инициировало программу «Безопасные гены»: сотрудничество семи ведущих мировых экспертов в области редактирования генов, направленное на поиск разнообразных противоядий от CRISPR и улучшение возможностей редактирования генов в пространстве и времени.

Смысл не в том, чтобы подпитывать общественный страх перед мощным инструментом; скорее, нужно заблаговременно увидеть потенциальные опасности и найти профилактические меры или контрмеры. Если CRISPR — это биологический ящик Пандоры, он уже открыт: в клиниках начались испытания CRISPR на людях; в лаборатории технология превращается в генные драйвы, способные уничтожить целые виды. Цель программы Safe Genes — найти способ, либо много способов снова захлопнуть этот ящик.

На прошлой неделе поиски антидота CRISPR стали еще активнее. Команда во главе с доктором Амитом Чоудхари из Института Брода при Массачусетском технологическом институте, членом Safe Genes, разработала платформу «скрининга» для быстрого просеивания боле 10 000 мелких химических веществ, которые снижают активность ножниц Cas9.

Команда доработала структуру нескольких многообещающих кандидатов, чтобы еще больше повысить их способности противодействовать CRISPR, и создала две молекулы-антидота, которые препятствуют связыванию Cas9 с мишенью ДНК и ее разрезанию. Во время испытаний на клетках человека в чашках Петри молекулы проплывали через клеточные мембраны и надежно уничтожали активность CRISPR в течение нескольких минут.

Эти препараты — самые первые кандидаты — и они могут быть еще токсичнее, чем даже CRISPR, пустившийся вразнос внутри организма. Ученым придется проверить их на животных, чтобы оценить эффективность и безопасность.

Но небольшие препараты, противодействующие CRISPR, даже самые первые, доказывают, что титана CRISPR можно остановить. С платформой скрининга появляеся шанс найти еще более мощные кнопки «отмены»: химические вещества, которые однажды могут превратиться в уколы или пилюли, блокирующие нежелательную активность по редактированию генов, в медицинской сфере или даже в биологическом оружии.

«Эти результаты закладывают основу для точного химического контроля активности CRISPR/Cas9, прокладывая путь к безопасному использованию таких технологий», говорит Чоудхари.

Что уже доступно на текущий момент?

Чоудхари не единственный из команды Safe Genes, кто ищет молекулы анти-CRISPR.

В 2013 году другой член проекта, доктор Джозеф Бонди-Деноми из Калифорнийского университета в Сан-Франциско помог найти первые препараты анти-CRISPR: большие, неуклюжие белки, которые блокируют ножницы Cas9, не позволяя им узнать молекулы ДНК или связаться с ними. Его гениальная идея состояла в том, чтобы вернуться к естественным корням CRISPR как системы бактериальной иммунной защиты от вирусов.

По своей сути, CRISPR позволяет бактериям хранить «фотокарточку преступника» — вирусной ДНК — в своем собственном геноме, так что когда вирус снова атакует, ножницы Cas могут разорвать вирус на кусочки. Но фаги тоже не промахи. В эволюционной гонке они также выработали гены, создающие белки анти-CRISPR, чтобы противодействовать иммунной защите бактерий.

Обратившись к биологии фаговых анти-CRISPR в 2012 году, Бонди-Деноми обнаружил несколько новых белков, которые широко ингибируют активность Cas12a — альтернативу Cas9, которая набирает популярность в качестве диагностического инструмента. Работающая отдельно Дженнифер Дудна из Калифорнийского университета в Беркли, один из первых открывателей CRISPR и также член проекта, использовала биоинформатику, чтобы выследить горстку Cas12a-убийц, которые блокируют активность по редактированию генов в выращенных клетках человека.

В то время Дудна сказала, что эти результаты «прокладывают прямой путь к открытию еще большего количества анти-CRISPR из мира микробов».

Крошечные огоньки

И все же, белки анти-CRISPR не особо полезные выключатели в реальном мире.

С белками сложно: они большие и неуклюжие, поэтому не могут проникать в клетки и вгрызаться в механизмы CRISPR. Они чувствительны к изменениям температуры и пищеварения и не особо долго живут внутри тела. Многие из них становятся привлекательной добычей для нашей иммунной системы, которая может запустить раздражающие — если не опасные — аллергические реакции.

Маленькие молекулы, как правило, таких проблем не имеют. Они быстрые, дешевые и их эффекты обратимы. Не хотите мешать CRISPR? Просто подождите, пока молекулы вымоются. Но эффективные очень трудно найти.

И вот здесь поможет платформа скрининга Чоудхари.

Высокопроизводительная система просеивает десятки тысяч химических веществ, используя два теста, в поисках обещающих. Сперва она мониторит сегменты ДНК, привязываясь к ножницам Cas9 при помощи неоновых огоньков. ДНК размечается флуоресцентными «лампочками», которые меняют поляризацию при связывании с Cas9, подобно тому, как поляризационные очки меняются на солнечном свете. Это позволяет команде быстро отслеживать, нарушает ли молекула связь Cas9 с ДНК.

Во-вторых, система использует автоматические микроскопы, которые ищут флуоресцентные сигналы в клетках, приобретенные или утраченные в процессе активности Cas9. В одной из работ ученые использовали клетки, которые обычно светятся зеленым, пока Cas9 не вырезает ген. Потенциальный препарат анти-CRISPR позволит клеткам оставаться зеленым даже в присутствии дозы CRISPR.

В процессе работы ученые выявили молекулу BRD0539, которая не позволяет Cas9 связываться с целевой последовательностью ДНК. Действия препарата были совершенно предсказуемы: чем выше доза, тем сильнее он подавлял активность CRISPR.

Эти результаты уже помогают снизить побочные эффекты CRISPR в терапевтических условиях. В клетках доза такого препарата быстро снижала способность разрезания Cas9 вдвое, что, в свою очередь, снижало непреднамеренное вырезание HBB — гена, вовлеченного в серповидноклеточную анемию — в пять раз.

Нетрудно представить себе будущее, в котором вы можете выпить таблетку из BRD0539 — или более мощный эквивалент следующего поколения — чтобы временно снизить или остановить действие CRISPR, прежде чем он пустится вразнос в вашем теле. Препарат, будучи небольшой молекулой, остается стабильным в вашей крови и легко проникает в ваши клетки, выступая в качестве тормоза, когда CRISPR слишком силен.

Как вы думаете, станет ли CRISPR ядерной бомбой в мире биологии? Поделитесь с нами в нашем чате в Телеграме.

Источник: hi-news.ru

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