Инфекция и иммунитет. 2022; 12: 9-20
Молекулярные диагностические платформы, созданные на базе систем CRISPR/Cas
https://doi.org/10.15789/2220-7619-CCB-1843Аннотация
Последние несколько лет системы CRISPR/Cas активно исследуются и используются для широкого спектра прикладных задач. Многообразие вариантов их применения обусловлено способностью нуклеаз типа Cas направленно расщеплять заданные последовательности нуклеиновых кислот. При этом исследователь может задавать необходимую последовательность направляющих элементов cистемы, в роли которых выступают так называемые единые гидовые РНК, что позволяет системе воздействовать на определенные мишени. Такое свойство и стало одной из причин интереса к системам CRISPR/Cas. Одним из первых направлений применения данных систем было использование их для геномного редактирования. В дальнейшем список потенциальных возможностей расширился: например, CRISPR/Cas можно задействовать в генотерапии и эпигенетических исследованиях. Из единых гидовых РНК могут быть составлены библиотеки, выступающие основой для создания вирусных векторов с последующим трансдуцированием бактериальных клеток и нокаутированием указанных мишеней при помощи cas-белков. Такой подход позволяет осуществлять поиск бактериальных генов, ответственных за устойчивость или чувствительность к различным препаратам. Использование этих систем в молекулярной диагностике инфекционных заболеваний считается одним из наиболее многообещающих направлений. Диагностика при помощи CRISPR/Cas позволяет обнаруживать в образцах даже небольшие концентрации патогенных организмов за счет детекции их нуклеотидных последовательностей. При этом такие анализы оказываются точными, быстрыми и несложными в применении, а для функционирования ряда платформ даже не требуется дорогостоящее оборудование, поскольку уже разработаны методы быстрой и простой пробоподготовки, а современные подходы преамплификации позволяют уйти от использования термоциклических аппаратов. Примечательно, что уже открыто огромное число естественных систем CRISPR/Cas различных типов. Такое изобилие способствует разработке разнообразных искусственных систем, каждая из которых обладает своими особенными характеристиками. На их базе создается и множество диагностических платформ, различающихся по свойствам, что позволяет исследователям и медицинским работникам подбирать наилучший метод для решения определенных задач. Для выбора подходящей платформы важно иметь представление об устройстве и функционировании систем CRISPR/Cas, а следовательно, необходима актуальная классификация систем, на базе которой, в свою очередь, уже удобно оценивать само многообразие платформ молекулярной диагностики и представлять типовые характеристики и нюансы устройства для каждого метода. Таким образом, данный обзор, посвященный преимущественно платформам молекулярной диагностики инфекционных заболеваний, также затрагивает вопросы функционирования, устройств и классификации систем CRISPR/Cas.
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Russian Journal of Infection and Immunity. 2022; 12: 9-20
CRISPR/Cas-based diagnostic platforms
https://doi.org/10.15789/2220-7619-CCB-1843Abstract
Over the last few years, CRISPR/Cas systems have been extensively studied and used for a wide range of applied purposes. The variety of their applications is accounted for by the ability of Cas-type nucleases to targetly cleave specified nucleic acid sequences. In this case, the researcher might set the necessary sequence of the guiding elements in the CRISPR/Cas system, played by so-called single guide RNAs allowing it to act on select targets. This potential underlies one of the reasons for exerting interest in CRISPR/Cas systems. One of the first areas for applying these systems was its use for genomic editing. Later, the list of potential opportunities has been expanded: e.g., they can be used in gene therapy and epigenetic research. It is possible to create sgRNA libraries which might be used to create a pool of viral vectors applied for bacterial cell transformation with subsequent cas-protein transduction that cause target gene knockout. This approach allows finding genes responsible for resistance or sensibility to diverse substances. Using such systems in molecular diagnostics of infectious diseases is considered as one of the most promising directions allowing to detect even extremely low concentrations of pathogenic organisms in samples due to their specific nucleotide sequences. Simultaneously, such assays turn out to be accurate, rapid and easy to utilize. In addition, some platforms may work without using expensive equipment, because methods for fast and simple sample preparation have already been developed, whereas modern preamplification approaches allow to avoid applying thermocycling devices. Interestingly, a great amount of diverse types of natural CRISPR/Cas systems have been already discovered. Such abundance promotes development of multiple artificial systems, each of which exerting own unique characteristics. Therefore, a variety of diagnostic platforms with different properties are created on their basis that allows researchers and physicians to choose an optimal approach for performing specific tasks. For this reason, insights into structure and operation of CRISPR/Cas systems are necessary for selecting a suitable platform. The current classification of systems is based on such principles serving as the basis, in turn, for convenient evaluation of the very variety of molecular diagnostics platforms and presentation of the typical technical characteristics and nuances for each method. Thus, this review, which is mainly devoted to the platforms for molecular diagnostics of infectious diseases, also touches upon the issues of functioning, devices, and classification of CRISPR/Cas systems.
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