Cas13a: purification and use for detection of viral RNA

Savinova, A.S.; Koptev, E.Y.; Usachev, E.V.; Tkachuk, A.P.; Guschin, V.A.

Авторы: Savinova A.S., Koptev E.Yu, Usachev E.V., Tkachuk A.P., Guschin V.A.
Журнал: Bulletin of RSMU
Номер: 2
Год издания: 2018
Издательство: Pirogov Russian National Research Medical University
Местоположение издательства: Moscow
Первая страница: 21
Последняя страница: 25
DOI: 10.24075/brsmu.2018.021
Аннотация: The use of CRIPSR-Cas systems in genome editing has recently become one of the major research areas. Meanwhile, CAS proteins can be employed to develop novel techniques for molecular diagnostics. Traditional approaches to the identification of microorganisms have a few drawbacks: they are time-consuming (microbiological methods), insufficiently sensitive (immunoassays), expensive or labor-intensive (PCR, sequencing). The aim of this work was to obtain a functionally active Cas13a protein that could be used as a diagnostic tool and study its behavior under different conditions and at various target concentrations. We constructed an expression vector with the cas13a gene of Leptotrichia wadei under the control of T7 promoter. We obtained a functionally active Cas13a RNAse with pre-programmed activity, guide RNA, and a fragment of influenza B RNA sequence serving as a target. The functional activity of Cas13 RNAse was assessed by fluorescence in the reaction mix containing guide RNA, target RNA, and a molecular RNA beacon. The obtained protein Cas13a was able to specifically recognize the target and did not exhibit any non-specific RNAse activity. This study can become a basis for developing a novel, rapid, specific and sensitive method for pathogen detection.
Добавил в систему: Гущин Владимир Алексеевич

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