CRISPR–Cas molecular beacons as tool for studies of assembly of CRISPR–Cas effector complexes and their interactions with DNA

Vladimir Mekler, Konstantin Kuznedelov, Leonid Minakhin, Karthik Murugan, Dipali G. Sashital, Konstantin Severinov

    Результат исследований: Глава в книге, отчете, сборнике статейГлаварецензирование

    4 Цитирования (Scopus)

    Аннотация

    CRISPR–Cas systems protect prokaryotic cells from invading phages and plasmids by recognizing and cleaving foreign nucleic acid sequences specified by CRISPR RNA spacer sequences. Several CRISPR–Cas systems have been widely used as tool for genetic engineering. In DNA-targeting CRISPR–Cas nucleoprotein effector complexes, the CRISPR RNA forms a hybrid with the complementary strand of foreign DNA, displacing the noncomplementary strand to form an R-loop. The DNA interrogation and R-loop formation involve several distinct steps the molecular details of which are not fully understood. This chapter describes a recently developed fluorometric Cas beacon assay that may be used for measuring of specific affinity of various CRISPR–Cas complexes for unlabeled target DNA and model DNA probes. The Cas beacon approach also can provide a sensitive method for monitoring the kinetics of assembly of CRISPR–Cas complexes.

    Язык оригиналаАнглийский
    Название основной публикацииMethods in Enzymology
    РедакторыScott Bailey
    ИздательAcademic Press Inc.
    Страницы337-363
    Число страниц27
    ISBN (печатное издание)9780128167601
    DOI
    СостояниеОпубликовано - 1 янв. 2019

    Серия публикаций

    НазваниеMethods in Enzymology
    Том616
    ISSN (печатное издание)0076-6879
    ISSN (электронное издание)1557-7988

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