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

    Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

    3 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Title of host publicationMethods in Enzymology
    EditorsScott Bailey
    PublisherAcademic Press Inc.
    Pages337-363
    Number of pages27
    ISBN (Print)9780128167601
    DOIs
    Publication statusPublished - 1 Jan 2019

    Publication series

    NameMethods in Enzymology
    Volume616
    ISSN (Print)0076-6879
    ISSN (Electronic)1557-7988

    Keywords

    • Cas12a
    • Cas9
    • CRISPR–Cas
    • Fluorescence

    Fingerprint

    Dive into the research topics of 'CRISPR–Cas molecular beacons as tool for studies of assembly of CRISPR–Cas effector complexes and their interactions with DNA'. Together they form a unique fingerprint.

    Cite this