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 proceedingChapter

2 Scopus citations

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

Publication series

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

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Keywords

  • CRISPR–Cas
  • Cas12a
  • Cas9
  • 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

    Mekler, V., Kuznedelov, K., Minakhin, L., Murugan, K., Sashital, D. G., & Severinov, K. (2019). CRISPR–Cas molecular beacons as tool for studies of assembly of CRISPR–Cas effector complexes and their interactions with DNA. In S. Bailey (Ed.), Methods in Enzymology (pp. 337-363). (Methods in Enzymology; Vol. 616). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2018.10.026