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Updated: Jan 30, 2026

Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
CRISPR-Cas molecular beacons as tool for studies of assembly of CRISPR-Cas effector complexes and their interactions
Vladimir Mekler1, Konstantin Kuznedelov1, Leonid Minakhin1
1Waksman Institute of Microbiology and Department of Molecular Biology and Biochemistry, Rutgers, State University of New Jersey, Piscataway, NJ, United States.
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.
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