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

Methods Enzymol. 2019:616:337-363. doi: 10.1016/bs.mie.2018.10.026. Epub 2018 Dec 1.

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.

Keywords: CRISPR–Cas; Cas12a; Cas9; Fluorescence.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteria / metabolism*
  • Bacterial Proteins / metabolism*
  • CRISPR-Associated Proteins / metabolism*
  • DNA / metabolism*
  • DNA Probes / metabolism
  • Fluorometry / methods
  • RNA, Guide, CRISPR-Cas Systems / metabolism

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • DNA Probes
  • RNA, Guide, CRISPR-Cas Systems
  • DNA