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

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.

journal_name

Methods Enzymol

journal_title

Methods in enzymology

authors

Mekler V,Kuznedelov K,Minakhin L,Murugan K,Sashital DG,Severinov K

doi

10.1016/bs.mie.2018.10.026

subject

Has Abstract

pub_date

2019-01-01 00:00:00

pages

337-363

eissn

0076-6879

issn

1557-7988

pii

S0076-6879(18)30441-5

journal_volume

616

pub_type

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