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 Enzymoljournal_title
Methods in enzymologyauthors
Mekler V,Kuznedelov K,Minakhin L,Murugan K,Sashital DG,Severinov Kdoi
10.1016/bs.mie.2018.10.026subject
Has Abstractpub_date
2019-01-01 00:00:00pages
337-363eissn
0076-6879issn
1557-7988pii
S0076-6879(18)30441-5journal_volume
616pub_type
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