Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes

Mark D Szczelkun, Maria S Tikhomirova, Tomas Sinkunas, Giedrius Gasiunas, Tautvydas Karvelis, Patrizia Pschera, Virginijus Siksnys, Ralf Seidel

Research output: Contribution to journalArticle (Academic Journal)peer-review

223 Citations (Scopus)
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Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems protect bacteria and archaea from infection by viruses and plasmids. Central to this defense is a ribonucleoprotein complex that produces RNA-guided cleavage of foreign nucleic acids. In DNA-targeting CRISPR-Cas systems, the RNA component of the complex encodes target recognition by forming a site-specific hybrid (R-loop) with its complement (protospacer) on an invading DNA while displacing the noncomplementary strand. Subsequently, the R-loop structure triggers DNA degradation. Although these reactions have been reconstituted, the exact mechanism of R-loop formation has not been fully resolved. Here, we use single-molecule DNA supercoiling to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade- and Cas9-based CRISPR-Cas systems. We find that the protospacer adjacent motif (PAM) affects primarily the R-loop association rates, whereas protospacer elements distal to the PAM affect primarily R-loop stability. Furthermore, Cascade has higher torque stability than Cas9 by using a conformational locking step. Our data provide direct evidence for directional R-loop formation, starting from PAM recognition and expanding toward the distal protospacer end. Moreover, we introduce DNA supercoiling as a quantitative tool to explore the sequence requirements and promiscuities of orthogonal CRISPR-Cas systems in rapidly emerging gene-targeting applications.

Original languageEnglish
Pages (from-to)9798-9803
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number27
Early online date27 May 2014
Publication statusPublished - 8 Jul 2014

Bibliographical note

Published online before print May 27, 2014


  • magnetic tweezers
  • genome engineering
  • crRNA

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