title: Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes.
abstract: 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.
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom; mark.szczelkun@bristol.ac.uk siksnys@ibt.lt ralf.seidel@uni-muenster.de.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Szczelkun
authors.affiliation: Institute for Molecular Cell Biology, Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany;Biotechnology Center, Technische Universität Dresden, 01062 Dresden, Germany; and.
authors.forename: Maria S
authors.initials: MS
authors.lastname: Tikhomirova
authors.affiliation: Department of Protein-Nucleic Acid Interactions, Institute of Biotechnology, Vilnius University, LT-02241 Vilnius, Lithuania.
authors.forename: Tomas
authors.initials: T
authors.lastname: Sinkunas
authors.affiliation: Department of Protein-Nucleic Acid Interactions, Institute of Biotechnology, Vilnius University, LT-02241 Vilnius, Lithuania.
authors.forename: Giedrius
authors.initials: G
authors.lastname: Gasiunas
authors.affiliation: Department of Protein-Nucleic Acid Interactions, Institute of Biotechnology, Vilnius University, LT-02241 Vilnius, Lithuania.
authors.forename: Tautvydas
authors.initials: T
authors.lastname: Karvelis
authors.affiliation: Biotechnology Center, Technische Universität Dresden, 01062 Dresden, Germany; and.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Pschera
authors.affiliation: Department of Protein-Nucleic Acid Interactions, Institute of Biotechnology, Vilnius University, LT-02241 Vilnius, Lithuania mark.szczelkun@bristol.ac.uk siksnys@ibt.lt ralf.seidel@uni-muenster.de.
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
authors.affiliation: Institute for Molecular Cell Biology, Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany;Biotechnology Center, Technische Universität Dresden, 01062 Dresden, Germany; and mark.szczelkun@bristol.ac.uk siksnys@ibt.lt ralf.seidel@uni-muenster.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Seidel
mesh_terms: D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000235:genetics; D004278:DNA, Superhelical / Q000737:chemistry; D009154:Mutation; D011487:Protein Conformation; D058727:RNA, Small Untranslated
chemical_list: D064130:CRISPR-Associated Proteins; D004278:DNA, Superhelical; D058727:RNA, Small Untranslated
keywords: crRNA; genome engineering; magnetic tweezers