title: Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection.
abstract: CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers) by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM). Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification.
authors.affiliation: Institute for Molecular Cell Biology, Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany.
authors.forename: Marius
authors.initials: M
authors.lastname: Rutkauskas
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania.
authors.forename: Tomas
authors.initials: T
authors.lastname: Sinkunas
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania.
authors.forename: Inga
authors.initials: I
authors.lastname: Songailiene
authors.affiliation: Institute for Molecular Cell Biology, Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany.
authors.forename: Maria S
authors.initials: MS
authors.lastname: Tikhomirova
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania. Electronic address: siksnys@ibt.lt.
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
authors.affiliation: Institute for Molecular Cell Biology, Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany. Electronic address: ralf.seidel@uni-muenster.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Seidel