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6 results on '"Ralf Seidel"'

1. Decision-Making in Cascade Complexes Harboring crRNAs of Altered Length

Author

Inga Songailiene, Marius Rutkauskas, Tomas Sinkunas, Elena Manakova, Sabine Wittig, Carla Schmidt, Virginijus Siksnys, and Ralf Seidel

Subjects
Biology (General), QH301-705.5
Abstract

Summary: The multi-subunit type I CRISPR-Cas surveillance complex Cascade uses its crRNA to recognize dsDNA targets. Recognition involves DNA unwinding and base-pairing between the crRNA spacer region and a complementary DNA strand, resulting in formation of an R-loop structure. The modular Cascade architecture allows assembly of complexes containing crRNAs with altered spacer lengths that promise increased target specificity in emerging biotechnological applications. Here we produce type I-E Cascade complexes containing crRNAs with up to 57-nt-long spacers. We show that these complexes form R-loops corresponding to the designed target length, even for the longest spacers tested. Furthermore, the complexes can bind their targets with much higher affinity compared with the wild-type form. However, target recognition and the subsequent Cas3-mediated DNA cleavage do not require extended R-loops but already occur for wild-type-sized R-loops. These findings set important limits for specificity improvements of type I CRISPR-Cas systems. : Songailiene et al. show that engineered versions of Cascade with elongated crRNAs can base-pair with target DNA over 57 bp. However, target recognition only requires wild-type-sized base-pairing of ∼30-bp length. These findings define constraints for improving the specificity of these complexes in biotechnological applications. Keywords: Cascade, CRISPR, type I-E, single molecule, crRNA, R-loop, Streptococcus thermophilus, Cse2, Cse1, genome engineering

Published
2019
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2. Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection

Author

Marius Rutkauskas, Tomas Sinkunas, Inga Songailiene, Maria S. Tikhomirova, Virginijus Siksnys, and Ralf Seidel

Subjects
Biology (General), QH301-705.5
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.

Published
2015
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4. Adressen

Author

Wielant Machleidt, Ulrike Kluge, Marcel G. Sieberer, Andreas Heinz, Agorastos Agorastos, Marion C. Aichberger, Carina Aichinger, Hans-Jörg Assion, Malek Bajbouj, Helene Basu, Katharina Behrens, Isaac Bermejo Bragado, Ursula Boos-Nünning, Theda Borde, Ronald Bottlender, Elmar Brähler, Jan Bulla, Ronald Burian, Matthias David, Claire Deery, Teresa-Marie Deffner, Cüneyt Demiralay, Albert Diefenbacher, Thomas Elbert, Monika Englisch, Thomas Eppenstein, Yesim Erim, Naika Foroutan, Natalie Gies-Powroznik, Heide Glaesmer, Iris Tatjana Graef-Calliess, Ali Kemal Gün, Stefan Gutwinski, Ferdinand Haenel, Eric Hahn, Martin Härter, Thomas Heise, Klaus Hoffmann, Julia Huemer, Serhat Karakayali, Doron Kiesel, Jan Ilhan Kizilhan, Eckhardt Koch, Jose Marie Koussemou, Christian Krauth, Bernhard Küchenhoff, Ruth Kutalek, Friedrich Leidinger, Ulrike von Lersner, Marianne Leuzinger-Bohleber, Jutta Lindert, Philipp A. Martius, Lea Mascarell-Maricic, Inge Missmahl, Eva Morawa, Christine Morgenroth, Martin Möckel, Mike Mösko, Daniel J. Müller, Oskar Negt, Frank Neuner, Cornelia Oestereich, Isgard Ohls, Torsten Passie, Simone Penka, Jens Plag, Stefan Priebe, Armin Prinz, Michael A. Rapp, Hartmut Reinbold, Ramazan Salman, Hannes Schammann, Maggie Schauer, Renate Schepker, Meryam Schouler-Ocak, Susanne Schröder, Ralf Seidel, Anna Sieben, Rainer G. Siefen, Thomas Stamm, Barbara Stöckigt, Thomas Stompe, Jürgen Straub, Andreas Ströhle, Igor Sutej, Thi Minh Tam Ta, Madlen Trenker, Dita Vogel, Waldemar Vogelgesang, Sabine Völkl-Kernstock, Ulla Walter, Aylin Yanık-Şenay, Katarzyna Zentner, and Andrea Zielke-Nadkarni

Published
2018

5. Single-Molecule Insight Into Target Recognition by CRISPR–Cas Complexes

Author

Marius Rutkauskas, Andrey Krivoy, Mark D. Szczelkun, Christophe Rouillon, and Ralf Seidel

Subjects
0301 basic medicine, Trans-activating crRNA, Magnetic tweezers, Base pair, Cas9, Nanotechnology, Computational biology, Biology, 03 medical and health sciences, Protospacer adjacent motif, 030104 developmental biology, 0302 clinical medicine, CRISPR, DNA supercoil, 030217 neurology & neurosurgery, Ribonucleoprotein
Abstract

Ribonucleoprotein (RNP) complexes from CRISPR-Cas systems have attracted enormous interest since they can be easily and flexibly reprogrammed to target any desired locus for genome engineering and gene regulation applications. Basis for the programmability is a short RNA (crRNA) inside these complexes that recognizes the target nucleic acid by base pairing. For CRISPR-Cas systems that target double-stranded DNA this results in local DNA unwinding and formation of a so-called R-loop structure. Here we provide an overview how this target recognition mechanism can be dissected in great detail at the level of a single molecule. Specifically, we demonstrate how magnetic tweezers are applied to measure the local DNA unwinding at the target in real time. To this end we introduce the technique and the measurement principle. By studying modifications of the consensus target sequence, we show how different sequence elements contribute to the target recognition mechanism. From these data, a unified target recognition mechanism can be concluded for the RNPs Cascade and Cas9 from types I and II CRISPR-Cas systems. R-loop formation is hereby initiated on the target at an upstream element, called protospacer adjacent motif (PAM), from which the R-loop structure zips directionally toward the PAM-distal end of the target. At mismatch positions, the R-loop propagation stalls and further propagation competes with collapse of the structure. Upon full R-loop zipping conformational changes within the RNPs trigger degradation of the DNA target. This represents a shared labor mechanism in which zipping between nucleic acid strands is the actual target recognition mechanism while sensing of the R-loop arrival at the PAM-distal end just verifies the success of the full zipping.

Published
2017

6. Autorinnen und Autoren

Author

Agorastos Agorastos, Marion Aichberger, Hans-Jörg Assion, Nora Balke, Helene Basu, Katharina Behrens, Isaac Bermejo, Ursula Boos-Nünning, Theda Borde, Ronald Bottlender, Elmar Brähler, Ronald Burian, Iris Tatjana Calliess, Renate Csellich-Ruso, Matthias David, Cüneyt Demiralay, Albert Diefenbacher, Monika Englisch, Thomas Eppenstein, Max H. Friedrich, P.H. Heide Glaesmer, Julia Göbber, Ali Kemal Gün, Harald Gündel, Ton Haans, Ferdinand Haenel, Martin Härter, Hannah Heimann, Jakob Hein, Andreas Heinz, Thomas Heise, Theresa-Maria Hloucal, Klaus Hoffmann, Julia Huemer, Aysun Kamaci, Doron Kiesel, Jan Ilhan Kizilhan, Ulrike Kluge, Eckhardt Koch, Christian Krauth, Bernhard Küchenhoff, Ruth Kutalek, Johan Lansen, Friedrich Leidinger, Jutta Lindert, Wielant Machleidt, Amanda Heredia Montesinos, Christine Morgenroth, Mike Mösko, Daniel J. Müller, Jürgen Münch, Oskar Negt, Cornelia Oestereich, Metin Özek†, Torsten Passie, Simone Penka, Stefan Priebe, Armin Prinz, Christian Pross, Michael A. Rapp, Hartmut Reinbold, Ulrich Reitemeier, Robert Rohner, Wulf Rössler, Daniela Ruf, Ramazan Salman, Renate Schepker, Meryam Schouler-Ocak, Susanne Schröder, Ralf Seidel, Anna Sieben, Rainer G. Siefen, Marcel Sieberer, Thomas Stamm, Barbara Stöckigt, Jürgen Straub, Andreas Ströhle, Dita Vogel, Sabine Völkl-Kernstock, Ulla Walter, Naoki Watanabe, Albina Zeef, Katarzyna Zentner, Marc Ziegenbein, and Andrea Zielke-Nadkarni

Published
2011

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