Two distinct DNA binding modes guide dual roles of a CRISPR-cas protein complex

Timothy R. Blosser, Luuk Loeff, Edze R. Westra, Marnix Vlot, Tim Künne, Małgorzata Sobota, Cees Dekker, Stan J.J. Brouns, Chirlmin Joo

Research output: Contribution to journalArticlepeer-review

78 Scopus citations


Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference byflagging cognate invader DNA for destruction, recent evidence has implicated their involvement innew CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E.coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled forhigh fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation ofbona fide targets and priming of mutated DNA targets.

Original languageEnglish
Pages (from-to)60-70
Number of pages11
JournalMolecular Cell
Issue number1
StatePublished - 2 Apr 2015

Bibliographical note

Funding Information:
The authors would like to thank K. Datsenko, K. Semenova, and K. Severinov for providing strain KD263; S. Bailey for providing the Cas3 overexpression construct; S. Sternberg for helpful advice; and C. Buyzerd for optimizing the biotinylation of Cascade. We thank A. Haagsma, M. Docter, P. Tuliniski, and I. Geuzebroek for their technical support. This work was supported by the Netherlands Organization for Scientific Research VIDI Grant 864.11.005 and the Beijerinck premium from the Royal Netherlands Academy of Arts and Sciences (to S.J.J.B.). T.R.B was supported by a Marie Curie International Incoming Fellowship within the seventh European Community Framework Programme. C.J. was funded by the Open Program of the Division for Earth and Life Sciences (822.02.008) of the Netherlands Organization for Scientific Research and by European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement number 309509. We thank I. Heo, M. Szczepaniak, and P. Tulinski for critically reading this manuscript.

Publisher Copyright:
© 2015 Elsevier Inc.


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