Print Email Facebook Twitter Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation Title Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation Author Künne, Tim (Wageningen University & Research) Kieper, S.N. (Wageningen University & Research) Bannenberg, Jasper W. (Wageningen University & Research) Vogel, Anne I M (Wageningen University & Research) Miellet, Willem R. (Wageningen University & Research) Klein, M. (TU Delft BN/Martin Depken Lab) Depken, S.M. (TU Delft BN/Martin Depken Lab) Suarez-Diez, Maria (Wageningen University & Research) Brouns, S.J.J. (TU Delft BN/Stan Brouns Lab; Wageningen University & Research) Date 2016-09-01 Abstract Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3′ ends. The Cas1-2 complex further processes these fragments and integrates them sequence-specifically into CRISPR repeats by coupling of a 3′ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers. Subject adaptive immunityCas1Cas2Cas3CascadeCRISPR-Casinterferencephage resistanceprimingspacer acquisition To reference this document use: http://resolver.tudelft.nl/uuid:fb0460bd-5b12-41a2-849e-7161bd9cd1d5 DOI https://doi.org/10.1016/j.molcel.2016.07.011 Embargo date 2017-08-18 ISSN 1097-2765 Source Molecular Cell, 63 (5), 852-864 Bibliographical note Accepted Author Manuscript Part of collection Institutional Repository Document type journal article Rights © 2016 Tim Künne, S.N. Kieper, Jasper W. Bannenberg, Anne I M Vogel, Willem R. Miellet, M. Klein, S.M. Depken, Maria Suarez-Diez, S.J.J. Brouns Files PDF Kunne_2016_MolCell.pdf 9.78 MB Close viewer /islandora/object/uuid:fb0460bd-5b12-41a2-849e-7161bd9cd1d5/datastream/OBJ/view