title: Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation.
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.
authors.affiliation: Laboratory of Microbiology, Wageningen University, 6708 WE Wageningen, the Netherlands.
authors.forename: Tim
authors.initials: T
authors.lastname: Künne
authors.affiliation: Laboratory of Microbiology, Wageningen University, 6708 WE Wageningen, the Netherlands.
authors.forename: Sebastian N
authors.initials: SN
authors.lastname: Kieper
authors.affiliation: Laboratory of Microbiology, Wageningen University, 6708 WE Wageningen, the Netherlands.
authors.forename: Jasper W
authors.initials: JW
authors.lastname: Bannenberg
authors.affiliation: Laboratory of Microbiology, Wageningen University, 6708 WE Wageningen, the Netherlands.
authors.forename: Anne I M
authors.initials: AI
authors.lastname: Vogel
authors.affiliation: Laboratory of Microbiology, Wageningen University, 6708 WE Wageningen, the Netherlands.
authors.forename: Willem R
authors.initials: WR
authors.lastname: Miellet
authors.affiliation: Kavli Institute of Nanoscience and Department of BioNanoscience, Delft University of Technology, 2629 HZ, Delft, the Netherlands.
authors.forename: Misha
authors.initials: M
authors.lastname: Klein
authors.affiliation: Kavli Institute of Nanoscience and Department of BioNanoscience, Delft University of Technology, 2629 HZ, Delft, the Netherlands.
authors.forename: Martin
authors.initials: M
authors.lastname: Depken
authors.affiliation: Laboratory of Systems and Synthetic Biology, Wageningen University, 6708 WE Wageningen, the Netherlands.
authors.forename: Maria
authors.initials: M
authors.lastname: Suarez-Diez
authors.affiliation: Laboratory of Microbiology, Wageningen University, 6708 WE Wageningen, the Netherlands; Kavli Institute of Nanoscience and Department of BioNanoscience, Delft University of Technology, 2629 HZ, Delft, the Netherlands. Electronic address: stanbrouns@gmail.com.
authors.forename: Stan J J
authors.initials: SJ
authors.lastname: Brouns
mesh_terms: D001665:Binding Sites; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D053837:DNA Cleavage; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D004247:DNA; D004265:DNA Helicases; C574631:ygcB protein, E coli
keywords: CRISPR-Cas; Cas1; Cas2; Cas3; Cascade; adaptive immunity; interference; phage resistance; priming; spacer acquisition