title: Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery.
abstract: CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR-Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR-Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR-Cas systems.
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo, Russia Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Daria
authors.initials: D
authors.lastname: Vorontsova
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Kirill A
authors.initials: KA
authors.lastname: Datsenko
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo, Russia Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Sofia
authors.initials: S
authors.lastname: Medvedeva
authors.affiliation: Department of Molecular Genetics and Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
authors.forename: Joseph
authors.initials: J
authors.lastname: Bondy-Denomy
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo, Russia Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Ekaterina E
authors.initials: EE
authors.lastname: Savitskaya
authors.affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Ksenia
authors.initials: K
authors.lastname: Pougach
authors.affiliation: Moscow State University, Moscow, Russia.
authors.forename: Maria
authors.initials: M
authors.lastname: Logacheva
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Blake
authors.initials: B
authors.lastname: Wiedenheft
authors.affiliation: Department of Molecular Genetics and Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
authors.forename: Alan R
authors.initials: AR
authors.lastname: Davidson
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo, Russia Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia severik@waksman.rutgers.edu.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
authors.affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA semenova@waksman.rutgers.edu.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Semenova
mesh_terms: D000222:Adaptation, Physiological; D001435:Bacteriophages / Q000235:genetics; D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D016680:Genome, Bacterial; D010957:Plasmids / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000821:virology; D012329:RNA, Bacterial / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D014764:Viral Proteins; D004247:DNA; D003850:Deoxyribonuclease I