title: Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation.
abstract: The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multisubunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.
authors.affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Kuznedelov
authors.affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Mekler
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
authors.forename: Sofia
authors.initials: S
authors.lastname: Lemak
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Monika
authors.initials: M
authors.lastname: Tokmina-Lukaszewska
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: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Ishita
authors.initials: I
authors.lastname: Jain
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia.|Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Savitskaya
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA.
authors.forename: John
authors.initials: J
authors.lastname: Mallon
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia.|NCBI, NLM, NIH, Bethesda, MD 20894, USA.
authors.forename: Sergey
authors.initials: S
authors.lastname: Shmakov
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Bothner
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Bailey
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Yakunin
authors.affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA severik@waksman.rutgers.edu.|Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia.|Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.|Peter the Great St Petersburg State Polytechnic University, St. Petersburg, 195251, Russia.
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; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D034622:RNA Interference; D012329:RNA, Bacterial / Q000502:physiology
chemical_list: D012329:RNA, Bacterial