title: The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers.
abstract: Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of this complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Finally, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Luo
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Ryan N
authors.initials: RN
authors.lastname: Jackson
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Denny
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 Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Maksimchuk
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Wayne
authors.initials: W
authors.lastname: Lin
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 Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Blake
authors.initials: B
authors.lastname: Wiedenheft
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA cbeisel@ncsu.edu.
authors.forename: Chase L
authors.initials: CL
authors.lastname: Beisel
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020868:Gene Silencing; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry* / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D012329:RNA, Bacterial; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA