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Title: The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers

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. Lastly, 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:
 [1] ;  [2] ;  [1] ;  [2] ;  [1] ;  [2] ;  [2] ;  [2] ;  [1]
  1. North Carolina State Univ., Raleigh, NC (United States)
  2. Montana State Univ., Bozeman, MT (United States)
Publication Date:
Grant/Contract Number:
SC0012518
Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 44; Journal Issue: 15; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Research Org:
Univ. of Colorado, Boulder, CO (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; dna; rna; escherichia coli; surveillance; medical; stoichiometry; spacer device; complex; crispr
OSTI Identifier:
1360145