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Nucleic Acids Res. 2016 Sep 6;44(15):7385-94. doi: 10.1093/nar/gkw421. Epub 2016 May 12.

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

Author information

1
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
2
Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
3
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA.
4
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA cbeisel@ncsu.edu.

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.

PMID:
27174938
PMCID:
PMC5009729
DOI:
10.1093/nar/gkw421
[Indexed for MEDLINE]
Free PMC Article

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