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Nucleic Acids Res. 2016 Jul 8;44(12):5872-82. doi: 10.1093/nar/gkw469. Epub 2016 May 23.

Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system.

Author information

1
Prokaryotic Small RNA Biology Group, Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg, Germany.
2
LOEWE Center for Synthetic Microbiology, Philipps University Marburg, D-35043 Marburg, Germany Department of Chemistry, Philipps University Marburg, D-35043 Marburg, Germany.
3
Bioanalytics Research Group, Department of Clinical Chemistry, University Medical Centre, D-37075 Göttingen, Germany.
4
Bioanalytics Research Group, Department of Clinical Chemistry, University Medical Centre, D-37075 Göttingen, Germany Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
5
Prokaryotic Small RNA Biology Group, Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg, Germany LOEWE Center for Synthetic Microbiology, Philipps University Marburg, D-35043 Marburg, Germany lennart.randau@mpi-marburg.mpg.de.

Abstract

Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity.

PMID:
27216815
PMCID:
PMC4937334
DOI:
10.1093/nar/gkw469
[Indexed for MEDLINE]
Free PMC Article

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