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Journal Article

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

MPS-Authors
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Sharma,  K.
Research Group of Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry, Max Planck Society;

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Urlaub,  H.
Research Group of Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry, Max Planck Society;

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Fulltext (public)

2331939.pdf
(Publisher version), 6MB

Supplementary Material (public)

2331939_Suppl_1.pdf
(Supplementary material), 4MB

2331939_Suppl_2.zip
(Supplementary material), 354KB

Citation

Gleditzsch, D., Müller-Esparza, H., Pausch, P., Sharma, K., Dwarakanath, S., Urlaub, H., et al. (2016). Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system. Nucleic Acids Research, 44(12), 5872-5882. doi: 10.1093/nar/gkw469.


Cite as: http://hdl.handle.net/11858/00-001M-0000-002B-2E20-7
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.