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CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III

Nature volume 471, pages 602–607 (2011)Cite this article

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Abstract

CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.

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Figure 1: A newly identified tracrRNA is required for crRNA maturation in S. pyogenes.
Figure 2: Co-processing of tracrRNA and pre-crRNA requires both endogenous RNase III and Csn1 in vivo.
Figure 3: tracrRNA directs pre-crRNA cleavage by RNase III in vitro.
Figure 4: Model for tracrRNA-mediated crRNA maturation involving RNase III and Csn1.
Figure 5: Both tracrRNA and pre-crRNA confer immunity against acquisition of a protospacer gene derived from a lysogenic phage.
Figure 6: tracrRNA-mediated crRNA maturation is conserved among different bacterial species.

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Acknowledgements

We thank D. Veit for technical help. This work was funded by the European Community (FP6, BACRNAs-018618; E.C.), the Austrian Science Fund (FWF, P17238-B09 (E.C.) and W1207-B09 (E.C., K.C.)), the Austrian Agency for Research Promotion (FFG, 812138-SCK/KUG; E.C.), the Theodor Körner Fonds (E.C.), Umeå University (E.C.), the Swedish Research Council (E.C.), IMPRS-IDI (Y.C.), the German Research Council (DFG Priority Program “Sensory and Regulatory RNAs in Prokaryotes” SPP1258, Vo875/4; J.V.), and the German Ministry of Education and Research (BMBF, 01GS0806/JV-BMBF-01 and 0315836; J.V.).

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Author notes

  1. Krzysztof Chylinski and Cynthia M. Sharma: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Umeå University, S-90187 Umeå, Sweden,

    Elitza Deltcheva, Krzysztof Chylinski & Emmanuelle Charpentier

  2. Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria,

    Elitza Deltcheva, Krzysztof Chylinski, Karine Gonzales, Zaid A. Pirzada, Maria R. Eckert & Emmanuelle Charpentier

  3. ZINF Research Center for Infectious Diseases, University of Würzburg, D-97080 Würzburg, Germany,

    Cynthia M. Sharma, Yanjie Chao & Jörg Vogel

  4. RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, D-97080 Würzburg, Germany,

    Yanjie Chao & Jörg Vogel

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  1. Elitza Deltcheva
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Contributions

E.D., K.C., C.M.S., K.G., J.V. and E.C. designed the research; E.D., K.C., C.M.S., K.G., Z.A.P., Y.C. and M.R.E. conducted the experiments; E.D., K.C., C.M.S., K.G., J.V. and E.C. analysed and interpreted the data; E.C. wrote the paper which E.D., K.C., C.M.S. and J.V. commented on, and supervised the project. Author information and raw data are available from E.D., K.C., C.M.S., J.V. and E.C.

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Correspondence to Emmanuelle Charpentier.

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Deltcheva, E., Chylinski, K., Sharma, C. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011). https://doi.org/10.1038/nature09886

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