Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs.

Small RNAs target invaders for silencing in the CRISPR-Cas pathways that protect bacteria and archaea from viruses and plasmids. The CRISPR RNAs (crRNAs) contain sequence elements acquired from invaders that guide CRISPR-associated (Cas) proteins back to the complementary invading DNA or RNA. Here, we have analyzed essential features of the crRNAs associated with the Cas RAMP module (Cmr) effector complex, which cleaves targeted RNAs. We show that Cmr crRNAs contain an 8 nucleotide 5' sequence tag (also found on crRNAs associated with other CRISPR-Cas pathways) that is critical for crRNA function and can be used to engineer crRNAs that direct cleavage of novel targets. We also present data that indicate that the Cmr complex cleaves an endogenous complementary RNA in Pyrococcus furiosus, providing direct in vivo evidence of RNA targeting by the CRISPR-Cas system. Our findings indicate that the CRISPR RNA-Cmr protein pathway may be exploited to cleave RNAs of interest.

[1]  Shiraz A. Shah,et al.  CRISPR-based immune systems of the Sulfolobales: complexity and diversity. , 2011, Biochemical Society transactions.

[2]  R. Terns,et al.  Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. , 2008, Genes & development.

[3]  Shiraz A. Shah,et al.  CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties , 2009, Molecular microbiology.

[4]  L. Schouls,et al.  Identification of genes that are associated with DNA repeats in prokaryotes , 2002, Molecular microbiology.

[5]  Stan J. J. Brouns,et al.  Evolution and classification of the CRISPR–Cas systems , 2011, Nature Reviews Microbiology.

[6]  G Vergnaud,et al.  CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. , 2005, Microbiology.

[7]  Albert J R Heck,et al.  Structural basis for CRISPR RNA-guided DNA recognition by Cascade , 2011, Nature Structural &Molecular Biology.

[8]  N. Grishin,et al.  A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action , 2006, Biology Direct.

[9]  Shiraz A. Shah,et al.  CRISPR/Cas and Cmr modules, mobility and evolution of adaptive immune systems. , 2011, Research in microbiology.

[10]  Shiraz A. Shah,et al.  Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism. , 2009, Biochemical Society transactions.

[11]  Anders F. Andersson,et al.  Virus Population Dynamics and Acquired Virus Resistance in Natural Microbial Communities , 2008, Science.

[12]  R. Barrangou,et al.  CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes , 2007, Science.

[13]  R. Garrett,et al.  Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers , 2011, Molecular microbiology.

[14]  R. Barrangou,et al.  CRISPR/Cas, the Immune System of Bacteria and Archaea , 2010, Science.

[15]  Konstantin Severinov,et al.  Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence , 2011, Proceedings of the National Academy of Sciences.

[16]  Jennifer A. Doudna,et al.  Sequence- and Structure-Specific RNA Processing by a CRISPR Endonuclease , 2010, Science.

[17]  Rolf Wagner,et al.  Identification and characterization of E. coli CRISPR‐cas promoters and their silencing by H‐NS , 2010, Molecular microbiology.

[18]  J. García-Martínez,et al.  Short motif sequences determine the targets of the prokaryotic CRISPR defence system. , 2009, Microbiology.

[19]  J. Vogel,et al.  CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III , 2011, Nature.

[20]  Daniel H. Haft,et al.  A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes , 2005, PLoS Comput. Biol..

[21]  L. Marraffini,et al.  CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA , 2008, Science.

[22]  Terrence S. Furey,et al.  The UCSC Genome Browser Database , 2003, Nucleic Acids Res..

[23]  A. MacMillan,et al.  Recognition and maturation of effector RNAs in a CRISPR interference pathway , 2011, Nature Structural &Molecular Biology.

[24]  Peter C. Fineran,et al.  Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum , 2011, RNA biology.

[25]  H. Deveau,et al.  CRISPR/Cas system and its role in phage-bacteria interactions. , 2010, Annual review of microbiology.

[26]  Andrea Manica,et al.  In vivo activity of CRISPR‐mediated virus defence in a hyperthermophilic archaeon , 2011, Molecular microbiology.

[27]  R. Terns,et al.  Binding and cleavage of CRISPR RNA by Cas6. , 2010, RNA.

[28]  M. F. White,et al.  Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE)* , 2011, The Journal of Biological Chemistry.

[29]  Philippe Horvath,et al.  Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus , 2007, Journal of bacteriology.

[30]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[31]  Stan J. J. Brouns,et al.  RNA in defense: CRISPRs protect prokaryotes against mobile genetic elements. , 2012, Cold Spring Harbor perspectives in biology.

[32]  Stan J. J. Brouns,et al.  Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes , 2008, Science.

[33]  R. Terns,et al.  Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. , 2008, RNA.

[34]  Stan J. J. Brouns,et al.  CRISPR-based adaptive and heritable immunity in prokaryotes. , 2009, Trends in biochemical sciences.

[35]  B. Graveley,et al.  RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex , 2009, Cell.

[36]  R. Terns,et al.  CRISPR-based adaptive immune systems. , 2011, Current opinion in microbiology.

[37]  R. Terns,et al.  Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage. , 2011, Structure.

[38]  Erik J. Sontheimer,et al.  Self vs. non-self discrimination during CRISPR RNA-directed immunity , 2009, Nature.

[39]  Philippe Horvath,et al.  The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA , 2010, Nature.

[40]  Fedor V. Karginov,et al.  The CRISPR system: small RNA-guided defense in bacteria and archaea. , 2010, Molecular cell.

[41]  Albert J R Heck,et al.  RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions , 2011, Proceedings of the National Academy of Sciences.

[42]  Dipali G. Sashital,et al.  An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3 , 2011, Nature Structural &Molecular Biology.