Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

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

[2]  M. Heel,et al.  Single-particle electron cryo-microscopy: towards atomic resolution , 2000, Quarterly Reviews of Biophysics.

[3]  Dmitri I. Svergun,et al.  Uniqueness of ab initio shape determination in small-angle scattering , 2003 .

[4]  T. Cech,et al.  Method for determining RNA 3' ends and application to human telomerase RNA. , 1996, Nucleic acids research.

[5]  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.

[6]  Dmitri I. Svergun,et al.  Determination of the regularization parameter in indirect-transform methods using perceptual criteria , 1992 .

[7]  S. Ehrlich,et al.  Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. , 2005, Microbiology.

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

[9]  D. Hornby,et al.  Enrichment and analysis of RNA centered on ion pair reverse phase methodology. , 2006, RNA.

[10]  Dmitri I. Svergun,et al.  PRIMUS: a Windows PC-based system for small-angle scattering data analysis , 2003 .

[11]  Wilko Keegstra,et al.  Automation of specimen selection and data acquisition for protein electron crystallography , 1998 .

[12]  K. Zhou,et al.  Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. , 2009, Structure.

[13]  G J Kleywegt,et al.  Detection, delineation, measurement and display of cavities in macromolecular structures. , 1994, Acta crystallographica. Section D, Biological crystallography.

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

[15]  J. Mccammon,et al.  Situs: A package for docking crystal structures into low-resolution maps from electron microscopy. , 1999, Journal of structural biology.

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

[17]  P. Cramer,et al.  Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution , 2001, Science.

[18]  H. Ochman,et al.  Standard reference strains of Escherichia coli from natural populations , 1984, Journal of bacteriology.

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

[20]  M. Lieber,et al.  Analysis of non-B DNA structure at chromosomal sites in the mammalian genome. , 2006, Methods in enzymology.

[21]  Thomas Tuschl,et al.  Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein , 2005, Nature.

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

[23]  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.

[24]  Eugene Berezikov,et al.  A Role for Piwi and piRNAs in Germ Cell Maintenance and Transposon Silencing in Zebrafish , 2007, Cell.

[25]  G. Krauss,et al.  Characterization of the endonuclease SSO2001 from Sulfolobus solfataricus P2 , 2009, FEBS letters.

[26]  Dmitri I. Svergun,et al.  Electronic Reprint Applied Crystallography Dammif, a Program for Rapid Ab-initio Shape Determination in Small-angle Scattering Applied Crystallography Dammif, a Program for Rapid Ab-initio Shape Determination in Small-angle Scattering , 2022 .

[27]  V. Kunin,et al.  Evolutionary conservation of sequence and secondary structures in CRISPR repeats , 2007, Genome Biology.

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

[29]  A. Hüttenhofer,et al.  Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[30]  T. Hsieh,et al.  Preferential cleavage of plasmid-based R-loops and D-loops by Drosophila topoisomerase IIIβ , 2002, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[33]  T. Itoh,et al.  Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[34]  P. Cramer,et al.  Structural biology of RNA polymerase III: mass spectrometry elucidates subcomplex architecture. , 2007, Structure.

[35]  Xuemei Chen,et al.  Methylation Protects miRNAs and siRNAs from a 3′-End Uridylation Activity in Arabidopsis , 2005, Current Biology.

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

[37]  R. W. Davis,et al.  Hybridization of RNA to double-stranded DNA: formation of R-loops. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[38]  F. J. Luque,et al.  Structure, recognition properties, and flexibility of the DNA.RNA hybrid. , 2005, Journal of the American Chemical Society.

[39]  Albert J R Heck,et al.  Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry. , 2006, Analytical chemistry.

[40]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.

[41]  Philippe Horvath,et al.  Diversity, Activity, and Evolution of CRISPR Loci in Streptococcus thermophilus , 2007, Journal of bacteriology.

[42]  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..

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

[44]  Daniel Barsky,et al.  Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3 , 2008, Proceedings of the National Academy of Sciences.

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

[46]  T. Baker,et al.  Transcriptional activation of initiation of replication from the E. coli chromosomal origin: An RNA-DNA hybrid near oriC , 1988, Cell.

[47]  A. Heck,et al.  The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionization orthogonal time-of-flight instrument. , 2001, Rapid communications in mass spectrometry : RCM.

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

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

[50]  M. Lieber,et al.  R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells , 2003, Nature Immunology.

[51]  A. Heck.  Native mass spectrometry: a bridge between interactomics and structural biology , 2008, Nature Methods.

[52]  L. Marraffini,et al.  CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea , 2010, Nature Reviews Genetics.

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

[54]  D. Clayton,et al.  Initiation of Mitochondrial DNA Replication by Transcription and R-loop Processing* , 1998, The Journal of Biological Chemistry.

[55]  S. Yokoyama,et al.  X‐ray crystal structure of a CRISPR‐associated protein, Cse2, from Thermus thermophilus HB8 , 2008, Proteins.

[56]  J. García-Martínez,et al.  Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements , 2005, Journal of Molecular Evolution.

[57]  D. Hornby,et al.  Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA:RNA interactions. , 2009, Journal of chromatography. A.

[58]  Jeffrey E. Barrick,et al.  Genome evolution and adaptation in a long-term experiment with Escherichia coli , 2009, Nature.

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

[60]  D. Wigley,et al.  Structure and mechanism of helicases and nucleic acid translocases. , 2007, Annual review of biochemistry.

[61]  Conrad C. Huang,et al.  Software extensions to UCSF chimera for interactive visualization of large molecular assemblies. , 2005, Structure.

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