Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity

The initial stage of CRISPR–Cas immunity involves the integration of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved among all CRISPR–Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å-resolution crystal structure of the Cas1–Cas2 complex. Mutations that perturb Cas1–Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Active site mutants of Cas2, unlike those of Cas1, can still acquire new spacers, thus indicating a nonenzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1–Cas2 complexes specify sites of CRISPR spacer integration.

[1]  E. Koonin,et al.  Live virus-free or die: coupling of antivirus immunity and programmed suicide or dormancy in prokaryotes , 2012, Biology Direct.

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

[3]  S. Shuman,et al.  Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris. , 2010, Acta crystallographica. Section F, Structural biology and crystallization communications.

[4]  E. Koonin,et al.  A Novel Family of Sequence-specific Endoribonucleases Associated with the Clustered Regularly Interspaced Short Palindromic Repeats* , 2008, Journal of Biological Chemistry.

[5]  M. DeLisa,et al.  Double-stranded Endonuclease Activity in Bacillus halodurans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas2 Protein* , 2012, The Journal of Biological Chemistry.

[6]  P. Evans,et al.  Scaling and assessment of data quality. , 2006, Acta crystallographica. Section D, Biological crystallography.

[7]  K. Severinov,et al.  High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli , 2013, RNA biology.

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

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

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

[11]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[12]  Stan J. J. Brouns,et al.  CRISPR Interference Directs Strand Specific Spacer Acquisition , 2012, PloS one.

[13]  Rotem Sorek,et al.  CRISPR-mediated adaptive immune systems in bacteria and archaea. , 2013, Annual review of biochemistry.

[14]  P. Schuck,et al.  Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. , 2000, Biophysical journal.

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

[16]  Arthur J. Rowe,et al.  Analytical ultracentrifugation in biochemistry and polymer science , 1992 .

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

[18]  N. Grishin,et al.  PROMALS3D: a tool for multiple protein sequence and structure alignments , 2008, Nucleic acids research.

[19]  B. Tjaden,et al.  Characterization of the CRISPR/Cas Subtype I-A System of the Hyperthermophilic Crenarchaeon Thermoproteus tenax , 2012, Journal of bacteriology.

[20]  U. Qimron,et al.  Experimental definition of a clustered regularly interspaced short palindromic duplicon in Escherichia coli. , 2012, Journal of molecular biology.

[21]  Konstantin Severinov,et al.  Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system , 2012, Nature Communications.

[22]  Jesús García-Martínez,et al.  CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli , 2013, RNA biology.

[23]  Andrew Emili,et al.  A dual function of the CRISPR–Cas system in bacterial antivirus immunity and DNA repair , 2011, Molecular microbiology.

[24]  J. Berger,et al.  Structure of human cGAS reveals a conserved family of second-messenger enzymes in innate immunity. , 2013, Cell reports.

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

[26]  Geoffrey J. Barton,et al.  Jalview Version 2—a multiple sequence alignment editor and analysis workbench , 2009, Bioinform..

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

[28]  Peter C. Fineran,et al.  In Vivo Protein Interactions and Complex Formation in the Pectobacterium atrosepticum Subtype I-F CRISPR/Cas System , 2012, PloS one.

[29]  U. Qimron,et al.  Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli , 2012, Nucleic acids research.

[30]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[31]  Jeong-Sun Kim,et al.  Crystal structure of Cas1 from Archaeoglobus fulgidus and characterization of its nucleolytic activity. , 2013, Biochemical and biophysical research communications.

[32]  P. Brown,et al.  Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation. , 2006, Biophysical journal.

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

[34]  R F Standaert,et al.  Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. , 1993, Journal of molecular biology.

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

[36]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

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

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

[39]  Dipali G. Sashital,et al.  Mechanism of foreign DNA selection in a bacterial adaptive immune system. , 2012, Molecular cell.

[40]  G. Krauss,et al.  SSO1450 – A CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA , 2009, FEBS letters.

[41]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

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

[43]  Jennifer A. Doudna,et al.  Structures of the RNA-guided surveillance complex from a bacterial immune system , 2011, Nature.

[44]  J. García-Martínez,et al.  Diversity of CRISPR loci in Escherichia coli. , 2010, Microbiology.