Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity

Immune systems must recognize and destroy different pathogens that threaten the host. CRISPR-Cas immune systems protect prokaryotes from viral and plasmid infection utilizing small CRISPR RNAs that are complementary to the invader's genome and specify the targets of RNA-guided Cas nucleases. Type III CRISPR-Cas immunity requires target transcription, and whereas genetic studies demonstrated DNA targeting, in vitro data have shown crRNA-guided RNA cleavage. The molecular mechanism behind these disparate activities is not known. Here, we show that transcription across the targets of the Staphylococcus epidermidis type III-A CRISPR-Cas system results in the cleavage of the target DNA and its transcripts, mediated by independent active sites within the Cas10-Csm ribonucleoprotein effector complex. Immunity against plasmids and DNA viruses requires DNA, but not RNA, cleavage activity. Our studies reveal a highly versatile mechanism of CRISPR immunity that can defend microorganisms against diverse DNA and RNA invaders.

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

[2]  S. Horinouchi,et al.  Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies , 1982, Journal of bacteriology.

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

[4]  W. Schaffner,et al.  Population snapshot of emergent Streptococcus pneumoniae serotype 19A in the United States, 2005. , 2008, The Journal of infectious diseases.

[5]  Luciano A. Marraffini,et al.  Genetic Characterization of Antiplasmid Immunity through a Type III-A CRISPR-Cas System , 2013, Journal of bacteriology.

[6]  Ceslovas Venclovas,et al.  Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus. , 2014, Molecular cell.

[7]  Rodolphe Barrangou,et al.  CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. , 2014, Molecular cell.

[8]  Q. She,et al.  An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference , 2014, Nucleic acids research.

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

[10]  S. Kowalczykowski,et al.  Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme. , 1992, The Journal of biological chemistry.

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

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

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

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

[15]  Luciano A. Marraffini,et al.  Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting , 2014, Nature.

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

[17]  L. Marraffini,et al.  Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site , 2011, Proceedings of the National Academy of Sciences.

[18]  Identification and purification of a single-stranded-DNA-specific exonuclease encoded by the recJ gene of Escherichia coli. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[19]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[20]  Jing Zhang,et al.  Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. , 2012, Molecular cell.

[21]  U. Qimron,et al.  The Escherichia coli CRISPR System Protects from λ Lysogenization, Lysogens, and Prophage Induction , 2010, Journal of bacteriology.

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

[23]  S. Horinouchi,et al.  Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance , 1982, Journal of bacteriology.

[24]  M. Kashlev,et al.  Crucial role of the RNA:DNA hybrid in the processivity of transcription. , 1998, Molecules and Cells.

[25]  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.

[26]  M. O'Reilly,et al.  The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage , 1983, Nature.

[27]  Konstantin Severinov,et al.  CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. , 2012, Molecular cell.

[28]  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.

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

[30]  Eugene V Koonin,et al.  Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems , 2011, Biology Direct.

[31]  J. Heitman,et al.  DNA nicks inflicted by restriction endonucleases are repaired by a RecA‐ and RecB‐dependent pathway in Escherichia coli , 1999, Molecular microbiology.

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

[33]  J. Oost,et al.  Unravelling the structural and mechanistic basis of CRISPR–Cas systems , 2014, Nature Reviews Microbiology.

[34]  Wenyan Jiang,et al.  A Ruler Protein in a Complex for Antiviral Defense Determines the Length of Small Interfering CRISPR RNAs , 2013, The Journal of Biological Chemistry.

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

[36]  Clement S. Chu,et al.  A New Class of Bacterial RNA Polymerase Inhibitor Affects Nucleotide Addition , 2003, Science.

[37]  Albert J R Heck,et al.  RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus. , 2014, Molecular cell.

[38]  Xu Peng,et al.  A novel interference mechanism by a type IIIB CRISPR‐Cmr module in Sulfolobus , 2013, Molecular microbiology.

[39]  L. Tang,et al.  Staphylococcus epidermidis Csm1 is a 3′–5′ exonuclease , 2013, Nucleic acids research.

[40]  James C. Hu.  Faculty Opinions recommendation of Self versus non-self discrimination during CRISPR RNA-directed immunity. , 2010 .

[41]  L. Aravind,et al.  Presence of a classical RRM-fold palm domain in Thg1-type 3'- 5'nucleic acid polymerases and the origin of the GGDEF and CRISPR polymerase domains , 2010, Biology Direct.