Unexpectedly broad target recognition of the CRISPR-mediated virus defence system in the archaeon Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus carries an extensive array of clustered regularly interspaced short palindromic repeats (CRISPR) systems able to mediate DNA degradation of invading genetic elements when complementarity to the small CRISPR-derived (cr)RNAs is given. Studying virus defence in vivo with recombinant viral variants, we demonstrate here that an unexpectedly high number of mutations are tolerated between the CRISPR-derived guide RNAs (crRNAs) and their target sequences (protospacer). Up to 15 mismatches in the crRNA still led to ∼50% of DNA degradation, when these mutations were outside the ‘seed’ region. More than 15 mutations were necessary to fully abolished interference. Different from other CRISPR systems investigated in vivo, mutations outside the protospacer region indicated no need for a protospacer adjacent motif sequence to confer DNA interference. However, complementarity of only 3 nucleotides between the repeat-derived 5′ handle of the crRNA and nucleotides adjacent to the protospacer enabled self-recognition, i.e. protection of the host locus. Our findings show commonalities and differences among the various CRISPR-mediated defence systems and suggest that they should not merely be perceived as a ‘first-barrier-defence system’ but may be considered to have a broader mechanism that allows host cells to cope with viruses keeping them at reduced levels.

[1]  G. Hong,et al.  Nucleic Acids Research , 2015, Nucleic Acids Research.

[2]  C. Schleper,et al.  CRISPR-mediated defense mechanisms in the hyperthermophilic archaeal genus Sulfolobus , 2013, RNA biology.

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

[4]  Shiraz A. Shah,et al.  Protospacer recognition motifs Mixed identities and functional diversity , 2013 .

[5]  Rolf Backofen,et al.  Characterization of CRISPR RNA processing in Clostridium thermocellum and Methanococcus maripaludis , 2012, Nucleic acids research.

[6]  Friedhelm Pfeiffer,et al.  An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA* , 2012, The Journal of Biological Chemistry.

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

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

[9]  R. Garrett,et al.  Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus , 2011, Nucleic acids research.

[10]  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.

[11]  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.

[12]  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.

[13]  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.

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

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

[16]  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.

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

[18]  Marko Djordjevic,et al.  Transcription, processing and function of CRISPR cassettes in Escherichia coli , 2010, Molecular microbiology.

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

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

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

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

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

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

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

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

[27]  S. Albers,et al.  Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea , 2007, Nucleic acids research.

[28]  Ibtissem Grissa,et al.  The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats , 2007, BMC Bioinformatics.

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

[30]  R. Garrett,et al.  Viruses of the Archaea: a unifying view , 2006, Nature Reviews Microbiology.

[31]  R. Garrett,et al.  A putative viral defence mechanism in archaeal cells. , 2006, Archaea.

[32]  N. Kurosawa,et al.  Homologous recombination of exogenous DNA with the Sulfolobus acidocaldarius genome: properties and uses. , 2005, FEMS microbiology letters.

[33]  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..

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

[35]  C. Schleper,et al.  A reporter gene system for the hyperthermophilic archaeon Sulfolobus solfataricus based on a selectable and integrative shuttle vector , 2003, Molecular microbiology.

[36]  R. Garrett,et al.  Genetic profile of pNOB8 from Sulfolobus: the first conjugative plasmid from an archaeon , 1998, Extremophiles.

[37]  D. Grogan,et al.  Phenotypic characterization of the archaebacterial genus Sulfolobus: comparison of five wild-type strains , 1989, Journal of bacteriology.