Research > Ongoing Projects > CRISPR
CRISPR-Mediated Virus Defense in Hyperthermophilic Archaea
Sequence analyses of complete bacterial and archaeal genomes have led to the discovery of Clustered Regularly Interspaced Short Palindromic Repeats (in short CRISPR). The potential function of these repeats and their intervening short spacer sequences as well as the function of their associated (Cas-) proteins as constituents of an immune defense system against viruses and other genetic elements, has only recently been recognized. Although CRISPR/Cas systems are found widespread in bacterial and archaeal genomes and exhibit considerable diversity, little insights into the action of most of the CRISPR modules have been obtained in particular in Archaea due to the lack of suitable in vivo test systems.
We have recently demonstrated CRISPR/Cas-based immune defense in the hyperthermophilic archaeon Sulfolobus solfataricus. Recombinant variants of the SSV1 virus containing a gene of the conjugative plasmid pNOB8 that represents a target for a corresponding CRISPR spacer in the chromosome were tested in transfection experiments. Almost 100% immunity against the recombinant virus was observed when the chromosomal CRISPR spacer matched perfectly to the protospacer. Different from bacterial systems immunity was still detected, albeit at decreased levels, when mutations distinguished target and spacer. CRISPR/Cas targeting was independent of the transcription of the target gene. Furthermore, a mini CRISPR locus introduced on the viral DNA with spacers targeting the (non-essential) chromosomal beta-galactosidase gene was unstable in host cells and triggered recombination with the indigenous CRISPR locus. Our experiments demonstrate in vivo activity of CRISPR/Cas in archaea for the first time and suggest that – unlike the recently demonstrated in vitro cleavage of RNA in Pyrococcus - DNA is targeted in this archaeon.
In the ongoing project, we further characterize this archaeal CRISPR-mediated virus defense system.
Reference: In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon. Manica A, Zebec Z, Teichmann D, Schleper C. (2011) Mol Microbiol. 80(2):481-91.
Participants: Christa Schleper, Ziga Zebec, Andrea Manica, Julia Steinkellner, Daniela Teichmann
Zebec Z, Manica A, Zhang J, White M, Schleper C (2014) CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus. Nucleic Acids Research. first published online March 6, 2014 doi:10.1093/nar/gku161.
Manica A, Zebec Z, Steinkellner J, Schleper C (2013) Unexpectedly broad target recognition of the CRISPR-mediated virus defence system in the archaeon Sulfolobus solfataricus. Nucleic Acids Research 41(22): 10509-10517. doi: 10.1093/nar/gkt767.
Märtens B, Manoharadas S, Hasenöhrl D, Manica A, Bläsi U (2013) Antisense regulation by transposon-derived RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus. Embo Reports 14(6): 527-533.
Manica A, Schleper C (2013) CRISPR-mediated defense mechanisms in the hyperthermophilic archaeal genus Sulfolobus. RNA Biology 10(5): 671-678. doi: 10.4161/rna.24154.
Manica A, Zebec Z, Teichmann D, Schleper C (2011) In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon. Molecular Microbiology 80(2): 481-491.