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The CRISPR-Cas system in Sulfolobus islandicus targets invaders using crRNA. This study reveals Cmr-α

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • CRISPR-Cas systems provide adaptive immunity in prokaryotes.
  • The Type III-B Cmr-α system from Sulfolobus islandicus targets both RNA and DNA invaders.
  • DNA targeting by Cmr-α is linked to protospacer transcription directionality.

Purpose of the Study:

  • To elucidate the mechanism of DNA targeting by the S. islandicus Type III-B Cmr-α system.
  • To characterize the in vitro activity of the purified Cmr-α effector complex.

Main Methods:

  • Purification of the native Cmr-α effector complex from S. islandicus.
  • In vitro biochemical assays to assess RNA and ssDNA cleavage activities.
  • Mutational analysis of key protein domains (HD, GGDD) and invader plasmid assays.

Main Results:

  • Cmr-α cleaves target RNAs and ssDNA, with ssDNA cleavage activated by target RNA.
  • ssDNA cleavage is dependent on specific mismatches between crRNA and target RNA.
  • Mutations in the HD domain or GGDD motif of Cmr-2α attenuate in vivo DNA interference, while HD mutations abolish in vitro DNase activity.
  • Activated Cmr-α exhibits potent DNase activity against excess DNA substrates.

Conclusions:

  • The Cmr-α complex possesses both RNA and DNA cleavage activities, with DNA targeting being RNA-activated.
  • Specific crRNA-target RNA interactions are crucial for ssDNA cleavage.
  • The HD domain and GGDD motif are essential for Cmr-α's DNA interference function.
  • The potent DNase activity of Cmr-α could be harnessed for antiviral strategies.