Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation

Nat Struct Mol Biol. 2014 Sep;21(9):771-7. doi: 10.1038/nsmb.2875. Epub 2014 Aug 17.


CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3'-to-5' ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinomycetales / chemistry
  • Actinomycetales / enzymology*
  • Actinomycetales / metabolism
  • Adenosine Triphosphate / metabolism
  • Base Sequence
  • CRISPR-Associated Proteins / chemistry
  • CRISPR-Associated Proteins / metabolism*
  • Crystallography, X-Ray
  • DNA Helicases / chemistry
  • DNA Helicases / metabolism*
  • DNA, Single-Stranded / chemistry
  • DNA, Single-Stranded / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation


  • CRISPR-Associated Proteins
  • DNA, Single-Stranded
  • Adenosine Triphosphate
  • DNA Helicases

Associated data

  • PDB/4QQW
  • PDB/4QQX
  • PDB/4QQY
  • PDB/4QQZ