Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System

Cell. 2015 Nov 5;163(4):854-65. doi: 10.1016/j.cell.2015.10.003.


CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.

Publication types

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

MeSH terms

  • Bacteriophage lambda / genetics*
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems*
  • DNA Helicases / metabolism*
  • DNA, Viral / metabolism*
  • Endodeoxyribonucleases / metabolism*
  • Endonucleases / metabolism*
  • Escherichia coli / genetics*
  • Escherichia coli / immunology
  • Escherichia coli / metabolism
  • Escherichia coli / virology*
  • Escherichia coli Proteins / metabolism*
  • Models, Biological
  • Repetitive Sequences, Nucleic Acid


  • CRISPR-Associated Proteins
  • DNA, Viral
  • Escherichia coli Proteins
  • Cas2 protein, E coli
  • Endodeoxyribonucleases
  • Endonucleases
  • YgbT protein, E coli
  • DNA Helicases
  • ygcB protein, E coli