Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays

Mol Cell. 2018 Apr 5;70(1):48-59.e5. doi: 10.1016/j.molcel.2018.03.003. Epub 2018 Mar 27.


CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration.

Keywords: CRISPR-Cas; Cas1-Cas2; Cas4; DNA cleavage; DNA integration; negative stain EM.

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

  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / immunology
  • CRISPR-Associated Protein 9 / isolation & purification
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Associated Proteins / immunology
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA, Bacterial / genetics*
  • DNA, Bacterial / immunology
  • DNA, Bacterial / metabolism
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Gene Editing / methods*
  • Models, Molecular
  • Multienzyme Complexes
  • Nucleic Acid Conformation
  • Protein Conformation
  • Protein Subunits
  • Substrate Specificity


  • CRISPR-Associated Proteins
  • DNA, Bacterial
  • Escherichia coli Proteins
  • Multienzyme Complexes
  • Protein Subunits
  • CRISPR-Associated Protein 9
  • Endodeoxyribonucleases
  • YgbT protein, E coli