A Functional Mini-Integrase in a Two-Protein-type V-C CRISPR System

Mol Cell. 2019 Feb 21;73(4):727-737.e3. doi: 10.1016/j.molcel.2018.12.015. Epub 2019 Jan 29.


CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.

Keywords: CRISPR; integrase; protein-DNA recognition; spacer acquisition.

Publication types

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

MeSH terms

  • Base Pairing
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA, Bacterial / genetics*
  • DNA, Bacterial / metabolism
  • Endodeoxyribonucleases / genetics*
  • Endodeoxyribonucleases / metabolism
  • Endonucleases / genetics*
  • Endonucleases / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Escherichia coli Proteins / genetics*
  • Escherichia coli Proteins / metabolism
  • Gene Editing / methods*
  • Gene Expression Regulation, Bacterial
  • Integrases / genetics*
  • Integrases / metabolism
  • Nucleotide Motifs
  • Substrate Specificity


  • CRISPR-Associated Proteins
  • DNA, Bacterial
  • Escherichia coli Proteins
  • Integrases
  • Cas2 protein, E coli
  • Endodeoxyribonucleases
  • Endonucleases
  • YgbT protein, E coli