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

Addison V Wright1, Joy Y Wang2, David Burstein3

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Molecular Cell
|February 3, 2019
PubMed
Summary

CRISPR-Cas immunity uses Cas1 and Cas2 proteins to integrate foreign DNA. A novel mini-integrase, comprising only the Cas1 protein, efficiently integrates short DNA fragments, suggesting an ancestral role for Cas1 before Cas2 evolved.

Keywords:
CRISPRintegraseprotein-DNA recognitionspacer acquisition

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Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • CRISPR-Cas immunity involves integrating foreign DNA into the host genome at CRISPR loci.
  • Cas1 and Cas2 proteins typically form the integration complex for DNA acquisition.
  • Some CRISPR systems (Type V-C, V-D) lack Cas2 and feature short repeats/spacers.

Purpose of the Study:

  • To investigate the DNA integration mechanism in CRISPR systems lacking Cas2.
  • To characterize the function of the Type V-C Cas1 protein as a mini-integrase.
  • To understand the evolutionary origins of CRISPR-Cas integration machinery.

Main Methods:

  • Biochemical assays to test DNA integration activity of the Type V-C Cas1 protein.
  • Analysis of DNA fragment length preference during integration.
  • Structural analysis to determine the oligomeric state of Cas1 during integration.

Main Results:

  • The Type V-C Cas1 protein alone acts as a mini-integrase, catalyzing DNA integration.
  • This mini-integrase shows a preference for short DNA fragments (17-19 bp).
  • Cas1 proteins form a tetramer during the integration process, utilizing an internal ruler mechanism.

Conclusions:

  • A minimal integrase, consisting of Cas1 alone, can facilitate DNA integration with a bias for shorter sequences.
  • This finding supports a model where Cas1 possesses inherent capabilities for integration, potentially predating the involvement of Cas2.
  • The mini-integrase mechanism offers insights into the evolution of CRISPR-Cas systems and their DNA acquisition strategies.