Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response

Mol Cell. 2017 Jan 5;65(1):168-175. doi: 10.1016/j.molcel.2016.11.031. Epub 2016 Dec 22.


CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, that increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application.

Keywords: CRISPR-Cas; Cas9; adaptation; adaptive immunity; bacteriophage; horizontal gene transfer; spacer acquisition.

MeSH terms

  • Adaptive Immunity*
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / immunology
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Protein 9
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Associated Proteins / immunology
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems / immunology*
  • Clustered Regularly Interspaced Short Palindromic Repeats / immunology*
  • DNA, Intergenic / genetics*
  • DNA, Intergenic / immunology
  • DNA, Intergenic / metabolism
  • DNA, Viral / genetics*
  • DNA, Viral / immunology
  • DNA, Viral / metabolism
  • Endonucleases / genetics*
  • Endonucleases / immunology
  • Endonucleases / metabolism
  • Genotype
  • High-Throughput Nucleotide Sequencing
  • Host-Pathogen Interactions
  • Mutation*
  • Phenotype
  • Staphylococcus aureus / enzymology
  • Staphylococcus aureus / genetics
  • Staphylococcus aureus / immunology
  • Staphylococcus aureus / virology
  • Substrate Specificity
  • Time Factors


  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • DNA, Intergenic
  • DNA, Viral
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases