Molecular Cell
Volume 65, Issue 1, 5 January 2017, Pages 168-175
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Short Article
Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response

https://doi.org/10.1016/j.molcel.2016.11.031Get rights and content
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Highlights

  • A library of cas9 alleles was screened for clones with enhanced CRISPR-Cas immunity

  • The I473F substitution in Cas9 enhances the CRISPR immune response by 100-fold

  • This phenotype is due to an increase of the rate of spacer acquisition

  • Cas9 is involved in spacer acquisition, and the I473F mutation is a tool to study it

Summary

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
spacer acquisition
adaptation
bacteriophage
adaptive immunity
horizontal gene transfer

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Present address: Synthetic Biology Group, Microbiology Department, Institut Pasteur, 75015 Paris, France

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