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

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

Author information

1
Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA.
2
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
3
Department of Physics, University of Virginia, Charlottesville, VA 22904, USA.
4
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
5
Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA. Electronic address: marraffini@rockefeller.edu.

Abstract

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

PMID:
28017588
PMCID:
PMC5218886
DOI:
10.1016/j.molcel.2016.11.031
[Indexed for MEDLINE]
Free PMC Article

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