title: Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response.
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.
authors.affiliation: Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Heler
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Addison V
authors.initials: AV
authors.lastname: Wright
authors.affiliation: Department of Physics, University of Virginia, Charlottesville, VA 22904, USA.
authors.forename: Marija
authors.initials: M
authors.lastname: Vucelja
authors.affiliation: Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA.
authors.forename: David
authors.initials: D
authors.lastname: Bikard
authors.affiliation: 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.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
authors.affiliation: Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA. Electronic address: marraffini@rockefeller.edu.
authors.forename: Luciano A
authors.initials: LA
authors.lastname: Marraffini
mesh_terms: D056704:Adaptive Immunity; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000276:immunology*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000276:immunology*; D021901:DNA, Intergenic / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D054884:Host-Pathogen Interactions; D009154:Mutation; D010641:Phenotype; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics / Q000276:immunology / Q000821:virology; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D021901:DNA, Intergenic; D004279:DNA, Viral; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR-Cas; Cas9; adaptation; adaptive immunity; bacteriophage; horizontal gene transfer; spacer acquisition