title: CRISPR interference and priming varies with individual spacer sequences.
abstract: CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring 'spacer' sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid 'primed' adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR-Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed 'naïve' adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for interference and priming, and reveal that priming is a major pathway for adaptation during initial infection.
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011, USA.
authors.forename: Chaoyou
authors.initials: C
authors.lastname: Xue
authors.affiliation: Genome Informatics Facility, Office of Biotechnology, Iowa State University Ames, IA 50011, USA.
authors.forename: Arun S
authors.initials: AS
authors.lastname: Seetharam
authors.affiliation: Institutes of Gene Biology and Molecular Genetics, Russian Academy of Sciences, Moscow 119991, Russia Skolkovo Institute of Science and Technology, Skolkovo, Russia, Moscow, Russia Peter the Great Polytechnical University, St. Petersburg, Russia.
authors.forename: Olga
authors.initials: O
authors.lastname: Musharova
authors.affiliation: Institutes of Gene Biology and Molecular Genetics, Russian Academy of Sciences, Moscow 119991, Russia Skolkovo Institute of Science and Technology, Skolkovo, Russia, Moscow, Russia Peter the Great Polytechnical University, St. Petersburg, Russia Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, the Netherlands.
authors.forename: Stan J J
authors.initials: SJ
authors.lastname: Brouns
authors.affiliation: Genome Informatics Facility, Office of Biotechnology, Iowa State University Ames, IA 50011, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Severin
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011, USA sashital@iastate.edu.
authors.forename: Dipali G
authors.initials: DG
authors.lastname: Sashital
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D009154:Mutation
chemical_list: D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; D004706:Endodeoxyribonucleases