title: High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli.
abstract: In Escherichia coli, the acquisition of new CRISPR spacers is strongly stimulated by a priming interaction between a spacer in CRISPR RNA and a protospacer in foreign DNA. Priming also leads to a pronounced bias in DNA strand from which new spacers are selected. Here, ca. 200,000 spacers acquired during E. coli type I-E CRISPR/Cas-driven plasmid elimination were analyzed. Analysis of positions of plasmid protospacers from which newly acquired spacers have been derived is inconsistent with spacer acquisition machinery sliding along the target DNA as the primary mechanism responsible for strand bias during primed spacer acquisition. Most protospacers that served as donors of newly acquired spacers during primed spacer acquisition had an AAG protospacer adjacent motif, PAM. Yet, the introduction of multiple AAG sequences in the target DNA had no effect on the choice of protospacers used for adaptation, which again is inconsistent with the sliding mechanism. Despite a strong preference for an AAG PAM during CRISPR adaptation, the AAG (and CTT) triplets do not appear to be avoided in known E. coli phages. Likewise, PAM sequences are not avoided in Streptococcus thermophilus phages, indicating that CRISPR/Cas systems may not have been a strong factor in shaping host-virus interactions.
authors.affiliation: Institute of Molecular Genetics of the Russian Academy of Sciences, Moscow, Russia.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Savitskaya
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Semenova
authors.forename: Vladimir
authors.initials: V
authors.lastname: Dedkov
authors.forename: Anastasia
authors.initials: A
authors.lastname: Metlitskaya
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
mesh_terms: D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003090:Coliphages / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D021901:DNA, Intergenic; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing; D008969:Molecular Sequence Data; D010957:Plasmids; D017108:Streptococcus Phages / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D021901:DNA, Intergenic
keywords: CRISPR adaptation; CRISPR/Cas systems; Escherichia coli; bacteriophage; high-throughput sequencing