High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• CRISPR-Cas systems provide adaptive immunity in bacteria against foreign genetic elements.
• In Escherichia coli, CRISPR spacer acquisition is enhanced by priming, which involves CRISPR RNA binding to foreign DNA.
• This priming mechanism also dictates a bias in the DNA strand from which new spacers are selected.

Purpose of the Study:

• To investigate the mechanism underlying strand bias during primed spacer acquisition in E. coli.
• To analyze the role of protospacer adjacent motifs (PAMs) in spacer acquisition.
• To assess the evolutionary impact of CRISPR-Cas systems on host-virus interactions.

Main Methods:

• Analysis of approximately 200,000 CRISPR spacers acquired during plasmid elimination mediated by the E. coli type I-E CRISPR-Cas system.
• Examination of the positions of protospacers on plasmids from which new spacers were derived.
• Assessment of the influence of protospacer adjacent motif (PAM) sequences on spacer acquisition.

Main Results:

• Spacer acquisition positions were inconsistent with a DNA sliding mechanism driving strand bias.
• A strong preference for the AAG PAM was observed, but introducing more AAG sites did not alter protospacer selection.
• Neither E. coli phages nor Streptococcus thermophilus phages avoid AAG or CTT PAM sequences.

Conclusions:

• The mechanism of primed spacer acquisition in E. coli does not primarily rely on the Cas machinery sliding along target DNA.
• PAM sequence availability does not significantly influence protospacer choice during adaptation.
• CRISPR-Cas systems may not have been a major evolutionary force shaping interactions between E. coli and its phages or S. thermophilus and its phages.