Systematic analysis of Type I‐E Escherichia coli CRISPR‐Cas PAM sequences ability to promote interference and primed adaptation

• Published in: Molecular microbiology Vol. 111; no. 6; pp. 1558 - 1570
• Main Authors: Musharova, Olga, Sitnik, Vasily, Vlot, Marnix, Savitskaya, Ekaterina, Datsenko, Kirill A., Krivoy, Andrey, Fedorov, Ivan, Semenova, Ekaterina, Brouns, Stan J. J., Severinov, Konstantin
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.06.2019; John Wiley and Sons Inc
• Subjects: Adaptation; Bacteria; CRISPR; CRISPR-Cas Systems; DNA, Intergenic; E coli; Escherichia coli; Escherichia coli - genetics; Laboratorium voor Microbiologie; Levels; Microbiological Laboratory; Microbiologie; Microbiology; Ribonucleic acid; RNA; Spacer; Spacers; Support interference
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.14237

Summary CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR‐Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR‐Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I‐E CRISPR‐Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition. All possible of 64 PAM combinations were tested. 36 trinucleotides were completely unable to support interference. The remaining 28 PAM trinucleotides fell into two groups: those that supported fast interference and those that supported intermediate, delayed interference that was apparently countered by plasmid copy number maintenance mechanisms over extended periods of time. PAM variants that did not lead to CRISPR interference also did not support primed adaptation. PAM variants supporting intermediate‐rate interference caused strong priming.