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

Olga Musharova,Ekaterina Savitskaya,Marnix Vlot,Ivan Fedorov,Ekaterina Semenova,Andrey Krivoy,Stan J J Brouns,Vasily Sitnik,Kirill A Datsenko,Konstantin Severinov

doi:10.1111/mmi.14237

Abstract

SummaryCRISPR 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.

Highlights

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated genes) systems provide prokaryotes with resistance against mobile genetic elements (MGEs), such as plasmids and bacteriophages (Barrangou et al, 2007; Brouns et al, 2008; Marraffini and Sontheimer, 2008)
CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors
Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference

Summary

Introduction

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated genes) systems provide prokaryotes with resistance against mobile genetic elements (MGEs), such as plasmids and bacteriophages (Barrangou et al, 2007; Brouns et al, 2008; Marraffini and Sontheimer, 2008). All CRISPR-Cas systems share a common defensive strategy and operate through three stages: adaptation, expression and interference (Makarova et al, 2015; Shmakov et al, 2017). Individual crRNAs bind to Cas proteins and the resulting effector complexes recognize protospacers complementary to crRNA spacer segments (Brouns et al, 2008; Jore et al, 2011; Wiedenheft et al, 2011; Szczelkun et al, 2014). This recognition leads to destruction of protospacer-containing DNA.

Results

Discussion

Conclusion