Genome Maintenance Proteins Modulate Autoimmunity Mediated Primed Adaptation by the Escherichia coli Type I-E CRISPR-Cas System.

Elena Kurilovich,Ekaterina Savitskaya,Natalia Morozova,Ivana Ivancic-Bace,Konstantin Severinov,Anastasia Metlitskaya,Anna Shiriaeva

doi:10.3390/genes10110872

Abstract

Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation, CRISPR-Cas systems acquire immunity by incorporating short fragments of invaders’ genomes into CRISPR arrays. The acquisition of fragments of host genomes leads to autoimmunity and may drive chromosomal rearrangements, negative cell selection, and influence bacterial evolution. In this study, we investigated the role of proteins involved in genome stability maintenance in spacer acquisition by the Escherichia coli type I-E CRISPR-Cas system targeting its own genome. We show here, that the deletion of recJ decreases adaptation efficiency and affects accuracy of spacers incorporation into CRISPR array. Primed adaptation efficiency is also dramatically inhibited in double mutants lacking recB and sbcD but not in single mutants suggesting independent involvement and redundancy of RecBCD and SbcCD pathways in spacer acquisition. While the presence of at least one of two complexes is crucial for efficient primed adaptation, RecBCD and SbcCD affect the pattern of acquired spacers. Overall, our data suggest distinct roles of the RecBCD and SbcCD complexes and of RecJ in spacer precursor selection and insertion into CRISPR array and highlight the functional interplay between CRISPR-Cas systems and host genome maintenance mechanisms.

Highlights

Arrays of clustered regularly interspaced short palindromic repeats (CRISPR) separated by variable spacers and cas (CRISPR-associated) genes constitute CRISPR-Cas adaptive immunity systems used by a wide range of bacteria and archaea to defend against invading DNA or RNA [1,2,3]
E. coli has been thoroughly described. It is based on a strain KD403, which contains a CRISPR array spacer targeting a non-essential yihN gene in the E. coli genome, and cas genes responsible for CRISPR interference and adaptation under control of inducible promoters (Figure 1A)
In naïve CRISPR adaptation, the efficiency of prespacer selection varies for different genomic regions and is stimulated by the breaks in genomic DNA [53]

Summary

Introduction

Arrays of clustered regularly interspaced short palindromic repeats (CRISPR) separated by variable spacers and cas (CRISPR-associated) genes constitute CRISPR-Cas adaptive immunity systems used by a wide range of bacteria and archaea to defend against invading DNA or RNA [1,2,3]. When targeting the host genome, CRISPR-Cas systems induce chromosomal rearrangements and/or cell death and in this way may drive bacterial evolution [4,5,6]. The Cas effector complexes consist of Cas protein(s) and crRNA, which is a product of CRISPR array transcription and processing. The effector complex recognizes and channels for degradation nucleic acids based on complementarity of a crRNA spacer and a region of a target called “protospacer” [9]. Fragments of DNA inside the cell, including those of bacteriophage genomes and plasmids as well as host DNA, may serve as “prespacers” and be channeled for integration into

Methods

Results

Conclusion