Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition

Edze R Westra,Blake Wiedenheft,Kirill A Datsenko,Ekaterina Semenova,Stan J J Brouns,Ryan N Jackson,Konstantin Severinov,Patrick H Viollier

doi:10.1371/journal.pgen.1003742

Abstract

Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.

Highlights

There are several prokaryotic defense systems that confer innate immunity against invading mobile genetic elements, such as receptor masking, blocking DNA injection, restriction/ modification (R-M) and abortive infection
CRISPR-Cas of Staphylococcus epidermidis (Type III-A system) is inhibited when invader sequences are flanked by repeat sequences, and this prevents targeting of the CRISPR locus on the host genome
We demonstrate that Escherichia coli CRISPR-Cas (Type I-E system) is not inhibited by repeat sequences

Summary

Introduction

There are several prokaryotic defense systems that confer innate immunity against invading mobile genetic elements, such as receptor masking, blocking DNA injection, restriction/ modification (R-M) and abortive infection (reviewed in [1,2,3]). Half of the bacteria, and most of the archaea, contain CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) defense systems, unique in being the only adaptive line of prokaryotic defense (reviewed in [4,5,6,7]). CRISPR-Cas systems provide adaptive immunity to the host by incorporating invader DNA sequences into chromosomal CRISPR loci [8,9,10,11]. Adjacent to a CRISPR locus, a set of cas genes can often be found that encode the protein machinery essential for CRISPR-immunity. In Type II CRISPR-Cas systems the pre-crRNA is processed by RNase III [21]. The processed crRNA molecules remain bound to one or more Cas proteins to guide recognition and cleavage of complementary nucleic acid sequences [22,23,24,25,26,27]

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Conclusion