Linear display of pRSF-1b and locations of protospacers.

<p>The (+) and (−) strands and corresponding protospacers are coloured red and black, respectively. Kanamycin marker (Kan), Origin of replication (Ori) and <i>lacI</i> (LacI) are shown as arrows. Protospacers have an AAG PAM unless indicated otherwise.</p

PAM and repeat-end correlation.

<p>(A): PAMs of observed spacers and the co-occurring trinucleotide repeat-ends associated with these spacers. Notice that the spacer-proximal nucleotide of the repeat end is identical to the protospacer-proximal nucleotide of the PAM. (B): Schematic of the proposed mechanism for spacer acquisition during CRISPR adaptation. A protospacer with specific PAM is selected after which it is processed into the pre-spacer (at least 33–34 bp), which contains the last nucleotide of the PAM (the pre-spacer could be single-stranded or double-stranded). The pre-spacer is than integrated at the leader proximal end of the CRISPR locus. The nucleotide derived from the PAM forms the last nucleotide of the repeat. (C): R-loop formation by mature crRNA (61 nucleotides) during CRISPR interference. Notice that the last nucleotide of the repeat (the nucleotide derived from the PAM) is complementary to the target DNA sequence. It remains unknown whether base-pairing between these nucleotides is important for interference.</p

Model of the strand specific positive feedback loop.

<p>Cells with a spacer against a known and actively present invader DNA produce targeting Cascade complexes in the expression stage. In the interference stage, Cascade binds the target dsDNA after which the target is cleaved and degraded by Cas3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888-Westra1" target="_blank">[15]</a>. DNA degradation products generated by Cascade and Cas3 (which could be ssDNA or dsDNA) act as precursors for new spacers in the adaptation phase in a strand-specific manner. By integration of these strand-specific precursors, the spacer repertoire against an actively present invader is expanded, completing the positive feedback loop.</p

Effect of integrated spacers on retransformation efficiency.

<p>Transformation efficiencies of various PIMs and the control (Wild type <i>E. coli</i> K12 W3110) are given in a logarithmic scale as colony forming units (CFU) per µg of pRSF-1b plasmid DNA. For each PIM, the number of spacers integrated in either CRISPR locus 2.1 or 2.3 is given. All spacers have an AAG PAM, unless indicated otherwise. The exact spacer composition of each PIM is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888.s001" target="_blank">Table S1</a>.</p

Graphical representation of AG and GC contents of each observed and possible spacer.

<p>Observed spacers (⧫) are spacers integrated in CRISPR loci 2.1 and 2.3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888.s001" target="_blank">Table S1</a>). These spacers are 32 or 33-mers with various PAMs. Possible spacers (Ο) are all 32-mers found on pRSF-1b directly downstream of an AAG PAM.</p

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

<div><p>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 <i>Staphylococcus epidermidis</i> 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 <i>Escherichia coli</i> 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.</p></div

Synonymous mutations of the crRNA and the PAM do not affect self versus non-self discrimination.

<p><b>A</b>) Infectivity of a library of M13 phage containing PAM mutants adjacent to the g8 protospacer was tested against cells expressing Cascade, Cas3 and g8 CRISPR containing mutations at the −1, −2 and −3 nucleotides of the CRISPR repeats. PAM mutations are shown on the left, with the PAM sequences indicated in the 3′ to 5′ direction. CRISPR repeat mutations at positions −1, −2 and −3 are indicated on the top in the 5′ to 3′ direction. Underscored sequences have been tested for binding affinity by EMSA. Base pairing potential between the PAM positions and the repeat is indicated using numbers (0–7) that correspond to a base pairing pattern that is shown in the panel on the right. A 0 signifies no base pairing, a 1 signifies base pairing at the −3 position, a 2 signifies base pairing at the −2 position, etc. Black circles with white digits indicate resistance against phage infection (e.o.p.<10⁻⁴), grey circles indicate partial resistance (e.o.p.∼10⁻²) and red digits without circle indicate susceptibility to phage infection (e.o.p. = 1), as determined by phage spot assays. Letters B, C, D, E indicate combinations shown in detail in the corresponding panels. <b>B</b>) Combination of g8^C-3AC-2T CRISPR and M13 phage with CAT PAM, gives rise to full base pairing and a lack of resistance (red font). <b>C</b>) Combination of g8^C-3AC-2T CRISPR and M13 phage with CTC PAM, gives rise to only base pairing at the −1 position and yields a lack of resistance (red font). <b>D</b>) Combination of g8^C-3AC-2A CRISPR and M13 phage with CTT PAM gives rise to full base pairing and a lack of resistance (red font). <b>E</b>) Combination of g8^C-3AC-2A CRISPR and M13 phage with CTC PAM gives rise to only two potential base pairs at the −1 and −2 positions and yields a lack of resistance (red font). Note in (A–E) PAMs are oriented in 3′ to 5′direction to display base pairing potential with the last three nucleotides of the crRNA repeat.</p

Potential base pairing between the crRNA repeat regions and protospacer flanking regions does not affect CRISPR-interference.

<p><b>A</b>) Model of the R-loop formed by Cascade during dsDNA binding. <b>B</b>) Cells expressing WT g8-Cascade and Cas3 are resistant to plasmids containing the CAT PAM adjacent to the g8 protospacer (black bars, transformation efficiency 6.7±1.5×10⁵ cfu/µg DNA for plasmid pWUR690 and 6.8±0.9×10⁵ cfu/µg DNA for plasmid pWUR688), but are susceptible to plasmid transformation when the g8 protospacer is flanked by a CGG PAM, which is fully complementary to the 5′-handle (red bars, transformation efficiency 4.2±0.9×10⁸ cfu/µg DNA for plasmid pWUR687 and 4.5±0.8×10⁸ cfu/µg DNA for plasmid pWUR689). Transformation efficiency for a control pUC19 plasmid is 6.2±1.1×10⁸ cfu/µg DNA. The histogram shows the <i>in vitro</i> binding affinity of purified WT g8-Cascade for dsDNA containing the g8 protospacer flanked by sequences with a varying base pairing potential, as shown on the right. Asterisks indicate that the Kd value is >>1000 nM and the error bars represent the standard deviation of the mean.</p

Base pairing at the −2 and −3 positions does not interfere with CRISPR-immunity.

<p><b>A</b>) Cells expressing g8^C-2A-Cascade and Cas3 are partially resistant to M13 phage containing the CTT PAM adjacent to the g8 protospacer (white font/grey bar, e.o.p.∼10⁻²), but not when containing the CAT, CCT, CTC or CGT PAM (red font/red bars). Note that in the figure the PAMs are oriented in 3′ to 5′direction to display base pairing potential with the last three nucleotides of the crRNA repeat. The <i>in vitro</i> binding affinity of purified WT g8^C-2A-Cascade for dsDNA containing the g8 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. <b>B</b>) Assays as in (A) using cells expressing the g8^C-3G CRISPR, Cascade and Cas3, show that cells are not resistant to M13 phage containing the CAT, CTT, CCT, CTC or CGT PAMs adjacent to the g8 protospacer (red font/red bars). The <i>in vitro</i> binding affinity of purified WT g8^C-3G-Cascade for dsDNA containing the M13 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. Asterisks indicate that the Kd value is >>1000 nM and the error bars represent the standard deviation of the mean.</p

Model of self versus non-self discrimination by Type III-A systems and target versus non-target discrimination by Type I-E systems.

<p><b>A</b>) Model of the mechanism employed by Type III-A systems. Type III-A systems presumably target DNA through R-loop formation. The crRNA (here depicted as the 37 nt species) could be part of a ribonucleoprotein complex consisting of Cas proteins and a single crRNA (Csm complex). R-loop formation and subsequent interference is inhibited when base pairing occurs between the −2, −3 and −4 positions of the repeat sequence at the 5′ end of the crRNA and the sequence flanking the 3′ end of the protospacer. How this base pairing is monitored is currently unknown, but it might involve a Cas protein. <b>B</b>) Model of target versus non-target discrimination by Type I-E systems. Type I-E systems target DNA through R-loop formation. The 61 nt crRNA is part of the Cascade ribonucleoprotein complex. R-loop formation and subsequent Cas3-mediated cleavage of the target DNA are activated when a PAM is present at the −1, −2 and −3 positions, in the sequence flanking the 3′ end of the protospacer. The presence of a PAM is monitored through the Cse1 subunit.</p