Challenges associated with CRISPR-Cas antimicrobials and potential routes to overcome them.

<p>A summary of the obstacles associated with using CRISPR-Cas–based antimicrobials in complex environmental populations of bacteria is shown. These include ensuring effective delivery of constructs (top left), routes of resistance evolution to these novel antimicrobials (top right), the species diversity and spatial complexity of bacterial communities (bottom left), and uncertainty in usage guidelines and stakeholder support (bottom right). AMR, antimicrobial resistance; CRISPR-Cas, clustered regularly interspaced short palindromic repeats-CRISPR–associated.</p

Genes differentially expressed in HB27+P compared to HB27.

<p>*: Fold-change increase in RNA levels in HB27+P compared to HB27. For all changes <i>P</i><0.02.</p><p>**: Function predicted based on domains and similarity to other genes.</p><p>Genes differentially expressed in HB27+P compared to HB27.</p

Effects of <i>Tt</i>Ago on plasmid DNA and plasmid encoded RNA.

<p>A, Schematic representation of the <i>Escherichia coli-T</i>. <i>thermophilus</i> shuttle vector pMKPnqosGFP. Ori/MCS indicates the <i>E</i>. <i>coli</i> origin of replication (Ori) and a multiple cloning site (MCS). Note that cloning of this plasmid resulted in insertion of (incomplete) TTC1921 and TTHV050 genes. B, Relative plasmid content of <i>T</i>. <i>thermophilus</i> strains HB27 and HB27Δ<i>ago</i> transformed with pMKPnqosGFP. Plasmid content was calculated from the complete DNA isolated from biological triplicates at an OD_{600 nm} of 0.5. C, Gene expression of plasmid encoded genes. Expression values are given in Fragments Per Kilobase of exon per Million fragments mapped (FPKM).</p

CRISPR loci and cas genes encoded by <i>T</i>. <i>thermophilus</i> HB27.

<p>A, Schematic representation of CRISPR loci and <i>cas</i> genes encoded on mega-plasmid pTT27 and the <i>T</i>. <i>thermophilus</i> HB27 chromosome. Encoded protein and KEGG annotation are given below each gene. Note that size of illustrated genes do not correspond to their actual size. CRISPR loci with type I and III repeats are colored gray and black, respectively. Repeat types are based on [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124880#pone.0124880.ref028" target="_blank">28</a>] and should not be confused with CRISPR-Cas Types [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124880#pone.0124880.ref029" target="_blank">29</a>]. Transp: Transposase. Hyp: Hypothetical protein. B, Characteristics of CRISPR loci encoded by <i>T</i>. <i>thermophilus</i> HB27. Fold change in CRISPR RNA levels is shown for HB27+P compared to HB27.</p

Effects of Argonaute on Gene Expression in <i>Thermus thermophilus</i>

<div><p>Background</p><p>Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium <i>Thermus thermophilus</i> (<i>Tt</i>Ago) targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, <i>Tt</i>Ago can cleave single and double stranded DNAs. Although <i>Tt</i>Ago additionally has been demonstrated to cleave RNA targets complementary to its DNA guide <i>in vitro</i>, RNA targeting by <i>Tt</i>Ago has not been demonstrated <i>in vivo</i>.</p><p>Methods</p><p>To investigate if <i>Tt</i>Ago also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four <i>T</i>. <i>thermophilus</i> strain HB27 variants: wild type, <i>Tt</i>Ago knockout (Δ<i>ago</i>), and either strain transformed with a plasmid. Additionally we determined the effect of <i>Tt</i>Ago on expression of plasmid-encoded RNA and plasmid DNA levels.</p><p>Results</p><p>In the absence of exogenous DNA (plasmid), <i>Tt</i>Ago presence or absence had no effect on gene expression levels. When plasmid DNA is present, <i>Tt</i>Ago reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that <i>Tt</i>Ago interferes with plasmid DNA, but not with plasmid-encoded RNA. Interestingly, <i>Tt</i>Ago presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of <i>Tt</i>Ago directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that <i>Tt</i>Ago-mediated interference with plasmid DNA stimulates CRISPR adaptation.</p></div

Δ<i>ago</i> result in stochastic changes in gene expression in <i>T</i>. <i>thermophilus</i> strains.

<p>A, Schematic representation of the gene regions encoding <i>Tt</i>Ago (TT_P0026) of <i>T</i>. <i>thermophilus</i> strain HB27 and HB27Δ<i>ago</i>. B, Schematic representation of the gene regions encoding TTB068 in <i>T</i>. <i>thermophilus</i> strain HB8. As no information on how the HB8 <i>ago</i> knockout was generated is available [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124880#pone.0124880.ref021" target="_blank">21</a>], HB8Δ<i>ago</i> is not displayed. HB8 genes colored grey and white are homologous to the HB27 genes indicated in Fig 1A. C, Expression of genes located near <i>ago</i> (TT_P0026) on the genome. Expression values are given in Fragments Per Kilobase of exon per Million fragments mapped (FPKM). D, Overlap in >2-fold up-regulated (▲) and >2-fold down-regulated (▼) homologous genes in HB27Δ<i>ago</i> relative to HB27, and HB8Δ<i>ago</i> relative to HB8.</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

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 −1 position is not required for CRISPR-interference.

<p><b>A</b>) Model of the R-loop formed by Cascade during dsDNA binding. The nucleotide adjacent to the spacer sequence (the −1 position) has the potential to base pair with the first nucleotide of the PAM in the target strand of the DNA. <b>B</b>) Cells expressing WT g8-Cascade and Cas3 are resistant to M13 phage containing the CAT, CTT, CCT or CTC PAMs adjacent to the M13 protospacer (white font/black bars, e.o.p.<10⁻⁴), but not when containing the CGT PAM (red font/red bars, e.o.p. = 1). 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-Cascade for dsDNA containing the g8 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. <b>C</b>) Assays as in (B) using cells expressing the g8^G-1T CRISPR, Cascade and Cas3, show that cells are resistant to M13 phage containing the CAT, CTT, CCT or CTC PAMs adjacent to the g8 protospacer (white font/black bars), but not when containing the CGT PAM (red font/red bars). The <i>in vitro</i> binding affinity of purified WT g8^G-1T-Cascade for dsDNA containing the g8 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. In (B) and (C) error bars represent the standard deviation of the mean.</p

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