Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands

<div><p>In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In <i>Pectobacterium atrosepticum</i> the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the <i>cas</i> operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.</p></div

Chromosomal targeting results in cell elongation indicative of DNA damage.

<p>Plasmids containing no spacers (control; pC1-16) or one spacer targeting <i>expI</i> (anti-<i>expI</i>; pE1-16) were repressed or induced in the WT and then visualised by (A) LIVE/DEAD staining and fluorescence microscopy (white scale bar; 20 µm; all images to scale) or (B) transmission electron microscopy (TEM) (black scale bars; 2 µm) (C) Quantification of cell lengths of 60 cells from each treatment as assessed by TEM. ns, not significant; *, p-value of <0.0001 when assessed by unpaired two-tailed t-test (PRISM).</p

A single nucleotide PAM mutation enables escape from native CRISPR/Cas targeting.

<p>(A) <i>P. atrosepticum</i> CRISPR/Cas genomic organisation. The <i>cas1</i> and the <i>cas2</i>-<i>cas3</i> hybrid genes are shown in blue, <i>csy1</i>-<i>3</i> in pale blue, <i>cas6f</i> in orange and the CRISPRs as grey arrows in the direction of transcription. CRISPR2 and 3 are separated by a toxin/antitoxin system (white arrows). (B) Spacer 6 of CRISPR2 (from leader) contains deviations from the repeat consensus (shown in blue) and (C) has a 100% match to a protospacer (red) within <i>eca0560</i> in HAI2 in the <i>P. atrosepticum</i> genome. The protospacer matching spacer 6 contains a non-consensus 5′-protospacer-TG-3′ PAM (green). (D) Toxicity assays in the WT with plasmids containing consensus repeats and either no spacer (pC1-16), a single native spacer 6 (5′-protospacer-TG-3′ PAM; pTraGS6-16) or an engineered spacer (5′-protospacer-GG-3′ PAM; pTraG1-16) that targets <i>eca0560</i>. The protospacer locations in <i>eca0560</i> of the native (pTraGS6-16) and engineered (pTraG1-16) spacers are shown below in red. (E) Sequence and pairing of the engineered anti-<i>eca0560</i> spacer (in pTraG1-16) with the consensus 5′-protospacer-GG-3′ PAM (green).</p

An engineered CRISPR plasmid with spacers targeting the chromosome displays Cas–dependent toxicity.

<p>(A) Strategy for chromosomal targeting (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003454#s4" target="_blank">Materials and Methods</a>). Protospacers were PCR-amplified from target DNA with full or partial repeats and BbsI sites on the primers. PCR products were digested with BbsI and cloned into BbsI-digested plasmids containing a leader sequence and one repeat. This process was repeated to generate multiple spacer inserts. These arrays generate crRNAs that target the template DNA strand but not the mRNA. (B) Transformation plates of <i>P. atrosepticum</i> WT or a Δ<i>cas</i> mutant (PCF80) after growth for 36 h on LBA containing Ap. Plasmids transformed contained no spacers (pC1-780), 3 scrambled spacers (pS3-780) and 3 anti-<i>expI</i> spacers (pE3-780). Representatives are shown from experiments performed at least in triplicate.</p

CRISPR/Cas–mediated chromosomal targeting causes rapid genome evolution.

<p>(A) Schematic of HAI2 inserted in the <i>P. atrosepticum</i> genome. The <i>attL</i> and <i>attR</i> sites are indicated by black and white boxes respectively, <i>eca0560</i> and <i>eca0573</i> are shown as white arrows and the protospacer in <i>eca0560</i> is indicated in grey and the Km^R marker in <i>eca0573</i> is depicted in black. Excision of HAI2 results in the circularised form, pHAI2, which contains the <i>attP</i> site and results in the generation of the <i>attB</i> site located within the phe-tRNA gene in the genome. In the absence of the circularised pHAI2 form, the strain is designated <b>Δ</b>HAI2. Primers used for strain confirmation in (B) are shown as black arrows and the <i>cas1</i> gene was used as a positive control. (B) PCR results for representative class I and class II mutants and the WT. PCR was performed for <i>cas1</i>, <i>eca0560</i>, <i>attR</i>, <i>attL</i>, <i>attP</i> and <i>attB</i> and with primers shown in part (A). Schematic representations of (C) the class I mutants (designated <b>Δ</b>HAI2) that have precisely lost the 97,875 bp island and (D) the class II mutants 2, 5 and 14 containing a 40,227 bp deletion between TTGGCAC sequences in both <i>eca0522</i> and internal to the Km^R insertion in <i>eca0573</i>. (E) Scale genetic map of the 7 classified HAI2 class II mutants defining the deleted regions. Black bars indicate the presence of the gene as specified. The gray vertical line represents the CRISPR-targeted <i>eca0560</i> gene. The star represents three accurately sequenced junctions. The blue arrow depicts the site of Km insertion within the chromosome. Class II mutants are numbered as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003454#pgen.1003454.s004" target="_blank">Figure S4</a> and their PCR profiles are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003454#pgen.1003454.s005" target="_blank">Figure S5</a>.</p

Protospacer, PAM, and repeat mutants can escape toxicity.

<p>(A) Predicted pairing between an <i>expI</i> crRNA and the <i>expI</i> protospacer (PAM is green, spacer is blue, protospacer is red and seed sequence is bold and underlined). (B) Left; protospacer sequences in WT, Δ<i>expI</i> (PCF81), Δ<i>expI</i> with a single WT (RBV01), C3T (RBV04), C6T (RBV03) or G-1T (PAM) (RBV02) <i>expI</i> protospacer. Right; toxicity assays of a single anti-<i>expI</i> spacer (<i>expI</i>; pE1-16; white bars) or a plasmid with no spacers (none; pC1-16; black bar) when expressed in the backgrounds shown on the left. (C) Predicted folds and position of mutations for WT, G20A, C18A and C18A/G8U single CRISPR repeats. The black triangle represents the site of cleavage by Cas6f. (D) Toxicity assays of plasmids expressing a single anti-<i>expI</i> spacer flanked by either WT (pE1-16), G20A (pE1-16 G20A), C18A (pE1-16 C18A) and C18A/G8U repeats (pE1-16 C18A/G8U) in WT <i>P. atrosepticum</i>.</p