Impact of Different Target Sequences on Type III CRISPR-Cas Immunity

International audienceClustered regularly interspaced short palindromic repeat (CRISPR) loci encode an adaptive immune system of prokaryotes. Within these loci, sequences intercalated between repeats known as "spacers" specify the targets of CRISPR immunity. The majority of spacers match sequences present in phages and plasmids; however, it is not known whether there are differences in the immunity provided against these diverse invaders. We studied this issue using the Staphylococcus epidermidis CRISPR system, which harbors spacers matching both phages and plasmids. We determined that this CRISPR system provides similar levels of defense against the conjugative plasmid pG0400 and the bacteriophage CNPX. However, whereas antiplasmid immunity was very sensitive to the introduction of mismatches in the target sequence, mutations in the phage target were largely tolerated. Placing the phage and plasmid targets into a vector that can be both conjugated and transduced, we demonstrated that the route of entry of the target has no impact on the effect of the mismatches on immunity. Instead, we established that the specific sequences of each spacer/target determine the susceptibility of the S. epidermidis CRISPR system to mutations. Therefore, spacers that are more resistant to mismatches would provide long-term immunity against phages and plasmids that otherwise would escape CRISPR targeting through the accumulation of mutations in the target sequence. These results uncover an unexpected complexity in the arms race between CRISPR-Cas systems and prokaryotic infectious genetic elements. IMPORTANCE:CRISPR-Cas loci protect bacteria and archaea from both phage infection and plasmid invasion. These loci harbor short sequences of phage and plasmid origin known as "spacers" that specify the targets of CRISPR-Cas immunity. The presence of a spacer sequence matching a phage or plasmid ensures host immunity against infection by these genetic elements. In turn, phages and plasmids constantly mutate their targets to avoid recognition by the spacers of the CRISPR-Cas immune system. In this study, we demonstrated that different spacer sequences vary in their ability to tolerate target mutations that allow phages and plasmids to escape from CRISPR-Cas immunity. These results uncover an unexpected complexity in the arms race between CRISPR-Cas systems and prokaryotic infectious genetic elements

Dealing with the Evolutionary Downside of CRISPR Immunity: Bacteria and Beneficial Plasmids

<div><p>The immune systems that protect organisms from infectious agents invariably have a cost for the host. In bacteria and archaea CRISPR-Cas loci can serve as adaptive immune systems that protect these microbes from infectiously transmitted DNAs. When those DNAs are borne by lytic viruses (phages), this protection can provide a considerable advantage. CRISPR-Cas immunity can also prevent cells from acquiring plasmids and free DNA bearing genes that increase their fitness. Here, we use a combination of experiments and mathematical-computer simulation models to explore this downside of CRISPR-Cas immunity and its implications for the maintenance of CRISPR-Cas loci in microbial populations. We analyzed the conjugational transfer of the staphylococcal plasmid pG0400 into <i>Staphylococcus epidermidis</i> RP62a recipients that bear a CRISPR-Cas locus targeting this plasmid. Contrary to what is anticipated for lytic phages, which evade CRISPR by mutations in the target region, the evasion of CRISPR immunity by plasmids occurs at the level of the host through loss of functional CRISPR-Cas immunity. The results of our experiments and models indicate that more than 10⁻⁴ of the cells in CRISPR-Cas positive populations are defective or deleted for the CRISPR-Cas region and thereby able to receive and carry the plasmid. Most intriguingly, the loss of CRISPR function even by large deletions can have little or no fitness cost in vitro. These theoretical and experimental results can account for the considerable variation in the existence, number and function of CRISPR-Cas loci within and between bacterial species. We postulate that as a consequence of the opposing positive and negative selection for immunity, CRISPR-Cas systems are in a continuous state of flux. They are lost when they bear immunity to laterally transferred beneficial genes, re-acquired by horizontal gene transfer, and ascend in environments where phage are a major source of mortality.</p></div

Different possibilities for the transfer of a beneficial plasmid into cells encoding CRISPR immunity against it.

<p><i>S. epidermidis</i> RP62a contains a CRISPR-Cas system with a spacer (pink) that produces crRNAs that match and target the <i>nickase</i> (<i>nes</i>) gene (also in pink) of staphylococcal conjugative plasmids, including pG0400. There are at least four different mechanisms that will allow the transfer of the plasmid in spite of CRISPR immunity: (i) mutation of the plasmid target (yellow), (ii) mutation or deletion of the anti-plasmid spacer, (iii) loss-of-function mutation of the <i>cas</i> genes required for immunity or partial or complete deletion of the CRISPR-Cas locus, or (iv) partial immunity that leads to tolerance of the plasmid.</p

Fluctuation experiment (cfu/ml).

(a)<p>All experiments were performed using an aliquot of the same <i>S. aureus</i> RN4220/pG0400 donor culture.</p>(b)<p>The average of two cfu counts of recipients (<i>S. epidermidis</i> RP62a) obtained after conjugation is reported. Donors were not enumerated.</p>(c)<p>Jackpot</p>(d)<p>Mean, median, variance and variance/mean values were re-calculated omitting the jackpot cfu (907 cfu/ml).</p

Simulation of plasmid competition with CRISPR-mediated immunity.

<p>Changes in the densities of different populations over time are plotted. Standard parameters (defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003844#pgen.1003844.s008" target="_blank">Supplementary Text 2</a>) are: ν = 1.4, <i>e</i> = 5×10⁻⁷, <i>k</i> = 1, γ = 10⁻¹⁴, initial values, R = 2500, CP = 200, D1 = 100, CN = D1 = D2 = T1 = T2 = 0. (A) Same rate of CRISPR loss (µ) and plasmid escape mutations (ν), µ = ν = 10⁻⁷. (B) High rate of plasmid escape mutants, µ = 10⁻⁷, ν = 3×10⁻⁴. (C) High rate of CRISPR loss or deletion mutations, µ = 3×10⁻⁴, ν = 10⁻⁷.</p

Genotype of transconjugants that escape CRISPR immunity.

(a)<p>Transconjugants obtained in two independent experiments (R and B) are shown. Transconjugants R21 and R26 are not reported as genotypic analysis indicated these were <i>S. aureus</i> RN4220/pG0400 donors that acquired neomycin resistance. Highlighted and underlined are transconjugants that were chosen for the pairwise competition experiments estimating fitness.</p>(b)<p>The number indicates the adenine residue deleted relative to the start codon of the gene.</p>(c)<p>The number indicates the position of insertion of the IS<i>256</i> element relative to the start codon of the gene. Direct, <i>d</i>, or inverted, <i>i</i>, refer to the orientation of the transposase gene of the IS<i>256</i> element with respect to the direction of transcription of the <i>cas</i>/<i>csm</i> operon.</p>(d)<p>The numbers indicate the chromosomal coordinates of the <i>S. epidermidis</i> RP62a genome that are deleted. The elements that are believed to have mediated the deletion are shown in parenthesis.</p>(e)<p>Deletion of the adenine in the first position of the <i>spc1</i> sequence.</p>(f)<p>The nucleotide mutation followed by the amino acid mutation are indicated, the numbers indicated nucleotide or amino acid position of the gene or encoded protein, relative to the start codon or initial methionine residue, respectively.</p>(g)<p>Highlighted and underlined are transconjugants that were chosen for the pairwise competition experiments estimating fitness.</p

CRISPR escapers accumulate deletions of the CRISPR/Cas region.

<p>Schematic representation of the deletions on the <i>S. epidermidis</i> RP62a genome. Wild-type sequences are shown in green, deletions mediated by IS<i>431</i> in pink, by IS<i>256</i> in yellow, by Tn<i>554</i> in orange, by recombination between SERP2353 and SERP2491 (96% identical at the nt level) or SERP2409 and SERP2493 (98% identical at the nt level) in light blue or brown, respectively, and by the excision of the SCC<i>mec</i> cassette in violet. Numbers represent genomic coordinates in kb.</p

Different mutations eliminate CRISPR immunity against conjugation in <i>S. epidermidis</i>.

<p>(<b>A</b>) Summary of the different mutations found in this study and their proportions. (<b>B</b>) Distribution of mutations within the CRISPR-Cas locus. <i>S. epidermidis</i> RP62a harbors a CRISPR-Cas system containing four repeats (white boxes), three spacers (colored, numbered boxes) and nine <i>cas</i>/<i>csm</i> genes. Mutations found in CRISPR escapers include deletions in the repeat-spacer region (brackets), transposon insertions (red arrowheads; top, direct insertion; bottom, inverted) and single nucleotide deletions or substitutions (asterisks). Arrows indicate primers used to analyze transconjugants. (<b>C</b>) PCR analysis of the CRISPR array of transconjugants using primers L50/L6. Deletion of 1, 2 and 3 spacers observed in escapers R23, R10 and R2, respectively, is shown. M, DNA marker. <i>wt</i>, amplification using wild-type template DNA. (<b>D</b>) PCR analysis of the <i>cas</i> gene region of escapers using primers L23/L106. IS<i>256</i> transposon insertions into <i>csm5</i>, <i>csm6</i> and <i>cas6</i> observed in escapers R60, B15 and R36, respectively, are shown. M, DNA marker. <i>wt</i>, amplification using wild-type template DNA.</p