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

Abstract

Clustered 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.

FIG 1

The CRISPR-Cas system of S. epidermidis and its targets. (A) Organization of the type III-A CRISPR system in S. epidermidis RP62A. This system contains 9 CRISPR-associated (cas and csm) genes, 4 direct repeats (blue boxes), and 3 spacers (yellow, green, and purple boxes). The first spacer targets the nickase gene present in staphylococcal conjugative plasmids; the second matches the cn20 gene of the CNPH82 S. epidermidis phage. “L” indicates the leader sequence, which contains the promoter elements for expression of the repeat-spacer region. DB5 is a derivative of S. epidermidis RP62A that lacks the repeat-spacer array but that harbors the leader and CRISPR-associated genes. To restore CRISPR-Cas immunity in this strain, the full repeat-spacer array of S. epidermidis RP62a was cloned into the staphylococcal plasmid pC194, generating the complementing plasmid pCRISPR. WT, wild type. (B) The cn20 target contains a single mismatch in position 24, which was eliminated by introducing a compensatory mutation in the spc2 sequence of pCRISPR.

FIG 2

Tolerance of crRNA:target mismatches during antiplasmid and antiphage type III-A CRISPR-Cas immunity. (A) Mismatch mutations in spc2 (mutations in red) within different versions of the pCRISPR complementing plasmid are shown. Each of these plasmids was introduced into S. epidermidis DB5 to generate different hosts for CNPX infection. Phage propagation in each host is measured as the number of PFU per milliliter obtained after plaquing serial dilutions of the phage stock into lawns of host bacteria. Three independent infection experiments were performed (means ± standard deviations [SD] are reported). The limit of detection of this assay is 100 PFU/ml. (B) Mismatch mutations in spc1 (mutations in red) within different versions of the pCRISPR complementing plasmid are shown. Each of these plasmids was introduced into S. epidermidis DB5 to generate different recipient strains for conjugation assay testing of the transfer of pG0400. Conjugation is measured as the number of CFU per milliliter of recipients and transconjugants obtained after three independent filter matings of donor and recipient strains (means ± SD are reported). The limit of detection of this assay is 100 CFU/ml.

FIG 3

Mismatches in positions 2 to 4 affect CRISPR immunity against conjugation and but not against phage infection. (A) Fitness of different transconjugants was measured by pairwise competition experiments. The change in the relative frequency of plasmid-bearing cells (y axis) is plotted against the number of transfers (one transfer per day; x axis). The top left panel shows the predicted changes in frequency for different selection coefficients, s. These are calculated from the equation dq/dt = −q(1 − q)s, where q is the relative frequency of the plasmid bearing cells and s is the selection coefficient (an s value of >0 indicates that the plasmid-bearing cells are at a disadvantage and an s value of <0 that the plasmid-bearing cells have an advantage). We are assuming 1/100 dilutions or t = 6.64 generations in each transfer. In all panels, the growth of transconjugants carrying pG0400 and pΔCRISPR, pCRISPR^spc1(2–4), or pCRISPR^{spc1(12–19)} was compared to growth of wild-type S. epidermidis RP62a. The black line and circles indicate the average change in relative frequency (the values for each of three independent experiments are shown as colored symbols). (B) Analysis of phage propagation measured as the efficiency of infection (ECOI). The ECOI value obtained for the propagation in sensitive cells (carrying pΔCRISPR) was set as 100% and was used to determine the value for the immune cells carrying versions of pCRISPR harboring different mutations in spc2. Experiments were performed in triplicate, and means ± SD are reported.

FIG 4

Design of a plasmid that can be transferred to cells via conjugation or CNPX-mediated transduction. (A) Schematic of transduction and conjugation of pC221-derived plasmids into a host carrying a targeting CRISPR-Cas system. (B) Mobilizable, tetracycline-resistant pC221-pT181 hybrid plasmid used in this study. It was created by fusion of the mobilization genes (orfA and orfB) and origin of transfer (ori^T) of pC221 with the tetracycline resistance (Tet^r) gene, replication gene (repC), and recombination gene (pre) of pT181, as well as its origin of replication gene (ori). The site of target insertion is shown. (C) Schematic of the pTgt plasmid series showing the insertion of the nes or cn20 targets in the leading or lagging strand, according to replication from the ori site of pT181.

FIG 5

Spacer-target mismatches eliminate immunity against the nes but not the cn20 target regardless of the mode of entry when the target is in the lagging strand. (A) Conjugation of pTgt1^Lag and pTgt2^Lag into recipient strains carrying mismatches in positions 2 to 4 within spc1 or spc2. CFU counts per milliliter of recipients and transconjugants were determined in three independent experiments, and means ± SD are reported. The limit of detection of this assay is 100 CFU/ml. (B) CNPX-mediated transduction of pTgt1^Lag or pTgt2^Lag into cells carrying mismatches in positions 2 to 4 within spc1 or spc2. CFU counts per milliliter of transductants were determined in three independent experiments, and means ± SD are reported. The limit of detection of this assay is 100 CFU/ml.

FIG 6

Spacer-target mismatches eliminate immunity against both the nes target and the cn20 target regardless of the mode of entry when the target is in the leading strand. (A) Conjugation of pTgt1^Lead and pTgt2^Lead into recipient strains carrying mismatches in positions 2 to 4 within spc1 or spc2. CFU counts per milliliter of recipients and transconjugants were determined in three independent experiments, and means ± SD are reported. The limit of detection of this assay is 100 CFU/ml. (B) CNPX-mediated transduction of pTgt1^Lead or pTgt2^Lead into cells carrying mismatches in positions 2 to 4 within spc1 or spc2. CFU counts per milliliter of transductants were determined in three independent experiments, and means ± SD are reported. The limit of detection of this assay is 100 CFU/ml. (C) RT-PCR was performed on RNA samples extracted from S. aureus RN4220 cells harboring pTgt1^Lead or pTgt1^Lag plasmids using primers that detect transcription of either the leading strand or lagging strand. PCRs of samples where reverse transcriptase was omitted are shown as controls.