CRISPRs: molecular signatures used for pathogen subtyping

Abstract

Rapid and accurate strain identification is paramount in the battle against microbial outbreaks, and several subtyping approaches have been developed. One such method uses clustered regular interspaced short palindromic repeats (CRISPRs), DNA repeat elements that are present in approximately half of all bacteria. Though their signature function is as an adaptive immune system against invading DNA such as bacteriophages and plasmids, CRISPRs also provide an excellent framework for pathogen tracking and evolutionary studies. Analysis of the spacer DNA sequences that reside between the repeats has been tremendously useful for bacterial subtyping during molecular epidemiological investigations. Subtyping, or strain identification, using CRISPRs has been employed in diverse Gram-positive and Gram-negative bacteria, including Mycobacterium tuberculosis, Salmonella enterica, and the plant pathogen Erwinia amylovora. This review discusses the several ways in which CRISPR sequences are exploited for subtyping. This includes the well-established spoligotyping methodologies that have been used for 2 decades to type Mycobacterium species, as well as in-depth consideration of newer, higher-throughput CRISPR-based protocols.

FIG 1

CRISPR-cas system. There are two CRISPR loci in Salmonella enterica and seven cas genes (light gray arrows). All CRISPR-Cas systems contain cas1 and cas2 (medium gray boxes). S. enterica has a type I CRISPR-Cas system of which cas3 is the signature gene (dark gray box). An AT-rich leader sequence is immediately upstream of each spacer array (white boxes; L). The direct repeats (DRs) are shown as black diamonds, and the terminal DR, which differs from the consensus DR, is shown as a white diamond. Spacers are shown as colored rectangles, and unique spacers are represented by unique colors. Below the spacer array, the sequence of two spacers and the respective three flanking DRs is shown with the DRs in black uppercase characters and the spacers color coded and in lowercase characters.

FIG 2

Spoligotyping. (A) Labeled primers (arrows; labeled primer indicated by asterisks [*]) complementary to the DRs are used to amplify the CRISPR spacer array and result in several PCR products of various lengths. (B) The mixture of labeled PCR products is hybridized to an array of probes, each of which corresponds to a unique spacer. The schematic shows unhybridized (top) and hybridized (bottom) membranes, with probes color coordinated for clarity. Positively hybridized probes are depicted with black borders. In this example, the hybridization pattern correlates to Strain a in panel C. (C) The spoligotype patterns show black boxes for a positive signal (spacer present) and white boxes for a negative signal (spacer absent). Different bacterial strains (Strain a, Strain b, and Strain c) can be identified on the basis of different hybridization profiles.

FIG 3

CRISPR-based typing. (A) Entire CRISPR spacer arrays are PCR amplified (the primers are indicated with red and blue arrows) and sequenced. Spacer sequence information is extracted, and unique spacers are represented as differently colored rectangles. A unique composition of spacers defines an allele, shown to the left of the array. In the example shown, where there are two CRISPR arrays, a CRISPR type is defined by a combination of two unique alleles as shown in the table. Polymorphisms between strains occur from loss of some spacers and/or duplication of others; for example, the light green spacer in CRISPR1 is duplicated in allele 5. (B) When a unique spacer (orange spacer; indicated by an arrow) is present in a pathogenic or predominant strain, these isolates can be easily identified using real-time PCR with fluorescent probes specific for that particular spacer, as shown for strain A. Since strain B does not contain that spacer, it would not be detected using real-time PCR. (C) Rapid CRISPR size typing screen based on comparative amplicon size analysis. Bacterial strains that differ in numbers of spacers can be easily visualized by analyzing the size difference after gel electrophoresis of the amplified PCR product.