Clustered regularly interspaced short palindromic repeats (CRISPRs) constitute a particular family of tandem repeats found in a wide range of prokaryotic genomes (half of eubacteria and almost all archaea). They consist of a succession of highly conserved regions (DR) varying in size from 23 to 47 bp, separated by similarly sized unique sequences (spacer) of usually viral origin. A CRISPR cluster is flanked on one side by an AT-rich sequence called the leader and assumed to be a transcriptional promoter. Recent studies suggest that this structure represents a putative RNA-interference-based immune system. Here we describe CRISPRFinder, a web service offering tools to (i) detect CRISPRs including the shortest ones (one or two motifs); (ii) define DRs and extract spacers; (iii) get the flanking sequences to determine the leader; (iv) blast spacers against Genbank database and (v) check if the DR is found elsewhere in prokaryotic sequenced genomes. CRISPRFinder is freely accessible at http://crispr.u-psud.fr/Server/CRISPRfinder.php.

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CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats.

Using in silico analysis we studied a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses. This family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non-repetitive sequences. To appreciate their characteri-stic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR). In most species with two or more CRISPR loci, these loci were flanked on one side by a common leader sequence of 300-500 b. The direct repeats and the leader sequences were conserved within a species, but dissimilar between species. The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR-associated (cas) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes. The cas genes were invariably located adjacent to a CRISPR locus, indicating that the cas genes and CRISPR loci have a functional relationship. The cas3 gene showed motifs characteristic for helicases of the superfamily 2, and the cas4 gene showed motifs of the RecB family of exonucleases, suggesting that these genes are involved in DNA metabolism or gene expression. The spatial coherence of CRISPR and cas genes may stimulate new research on the genesis and biological role of these repeats and genes.

https://onlinelibrary.wiley.com/doi/epdf/10.1046/j.1365-2958.2002.02839.x

Identification of genes that are associated with DNA repeats in prokaryotes.

Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.

Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.

Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%—and by 23% in combination with spoligotyping—among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold—by threefold in combination with spoligotyping—under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.

Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis

The remarkable repetitive elements called CRISPRs (clustered regularly interspaced short palindromic repeats) consist of repeats interspaced with non-repetitive elements or 'spacers' CRISPRs are present in both archaea and bacteria, in association with genes involved in DNA recombination and repair In the Yersinia pestis genome, three such elements are found at three distinct loci, one of them being highly polymorphic The authors have sequenced a total of 109 alleles of the three Y pestis CRISPRs and they describe 29 new spacers, most being specific to one isolate In nine strains of Yersinia pseudotuberculosis, 132 spacers were found, of which only three are common to Y pestis isolates In Y pestis of the Orientalis biovar investigated in detail here, deletion of motifs is observed but it appears that addition of new motifs to a common ancestral element is the most frequent event This takes place at the three different loci, although at a higher rate in one of the loci, and the addition of new motifs is polarized Interestingly, the most recently acquired spacers were found to have a homologue at another locus in the genome, the majority of these inside an inactive prophage This is believed to be the first time that the origin of the spacers in CRISPR elements has been explained The CRISPR structure provides a new and robust identification tool

CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.

Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 3 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrug-resistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.

Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.

Mycobacterium tuberculosis isolates from 167 patients attending three London hospitals were analyzed by two techniques for strain differentiation. A significant number of isolates that appeared identical with the recently developed spoligotyping system could be distinguished from each other by IS6110 restriction fragment length polymorphism analysis, with the latter technique demonstrating a generally higher level of discrimination. Spoligotyping, on the other hand, was particularly useful for analysis of isolates with low IS6110 copy numbers, and use of the two techniques in tandem provided an optimal approach to the identification of clusters with epidemiological evidence consistent with recent transmission. Spoligotyping can be applied directly to clinical samples by PCR and provides an important tool for the rapid detection of nosocomial transmission of individual strains.

Differentiation of Mycobacterium tuberculosis isolates by spoligotyping and IS6110 restriction fragment length polymorphism.

BACKGROUND—Over the last 10 years there has been a fourfold increase in cases of tuberculosis in Harare, Zimbabwe. The use of molecular epidemiology to understand tuberculosis transmission in this epidemic has been hampered by the availability of suitable culture facilities. A study was therefore undertaken to explore the potential of spoligotyping, a polymerase chain reaction based technique that does not require tuberculosis culture.
METHODS—Adults attending a chest clinic with clinical or radiological pulmonary tuberculosis and one smear positive sputum were enrolled over one month. Demographic, socioeconomic, and clinical data were gathered using a standardised questionnaire. Molecular fingerprinting of genomic DNA recovered from sputum was performed by spoligotyping.
RESULTS—Sixty one subjects (median age 28 years (range 18-73); 61% men) were recruited and 57 provided adequate sputum samples. Recent rural-urban migration or immigration was not common; 40% of subjects lived in crowded living conditions. DNA suitable for spoligotyping was recovered from 28 patients and 20 different genotypes of Mycobacterium tuberculosis were identified. Fifteen patients were infected with an M tuberculosis strain shared by one or more individuals. Patients infected with a shared spoligotype were not closely linked geographically within Harare, but were more likely to live in overcrowded conditions (69% versus 23%; odds ratio 6.85 (95% CI 1.2 to 47), p = 0.026). Analysis of the patients' original rural family homes revealed two geographically related spoligotype clusters.
CONCLUSIONS—Spoligotyping may yield valuable molecular typing information in populations where tuberculosis culture is not available. This novel technique requires further development and evaluation in larger epidemiological studies.

https://thorax.bmj.com/content/thoraxjnl/53/5/346.full.pdf

M Goyal

Papers

Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.

Differentiation of Mycobacterium tuberculosis isolates by spoligotyping and IS6110 restriction fragment length polymorphism.

Pulmonary tuberculosis in Harare, Zimbabwe: analysis by spoligotyping