The Open Microbiology Journal




ISSN: 1874-2858 ― Volume 13, 2019
REVIEW ARTICLE

Bacterial CRISPR Regions: General Features and their Potential for Epidemiological Molecular Typing Studies



Zahra Karimi, Ali Ahmadi, Ali Najafi*, Reza Ranjbar
Molecular Biology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

Abstract

Introduction:

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci as novel and applicable regions in prokaryotic genomes have gained great attraction in the post genomics era.

Methods:

These unique regions are diverse in number and sequence composition in different pathogenic bacteria and thereby can be a suitable candidate for molecular epidemiology and genotyping studies. Results:Furthermore, the arrayed structure of CRISPR loci (several unique repeats spaced with the variable sequence) and associated cas genes act as an active prokaryotic immune system against viral replication and conjugative elements. This property can be used as a tool for RNA editing in bioengineering studies.

Conclusion:

The aim of this review was to survey some details about the history, nature, and potential applications of CRISPR arrays in both genetic engineering and bacterial genotyping studies.

Keywords: CRISPR, Bioengineering, Genotyping, Epidemiological studies, Pathogenic bacteria, Prokaryotic immune system.


Article Information


Identifiers and Pagination:

Year: 2018
Volume: 12
First Page: 59
Last Page: 70
Publisher Id: TOMICROJ-12-59
DOI: 10.2174/1874285801812010059

Article History:

Received Date: 2/2/2018
Revision Received Date: 8/04/2018
Acceptance Date: 9/04/2018
Electronic publication date: 23/04/2018
Collection year: 2018

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© 2018 Karimi et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Molecular Biology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran; Tel: 00982182482569; E-mails: najafi74@bmsu.ac.ir , najafi74@yahoo.com




1. INTRODUCTION

Classification and molecular typing of pathogenic bacteria are an important issue in modern microbiology. The main purpose of microbial typing is to assess the relationships between microbial isolates These techniques have a the main role to determine the source and routes of infections, confirm or rule out outbreaks, to assess the cross-transmission of healthcare-associated pathogens, to recognize particularly virulent strains and finally to evaluate the effectiveness of control measures [1Ranjbar R, Karami A, Farshad S, Giammanco GM, Mammina C. Typing methods used in the molecular epidemiology of microbial pathogens: a how-to guide. New Microbiol 2014; 37(1): 1-15.[PMID: 24531166] ].

Actually, there is a wide variety of typing methods applied in studies of pathogenic bacteria to screening and microbial source tracking purposes. These techniques are classified into two groups, including traditional and molecular typing methods [2Pfaller MA. Molecular epidemiology in the care of patients. Arch Pathol Lab Med 1999; 123(11): 1007-10.[PMID: 10539897] ]. Traditional methods that consist of serotyping, phagetyping, and antibiogram typing methods have some weaknesses such as variable results, are highly time-consuming as well as have low sensitivity and specificity [3Shariat N, Dudley EG. CRISPRs: molecular signatures used for pathogen subtyping. Appl Environ Microbiol 2014; 80(2): 430-9.[http://dx.doi.org/10.1128/AEM.02790-13] [PMID: 24162568] ]. So, traditional methods have limitations that do not permit to understand about bacterial population genetics, evolution, and molecular epidemiology for more appropriate and accurate typing. Since 2000, high-throughput and high-resolution molecular typing methods are integrated profoundly with epidemiologic molecular studies, most of which are DNA-based and PCR-based methods [2Pfaller MA. Molecular epidemiology in the care of patients. Arch Pathol Lab Med 1999; 123(11): 1007-10.[PMID: 10539897] , 3Shariat N, Dudley EG. CRISPRs: molecular signatures used for pathogen subtyping. Appl Environ Microbiol 2014; 80(2): 430-9.[http://dx.doi.org/10.1128/AEM.02790-13] [PMID: 24162568] ].

PCR and similar nucleotide-based methods have become potentially powerful approaches in microbial detection as well as microbial typing because of their higher user-friendliness, rapidity, reproducibility, accuracy and affordability [4Ranjbar R, Mortazavi SM, Mehrabi Tavana A, Sarshar M, Najafi A, Soruri Zanjani R. Simultaneous Molecular Detection ofSalmonella entericaSerovars Typhi, Enteritidis, Infantis, and Typhimurium. Iran J Public Health 2017; 46(1): 103-11.[PMID: 28451535] , 5Ranjbar R, Naghoni A, Farshad S, et al. Use of TaqMan® real-time PCR for rapid detection of Salmonella enterica serovar Typhi. Acta Microbiol Immunol Hung 2014; 61(2): 121-30.[http://dx.doi.org/10.1556/AMicr.61.2014.2.3] [PMID: 24939681] ]. These techniques consist of analysis of plasmid profiles [6Ranjbar R, Owlia P, Saderi H, et al. Isolation of clinical strains of Pseudomonas aeruginosa harboring different plasmids. Pak J Biol Sci 2007; 10(17): 3020-2.[http://dx.doi.org/10.3923/pjbs.2007.3020.3022] [PMID: 19090223] , 7Farshad S, Ranjbar R, Japoni A, Hosseini M, Anvarinejad M, Mohammadzadegan R. Microbial susceptibility, virulence factors, and plasmid profiles of uropathogenic Escherichia coli strains isolated from children in Jahrom, Iran. Arch Iran Med 2012; 15(5): 312-6.[PMID: 22519382] ], Ribotyping [8Ranjbar R, Soltan Dallal MM, Talebi M, Pourshafie MR. Increased isolation and characterization of Shigella sonnei obtained from hospitalized children in Tehran, Iran. J Health Popul Nutr 2008; 26(4): 426-30.[PMID: 19069621] , 9Ranjbar R, Mammina C, Pourshafie MR, Soltan-Dallal MM. Characterization of endemic Shigella boydii strains isolated in Iran by serotyping, antimicrobial resistance, plasmid profile, ribotyping and pulsed-field gel electrophoresis. BMC Res Notes 2008; 1(1): 74.[http://dx.doi.org/10.1186/1756-0500-1-74] [PMID: 18755045] ], PFGE (Pulsed-Field Gel electrophoresis) [10Ranjbar R, Aleo A, Giammanco GM, Dionisi AM, Sadeghifard N, Mammina C. Genetic relatedness among isolates of Shigella sonnei carrying class 2 integrons in Tehran, Iran, 2002-2003. BMC Infect Dis 2007; 7(1): 62.[http://dx.doi.org/10.1186/1471-2334-7-62] [PMID: 17587439] , 11Pooideh M, Jabbarzadeh I, Ranjbar R, Saifi M. Molecular Epidemiology of Mycobacterium tuberculosis Isolates in 100 Patients With Tuberculosis Using Pulsed Field Gel Electrophoresis. Jundishapur J Microbiol 2015; 8(7): e18274.[PMID: 26396714] ], RFLP (Restriction Fragment Length Polymorphism) [12Farshad S, Ranjbar R, Hosseini M. Molecular genotyping of Shigella sonnei strains isolated from children with bloody diarrhea using pulsed field gel electrophoresis on the total genome and PCR-RFLP of IpaH and IpaBCD genes. Jundishapur J Microbiol 2014; 8(1): e14004.[http://dx.doi.org/10.5812/jjm.14004] [PMID: 25789126] , 13Arjomandzadegan M, Owlia P, Ranjbar R, et al. Prevalence of mutations at codon 463 of katG gene in MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus and application of the method in rapid diagnosis. Acta Microbiol Immunol Hung 2011; 58(1): 51-63.[http://dx.doi.org/10.1556/AMicr.58.2011.1.6] [PMID: 21450555] ], MLST (Multi Locus Sequence Typing) [14Ranjbar R, Elhaghi P, Shokoohizadeh L. Multilocus Sequence Typing of the Clinical Isolates ofSalmonella EntericaSerovar Typhimurium in Tehran Hospitals. Iran J Med Sci 2017; 42(5): 443-8.[PMID: 29234176] ], VNTR (Variable Number Tandem Repeat) [15Ranjbar R, Memariani M, Memariani H. Diversity of variable number tandem repeat loci in Shigella species isolated from pediatric patients. Int J Mol Cell Med 2015; 4(3): 174-81.[PMID: 26629486] , 16Ranjbar R, Memariani H, Sorouri R, Memariani M. Distribution of virulence genes and genotyping of CTX-M-15-producing Klebsiella pneumoniae isolated from patients with community-acquired urinary tract infection (CA-UTI). Microb Pathog 2016; 100: 244-9.[http://dx.doi.org/10.1016/j.micpath.2016.10.002] [PMID: 27725280] ], RAPD (Randomly Amplification of Polymorphic DNA) [17Pourshafie MR, Bakhshi B, Ranjbar R, et al. Dissemination of a single Vibrio cholerae clone in cholera outbreaks during 2005 in Iran. J Med Microbiol 2007; 56(Pt 12): 1615-9.[http://dx.doi.org/10.1099/jmm.0.47218-0] [PMID: 18033829] , 18Sadeghifard N, Ranjbar R, Zaeimi J. Antimicrobial susceptibility, plasmid profiles, and RAPD-PCR typing of Acinetobacter bacteria 2011.], AP-PCR (Arbitrary Pprimed PCR) [19Ranjbar R, et al. Molecular characterization of epidemic isolates of Vibrio cholerae O1 by arbitrarily primed PCR (AP-PCR). Iran J Public Health 2008; 37(2): 83-7.], Rep-PCR (Repetitive extragenic palindromic) [20Ranjbar R, Pezeshknejad P, Khamesipour F, Amini K, Kheiri R. Genomic fingerprints of Escherichia coli strains isolated from surface water in Alborz province, Iran. BMC Res Notes 2017; 10(1): 295.[http://dx.doi.org/10.1186/s13104-017-2575-z] [PMID: 28728566] ], ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus) [21Ranjbar R, Mirsaeed Ghazi F. Antibiotic sensitivity patterns and molecular typing of Shigella sonnei strains using ERIC-PCR. Iran J Public Health 2013; 42(10): 1151-7.[PMID: 26060624] -23Sabat AJ, Budimir A, Nashev D, et al. Overview of molecular typing methods for outbreak detection and epidemiological surveillance. Euro Surveill 2013; 18(4): 20380.[http://dx.doi.org/10.2807/ese.18.04.20380-en] [PMID: 23369389] ], Microarray [24Khakabimamaghani S, Najafi A, Ranjbar R, Raam M. GelClust: a software tool for gel electrophoresis images analysis and dendrogram generation. Comput Methods Programs Biomed 2013; 111(2): 512-8.[http://dx.doi.org/10.1016/j.cmpb.2013.04.013] [PMID: 23727299] , 25Jahandeh N, Ranjbar R, Behzadi P, Behzadi E. Uropathogenic Escherichia coli virulence genes: invaluable approaches for designing DNA microarray probes. Cent European J Urol 2015; 68(4): 452-8.[PMID: 26855801] ] and most recently, CRISPR regions analysis being used as a new and powerful method for molecular typing and genetic comparative analyses of bacterial strains. In this review, we aim to explain the nature, history, and epidemiological applications of CRISPR region analysis.

2. HISTORY OF CRISPR RESEARCH

In 1987, Ishino and colleagues recognized for the first time a new mysterious distinct class of interspaced Short Sequences Repeats (SSRs) downstream of the iap gene on the chromosome of Escherichia coli K12. These sequences comprised of repetitive motifs of 29 nucleotide identical direct repeats separated by variable 32 nucleotide spacer regions; But the biological role of these repetitive motifs remains unclear [26Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 1987; 169(12): 5429-33.[http://dx.doi.org/10.1128/jb.169.12.5429-5433.1987] [PMID: 3316184] , 27Al-Attar S, Westra ER, van der Oost J, Brouns SJ. Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes. Biol Chem 2011; 392(4): 277-89.[http://dx.doi.org/10.1515/bc.2011.042] [PMID: 21294681] ].

In 1992, Groenen and colleagues found 36 bp repeat units being separated by 35-41-nt spacers in the genome of Mycobacterium tuberculosis and named it “Direct Variable Repeats” [28Groenen PM, Bunschoten AE, van Soolingen D, van Embden JD. Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method. Mol Microbiol 1993; 10(5): 1057-65.[http://dx.doi.org/10.1111/j.1365-2958.1993.tb00976.x] [PMID: 7934856] ]. Subsequently, Mojica and coworkers identified the same repeats in Haloferax volcanii and Haloferax mediterranei and referred them as Tandem Repeats (TREPs) [29Mojica FJ, Ferrer C, Juez G, Rodríguez-Valera F. Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning. Mol Microbiol 1995; 17(1): 85-93.[http://dx.doi.org/10.1111/j.1365-2958.1995.mmi_17010085.x] [PMID: 7476211] ]. In 1997, Goyal and his team showed that the diversity of these spacers in M. tuberculosis could be used for a new specific genotyping method called Spoligotyping [30Goyal M, Saunders NA, van Embden JD, Young DB, Shaw RJ. Differentiation of Mycobacterium tuberculosis isolates by spoligotyping and IS6110 restriction fragment length polymorphism. J Clin Microbiol 1997; 35(3): 647-51.[PMID: 9041405] ]. In 2000, Mojica et al. surveyed the occurrence of TREPs in a number of eubacteria and archaea and suggested that these motifs constituted a new family of prokaryotic repeats. They coined a new acronym, SRSRs (Short Regularly Spaced Repeats), to appreciate the unique regularity of these repeats [31Mojica FJ, Díez-Villaseñor C, Soria E, Juez G. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol 2000; 36(1): 244-6.[http://dx.doi.org/10.1046/j.1365-2958.2000.01838.x] [PMID: 10760181] ]. Also, other names for the repeat/spacer arrays are SPacer Interspaced and Direct Repeats (SPIDRs) and Long Clustered Tandem Repeats (LCTRs) [32She Q, Singh RK, Confalonieri F, et al. The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc Natl Acad Sci USA 2001; 98(14): 7835-40.[http://dx.doi.org/10.1073/pnas.141222098] [PMID: 11427726] ]. Finally, At the same time with the discovery of the first core CAS genes, Jansen et al. introduced “CRISPR” as a fresh acronym [33Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 2002; 43(6): 1565-75.[http://dx.doi.org/10.1046/j.1365-2958.2002.02839.x] [PMID: 11952905] ].

Although the biological function of the CRISPR regions was unknown up to that time, several activities such as developmental regulation [34Thöny-Meyer L, Kaiser D. devRS, an autoregulated and essential genetic locus for fruiting body development in Myxococcus xanthus. J Bacteriol 1993; 175(22): 7450-62.[http://dx.doi.org/10.1128/jb.175.22.7450-7462.1993] [PMID: 7693658] ], replicon partitioning during cell division [29Mojica FJ, Ferrer C, Juez G, Rodríguez-Valera F. Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning. Mol Microbiol 1995; 17(1): 85-93.[http://dx.doi.org/10.1111/j.1365-2958.1995.mmi_17010085.x] [PMID: 7476211] ], and DNA repair [35Makarova KS, Aravind L, Grishin NV, Rogozin IB, Koonin EV. A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis. Nucleic Acids Res 2002; 30(2): 482-96.[http://dx.doi.org/10.1093/nar/30.2.482] [PMID: 11788711] ] were attributed to these arrayed sequences. In 2005, a profound change occurred in our understanding of the nature and function of the CRISPR regions when several independent research teams found independently that many CRISPR spacers were most often homologous to fragments of the mobile genetic elements (phages, plasmids, and transposons). It suggests that they were from the extrachromosomal origin and probably the memory of a novel prokaryotic immune system [36Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 2005; 60(2): 174-82.[http://dx.doi.org/10.1007/s00239-004-0046-3] [PMID: 15791728] -39Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 2005; 151(Pt 8): 2551-61.[http://dx.doi.org/10.1099/mic.0.28048-0] [PMID: 16079334] ]. Subsequently, the immune system was proposed to act by using the RNA interference (RNAi) principle [40Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 2006; 1: 7.[http://dx.doi.org/10.1186/1745-6150-1-7] [PMID: 16545108] ], based on the presence of some key elements that would carry out the necessary functionalities [41Barrangou R, Marraffini LA. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol Cell 2014; 54(2): 234-44.[http://dx.doi.org/10.1016/j.molcel.2014.03.011] [PMID: 24766887] ]. Two years later in 2007, the first experimental based evidence was provided by Barrangou et al [42Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007; 315(5819): 1709-12.[http://dx.doi.org/10.1126/science.1138140] [PMID: 17379808] ] for the association of the CRISPR regions with the adaptive immunity in Streptococcus thermophilus. He indicated that acquiring new spacers are accompanied with the ability to phage resistance in a CAS-dependent manner. Soon after, it was demonstrated in E. coli that the CRISPR involved immunity is mediated by small noncoding interfering CRISPR RNAs (crRNAs) that direct CRISPR-associated complex for antiviral defense (cascade) [43Brouns SJ, Jore MM, Lundgren M, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008; 321(5891): 960-4.[http://dx.doi.org/10.1126/science.1159689] [PMID: 18703739] , 44Barrangou R. CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip Rev RNA 2013; 4(3): 267-78.[http://dx.doi.org/10.1002/wrna.1159] [PMID: 23520078] ]. Furthermore, CRISPR mediated immunity against plasmid DNA was shown in Staphylococcus epidermidis and also supported their observation through Bioinformatics predictions that CRISPR/Cas system is an active prokaryotic immune system against phages and plasmids [45Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 2008; 322(5909): 1843-5.[http://dx.doi.org/10.1126/science.1165771] [PMID: 19095942] ].

In 2010, numerous consecutive studies were directed to the mechanism of CRISPR regions- conferred immunity and finally concluded that CRISPR/CAS system is an active prokaryotic system immune system against viral replication and conjugative elements [46He J, Deem MW. Heterogeneous diversity of spacers within CRISPR (clustered regularly interspaced short palindromic repeats). Phys Rev Lett 2010; 105(12): 128102.[http://dx.doi.org/10.1103/PhysRevLett.105.128102] [PMID: 20867676] ]. So, CRISPR is now considered as the hallmark of ingenious antiviral defense mechanism in prokaryotes. In 2011, three main types of classification of the different CRISPR-CAS system were suggested by Makarova et al in the base of the phylogeny and the presence of particular CAS proteins.

3. THE CRISPR REGIONS: A NEW CONCEPT

Throughout the course of evolution, prokaryotes have succeeded to use a number of innate defensive strategies against bacteriophages. An example of such systems is adsorption inhibition by which bacteria hide or modify their receptors to escape from recognition by viral particles [27Al-Attar S, Westra ER, van der Oost J, Brouns SJ. Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes. Biol Chem 2011; 392(4): 277-89.[http://dx.doi.org/10.1515/bc.2011.042] [PMID: 21294681] , 47Hyman P, Abedon ST. Bacteriophage host range and bacterial resistance. Adv Appl Microbiol 2010; 70: 217-48.[http://dx.doi.org/10.1016/S0065-2164(10)70007-1] [PMID: 20359459] ]. A new form of defense system has been discovered in some bacteria characterized by the presence of specific arrayed sequences named CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which provides a defense mechanism against viruses, plasmid, and transposon [27Al-Attar S, Westra ER, van der Oost J, Brouns SJ. Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes. Biol Chem 2011; 392(4): 277-89.[http://dx.doi.org/10.1515/bc.2011.042] [PMID: 21294681] , 48Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 2007; 8(1): 172.[http://dx.doi.org/10.1186/1471-2105-8-172] [PMID: 17521438] ].

There are two main classes of Short Sequences Repeats (SSRs): contiguous repeats and interspersed repeats [49van Belkum A, Scherer S, van Alphen L, Verbrugh H. Short-sequence DNA repeats in prokaryotic genomes. Microbiol Mol Biol Rev 1998; 62(2): 275-93.[PMID: 9618442] ]. CRISPR arrays comprise two main functional elements: highly conserved 23 to 55 nucleotides tandem short DNA repeats, and 26 to 72 nucleotides variable sequences called Spacers which separate the conserved regions [37Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 2005; 151(Pt 3): 653-63.[http://dx.doi.org/10.1099/mic.0.27437-0] [PMID: 15758212] , 42Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007; 315(5819): 1709-12.[http://dx.doi.org/10.1126/science.1138140] [PMID: 17379808] ]. The CAS genes are co-localized with the spacer arrays, but this confuses the origin of spacer sequences with CAS genes. The CRISPRs are varied in bacterial genomes from one strain to another and therefore, this heterogeneity may lead to phenotypic differences. These repetitive sequences are common in the genomes of prokaryotic organisms and are variable in length, sequence, and position and often are unique for a single strain, thereby making them as a good tool for the identification of specific strains of bacteria and molecular epidemiology studies.

3.1. The Composition of the CRISPR Region

The CRISPR loci contain three elements: Direct repeated sequences; Non-repetitive spacer sequences; and a leader sequence flanking at one end of the repeats. Also, CAS genes are associated with the CRISPR loci as regulatory elements [33Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 2002; 43(6): 1565-75.[http://dx.doi.org/10.1046/j.1365-2958.2002.02839.x] [PMID: 11952905] ]. The schematic image of the CRISPR loci was illustrated in (Fig. 1).

Fig. (1)
Schematic image of the CRISPR loci.


3.1.1. Direct Repeated Sequences

The CRISPR region varies between various organisms according to the number and also the size (from 23-55 base pairs in length). These repeats that are clustered into one or more loci on the chromosome are often partially palindromic with the ability to form hairpin structures. On average, the bacterial genome contains three CRISPR arrays, compared to five CRISPR arrays found in the archaeal genome [48Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 2007; 8(1): 172.[http://dx.doi.org/10.1186/1471-2105-8-172] [PMID: 17521438] ]. A considerable characteristic of the CRISPR arrays is the ability of their transcripts to form RNA secondary structures [50Kunin V, Sorek R, Hugenholtz P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol 2007; 8(4): R61.[http://dx.doi.org/10.1186/gb-2007-8-4-r61] [PMID: 17442114] ].

3.1.2. Non-Repetitive Spacer Sequences

The spacer sequences vary in size from 26-72 base pairs. The spacers have similar lengths within a particular CRISPR array, but two identical spacers are not found in the same array [51Lillestøl RK, Redder P, Garrett RA, Brügger K. A putative viral defence mechanism in archaeal cells. Archaea 2006; 2(1): 59-72.[http://dx.doi.org/10.1155/2006/542818] [PMID: 16877322] ]. Although it is believed that the spacer sequences have originated from foreign mobile genetic elements, just a small portion of all known spacer maps have been found to contain extrachromosomal sequences from phages and plasmids [52Shah SA, Hansen NR, Garrett RA. Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism. Biochem Soc Trans 2009; 37(Pt 1): 23-8.[http://dx.doi.org/10.1042/BST0370023] [PMID: 19143596] ]. Recent CRISPR analysis studies have shown a significant polymorphism in the number and type of spacer sequences within different strains of a particular species, thereby turning them into a suitable tool for epidemiological studies. Besides, there is an offer that CRISPRs can impact the autoimmunity by use of spacers that target self-genes so CRISPRs can incur an autoimmune fitness cost by incorporation of nucleic acids, that may justify the abundance of degraded CRISPR systems across prokaryote [53Stern A, Keren L, Wurtzel O, Amitai G, Sorek R. Self-targeting by CRISPR: gene regulation or autoimmunity? Trends Genet 2010; 26(8): 335-40.[http://dx.doi.org/10.1016/j.tig.2010.05.008] [PMID: 20598393] ].

3.1.3. Leader Sequences

The AT rich, non-coding stretch nucleotides are located on one side of the CRISPR loci at the 5ʹ end, upstream of the first CRISPR repeat and often downstream of the last cas gene [33Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 2002; 43(6): 1565-75.[http://dx.doi.org/10.1046/j.1365-2958.2002.02839.x] [PMID: 11952905] , 54Tang T-H, Bachellerie JP, Rozhdestvensky T, et al. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc Natl Acad Sci USA 2002; 99(11): 7536-41.[http://dx.doi.org/10.1073/pnas.112047299] [PMID: 12032318] ]. Leader sequences are highly similar within the same prokaryotic species but are so different in distantly related species [33Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 2002; 43(6): 1565-75.[http://dx.doi.org/10.1046/j.1365-2958.2002.02839.x] [PMID: 11952905] ]. Since regions being homologous to the 3'-end of the leader sequence were found in the precursor CRISPR RNA (pre-crRNA) of Pyrococcus furiosus, it is suggested that the transcription of the CRISPR arrays is initiated in the leader region [55Hale C, Kleppe K, Terns RM, Terns MP. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 2008; 14(12): 2572-9.[http://dx.doi.org/10.1261/rna.1246808] [PMID: 18971321] ]. Recently, the role of leader sequences as a promoter for transcription of the pre-crRNA has been demonstrated. Besides, it has also been suggested that the leader sequences could prepare a platform for binding of the CAS proteins required for integrating the spacers [36Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 2005; 60(2): 174-82.[http://dx.doi.org/10.1007/s00239-004-0046-3] [PMID: 15791728] , 46He J, Deem MW. Heterogeneous diversity of spacers within CRISPR (clustered regularly interspaced short palindromic repeats). Phys Rev Lett 2010; 105(12): 128102.[http://dx.doi.org/10.1103/PhysRevLett.105.128102] [PMID: 20867676] ]. This promoter was active both in vitro and in vivo and was able to form an open transcription initiation complex [56Pul U, Wurm R, Arslan Z, Geissen R, Hofmann N, Wagner R. Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol Microbiol 2010; 75(6): 1495-512.[http://dx.doi.org/10.1111/j.1365-2958.2010.07073.x] [PMID: 20132443] ]. Bioinformatics analysis has shown that CRISPR loci lacking leader sequences are unable to incorporate new spacers [46He J, Deem MW. Heterogeneous diversity of spacers within CRISPR (clustered regularly interspaced short palindromic repeats). Phys Rev Lett 2010; 105(12): 128102.[http://dx.doi.org/10.1103/PhysRevLett.105.128102] [PMID: 20867676] ] and to execute the CRISPR-Expression and Interference [45Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 2008; 322(5909): 1843-5.[http://dx.doi.org/10.1126/science.1165771] [PMID: 19095942] ]. The direct repeats and leader sequences are both conserved in a single bacterial species but are diverse between different species.

3.1.4. CAS Genes

CRISPR-Associated (CAS) genes have been only detected in CRISPR-containing prokaryotes and have invariably been located adjacent to the CRISPR loci [57Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science 2010; 327(5962): 167-70.[http://dx.doi.org/10.1126/science.1179555] [PMID: 20056882] ]. It is suggested that CRISPR loci and CAS genes have related functions, especially in DNA metabolism and/or gene expression [44Barrangou R. CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip Rev RNA 2013; 4(3): 267-78.[http://dx.doi.org/10.1002/wrna.1159] [PMID: 23520078] ].

Haft and coworkers in 2005 documented 45 CAS gene families constituting six core CAS families (cas1-cas6), among which two families (cas1 and cas2) are universal and present in all CAS subtype [58van der Oost J, Brouns SJ. RNAi: prokaryotes get in on the act. Cell 2009; 139(5): 863-5.[http://dx.doi.org/10.1016/j.cell.2009.11.018] [PMID: 19945373] , 59Haft DH, Selengut J, Mongodin EF, Nelson KE. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLOS Comput Biol 2005; 1(6): e60.[http://dx.doi.org/10.1371/journal.pcbi.0010060] [PMID: 16292354] ]. Cas1and cas2 families are implicated in the novel spacer acquisition, novel repeat synthesis, and repeat-spacer insertion at the leader end [44Barrangou R. CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip Rev RNA 2013; 4(3): 267-78.[http://dx.doi.org/10.1002/wrna.1159] [PMID: 23520078] ].

Furthermore, three CRISPR-CAS type systems have been recently recognized based on the phylogeny and also the molecular mechanism of action of cas genes, including type Ι, according to the presence of cas3 gene; type ΙΙ, for the presence of cas9 gene; and type ΙΙΙ, for the presence of cas10 gene [60Makarova KS, Haft DH, Barrangou R, et al. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 2011; 9(6): 467-77.[http://dx.doi.org/10.1038/nrmicro2577] [PMID: 21552286] , 61Louwen R, Staals RH, Endtz HP, van Baarlen P, van der Oost J. The role of CRISPR-Cas systems in virulence of pathogenic bacteria. Microbiol Mol Biol Rev 2014; 78(1): 74-88.[http://dx.doi.org/10.1128/MMBR.00039-13] [PMID: 24600041] ]. Although type Ι and type ΙΙΙ CRISPR-CAS system have some common features, it is not the case for the type ΙΙ system. Aside from a conserved set of cas genes (i.e., cas1, cas2, and cas9), three different subtypes have been identified, including type ΙΙ-A with an additional cas2, type ΙΙ-B type ΙΙ-C with an additional cas4, and type ΙΙ-C with no additional gene [60Makarova KS, Haft DH, Barrangou R, et al. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 2011; 9(6): 467-77.[http://dx.doi.org/10.1038/nrmicro2577] [PMID: 21552286] ].

Cas proteins comprise a highly genetically polymorphic and functionally diverse family that is involved in various stages of CRISPR-mediated immunity. Several different Cas protein families, being highly variable in number, distribution, and also organization has been classified [60Makarova KS, Haft DH, Barrangou R, et al. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 2011; 9(6): 467-77.[http://dx.doi.org/10.1038/nrmicro2577] [PMID: 21552286] , 62Marraffini LA, Sontheimer EJ. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 2010; 11(3): 181-90.[http://dx.doi.org/10.1038/nrg2749] [PMID: 20125085] ]. The most widely distributed functional domain that is characteristic of Cas proteins is the RNA Recognition Motif (RRM). Based on sequence analysis studies, nucleases, helicases, integrases, and polymerases domains have been predicted in Cas proteins, indicating their involvement in the nucleic acid metabolism [41Barrangou R, Marraffini LA. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol Cell 2014; 54(2): 234-44.[http://dx.doi.org/10.1016/j.molcel.2014.03.011] [PMID: 24766887] ]. The key conserved Cas proteins and their function are listed in Table (1).

Table 1
Key conserved Cas proteins and their function.


4. IN SILICO ANALYSIS OF CRISPR

4.1. CRISPR databases

Bioinformatics and their capabilities can accelerate molecular epidemiologic investigations in post genomics era. In this regard, several special CRISPR databases and tools have been developed during recent years. There are two general categories of CRISPR database: 1) Gene Editing based databases, and 2) Genotyping based databases. The second group (Genotyping based databases) is a database that deposits CRISPR sequences and their annotations. CRISPR Finder [63Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats 2007.[http://dx.doi.org/10.1093/nar/gkm360] ] and CRISPRdb [48Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 2007; 8(1): 172.[http://dx.doi.org/10.1186/1471-2105-8-172] [PMID: 17521438] ] are the best examples of Genotyping based databases that are specific resources for deposition of CRISPR loci and spacers in some prokaryotic genome. But, since Genotyping based databases do not cover all bacterial genomes, a flowchart designed here as (Fig. 2) could be used for discovering CRISPR regions in bacterial and archeal complete genomes [48Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 2007; 8(1): 172.[http://dx.doi.org/10.1186/1471-2105-8-172] [PMID: 17521438] ].

Fig. (2)
A flow chart for discovering CRISPR regions in the bacterial genome.


4.2. CRISPR Tools

Despite the presence of several various bioinformatic tools for identifying direct repeats in the genome, due to the importance and nature of CRISPR composition, specific software applications have been developed for discovering them in prokaryotic genomes and metagenomes including CRISPR Finder, CRISPR Recognition tool (CRT), PILER-CR, and CRISPI.

Among them, the CRISPR Finder is freely accessible at http://crispr.u-psud.fr/crispr/, a user-friendly web service that contains information about CRISPR systems in several bacterial genomes which can identify and extract CRISPR array and spacers. This program can find the largest number of possible CRISPRs, especially the shortest ones that only contain one or two spacers [63Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats 2007.[http://dx.doi.org/10.1093/nar/gkm360] ].

Fig. (3)
Development stages of the CRISPR/CAS system.


5. ROLE OF CRISPR-CAS SYSTEM

CRISPR arrays and cas genes are the two parts of the CRISPR-CAS immune system in bacteria and archaea, which provides adaptive immunity against foreign genetic elements. Conferring of immunity is the duty of spacers. These immune markers are transcribed and processed into small non-coding interfering CRISPR RNAs (crRNAs) that direct Cas proteins toward foreign nucleic acids for specific cleavage of homologous sequences [41Barrangou R, Marraffini LA. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol Cell 2014; 54(2): 234-44.[http://dx.doi.org/10.1016/j.molcel.2014.03.011] [PMID: 24766887] ]. In fact, it has been suggested that CRISPR-CAS system memorizes invaders by incorporating the new invader-derived DNA sequences into the CRISPR sequences and providing immunity to future attack by the same invader [40Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 2006; 1: 7.[http://dx.doi.org/10.1186/1745-6150-1-7] [PMID: 16545108] ]. Overall, CRISPR-CAS system functions in three steps: 1) Adaptation: new spacers are acquired from invader nucleic acids and integrated into the CRISPR loci; 2) Expression: CRISPR loci are transcribed and processed into small interfering crRNAs; and 3) Interference: cr-RNAs direct the CAS machinery to specifically cleave homologous invader nucleic acids [57Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science 2010; 327(5962): 167-70.[http://dx.doi.org/10.1126/science.1179555] [PMID: 20056882] ]. The response cascade of the CRISPR/CAS system is shown in (Fig. 3). In 2009, Zegans et al suggested that the CRISPR regions can change the lysogeny effects of Pseudomonas aeruginosa PA14 by restoring both the biofilm formation and swarming motility, which had been inactivated by lysogenic bacteriophage DMS3 [64Zegans ME, Wagner JC, Cady KC, Murphy DM, Hammond JH, O’Toole GA. Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa. J Bacteriol 2009; 191(1): 210-9.[http://dx.doi.org/10.1128/JB.00797-08] [PMID: 18952788] ]. Also, Edgar and Qimron have shown that the CRISPR system protects bacteria during the lysogenic cycle of phages [65Edgar R, Qimron U. The Escherichia coli CRISPR system protects from λ lysogenization, lysogens, and prophage induction. J Bacteriol 2010; 192(23): 6291-4.[http://dx.doi.org/10.1128/JB.00644-10] [PMID: 20889749] ].

5.1. CRISPR-Cas 9 Editing

The CRISPR-Cas 9 is a RNA-guided genome editing tool for genome editing purposes such as gene therapy studies and therapeutic purposes in cell lines or animal models.

It can act for the correction of causal mutations in monogenic disorders, or manipulate pathogen genomes such as HIV, or induce protective or therapeutic mutations in host tissues. Also, the potential of CRISPR-CAS 9 for deactivating oncogenic viruses and inducing oncosuppressor expressions for cancer gene therapy has been shown [66Xiao-Jie L, Hui-Ying X, Zun-Ping K, Jin-Lian C, Li-Juan J. CRISPR-Cas9: a new and promising player in gene therapy. J Med Genet 2015; 52(5): 289-96.[http://dx.doi.org/10.1136/jmedgenet-2014-102968] [PMID: 25713109] ].

6. OCCURRENCE AND EPIDEMIOLOGICAL APPLICATIONS OF CRISPR SYSTEM

During the past decade, studies have shown that CRISPR regions are a family of repetitive sequences that are present in the genome sequence of approximately 48% of bacteria and 90% of archaea. In contrast, they have not been identified in eukaryotic genomes and also viruses to date. Gram positive and negative bacteria found to harbor CRISPR regions are listed in Table (2) [33Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 2002; 43(6): 1565-75.[http://dx.doi.org/10.1046/j.1365-2958.2002.02839.x] [PMID: 11952905] ].

Table 2
Gram positive and negative bacteria harboring CRISPR regions.


Some bacterial strains may have spacer sequences with different numbers at the same CRISPR loci, offering a strain-specific polymorphism that can be used in phylogeny and epidemiological studies [63Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats 2007.[http://dx.doi.org/10.1093/nar/gkm360] ]. But, it is noteworthy that since some bacterial species (about 50%) did not acquire CRISPR loci in their genomes, and also some bacterial species acquired spacers at a higher rate, they could not be sub-grouped through the CRISPR based- molecular subtyping [3Shariat N, Dudley EG. CRISPRs: molecular signatures used for pathogen subtyping. Appl Environ Microbiol 2014; 80(2): 430-9.[http://dx.doi.org/10.1128/AEM.02790-13] [PMID: 24162568] ]. Spacer-oligonucleotide typing or “Spoligotyping” was the first use of spacer information for bacterial subtyping applications [28Groenen PM, Bunschoten AE, van Soolingen D, van Embden JD. Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method. Mol Microbiol 1993; 10(5): 1057-65.[http://dx.doi.org/10.1111/j.1365-2958.1993.tb00976.x] [PMID: 7934856] , 88Kamerbeek J, Schouls L, Kolk A, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997; 35(4): 907-14.[PMID: 9157152] ]. The principle is the PCR amplification of the CRISPR arrays with labeled primers that detect direct repeat sequences, followed by hybridization of the PCR products to a membrane containing probes bearing spacer DNA sequences. Because of strain-specificity, different hybridization patterns could distinguish between different strains [3Shariat N, Dudley EG. CRISPRs: molecular signatures used for pathogen subtyping. Appl Environ Microbiol 2014; 80(2): 430-9.[http://dx.doi.org/10.1128/AEM.02790-13] [PMID: 24162568] ]. For example, Simon Le Hello and coworkers in 2013 reported that CRISPR sequences are very applicable targets for subtyping of Salmonella enteritidis isolates. They concluded that since CRISPR spacer content can be easily obtained from short-read DNA sequences, it could be used to identify particular Salmonella and probably other bacterial pathogens [89Le Hello S, Maillard F, Mallet HP, et al. Salmonella enterica serotype enteritidis in French Polynesia, South Pacific, 2008-2013. Emerg Infect Dis 2015; 21(6): 1045-8.[http://dx.doi.org/10.3201/eid2106.141103] [PMID: 25988406] , 90Zheng J, Pettengill J, Strain E, et al. Genetic diversity and evolution of Salmonella enterica serovar Enteritidis strains with different phage types. J Clin Microbiol 2014; 52(5): 1490-500.[http://dx.doi.org/10.1128/JCM.00051-14] [PMID: 24574287] ]. Also, Touchon et al. analyzed the CRISPR loci of 51 complete genomes of Salmonella and Escherichia isolates and found two pairs of CRISPR loci in Escherichia and one single pair in salmonella. In general, different studies in recent years have shown controversial results for CRISPR loci based bacterial genotyping. For example, a study on Campylobacter jejuni isolates concluded that CRISPR based genotyping is not an appropriate approach for Campylobacter species, because some campylobacter species either lacked an amplifiable CRISPR locus or contained just a single DR [3Shariat N, Dudley EG. CRISPRs: molecular signatures used for pathogen subtyping. Appl Environ Microbiol 2014; 80(2): 430-9.[http://dx.doi.org/10.1128/AEM.02790-13] [PMID: 24162568] ]. Also, in two separated studies, it has been shown that CRISPR loci had a few changes in the genome of Shigella species [85Guo X, Wang Y, Duan G, et al. Detection and analysis of CRISPRs of Shigella. Curr Microbiol 2015; 70(1): 85-90.[http://dx.doi.org/10.1007/s00284-014-0683-8] [PMID: 25199561] ], and are not widely distributed in Klebsiella pneumoniae isolates too, thereby making CRISPR loci a poor genotyping marker for these two pathogens [86Ostria-Hernández ML, Sánchez-Vallejo CJ, Ibarra JA, Castro-Escarpulli G. Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae. BMC Res Notes 2015; 8(1): 332.[http://dx.doi.org/10.1186/s13104-015-1285-7] [PMID: 26238567] ]. Some studies, on the other hand, have found promising results, especially for Salmonella isolates. A study on Salmonella enterica serotypes typhi and paratyphi has shown that CRISPR regions could be used as an original target for the development of PCR assays specific for particular salmonella species [91Fabre L, Le Hello S, Roux C, Issenhuth-Jeanjean S, Weill FX. CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A. PLoS Negl Trop Dis 2014; 8(1): e2671.[http://dx.doi.org/10.1371/journal.pntd.0002671] [PMID: 24498453] ]; another study comparing the CRISPR based genotyping with PFGE and MLVA techniques observed high levels of correlation between their results [3Shariat N, Dudley EG. CRISPRs: molecular signatures used for pathogen subtyping. Appl Environ Microbiol 2014; 80(2): 430-9.[http://dx.doi.org/10.1128/AEM.02790-13] [PMID: 24162568] ]. Furthermore, some researchers have suggested that the CRISPR/CAS system may be an important tool for evolutionary dynamics investigations for Clostridium difficile isolates [71Hargreaves KR, Flores CO, Lawley TD, Clokie MR. Abundant and diverse clustered regularly interspaced short palindromic repeat spacers in Clostridium difficile strains and prophages target multiple phage types within this pathogen. MBio 2014; 5(5): e01045-13.[http://dx.doi.org/10.1128/mBio.01045-13] [PMID: 25161187] ].

Conclusion: The CRISPR loci have potential for broad genotyping of bacterial strains and could be further used in an epidemiological survey [41Barrangou R, Marraffini LA. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol Cell 2014; 54(2): 234-44.[http://dx.doi.org/10.1016/j.molcel.2014.03.011] [PMID: 24766887] ]. CRISPR features can, moreover, be used for host-virus environmental studies, offering a specific immunity against undesirable genetic elements, and increasing viral resistance in domesticated microbes [57Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science 2010; 327(5962): 167-70.[http://dx.doi.org/10.1126/science.1179555] [PMID: 20056882] ]. By use of CRISPR loci as a template for genotyping, we retrieve information more precisely to define the epidemic strains, because the spacers inserted in a polarized manner at the leader end of CRISPR loci can lead to provide a genetic basis for the detection of historical path of a strain, share ancestry between strains and establish phylogenetic relationships. So, these hypervariable loci of the CRISPR with novel Bioinformatics tools for processing of high-throughput sequencing data, and the visualization of complex datasets are broadly useful for epidemiological surveys.

CONSENT FOR PUBLICATION

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CONFLICT OF INTEREST

The author confirms that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

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REFERENCES

[1] Ranjbar R, Karami A, Farshad S, Giammanco GM, Mammina C. Typing methods used in the molecular epidemiology of microbial pathogens: a how-to guide. New Microbiol 2014; 37(1): 1-15.[PMID: 24531166]
[2] Pfaller MA. Molecular epidemiology in the care of patients. Arch Pathol Lab Med 1999; 123(11): 1007-10.[PMID: 10539897]
[3] Shariat N, Dudley EG. CRISPRs: molecular signatures used for pathogen subtyping. Appl Environ Microbiol 2014; 80(2): 430-9.[http://dx.doi.org/10.1128/AEM.02790-13] [PMID: 24162568]
[4] Ranjbar R, Mortazavi SM, Mehrabi Tavana A, Sarshar M, Najafi A, Soruri Zanjani R. Simultaneous Molecular Detection ofSalmonella entericaSerovars Typhi, Enteritidis, Infantis, and Typhimurium. Iran J Public Health 2017; 46(1): 103-11.[PMID: 28451535]
[5] Ranjbar R, Naghoni A, Farshad S, et al. Use of TaqMan® real-time PCR for rapid detection of Salmonella enterica serovar Typhi. Acta Microbiol Immunol Hung 2014; 61(2): 121-30.[http://dx.doi.org/10.1556/AMicr.61.2014.2.3] [PMID: 24939681]
[6] Ranjbar R, Owlia P, Saderi H, et al. Isolation of clinical strains of Pseudomonas aeruginosa harboring different plasmids. Pak J Biol Sci 2007; 10(17): 3020-2.[http://dx.doi.org/10.3923/pjbs.2007.3020.3022] [PMID: 19090223]
[7] Farshad S, Ranjbar R, Japoni A, Hosseini M, Anvarinejad M, Mohammadzadegan R. Microbial susceptibility, virulence factors, and plasmid profiles of uropathogenic Escherichia coli strains isolated from children in Jahrom, Iran. Arch Iran Med 2012; 15(5): 312-6.[PMID: 22519382]
[8] Ranjbar R, Soltan Dallal MM, Talebi M, Pourshafie MR. Increased isolation and characterization of Shigella sonnei obtained from hospitalized children in Tehran, Iran. J Health Popul Nutr 2008; 26(4): 426-30.[PMID: 19069621]
[9] Ranjbar R, Mammina C, Pourshafie MR, Soltan-Dallal MM. Characterization of endemic Shigella boydii strains isolated in Iran by serotyping, antimicrobial resistance, plasmid profile, ribotyping and pulsed-field gel electrophoresis. BMC Res Notes 2008; 1(1): 74.[http://dx.doi.org/10.1186/1756-0500-1-74] [PMID: 18755045]
[10] Ranjbar R, Aleo A, Giammanco GM, Dionisi AM, Sadeghifard N, Mammina C. Genetic relatedness among isolates of Shigella sonnei carrying class 2 integrons in Tehran, Iran, 2002-2003. BMC Infect Dis 2007; 7(1): 62.[http://dx.doi.org/10.1186/1471-2334-7-62] [PMID: 17587439]
[11] Pooideh M, Jabbarzadeh I, Ranjbar R, Saifi M. Molecular Epidemiology of Mycobacterium tuberculosis Isolates in 100 Patients With Tuberculosis Using Pulsed Field Gel Electrophoresis. Jundishapur J Microbiol 2015; 8(7): e18274.[PMID: 26396714]
[12] Farshad S, Ranjbar R, Hosseini M. Molecular genotyping of Shigella sonnei strains isolated from children with bloody diarrhea using pulsed field gel electrophoresis on the total genome and PCR-RFLP of IpaH and IpaBCD genes. Jundishapur J Microbiol 2014; 8(1): e14004.[http://dx.doi.org/10.5812/jjm.14004] [PMID: 25789126]
[13] Arjomandzadegan M, Owlia P, Ranjbar R, et al. Prevalence of mutations at codon 463 of katG gene in MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus and application of the method in rapid diagnosis. Acta Microbiol Immunol Hung 2011; 58(1): 51-63.[http://dx.doi.org/10.1556/AMicr.58.2011.1.6] [PMID: 21450555]
[14] Ranjbar R, Elhaghi P, Shokoohizadeh L. Multilocus Sequence Typing of the Clinical Isolates ofSalmonella EntericaSerovar Typhimurium in Tehran Hospitals. Iran J Med Sci 2017; 42(5): 443-8.[PMID: 29234176]
[15] Ranjbar R, Memariani M, Memariani H. Diversity of variable number tandem repeat loci in Shigella species isolated from pediatric patients. Int J Mol Cell Med 2015; 4(3): 174-81.[PMID: 26629486]
[16] Ranjbar R, Memariani H, Sorouri R, Memariani M. Distribution of virulence genes and genotyping of CTX-M-15-producing Klebsiella pneumoniae isolated from patients with community-acquired urinary tract infection (CA-UTI). Microb Pathog 2016; 100: 244-9.[http://dx.doi.org/10.1016/j.micpath.2016.10.002] [PMID: 27725280]
[17] Pourshafie MR, Bakhshi B, Ranjbar R, et al. Dissemination of a single Vibrio cholerae clone in cholera outbreaks during 2005 in Iran. J Med Microbiol 2007; 56(Pt 12): 1615-9.[http://dx.doi.org/10.1099/jmm.0.47218-0] [PMID: 18033829]
[18] Sadeghifard N, Ranjbar R, Zaeimi J. Antimicrobial susceptibility, plasmid profiles, and RAPD-PCR typing of Acinetobacter bacteria 2011.
[19] Ranjbar R, et al. Molecular characterization of epidemic isolates of Vibrio cholerae O1 by arbitrarily primed PCR (AP-PCR). Iran J Public Health 2008; 37(2): 83-7.
[20] Ranjbar R, Pezeshknejad P, Khamesipour F, Amini K, Kheiri R. Genomic fingerprints of Escherichia coli strains isolated from surface water in Alborz province, Iran. BMC Res Notes 2017; 10(1): 295.[http://dx.doi.org/10.1186/s13104-017-2575-z] [PMID: 28728566]
[21] Ranjbar R, Mirsaeed Ghazi F. Antibiotic sensitivity patterns and molecular typing of Shigella sonnei strains using ERIC-PCR. Iran J Public Health 2013; 42(10): 1151-7.[PMID: 26060624]
[22] Ranjbar R, Hosseini MJ, Kaffashian AR, Farshad S. An outbreak of shigellosis due to Shigella flexneri serotype 3a in a prison in Iran. Arch Iran Med 2010; 13(5): 413-6.[PMID: 20804308]
[23] Sabat AJ, Budimir A, Nashev D, et al. Overview of molecular typing methods for outbreak detection and epidemiological surveillance. Euro Surveill 2013; 18(4): 20380.[http://dx.doi.org/10.2807/ese.18.04.20380-en] [PMID: 23369389]
[24] Khakabimamaghani S, Najafi A, Ranjbar R, Raam M. GelClust: a software tool for gel electrophoresis images analysis and dendrogram generation. Comput Methods Programs Biomed 2013; 111(2): 512-8.[http://dx.doi.org/10.1016/j.cmpb.2013.04.013] [PMID: 23727299]
[25] Jahandeh N, Ranjbar R, Behzadi P, Behzadi E. Uropathogenic Escherichia coli virulence genes: invaluable approaches for designing DNA microarray probes. Cent European J Urol 2015; 68(4): 452-8.[PMID: 26855801]
[26] Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 1987; 169(12): 5429-33.[http://dx.doi.org/10.1128/jb.169.12.5429-5433.1987] [PMID: 3316184]
[27] Al-Attar S, Westra ER, van der Oost J, Brouns SJ. Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes. Biol Chem 2011; 392(4): 277-89.[http://dx.doi.org/10.1515/bc.2011.042] [PMID: 21294681]
[28] Groenen PM, Bunschoten AE, van Soolingen D, van Embden JD. Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method. Mol Microbiol 1993; 10(5): 1057-65.[http://dx.doi.org/10.1111/j.1365-2958.1993.tb00976.x] [PMID: 7934856]
[29] Mojica FJ, Ferrer C, Juez G, Rodríguez-Valera F. Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning. Mol Microbiol 1995; 17(1): 85-93.[http://dx.doi.org/10.1111/j.1365-2958.1995.mmi_17010085.x] [PMID: 7476211]
[30] Goyal M, Saunders NA, van Embden JD, Young DB, Shaw RJ. Differentiation of Mycobacterium tuberculosis isolates by spoligotyping and IS6110 restriction fragment length polymorphism. J Clin Microbiol 1997; 35(3): 647-51.[PMID: 9041405]
[31] Mojica FJ, Díez-Villaseñor C, Soria E, Juez G. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol 2000; 36(1): 244-6.[http://dx.doi.org/10.1046/j.1365-2958.2000.01838.x] [PMID: 10760181]
[32] She Q, Singh RK, Confalonieri F, et al. The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc Natl Acad Sci USA 2001; 98(14): 7835-40.[http://dx.doi.org/10.1073/pnas.141222098] [PMID: 11427726]
[33] Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 2002; 43(6): 1565-75.[http://dx.doi.org/10.1046/j.1365-2958.2002.02839.x] [PMID: 11952905]
[34] Thöny-Meyer L, Kaiser D. devRS, an autoregulated and essential genetic locus for fruiting body development in Myxococcus xanthus. J Bacteriol 1993; 175(22): 7450-62.[http://dx.doi.org/10.1128/jb.175.22.7450-7462.1993] [PMID: 7693658]
[35] Makarova KS, Aravind L, Grishin NV, Rogozin IB, Koonin EV. A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis. Nucleic Acids Res 2002; 30(2): 482-96.[http://dx.doi.org/10.1093/nar/30.2.482] [PMID: 11788711]
[36] Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 2005; 60(2): 174-82.[http://dx.doi.org/10.1007/s00239-004-0046-3] [PMID: 15791728]
[37] Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 2005; 151(Pt 3): 653-63.[http://dx.doi.org/10.1099/mic.0.27437-0] [PMID: 15758212]
[38] Brigulla M, Wackernagel W. Molecular aspects of gene transfer and foreign DNA acquisition in prokaryotes with regard to safety issues. Appl Microbiol Biotechnol 2010; 86(4): 1027-41.[http://dx.doi.org/10.1007/s00253-010-2489-3] [PMID: 20191269]
[39] Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 2005; 151(Pt 8): 2551-61.[http://dx.doi.org/10.1099/mic.0.28048-0] [PMID: 16079334]
[40] Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 2006; 1: 7.[http://dx.doi.org/10.1186/1745-6150-1-7] [PMID: 16545108]
[41] Barrangou R, Marraffini LA. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol Cell 2014; 54(2): 234-44.[http://dx.doi.org/10.1016/j.molcel.2014.03.011] [PMID: 24766887]
[42] Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007; 315(5819): 1709-12.[http://dx.doi.org/10.1126/science.1138140] [PMID: 17379808]
[43] Brouns SJ, Jore MM, Lundgren M, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008; 321(5891): 960-4.[http://dx.doi.org/10.1126/science.1159689] [PMID: 18703739]
[44] Barrangou R. CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip Rev RNA 2013; 4(3): 267-78.[http://dx.doi.org/10.1002/wrna.1159] [PMID: 23520078]
[45] Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 2008; 322(5909): 1843-5.[http://dx.doi.org/10.1126/science.1165771] [PMID: 19095942]
[46] He J, Deem MW. Heterogeneous diversity of spacers within CRISPR (clustered regularly interspaced short palindromic repeats). Phys Rev Lett 2010; 105(12): 128102.[http://dx.doi.org/10.1103/PhysRevLett.105.128102] [PMID: 20867676]
[47] Hyman P, Abedon ST. Bacteriophage host range and bacterial resistance. Adv Appl Microbiol 2010; 70: 217-48.[http://dx.doi.org/10.1016/S0065-2164(10)70007-1] [PMID: 20359459]
[48] Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 2007; 8(1): 172.[http://dx.doi.org/10.1186/1471-2105-8-172] [PMID: 17521438]
[49] van Belkum A, Scherer S, van Alphen L, Verbrugh H. Short-sequence DNA repeats in prokaryotic genomes. Microbiol Mol Biol Rev 1998; 62(2): 275-93.[PMID: 9618442]
[50] Kunin V, Sorek R, Hugenholtz P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol 2007; 8(4): R61.[http://dx.doi.org/10.1186/gb-2007-8-4-r61] [PMID: 17442114]
[51] Lillestøl RK, Redder P, Garrett RA, Brügger K. A putative viral defence mechanism in archaeal cells. Archaea 2006; 2(1): 59-72.[http://dx.doi.org/10.1155/2006/542818] [PMID: 16877322]
[52] Shah SA, Hansen NR, Garrett RA. Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism. Biochem Soc Trans 2009; 37(Pt 1): 23-8.[http://dx.doi.org/10.1042/BST0370023] [PMID: 19143596]
[53] Stern A, Keren L, Wurtzel O, Amitai G, Sorek R. Self-targeting by CRISPR: gene regulation or autoimmunity? Trends Genet 2010; 26(8): 335-40.[http://dx.doi.org/10.1016/j.tig.2010.05.008] [PMID: 20598393]
[54] Tang T-H, Bachellerie JP, Rozhdestvensky T, et al. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc Natl Acad Sci USA 2002; 99(11): 7536-41.[http://dx.doi.org/10.1073/pnas.112047299] [PMID: 12032318]
[55] Hale C, Kleppe K, Terns RM, Terns MP. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 2008; 14(12): 2572-9.[http://dx.doi.org/10.1261/rna.1246808] [PMID: 18971321]
[56] Pul U, Wurm R, Arslan Z, Geissen R, Hofmann N, Wagner R. Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol Microbiol 2010; 75(6): 1495-512.[http://dx.doi.org/10.1111/j.1365-2958.2010.07073.x] [PMID: 20132443]
[57] Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science 2010; 327(5962): 167-70.[http://dx.doi.org/10.1126/science.1179555] [PMID: 20056882]
[58] van der Oost J, Brouns SJ. RNAi: prokaryotes get in on the act. Cell 2009; 139(5): 863-5.[http://dx.doi.org/10.1016/j.cell.2009.11.018] [PMID: 19945373]
[59] Haft DH, Selengut J, Mongodin EF, Nelson KE. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLOS Comput Biol 2005; 1(6): e60.[http://dx.doi.org/10.1371/journal.pcbi.0010060] [PMID: 16292354]
[60] Makarova KS, Haft DH, Barrangou R, et al. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 2011; 9(6): 467-77.[http://dx.doi.org/10.1038/nrmicro2577] [PMID: 21552286]
[61] Louwen R, Staals RH, Endtz HP, van Baarlen P, van der Oost J. The role of CRISPR-Cas systems in virulence of pathogenic bacteria. Microbiol Mol Biol Rev 2014; 78(1): 74-88.[http://dx.doi.org/10.1128/MMBR.00039-13] [PMID: 24600041]
[62] Marraffini LA, Sontheimer EJ. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 2010; 11(3): 181-90.[http://dx.doi.org/10.1038/nrg2749] [PMID: 20125085]
[63] Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats 2007.[http://dx.doi.org/10.1093/nar/gkm360]
[64] Zegans ME, Wagner JC, Cady KC, Murphy DM, Hammond JH, O’Toole GA. Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa. J Bacteriol 2009; 191(1): 210-9.[http://dx.doi.org/10.1128/JB.00797-08] [PMID: 18952788]
[65] Edgar R, Qimron U. The Escherichia coli CRISPR system protects from λ lysogenization, lysogens, and prophage induction. J Bacteriol 2010; 192(23): 6291-4.[http://dx.doi.org/10.1128/JB.00644-10] [PMID: 20889749]
[66] Xiao-Jie L, Hui-Ying X, Zun-Ping K, Jin-Lian C, Li-Juan J. CRISPR-Cas9: a new and promising player in gene therapy. J Med Genet 2015; 52(5): 289-96.[http://dx.doi.org/10.1136/jmedgenet-2014-102968] [PMID: 25713109]
[67] Lyons C, Raustad N, Bustos MA, Shiaris M. Incidence of Type II CRISPR1-Cas Systems in Enterococcus Is Species-Dependent. PLoS One 2015; 10(11): e0143544.[http://dx.doi.org/10.1371/journal.pone.0143544] [PMID: 26600384]
[68] Shariat N, Timme RE, Pettengill JB, Barrangou R, Dudley EG. Characterization and evolution of Salmonella CRISPR-Cas systems. Microbiology 2015; 161(2): 374-86.[http://dx.doi.org/10.1099/mic.0.000005] [PMID: 28206902]
[69] Bikard D, Hatoum-Aslan A, Mucida D, Marraffini LA. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. Cell Host Microbe 2012; 12(2): 177-86.[http://dx.doi.org/10.1016/j.chom.2012.06.003] [PMID: 22901538]
[70] Zheng P-X, Chan YC, Chiou CS, et al. Clustered Regularly Interspaced Short Palindromic Repeats Are emm Type-Specific in Highly Prevalent Group A Streptococci. PLoS One 2015; 10(12): e0145223.[http://dx.doi.org/10.1371/journal.pone.0145223] [PMID: 26710228]
[71] Hargreaves KR, Flores CO, Lawley TD, Clokie MR. Abundant and diverse clustered regularly interspaced short palindromic repeat spacers in Clostridium difficile strains and prophages target multiple phage types within this pathogen. MBio 2014; 5(5): e01045-13.[http://dx.doi.org/10.1128/mBio.01045-13] [PMID: 25161187]
[72] Andersen JM, Shoup M, Robinson C, Britton R, Olsen KE, Barrangou R. CRISPR diversity and microevolution in Clostridium difficile. Genome Biol Evol 2016; 8(9): 2841-55.[http://dx.doi.org/10.1093/gbe/evw203] [PMID: 27576538]
[73] Briner AE, Barrangou R. Lactobacillus buchneri genotyping on the basis of clustered regularly interspaced short palindromic repeat (CRISPR) locus diversity. Appl Environ Microbiol 2014; 80(3): 994-1001.[http://dx.doi.org/10.1128/AEM.03015-13] [PMID: 24271175]
[74] Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. science, 2008; 1843-5.[http://dx.doi.org/10.1126/science.1165771]
[75] Di H, Ye L, Yan H, Meng H, Yamasak S, Shi L. Comparative analysis of CRISPR loci in different Listeria monocytogenes lineages. Biochem Biophys Res Commun 2014; 454(3): 399-403.[http://dx.doi.org/10.1016/j.bbrc.2014.10.018] [PMID: 25445602]
[76] Mokrousov I, Narvskaya O, Limeschenko E, Vyazovaya A. Efficient discrimination within a Corynebacterium diphtheriae epidemic clonal group by a novel macroarray-based method. J Clin Microbiol 2005; 43(4): 1662-8.[http://dx.doi.org/10.1128/JCM.43.4.1662-1668.2005] [PMID: 15814981]
[77] Mokrousov I, Limeschenko E, Vyazovaya A, Narvskaya O. Corynebacterium diphtheriae spoligotyping based on combined use of two CRISPR loci. Biotechnol J 2007; 2(7): 901-6.[http://dx.doi.org/10.1002/biot.200700035] [PMID: 17431853]
[78] Mokrousov I, Vyazovaya A, Kolodkina V, Limeschenko E, Titov L, Narvskaya O. Novel macroarray-based method of Corynebacterium diphtheriae genotyping: evaluation in a field study in Belarus. Eur J Clin Microbiol Infect Dis 2009; 28(6): 701-3.[http://dx.doi.org/10.1007/s10096-008-0674-4] [PMID: 19089478]
[79] de Cárdenas I, Fernández-Garayzábal JF, de la Cruz ML, Domínguez L, Ugarte-Ruiz M, Gómez-Barrero S. Efficacy of a typing scheme for Campylobacter based on the combination of true and questionable CRISPR. J Microbiol Methods 2015; 119: 147-53.[http://dx.doi.org/10.1016/j.mimet.2015.10.020] [PMID: 26518609]
[80] Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 2005; 151(Pt 3): 653-63.[http://dx.doi.org/10.1099/mic.0.27437-0] [PMID: 15758212]
[81] Díez-Villaseñor C, Almendros C, García-Martínez J, Mojica FJ. Diversity of CRISPR loci in Escherichia coli. Microbiology 2010; 156(5): 1351-61.[http://dx.doi.org/10.1099/mic.0.036046-0] [PMID: 28206910]
[82] Fabre L, Zhang J, Guigon G, et al. CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections. PLoS One 2012; 7(5): e36995.[http://dx.doi.org/10.1371/journal.pone.0036995] [PMID: 22623967]
[83] Li H, Li P, Xie J, et al. New clustered regularly interspaced short palindromic repeat locus spacer pair typing method based on the newly incorporated spacer for Salmonella enterica. J Clin Microbiol 2014; 52(8): 2955-62.[http://dx.doi.org/10.1128/JCM.00696-14] [PMID: 24899040]
[84] Le Hello S, Maillard F, Mallet HP, et al. Salmonella enterica serotype enteritidis in French Polynesia, South Pacific, 2008-2013. Emerg Infect Dis 2015; 21(6): 1045-8.[http://dx.doi.org/10.3201/eid2106.141103] [PMID: 25988406]
[85] Guo X, Wang Y, Duan G, et al. Detection and analysis of CRISPRs of Shigella. Curr Microbiol 2015; 70(1): 85-90.[http://dx.doi.org/10.1007/s00284-014-0683-8] [PMID: 25199561]
[86] Ostria-Hernández ML, Sánchez-Vallejo CJ, Ibarra JA, Castro-Escarpulli G. Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae. BMC Res Notes 2015; 8(1): 332.[http://dx.doi.org/10.1186/s13104-015-1285-7] [PMID: 26238567]
[87] Sola C. Clustured regularly interspersed short palindromic repeats (CRISPR) genetic diversity studies as a mean to reconstruct the evolution of the Mycobacterium tuberculosis complex. Tuberculosis (Edinb) 2015; 95(Suppl. 1): S159-66.[http://dx.doi.org/10.1016/j.tube.2015.02.029] [PMID: 25748060]
[88] Kamerbeek J, Schouls L, Kolk A, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997; 35(4): 907-14.[PMID: 9157152]
[89] Le Hello S, Maillard F, Mallet HP, et al. Salmonella enterica serotype enteritidis in French Polynesia, South Pacific, 2008-2013. Emerg Infect Dis 2015; 21(6): 1045-8.[http://dx.doi.org/10.3201/eid2106.141103] [PMID: 25988406]
[90] Zheng J, Pettengill J, Strain E, et al. Genetic diversity and evolution of Salmonella enterica serovar Enteritidis strains with different phage types. J Clin Microbiol 2014; 52(5): 1490-500.[http://dx.doi.org/10.1128/JCM.00051-14] [PMID: 24574287]
[91] Fabre L, Le Hello S, Roux C, Issenhuth-Jeanjean S, Weill FX. CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A. PLoS Negl Trop Dis 2014; 8(1): e2671.[http://dx.doi.org/10.1371/journal.pntd.0002671] [PMID: 24498453]

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