Clustered Regularly Interspaced Short Palindromic Repeats Sequences Research Articles

Genomic characterization and comparative genomic analysis of pathogenic Vibrio isolated from aquaculture-grown white-leg shrimp (Penaeus vannamei) in Guangdong and Jiangsu, China

The proliferation of opportunistic pathogens Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio owensii have impeded the rapid development of aquaculture farming. The study aimed to analyze the whole genome of pathogenic Vibrio strains associated with hepatopancreatic necrosis in Penaeus vannamei. The analysis included genomic characterization, virulence genes profiling, antibiotic resistance genes (ARGs) identification, antimicrobial resistance phenotypes, clustered regularly interspaced short palindromic repeats (CRISPR) and prophage prediction, multi-locus sequence typing, and pan-genome analysis of Vibrio isolates from Guangdong and Jiangsu provinces, China. Sequence analysis of the specimens revealed 29 V. parahaemolyticus isolates, three V. alginolyticus isolates, and one V. owensii isolate. Their genomes are 4.9–5.9 Mbp in size, with a GC content of 44.4–45.4%. Each Vibrio isolate harbored >120 virulence genes. Out of the 33 isolates, 13 (39%) demonstrated multidrug-resistant (MDR). Notably, at least 14 isolates exhibited heightened resistance to sulfamethoxazole, erythromycin, and amoxicillin, with a cumulative detection of 20 ARGs. Nevertheless, no significant correlation was found between the composition of ARGs and the observed antimicrobial resistance phenotypes. All Vibrio isolates differed in the presence of prophage and CRISPR sequences; and two of these prophages harbored virulence genes but not ARGs. The study identified 17 distinct sequence types (STs) in V. parahaemolyticus isolates, primarily ST415 (n = 5), while two Vibrio isolates were classified as ST3. Using the 117 V. parahaemolyticus strains available in the National Center for Biotechnology Information database as a reference, pan-genomic analysis and phylogenetic analysis of the core genome revealed that clinical isolates of the same STs clustered into the same clade, irrespective of isolation date and geographic origins. In summary, these data enhance our understanding of the molecular characteristics and epidemiological risk of Vibrio strains in southern China, especially regarding shrimp aquaculture.

CRISPR-Cas-Based Adaptive Immunity Mediates Phage Resistance in Periodontal Red Complex Pathogens.

Periodontal diseases are polymicrobial immune-inflammatory diseases that can severely destroy tooth-supporting structures. The critical bacteria responsible for this destruction include red complex bacteria such as Porphoromonas gingivalis, Tanerella forsythia and Treponema denticola. These organisms have developed adaptive immune mechanisms against bacteriophages/viruses, plasmids and transposons through clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas). The CRISPR-Cas system contributes to adaptive immunity, and this acquired genetic immune system of bacteria may contribute to moderating the microbiome of chronic periodontitis. The current research examined the role of the CRISPR-Cas system of red complex bacteria in the dysbiosis of oral bacteriophages in periodontitis. Whole-genome sequences of red complex bacteria were obtained and investigated for CRISPR using the CRISPR identification tool. Repeated spacer sequences were analyzed for homologous sequences in the bacteriophage genome and viromes using BLAST algorithms. The results of the BLAST spacer analysis for T. denticola spacers had a 100% score (e value with a bacillus phage), and the results for T. forsthyia and P. gingivalis had a 56% score with a pectophage and cellulophage (e value: 0.21), respectively. The machine learning model of the identified red complex CRISPR sequences predicts with area an under the curve (AUC) accuracy of 100 percent, indicating phage inhibition. These results infer that red complex bacteria could significantly inhibit viruses and phages with CRISPR immune sequences. Therefore, the role of viruses and bacteriophages in modulating sub-gingival bacterial growth in periodontitis is limited or questionable.

Comparative genomics reveals the diversity of CRISPR-Cas locus in Azotobacter organisms.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are known to provide adaptive immunity to bacteria against invading bacteriophages. In recent years, CRISPR-based technologies have been used for creating improved plant varieties; however, the indigenous CRISPR-Cas elements of plant growth-promoting bacteria are usually neglected. These indigenous genetic cassettes have evolved over millions of years and have shaped the bacterial genome. Therefore, these genetic loci can be used to study the adaptive capability of the bacteria in the environment. This study aims to bioinformatically analyze the genomes of a common free-living nitrogen-fixing Azotobacter spp. to assess their CRISPR-Cas diversity. Strains of Azotobacter vinelandii and Azotobacter chroococcum were found to harbor a large number of spacers. The phylogeny of different Cas and Cse1 proteins revealed a close evolutionary relationship among A. chroococcum B3, A. chroococcum NCIMB 8003 locus II, and A. vinelandii DJ locus I. The secondary structure of the hairpin loop of the repeat was also analyzed, and a correlation was derived between the structural stability of the hairpin loop and the number of spacers acquired by the CRISPR loci. These findings revealed the diversity and evolution of the CRISPR sequences and Cas proteins in Azotobacter species. Although the adaptive immune system of bacteria against bacteriophage, CRISPR-Cas, has been identified in many bacteria, studies of plant growth-promoting bacteria have been neglected. These indigenous CRISPRs have shaped the genome over millions of years and their study can lead to the understanding of the genome composition of these organisms. Our results strengthen the idea of using A. chroococcum and A. vinelandii as biofertilizer strains as they possess more spacers with highly stable repeat sequences, thereby imparting them higher chance of survival against mobile genetic elements like phages and plasmids.

CRISPR-CAS9 Sisteminde Kullanılan Polimer Bazlı Transfeksiyon Ajanları

Genome editing is a method used to make desired changes in the target gene. Today, various methods are used for genome-editing studies; among them, one of the most widely used methods is the clustered, regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated (Cas) genes and their corresponding CRISPR sequences constitute CRISPR-Cas systems. Due to its simplicity, it is likely that the CRISPR–Cas system could be used effectively in ex vivo gene therapy studies in humans. If this happens, the importance of CRISPR carrier systems will gradually increase. Viral and non-viral systems are used as delivery modalities in genome-editing studies. It has been proven that nanoparticles are the most promising tools for gene therapy due to their adjustable size, surface, shape, and biological behaviours. The polymeric carrier system has become the main non-viral substitute for gene delivery due to its reduced immunogenicity and pathogenicity. In this review, information about current studies related to polymeric carriers used in non-viral CRISPR delivery systems is presented.

Detection of Campylobacter jejuni diversity by clustered regularly interspaced short palindromic repeats (CRISPR) from an animal farm

Background Campylobacter jejuni is the leading bacterial pathogen that causes foodborne illness worldwide. Because of genetic diversity and sophisticated growth requirements of C. jejuni, several genotyping methods have been investigated to classify this bacterium during the outbreaks. One of such method is to use clustered regularly interspaced short palindromic repeats (CRISPR).ObjectivesThe goal of this study was to explore the diversity of C. jejuni isolates with CRISPR from an animal farm.MethodsSeventy‐seven C. jejuni isolates from an animal farm were used in this study. The day‐old broilers were reared with other poultry and farm animals, including layer hens, guinea hens, dairy goats and sheep. A small swine herd was also present on an adjacent, but separate plot of land. Isolation and identification of C. jejuni were performed according to the standard procedures. The CRISPR type 1 was PCR amplified from genomic DNA, and the amplicons were sequenced by the Sanger dideoxy method. The direct repeats (DRs) and spacers of the CRISPR sequences were identified using the CRISPRFinder.ResultsThe CRISPR sequences were detected in all 77 isolates. One type of DRs was identified in these 77 isolates. The lengths of the CRISPR locus ranged from 100 to 560 nucleotides, whereas the number of spacers ranged from one to eight. The distributions of the numbers of CRISPR spacers from different sources seemed to be random. Overall, 17 out of 77 (22%) C. jejuni isolates had two and five spacers, whereas 14 out of 77 (18%) isolates had three spaces in their genomes. By further analysis of spacer sequences, a total of 266 spacer sequences were identified in 77 C. jejuni isolates. By comparison with known published spacer sequences, we observed that 49 sequences were unique in this study. The CRISPR sequence combination of Nos. 16, 19, 48 and 57 was found among a total of 15 C. jejuni isolates containing various multi‐locus sequence typing (MLST) types (ST‐50, ST‐607, ST‐2231 and ST‐5602). No. 57 spacer sequence was unique from this study, whereas the other three (Nos. 16, 19 and 48) sequences were found in previous reports. Combination of Nos. 5, 9, 15, 30 and 45 was associated with ST‐353. To compare the CRISPR genotyping with other methods, the MLST was selected due to its high discriminatory power to differentiate isolates. Based on calculation of the Simpson's index of diversity, a combination of both methods had higher Simpson's index value than those for CRISPR or MLST, respectively.ConclusionsOur results suggest that the MLST from C. jejuni isolates can be discriminated based on the CRISPR unique spacer sequences and the numbers of spacers. In the future, investigation on the CRISPR resolution for C. jejuni identification in outbreaks is needed. A database that integrates both MLST sequences and CRISPR sequences and is searchable is greatly in demand for tracking outbreaks and evolution of this bacterium.

Bioinformatics investigation of CRISPR/Cas systems in Bifidobacterium longum

Bifidobacterium longum is one of the most important probiotics used in the food industries. These bacteria are also applied for the treatment or prevention of gastrointestinal and gynecological diseases as well the regulation of immune system. On the other hand, the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) systems has revolutionized gene therapy approaches and provided a novel tool for gene editing. Several investigators are seeking for potential CRISPR sequences as well the CRISPR-associated (Cas) proteins in various microorganism. Considering the importance of B. longum and its safe application in food industry, the aim of the present study is to investigate the presence and characteristics of CRISPRs in this bacterium via in silico study in 96 strains of the bacteria. According to the results obtained in this study, nine B. longum strains demonstrated high occurrence of CRISPR arrays. These arrays could probably form four RNA secondary structures. Since the bacterium B. longum have been widely used as a safe microorganism in foods, the first report on the probable presence of CRISPR sequences may provide the opportunity to introduce the bacteria as a gene editing tool.

Virulence Variation of Salmonella Gallinarum Isolates through SpvB by CRISPR Sequence Subtyping, 2014 to 2018.

Simple SummarySalmonella Gallinarum causes fowl typhoid in all ages of chickens, which results in economic loss of commercial chicken farms. The disease has been eradicated in many developed countries, but is still prevalent in Korea. In this study, we investigated virulence and genetic variation of S. Gallinarum from Korea, between 2014 and 2018. The results indicated that virulence was increased, which was associated with genetic change over time. Therefore, surveillance of genetic change associated with virulence increase is necessary for monitoring of S. Gallinarum isolates for dissemination.Salmonella Gallinarum is a Gram-negative bacteria that causes fowl typhoid, a septicemic disease with high morbidity and mortality that affects all ages of chickens. Although vaccines and antimicrobials have been used nationwide to eradicate the disease, the malady is still prevalent in Korea. In this study, we investigated the virulence and genetic variation of 116 S. Gallinarum isolates from laying hens between 2014 and 2018. A total of 116 isolates were divided into five Gallinarum Sequence Types (GST) through clustered regularly interspaced short palindromic repeats (CRISPR) subtyping method. The GSTs displayed changes over time. The 116 isolates showed no difference in virulence gene distribution, but the polyproline linker (PPL) length of the SpvB, one of the virulence factors of Salmonella spp., served as an indicator of S. Gallinarum pathogenicity. The most prevalent PPL length was 22 prolines (37.9%). The shortest PPL length (19 prolines) was found only in isolates from 2014 and 2015. However, the longest PPL length of 24 prolines appeared in 2018. This study indicates that PPLs of S. Gallinarum in Korea tend to lengthen over time, so the pathogenic potency of the bacteria is increasing. Moreover, the transition of GST was associated with PPL length extension over time. These results indicate that surveillance of changing GST and PPL length are necessary in the monitoring of S. Gallinarum isolates.

CRISPR Genome Editing Technology and its Application in Genetic Diseases: A Review.

Gene therapy has been a long lasting goal for scientists, and there are many optimal methods and tools to correct disease-causing mutations in humans. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been progressively adopted for the assessment a treatment of human diseases, including thalassemia, Parkinson's disease, cystic fibrosis, glaucoma, Huntington's disease, and Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS). CRISPR sequences belong to the bacterial immune system, which includes the nuclease Cas enzyme and an RNA sequence. The RNA sequence is unique and pathogen-specific, and identifies and binds to the DNA of invasive viruses, allowing the nuclease Cas enzyme to cut the identified DNA and destroy the invasive viruses. This feature provides the possibility to edit mutations in the DNA sequence of live cells by replacing a specific targeted RNA sequence with the RNA sequence in the CRISPR system. Previous studies have reported the improvement steps in confrontation with human diseases caused by single-nucleotide mutations using this system. In this review, we first introduce CRISPR and its functions and then elaborate on the use of CRISPR in the treatment of human diseases.

Genotyping of Campylobacter jejuni Isolates from Poultry by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

Campylobacter jejuni is the leading bacterial foodborne pathogen that causes human acute gastrointestinal illness worldwide. Due to its genetic diversity, fastidious growth and sophisticated biochemical requirements, classification of Campylobacter by traditional techniques is problematic. Several molecular typing methods have been explored in this bacterium. One such method is to use clustered regularly interspaced short palindromic repeats (CRISPR). These CRISPRs consist of a direct repeat interspaced with nonrepetitive spacer sequences. In this study, we applied this genotyping method to explore the genetic diversity of C. jejuni isolated from poultry sources. Ninety-nine C. jejuni isolates from poultry environments in four different US states were used. Genomic DNA of the isolates were extracted from cultures using a commercial kit. PCR primers and conditions for CRISPR type 1 amplification were described previously. The amplicons were purified and sequenced by the Sanger dideoxy sequencing method. The direct repeats (DR) and spacers of the CRISPR sequences were identified using the CRISPRFinder. The results show there were 21% isolates no detectable, 30% isolates questionable, and 49% isolates confirmed CRISPR, respectively. The lengths of CRISPR range from 100 to 695 nucleotides. One type of DR was found in CRISPR of these isolates. The number of spacers in CRISPR ranges from 1 to 10 with various sequences. A total of 55 distinctive spacer sequences were identified in 78 isolates. Among them, 33 sequences were found unique in this study. In addition, the CRISPR genotyping had higher the Simpson's index of diversity value than that from flaA nucleotide typing. The results of our study show the CRISPR genotyping on C. jejuni may be complementary to the other genotyping methods.

CRISPR/Cas9-Mediated Gene Knockout and Knockin Human iPSCs.

The realization of the full potential of human pluripotent stem cells (hPSCs), including human induced PSCs (iPSC), relies on the ability to precisely edit their genome in a locus-specific and multiplex manner. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) serve as a guide for the endonuclease Cas9 (CRISPR-associated protein 9) to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. CRISPR/Cas9-mediated editing has become the gold standard for precise genome manipulation as it offers a unique, versatile, and limitless tool for fast, robust, and efficient genome editing. Here, we provide a protocol to successfully generate gene knockout and/or knockin iPSCs. We include detailed information on the design of guide RNAs (gRNAs), T7 endonuclease assay to detect on-target CRISPR/Cas9 editing events, DNA electroporation of the iPSCs with a ribonucleoprotein complex, and single-cell cloning steps for the selection of the genome-edited iPSC clones.

Pangenome of Acinetobacter baumannii uncovers two groups of genomes, one of them with genes involved in CRISPR/Cas defence systems associated with the absence of plasmids and exclusive genes for biofilm formation.

Acinetobacter baumannii is an opportunistic bacterium that causes hospital-acquired infections with a high mortality and morbidity, since there are strains resistant to virtually any kind of antibiotic. The chase to find novel strategies to fight against this microbe can be favoured by knowledge of the complete catalogue of genes of the species, and their relationship with the specific characteristics of different isolates. In this work, we performed a genomics analysis of almost 2500 strains. Two different groups of genomes were found based on the number of shared genes. One of these groups rarely has plasmids, and bears clustered regularly interspaced short palindromic repeat (CRISPR) sequences, in addition to CRISPR-associated genes (cas genes) or restriction-modification system genes. This fact strongly supports the lack of plasmids. Furthermore, the scarce plasmids in this group also bear CRISPR sequences, and specifically contain genes involved in prokaryotic toxin–antitoxin systems that could either act as the still little known CRISPR type IV system or be the precursors of other novel CRISPR/Cas systems. In addition, a limited set of strains present a new cas9-like gene, which may complement the other cas genes in inhibiting the entrance of new plasmids into the bacteria. Finally, this group has exclusive genes involved in biofilm formation, which would connect CRISPR systems to the biogenesis of these bacterial resistance structures.

Exploring Clustered Regularly Interspaced Short Palindromic Repeats Diversity in Soil Bacteria

Clustered regularly interspaced short palindromic repeats (CRISPR) is a defense system found in bacteria (and archaea) against bacteriophages, viruses that infect bacteria. CRISPR DNA sequence consists of short, identical nucleotide repeats that are interspaced by spacers. The spacer sequences are records of viral infections experienced by the bacteria and play the role of an “immunization card”: if the spacer sequences match the infecting viral DNA, the bacteria will cut the viral DNA with CRISPR‐associated (Cas) proteins, eliminating the threat of viral infection. We speculate that the spacer sequences reflect the history of bacteriophage infections, which are unique to the environment bacteria reside in. Environmental factors, such as location and time, weather, local ecosystems, and pollutions, will alter the spacer sequences found in bacteria. This research focuses on investigating and discovering new spacer sequences from soil samples collected around NY/NJ metropolitan area, a historically heavily polluted urban ecosystem, at various locations, timepoints, and weather conditions. To accomplish this goal, the genomic DNA was extracted and purified from the collected soil samples. The CRISPR locus was amplified by using polymerase chain reaction (PCR) and then purified via gel electrophoresis. The purified CRISPR DNA was then ligated into a p‐Drive vector and transformed into M15 competent cells. The cells were cultured to amply the vector containing ligated CRISPR sequence, which was subsequently analyzed by aligning against CRISPR spacer database to identify new space sequence. We expect that the newly discovered spacer sequences reflect the history of viral infections unique to the regions investigated, which can potentially inform the understanding of health risks related to environmental bacteria and viruses.Support or Funding Information‐ U.S. Education Department Minority Science & Engineering Improvement Program grant # P120A160084‐ NIH AREA Grant # CA179410‐ USED Title III Part F, HSI‐STEM & Articulation Grant #P03C160155This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Analysis of genetic diversity of Xanthomonas oryzae pv. oryzae populations in Taiwan

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major rice disease. In Taiwan, the tropical indica type of Oryza sativa originally grown in this area is mix-cultivated with the temperate japonica type of O. sativa, and this might have led to adaptive changes of both rice host and Xoo isolates. In order to better understand how Xoo adapts to this unique environment, we collected and analyzed fifty-one Xoo isolates in Taiwan. Three different genetic marker systems consistently identified five groups. Among these groups, two of them had unique sequences in the last acquired ten spacers in the clustered regularly interspaced short palindromic repeats (CRISPR) region, and the other two had sequences that were similar to the Japanese isolate MAFF311018 and the Philippines isolate PXO563, respectively. The genomes of two Taiwanese isolates with unique CRISPR sequence features, XF89b and XM9, were further completely sequenced. Comparison of the genome sequences suggested that XF89b is phylogenetically close to MAFF311018, and XM9 is close to PXO563. Here, documentation of the diversity of groups of Xoo in Taiwan provides evidence of the populations from different sources and hitherto missing information regarding distribution of Xoo populations in East Asia.

An exciting time for genome editing: an interview with David R. Liu.

In 1987, several Osaka University researchers discovered a special kind of clustered DNA repeats in bacteria. Within a few years, two other groups independently discovered the same phenomenon but no one knew its function at the time. Only a small handful of scientists studied this property from its discovery in 1987 to 2005. It was then that the function of these DNA repeats, which were named Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), was finally elucidated. Researchers found that CRISPR, when combined with its CRISPR-associated partner (Cas), is crucial for the functioning of the bacterial adaptive immune system against viral phage infection. CRISPR sequences can be transcribed into targeting RNA molecules, and Cas enzymes are guided by these RNAs to cut specific viral DNA loci, rendering resistance against the viral infection.Scientists realized that this natural bacterial immune response system could be engineered to become a powerful genome editing tool. Prior to CRISPR, existing genome editing tools such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) relied solely upon protein–DNA interactions to target an enzyme to specific DNA sequences. The design, engineering and evolution of proteins for various DNA sequences is difficult and time-consuming. In contrast, the CRISPR-Cas system uses Watson–Crick base pairing between a guide RNA and the target DNA to localize the complex to specific DNA sequences. This feature enables users to simply change an RNA sequence to match a DNA target to reposition the whole complex.Since then, numerous talented scientists have headed into this field. Within a single decade, they have developed the CRISPR-Cas system into a powerful genome editing tool and applied it to the editing of microorganisms, plants, animals and even human embryos. David R. Liu, Professor of Harvard University and the Broad Institute, and an investigator of the Howard Hughes Medical Institute, is one of them. One of his major contributions to the field is the development of ‘base editing’. His group engineered the CRISPR system to transform it from being DNA scissors that cut DNA into specific DNA base pair rewriters that directly convert one base pair to a different base pair. This development opens the door to precision genome editing, raising the possibility of treating thousands of genetic diseases that are caused by single point mutations in the human genome. Here, David talks about this exciting time for genome editing.

Characterization and CRISPR-based genotyping of clinical trh-positive Vibrio parahaemolyticus

BackgroundVibrio parahaemolyticus is a causative agent of gastroenteritis. Most of the clinical isolates carry either tdh and/or trh genes which are considered as the major virulence genes of this pathogen. In this study, the clinical isolates of V. parahaemolyticus carrying trh gene (n = 73) obtained from 1886 to 2012 from various countries were investigated for the urease production, haemolytic activity, and biofilm formation. In addition, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)-based genotyping among these isolates was investigated.ResultsIn this study, no significant differences were observed in the urease production between tdh+trh1+ and tdh+trh2+ isolates (p = 0.063) and between the tdh−trh1+ and tdh−trh2+ isolates (p = 0.788). The isolates carrying only the trh gene showed variation in their haemolytic activity. The ratio of urease production and haemolytic activity between the trh1+ and trh2+ isolates and biofilm formation of trh+V. parahaemolyticus isolates were not significantly different. Sixteen of thirty-four tested isolates (47.0%) of trh+V. parahaemolyticus were positive for CRISPR detection. The discriminatory power index (DI) of CRISPR-virulence typing was higher than the DI obtained by CRISPR typing alone.ConclusionThe tdh and trh genes were not involved in urease production in the trh+V. parahaemolyticus, and variation of haemolytic activity detected in V. parahaemolyticus carrying only the trh gene might be correlated to the sequence variation within trh1 and trh2 genes. Additionally, biofilm production of V. parahaemolyticus was not associated with harboring of virulence genes. For genotyping, CRISPR sequences combined with virulence genes can be used as genetic markers to differentiate trh+V. parahaemolyticus strains.

Incomplete Selective Sweeps of Microcystis Population Detected by the Leader-End CRISPR Fragment Analysis in a Natural Pond.

The freshwater cyanobacterium Microcystis aeruginosa frequently forms toxic massive blooms and exists in an arms race with its infectious phages in aquatic natural environments, and as a result, has evolved extremely diverse and elaborate antiviral defense systems, including the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated genes (Cas) system. Here, to assess Microcystis population dynamics associated with exogenous mobile genetic elements such as phages and plasmids, we examined the temporal variation in CRISPR genotypes (CTs) by analyzing spacer sequences detected in a natural pond between June and October 2013 when a cyanobacterial bloom occurred. A total of 463,954 high-quality leader-end CRISPR sequences were obtained and the sequences containing spacers were classified into 31 previously reported CTs and 68 new CTs based on the shared order of the leader-end spacers. CT19 was the most dominant genotype (32%) among the 16 most common CTs, followed by CT52 (14%) and CT58 (9%). Spacer repertoires of CT19 showed mainly two different types; CT19origin, which was identical to the CT19 spacer repertoire of previously isolated strains, and CT19new+, which contained a new spacer at the leader-end of the CRISPR region of CT19origin, which were present in almost equal abundance, accounting for up to 99.94% of CT19 sequences. Surprisingly, we observed the spacer repertoires of the second to tenth spacers of CT19origin at the most leader-end of proto-genotype sequences of CT19origin. These were observed during the sampling in this study and our previous study at the same ecosystem in 2010 and 2011, suggesting these CTs persisted from 2011 to 2013 in spite of phage pressure. The leader-end variants were observed in other CT genotypes. These findings indicated an incomplete selective sweep of Microcystis populations. We explained the phenomenon as follow; the abundance of Microcystis varied seasonally and drastically, resulting that Microcystis populations experience a bottleneck once a year, and thereby founder effects following a bottleneck mean that older CTs have an equal chance of increasing in prevalence as the CTs generated following acquisition of newer spacers.

Global acquisition of genetic material from different bacteria into the staphylococcal cassette chromosome elements of a Staphylococcus epidermidis isolate

Staphylococcus epidermidis has been suggested as a main reservoir of methicillin resistance and virulence genes facilitating the evolution of Staphylococcus aureus as a successful pathogen. However, it remains a mystery where and how S. epidermidis obtains these numerous genes to serve as the reservoir. In this study, methicillin-resistant S. epidermidis isolate NW32 from a mastitic milk sample was sequenced and its staphylococcal cassette chromosome (SCC) elements were characterised. The SCC composite island covered 3.5% of the genome and consisted of three intact SCC elements carrying resistance genes against β-lactam antibiotics, several heavy metals and polyamines as well as genes for utilisation of sorbitol as a carbon source. Analysis of the postulated evolutionary route suggested that the three SCC elements were assembled from genetic material from various bacterial species (staphylococci, streptococci, salinicocci and Lysinibacillus) from three habitats (human, soil and cow) in different countries (Asia, North America, South America and Europe). We propose that the hsdS restriction-modification profile and the lack of CRISPR (clustered regularly interspaced short palindromic repeat) sequences in this bacterium may facilitate the genetic exchange of SCC elements among different staphylococcal species.

The emerging role of CRISPR-Cas9 in molecular oncology

It is not uncommon to be curious about the recent hype surrounding the new gene editing player, Cas9, which recognizes and holds into place DNA segments known as clustered regularly interspaced short palindromic repeats (CRISPR). Together, they are known as CRISPR-Cas9 or simply “CRISPR” for brevity. The binding of Cas9 causes the CRISPR sequences to become available for editing.

Genetic analysis of Salmonella enterica serovar Gallinarum biovar Pullorum based on characterization and evolution of CRISPR sequence

Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the cause of pullorum disease, characterized by white diarrhea, which leads to high mortality in poultry. In this study, we aimed to assess the genetic diversity of 655 S. Pullorum strains from 1962 to 2015 in China, Europe, and South America. A sequence typing scheme based on clustered regularly interspaced short palindromic repeats (CRISPR) was used to reveal the genetic relationships among these strains in this study. Overall, a total of 20 Pullorum sequence types (PSTs) of CRISPR were identified in the 655 isolates with PST7 (74%, 486/655) and PST3 (13%, 86/655) to be the most two frequent PSTs belonging to two different lineages, which confirmed the genetic conservation of S. Pullorum strains isolated from six provinces and two direct-controlled municipalities (Beijing and Shanghai) in China. However, the identification of seven new PSTs distributed in strains isolated since 2001 implied that genetic variation continues to develop in S. Pullorum. Interestingly, the whole-genome single-nucleotide polymorphism typing (WGST) of 96 strains out of the 655 isolates divided them into four lineages based on SNP analysis of core genomic sequence and exhibit good correspondence with the CRISPR subtyping method. Notably, 22 out of 26 isolates from Europe and South America were distributed in five distinctive PSTs (with no Chinese strains). Additionally, CRISPR data of spacers and their arrangement exhibit subtle but distinct specificity between different strains, and the dynamic adaptive nature of CRISPR loci provides critical insights into the evolution of S. Pullorum as the bacteria are influenced by their environment.

Comparative analysis of CRISPR-Cas systems of <em>Yersinia pestis</em> and <em>Escherichia coli</em> strains

The CRISPR-Cas system primarily refers to the clustered regularly interspaced short palindromic repeats (CRISPR) and its related protein enzymes (Cas) that confer adaptive protection against bacteriophages and other exogenous elements. The CRISPR-spacers that constitute the CRISPR-Cas systems have been reportedly found in Prokaryotes and Archea. This study analyzed and compared genome assemblies of E. coli and Yersinia pestis with the aid of bioinformatics tools (MinCED, MUSCLE alignment tool, nucleotide Basic Local Alignment Search tool (nBLAST), and prokaryotic database of the National Centre for Biotechnology Information (NCBI). The CRISPR-spacer analyses of meta-genomic sequences of 162 Escherichia coli and 121 Yersinia pestis strains showed the presence of 4 CRISPR classes (I, II, III, and IV) in E. coli and 3 CRISPR classes (I, II, and III) in Y. pestis, respectively. The result of MinCED-CRISPR analysis of CRISPR size of genome assemblies of E. coli and Y. pestis strains showed that E. coli CRISPR class I had the highest percentage value (51.9%) compared with E. coli class II (42%), Y. pestis CRISPR class I (40%), Y. pestis CRISPR class II (36%), Y. pestis CRISPR class III (24%), E. coliISPR class class III (4.8%) and E. coli class IV (1%). It further revealed that no CRISPR sequences were found in class IV of Y. pestis strains. The result of CRISPR-spacer contents also showed that the E. coli CRISPR class I showed the highest significant percentage value (54.6%) than Y. pestis CRISPR class I (36.93%), Y. pestis CRISPR class II (41.13%), Y. pestis CRISPR class III (21.93%), E. coli CRISPR class II (42.18%), E. coli class III (2.86%) and E. coli class IV (0.33%). It was observed that there was absence of CRISPR class IV in the genomic sequences of Y. pestis strains analyzed. The percentage distribution of exogenous spacers among E. coli CRISPR classes revealed that CRISPR class I showed 65.77% homologous sequence match with plasmids spacers, 51.96% bacteriophages spacers, 68.26% bacterial spacers, and 20.18% unknown target spacers, while CRISPR II had 79.82% homologous sequence match with unknown self-targets spacers, 49% bacteriophages spacers, 35%plasmids and 29.9% from other bacterial spacers available in NCBI databank. E. coli CRISPR class III showed no homologous spacers sequence match with unknown targets and bacteriophages but showed homologous sequence match with plasmid (0.19%) and other bacteria (1.83%).E. coli CRISPR class IV showed absence of homologous spacers matches against bacteriophages spacers from the NCBI databank. The spacer distribution among Y. pestis CRISPR classes revealed that the CRISPR class I showed a highest percentage value of homologous sequence match with 47.62% bacteriophages spacers, 38.7% plasmids, and 38.44% from other bacterial spacers, while the Y. pestis CRISPR class II showed spacers similarity with plasmids (25.45%) and bacteriophages (25.71%) and Y. pestis CRISPR III also showed percentage similarity sequence match with plasmids spacers(35.84%), bacteriophages spacers(26.67%) and 22.06% bacterial spacers available in NCBI databank. However, there was no homologous spacer sequence match contributed by unknown mobile genetic targets in all the Y. pestis CRISPR classes compared with the E. coli CRISPR classes. The evolutionary relationships of 22 representative strains of each bacterium were carefully selected on temporary relationship over a specific period [n < 10 years] and analyzed. The phylogenetic tree of E. coli revealed that 22 E. coli strains showed a common ancestral origin from E. coli BIDMC_74, and the E. coli BIDMC_74 strain was more closely related to E. coli strain IH57218 than E. coli str. HVH 50 and E. coli str. 122262 NODE_1 respectively. Phylogenetic analysis of all 22 Y. pestis showed a close relationship with one another, suggesting a common evolutionary relationship among them. The results obtained from this study give credence to show that the E.coli showed a significant CRISPR diversity than Y. pestis, in terms of its CRISPR class size, spacer’s contents, exogenous homologous sequence matches and phylogenetic relationships among its strains. This characteristic feature showed by E. coli strains could be attributed to increased homologous spacer acquisition from exogenous plasmids, bacteriophages, bacteria, and unknown targeting elements. The findings further suggest that the increased CRISPR diversity observed in the E. coli could be associated with increased exposure of its strains to these exogenous elements than Y. pestis strains due to bacteriophages infection, co-evolution and conjugation with exogenous mobile genetic elements (MGE).