CRISPR Arrays Research Articles

Tracking the progeny of bacterial persisters using a CRISPR-based genomic recorder

The rise of antimicrobial failure is a global emergency, and causes beyond typical genetic resistance must be determined. One probable factor is the existence of subpopulations of transiently growth-arrested bacteria, persisters, that endure antibiotic treatment despite genetic susceptibility to the drug. The presence of persisters in infected hosts has been successfully established, notably through the development of fluorescent reporters. It is proposed that infection relapse is caused by persisters resuming growth after cessation of the antibiotic treatment, but to date, there is no direct evidence for this. This is because no tool or reporter currently exists to track the extent to which infection relapse is initiated by regrowth of persisters in the host. Indeed, once they have transitioned out of the persister state, the progeny of persisters are genetically and phenotypically identical to susceptible bacteria in the population, making it virtually impossible to ascertain the source of relapse. We designed pSCRATCH (plasmid for Selective CRISPR Array expansion To Check Heritage), a molecular tool that functions to record the state of antibiotic persistence in the genome of Salmonella persisters. We show that pSCRATCH successfully marks persisters by adding spacers in their CRISPR arrays and the genomic label is stable in persister progeny after exit from persistence. We further show that in a Salmonella infection model the system enables the discrimination of treatment failure originating from persistence versus resistance. Thus, pSCRATCH provides proof of principle for stable marking of persisters and a prototype for applications to more complex infection models and other pathogens.

Deciphering the complete genome sequence of multidrug-resistant Escherichia coli strain Hakim RU_GHWS isolated from ladies' hall sewage water in Bangladesh.

This report details the genome sequence of Escherichia coli strain Hakim RU_GHWS, isolated from sewage water. The assembled genome comprises 5.022 Mb with 77.675× coverage, depicting an average GC content of 50.50%. This genome contains 10 CRISPR arrays, 14 prophages, 65 antibiotic resistance genes, and 28 virulence factor genes.

CRISPR RNA binding drives structural ordering that primes Cas7-11 for target cleavage.

Type III-E CRISPR-Cas effectors, of which Cas7-11 is the first, are single proteins that cleave target RNAs without nonspecific collateral cleavage, opening new possibilities for RNA editing. Biochemical experiments combined with amide hydrogen-deuterium exchange (HDX-MS) experiments provide a first glimpse of the conformational dynamics of apo Cas7-11. HDX-MS revealed the backbone comprised of the four Cas7 zinc-binding RRM folds are well-folded but insertion sequences are highly dynamic and fold upon binding crRNA. The crRNA causes folding of disordered catalytic loops and β-hairpins, stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. Target RNA binding causes only minor ordering around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot fully process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces target RNA cleavage. The insertion domain shows the most ordering upon binding of mature crRNA. Finally, we show a crRNA-induced conformational change in one of the TPR-CHAT binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding. The results provide the first glimpse of the apo state of Cas7-11 and reveal how its structure and function are regulated by crRNA binding.

SpacerPlacer: ancestral reconstruction of CRISPR arrays reveals the evolutionary dynamics of spacer deletions.

Bacteria employ CRISPR-Cas systems for defense by integrating invader-derived sequences, termed spacers, into the CRISPR array, which constitutes an immunity memory. While spacer deletions occur randomly across the array, newly acquired spacers are predominantly integrated at the leader end. Consequently, spacer arrays can be used to derive the chronology of spacer insertions. Reconstruction of ancestral spacer acquisitions and deletions could help unravel the coevolution of phages and bacteria, the evolutionary dynamics in microbiomes, or track pathogens. However, standard reconstruction methods produce misleading results by overlooking insertion order and joint deletions of spacers. Here, we present SpacerPlacer, a maximum likelihood-based ancestral reconstruction approach for CRISPR array evolution. We used SpacerPlacer to reconstruct and investigate ancestral deletion events of 4565 CRISPR arrays, revealing that spacer deletions occur 374times more frequently than mutations and are regularly deleted jointly, with an average of 2.7 spacers. Surprisingly, we observed a decrease in the spacer deletion frequency towards both ends of the reconstructed arrays. While the resulting trailer-end conservation is commonly observed, a reduced deletion frequency is now also detectable towards the variable leader end. Finally, our results point to the hypothesis that frequent loss of recently acquired spacers may provide a selective advantage.

New Viruses Infecting Hyperthermophilic Bacterium Thermus thermophilus.

Highly diverse phages infecting thermophilic bacteria of the Thermus genus have been isolated over the years from hot springs around the world. Many of these phages are unique, rely on highly unusual developmental strategies, and encode novel enzymes. The variety of Thermus phages is clearly undersampled, as evidenced, for example, by a paucity of phage-matching spacers in Thermus CRISPR arrays. Using water samples collected from hot springs in the Kunashir Island from the Kuril archipelago and from the Tsaishi and Nokalakevi districts in the Republic of Georgia, we isolated several distinct phages infecting laboratory strains of Thermus thermophilus. Genomic sequence analysis of 11 phages revealed both close relatives of previously described Thermus phages isolated from geographically distant sites, as well as phages with very limited similarity to earlier isolates. Comparative analysis allowed us to predict several accessory phage genes whose products may be involved in host defense/interviral warfare, including a putative Type V CRISPR-cas system.

Complete genome sequences of Clostridium perfringens CP201 isolated from an asymptomatic rhesus monkey (Macaca mulatta).

Clostridium perfringens is a leading cause of human gastroenteritis. The genome sequence of C. perfringens CP201 from an asymptomatic rhesus monkey, Macaca mulatta, consists of one chromosome (3,241,413 bp; 30.22% G + C) and one plasmid (92,983 bp; 28.43% G + C), encoding 2,935 genes, 94 tRNAs, 30 rRNAs, and 1 CRISPR array.

Genomic analysis of "Candidatus Liberibacter africanus" strain from Zimbabwe reveals unique virulence and prophage characteristics compared to "Ca. Liberibacter asiaticus".

Citrus Huanglongbing (HLB) is caused by the phloem-limited α-proteobacterium "Candidatus Liberibacter spp.", among which "Ca. Liberibacter africanus" (CLaf) have posed a significant threat to citrus production in Africa near a century. CLaf is closely related to the globally prevalent "Ca. Liberibacter asiaticus" (CLas), whereas little is known about the virulence of CLaf, primarily due to limited genome resources. In this study, we completed the whole-genome assembly and annotation of CLaf strain Zim (from Zimbabwe). Compared to CLas, a total of 102 CLaf unique genes were identified, including 14 potential Sec-dependent effectors (SDEs) genes, 29 phage-associated genes, and 59 genes with hypothetical function. Among 14 SDEs, V9J15_03810 was able to induce a significant hypersensitive response (HR) in Nicotiana benthamiana, indicating its potential as a virulence factor for CLaf. Genome analysis showed that CLaf strain Zim genome harbored a complete prophage region (named P-Zim-1, 42,208 bp). P-Zim-1 retained two immunosuppressive peroxidase genes (V9J15_02125 and V9J15_02130) homologous to CLas prophage SC1/SC2, whereas the lysogen-associated genes encoding integrase (V9J15_01970) and repressor (V9J15_02080) were homologous to the prophage of "Ca. Liberibacter solanacearum", the causal agent of potato zebra chip disease. In addition, P-Zim-1 carried a novel CRISPR/Cas system, including a CRISPR array (located within V9J15_02040, ranging from 443,643 to 443,897) and five CRISPR-related Cas proteins (V9J15_02005, 02010, 02015, 02025 and 02035). This study first characterized the unique genomic feature of CLaf related to virulence and prophage, which will facilitate future research on CLaf biology and African HLB management.

Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons

CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can’t acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π − π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.

Genome characterization of Leuconostoc pseudomesenteroides UTNElla29 isolated from Morus nigra (L.) fruits: A promising exopolysaccharides producing strain

Lactic acid bacteria produce various natural polysaccharides that are secreted into the cellular matrix and bonded to the cell surface or released into the surrounding environment. Due to their broad applications in the food industry, the search for novel polysaccharide producer strains remains of interest. In this study, we report the genome sequencing and characterization of Leuconostoc pseudomesenteroides strain UTNElla29 isolated from Morus nigra (L.) fruits. Using in silico different “genome mining” tools we prospect their genetic potential. The genome size was estimated at 2,272,560 bp and a GC content of 39.26%. Of the total proteins, 2170 matched in the EggNOG database. The key genes related to amino acid biosynthesis, carbohydrate metabolism, exopolysaccharide (EPS) production, were annotated. The genome harbors one intact prophage, one CRISPR array, and two putative gene sequences associated with CAS3 Type I, and no plasmids indicating its stability. Moreover, no acquired resistance genes, virulence and pathogenic factors, nor biogenic amines were predicted, suggesting its safety. In vitro preliminary studies showed that EPS yield from 0.4 to 10.2 (g/L) dry biomass and the production varied with the inoculum size and sugar type MRS containing medium. Furthermore, several polyketides and terpenes were predicted, indicating the possibility of generating a wide range of naturally compounds with broad biological functions. However, these promising results suggest that Ella29 is a beneficial strain to be further exploited in vitro for various technological use.

Genomic reconstruction of unclassified microorganisms: Analysis of CRISPR arrays and genes involved in defense mechanisms

The constant battle between bacteria and viruses has led to the development of sophisticated antiviral defense strategies by bacteria to defend themselves against phages. This study analyzed a marshland metagenome to identify and characterize bacterial antiviral defense systems and phage interactions. We assembled 210 metagenome-assembled genomes (MAGs) from environmental DNA extracted from Pallikaranai marshland soil and 37 unclassified MAGs were filtered. MIMAG standards were followed, 2 high-quality and 15 medium-quality unclassified MAGs were picked. MINCED was used to identify 137 CRISPR arrays in the quality MAGs, and ViroBLAST was used to identify the phages that interact with the bacteria. About 242 spacer sequences were extracted from the CRISPR arrays, of which 54 had significant matches in the ViroBLAST database. 7 unverified bacteriophage species were also detected in the MAGs. The viral group of Caudoviricetes phage elements were identified as a frequent genome terminal repeat. The PADLOC identified 11 genes involved as a defense system in the MAGs. The PD-T4-6 defense system was found to be prevalent in 15 different unclassified MAGs. This study presents valuable insights intothe adaptations of unclassified bacteria to bacteriophages, as well as the genes used by these bacteria as a defense mechanism.

Comparative Genomic Analysis of Campylobacter rectus and Closely Related Species.

Campylobacter rectus is a gram-negative, anaerobic bacterium strongly associated with periodontitis. It also causes various extraoral infections and is linked to adverse pregnancy outcomes in humans and murine models. C. rectus and related oral Campylobacters have been termed "emerging Campylobacter species" because infections by these organisms are likely underreported. Previously, no comparative methods have been used to analyze more than single C. rectus strains and until recently, very few C. rectus genomes have been publicly available. More sequenced genomes and comparative analyses are needed to study the genomic features and pathogenicity of this species. We sequenced eight new C. rectus strains and used comparative methods to identify regions of interest. An emphasis was put on the type III flagellar secretion system (T3SS), type IV secretion system (T4SS), and type VI secretion system (T6SS) because these protein complexes are important for pathogenesis in other Campylobacter species. RAST, BV-BRC, and other bioinformatics tools were used to assemble, annotate, and compare these regions in the genomes. The pan-genome of C. rectus consists of 2670 genes with core and accessory genomes of 1429 and 1241 genes, respectively. All isolates analyzed in this study have T3SS and T6SS hallmark proteins, while five of the isolates are missing a T4SS system. Twenty-one prophage clusters were identified across the panel of isolates, including four that appear intact. Overall, significant genomic islands were found, suggesting regions in the genomes that underwent horizontal gene transfer. Additionally, the high frequency of CRISPR arrays and other repetitive elements has led to genome rearrangements across the strains, including in areas adjacent to secretion system gene clusters. This study describes the substantial diversity present among C. rectus isolates and highlights tools/assays that have been developed to permit functional genomic studies. Additionally, we have expanded the studies on C. showae T4SS since we have two new C. showae genomes to report. We also demonstrate that unlike C. rectus , C showae does not demonstrate evidence of intact T6SS except for the strain CAM. The only strain of sequenced C. massilensis has neither T4SS or T6SS.

Draft genome sequence of multidrug-resistant Kurthia gibsonii strain Hakim RU_BHWE isolated from sewage water in Bangladesh.

We have sequenced the genome of Kurthia gibsonii strain Hakim RU_BHWE, isolated from sewage water. The assembled genome consists of 2.891 Mb with 58.6883× coverage, presenting an average GC content of 36.60%. This genome includes 8 CRISPR arrays, 3 prophages, 3 antibiotic resistance genes, and 12 virulence factor genes.

Ecological and evolutionary dynamics of CRISPR-Cas systems in Clostridium botulinum: Insights from genome mining and comparative analysis

Understanding the prevalence and distribution of CRISPR-Cas systems across different strains can illuminate the ecological and evolutionary dynamics of Clostridium botulinum populations. In this study, we conducted genome mining to characterize the CRISPR-Cas systems of C. botulinum strains. Our analysis involved retrieving complete genome sequences of these strains and assessing the diversity, prevalence, and evolution of their CRISPR-Cas systems. Subsequently, we performed an analysis of homology in spacer sequences from identified CRISPR arrays to investigate and characterize the range of targeted phages and plasmids. Additionally, we investigated the evolutionary trajectory of C. botulinum strains under selective pressures from foreign invasive DNA. Our findings revealed that 306 strains possessed complete CRISPR-Cas structures, comprising 58% of the studied C. botulinum strains. Secondary structure prediction of consensus repeats indicated that subtype II-C, with longer stems compared to subtypes ID and IB, tended to form more stable RNA secondary structures. Moreover, protospacer motif analysis demonstrated that strains with subtype IB CRISPR-Cas systems exhibited 5′-CGG-3′, 5′-CC-3′, and 5′-CAT-3′ motifs in the 3′ flanking regions of protospacers. The diversity observed in CRISPR-Cas systems indicated their classification into subtypes IB, ID, II-C, III-B, and III-D. Furthermore, our results showed that systems with subtype ID and III-D frequently harbored similar spacer patterns. Moreover, analysis of spacer sequences homology with phage and prophage genomes highlighted the specific activities exhibited by subtype IB and III-B against phages and plasmids, providing valuable insights into the functional specialization within these systems.

CRISPR-array-mediated imaging of non-repetitive and multiplex genomic loci in living cells.

Dynamic imaging of genomic loci is key for understanding gene regulation, but methods for imaging genomes, in particular non-repetitive DNAs, are limited. We developed CRISPRdelight, a DNA-labeling system based on endonuclease-deficient CRISPR-Cas12a (dCas12a), with an engineered CRISPR array to track DNA location and motion. CRISPRdelight enables robust imaging of all examined 12 non-repetitive genomic loci in different cell lines. We revealed the confined movement of the CCAT1 locus (chr8q24) at the nuclear periphery for repressed expression and active motion in the interior nucleus for transcription. We uncovered the selective repositioning of HSP gene loci to nuclear speckles, including a remarkable relocation of HSPH1 (chr13q12) for elevated transcription during stresses. Combining CRISPR-dCas12a and RNA aptamers allowed multiplex imaging of four types of satellite DNA loci with a single array, revealing their spatial proximity to the nucleolus-associated domain. CRISPRdelight is a user-friendly and robust system for imaging and tracking genomic dynamics and regulation.

Genome mining approach reveals the CRISPR-Cas characteristics and diversity of targeting phages in Lactobacillus iners strains

This study utilized a genomic approach to investigate CRISPR-Cas systems, focusing on Lactobacillus iners strains, which are crucial for bacterial defense against bacteriophage attacks. Genome sequences were analyzed to understand the diversity, occurrence, and evolution of these systems. Spacer sequences within CRISPR arrays were examined to identify targeted phages. The research explored the evolutionary paths of spaceromes within each CRISPR array subtype, considering acquisition and deletion events influenced by phage pressure. Results revealed that approximately 50 % of L. iners strains possess complete CRISPR-Cas systems, predominantly subtype II-A and I-E. Homology analysis indicated that subtype I-E systems target a wider range of foreign phages compared to subtype II-A systems. Overall, the findings underscore the varied components and structure of CRISPR-Cas systems in L. iners strains, highlighting their role as active immune defenses against phages and foreign DNA.

Methanotrophic Methanoperedens archaea host diverse and interacting extrachromosomal elements.

Methane emissions are mitigated by anaerobic methane-oxidizing archaea, including Methanoperedens. Some Methanoperedens host huge extrachromosomal genetic elements (ECEs) called Borgs that may modulate their activity, yet the broader diversity of Methanoperedens ECEs is understudied. Here we report small enigmatic linear ECEs, circular viruses and unclassified ECEs that are predicted to replicate within Methanoperedens. Linear ECEs have inverted terminal repeats, tandem repeats and coding patterns that are strongly reminiscent of Borgs, but they are only 52-145 kb in length. As they share proteins with Borgs and Methanoperedens, we refer to them as mini-Borgs. Mini-Borgs are genetically diverse and can be assigned to at least five family-level groups. We identify eight families of Methanoperedens viruses, some of which encode multi-haem cytochromes, and circular ECEs encoding transposon-associated TnpB genes with proximal population-heterogeneous CRISPR arrays. These ECEs exchange genetic information with each other and with Methanoperedens, probably impacting their archaeal host activity and evolution.

Draft genome sequence of carbapenems-resistant Acinetobacter baumannii Hakim RU_CBWP strain isolated from a pond surface water in Bangladesh.

We have revealed the genomic sequence of Acinetobacter baumannii strain Hakim RU_CBWP isolated from pond surface water. Our assembled genome covers 3.787 Mb with 45.5629× coverage, showcasing an average GC content of 38.60%. This genome contains two CRISPR arrays, 17 prophages, 22 antibiotic resistance genes, and 20 virulence factor genes.

Draft genome sequence of Escherichia coli MTR_GS_S1457 strain isolated from a soil sample of a vegetable garden in Gazipur, Bangladesh.

We announce the sequence of the Escherichia coli MTR_GS_S1457 strain isolated from a soil sample of a vegetable gardening system for the first time in Bangladesh. With a length of 4,918,647 bp, this strain contained one plasmid, two CRISPR arrays, 54 predicted antibiotic resistance genes, and 81 predicted virulence factor genes.

Characterization of Erwinia amylovora isolates from the northern region of Saudi Arabia, including CRISPR genotyping

AbstractFire blight, caused by the bacterium Erwinia amylovora, poses a global threat to the pome fruit industry, leading to reduced yields and significant economic losses. A comprehensive countrywide survey was carried out to determine the possible E. amylovora distribution in pome fruit‐growing regions. During the period 2020–2023, 85 isolates characteristic of E. amylovora were obtained from pear across 23 locations in three main pear‐producing regions (Al‐Jouf, Hail and Tabuk) in northern Saudi Arabia (SA). Isolates were identified using biochemical, molecular and pathogenicity assays. Molecular characterization of Saudi strains was achieved using partial 16S rRNA and rpoB gene sequencing, rep‐PCR (specifically, BOX‐PCR) and CRISPR array analysis. The CRISPR genotyping in particular, using an existing scheme of three CRISPR arrays, CR1, CR2 and CR3, provided consistent patterns of the Saudi isolates with high similarity to those of E. amylovora strains from the UK and France, hinting at a possible origin of the fire blight infections from these countries. Some variation was observed in patterns between local strains, highlighting the importance of continued surveillance and genetic studies for effective disease management strategies. Identification of fire blight in three regions over several years should serve as a warning to the Ministry of Environment, Water and Agriculture in Saudi Arabia, prompting the initiation of a more comprehensive survey and an active disease management programme. This programme should include early detection and advice to growers on prevention and control measures. This is the first scientific study on the occurrence and genetic variability of E. amylovora in Saudi Arabia.

Discarded diversity: Novel megaphages, auxiliary metabolic genes, and virally encoded CRISPR-Cas systems in landfills.

Viruses are the most abundant microbial entity on the planet, impacting microbial community structure and ecosystem services. Despite outnumbering Bacteria and Archaea by an order of magnitude, viruses have been comparatively underrepresented in reference databases. Metagenomic examinations have illustrated that viruses of Bacteria and Archaea have been specifically understudied in engineered environments. Here we employed metagenomic and computational biology methods to examine the diversity, host interactions, and genetic systems of viruses predicted from 27 samples taken from three municipal landfills across North America. We identified numerous viruses that are not represented in reference databases, including the third largest bacteriophage genome identified to date (~678 kbp), and note a cosmopolitan diversity of viruses in landfills that are distinct from viromes in other systems. Host-virus interactions were examined via host CRISPR spacer to viral protospacer mapping which captured hyper-targeted viral populations and six viral populations predicted to infect across multiple phyla. Virally-encoded auxiliary metabolic genes (AMGs) were identified with the potential to augment hosts' methane, sulfur, and contaminant degradation metabolisms, including AMGs not previously reported in literature. CRISPR arrays and CRISPR-Cas systems were identified from predicted viral genomes, including the two largest bacteriophage genomes to contain these genetic features. Some virally encoded Cas effector proteins appear distinct relative to previously reported Cas systems and are interesting targets for potential genome editing tools. Our observations indicate landfills, as heterogeneous contaminated sites with unique selective pressures, are key locations for diverse viruses and atypical virus-host dynamics.