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CRISPR locus occurrence and position relative to cas operons. a Histogram representing the frequency of genomes carrying the specified number of CRISPR loci. Histograms representing the relationship of the proportion of CRISPR arrays that are adjacent to the cas operon. b Number of CRISPR arrays encoded on a given chromosome. c Location of the CRISPR arrays relative to the cas operon
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Background
Much effort is underway to build and upgrade databases and tools related to occurrence, diversity, and characterization of CRISPR-Cas systems. As microbial communities and their genome complements are unearthed, much emphasis has been placed on details of individual strains and model systems within the CRISPR-Cas classification, and that...
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Context 1
... our data set, it was found that 82% of bacterial Class I CRISPR-Cas systems had only one set of cas genes as show in Fig. 5a. We next investigated patterns that may occur when multiple arrays occur in one chromosome. After iso- lating all systems that had only one set of cas genes, the rank correlation statistic was calculated for Class I and the two major types for the relationship between the number of spacers in the CRISPR array that is closest to the cas genes and the number of arrays in the genome. Spearman's rho can result in values along the spectrum between −1 and 1 where higher values indicates a stronger association between the two variables. Class I had a value of −0.85, Type I had a value of −0.79, and Type III had a value of −0.75. These values showed a strong association in the manner that as you increased the number of CRISPRs present in a genome that had only one CRISPR-Cas system, the CRISPR closest to the set of cas genes was less likely to be the largest CRISPR in the genome. A visual representa- tion for Class I can be seen in Fig. 5b. Therefore, it was concluded that CRISPRs tend to be larger when they are in closer proximity to a set of cas genes but as you increase the number of CRISPRs on a chromosome, then the probability of the largest CRISPRs being associated with those cas genes tends to for chromosomes with a single set of cas genes. Mechanistically, we hypothesize that this enables the host to control the expansion of one given locus, which would be advantageous transcriptionally, by having the ability to both initiate transcription from multiple loca- tions, and also limit the size of primary CRISPR ...
Context 2
... our data set, it was found that 82% of bacterial Class I CRISPR-Cas systems had only one set of cas genes as show in Fig. 5a. We next investigated patterns that may occur when multiple arrays occur in one chromosome. After iso- lating all systems that had only one set of cas genes, the rank correlation statistic was calculated for Class I and the two major types for the relationship between the number of spacers in the CRISPR array that is closest to the cas genes and the number of arrays in the genome. Spearman's rho can result in values along the spectrum between −1 and 1 where higher values indicates a stronger association between the two variables. Class I had a value of −0.85, Type I had a value of −0.79, and Type III had a value of −0.75. These values showed a strong association in the manner that as you increased the number of CRISPRs present in a genome that had only one CRISPR-Cas system, the CRISPR closest to the set of cas genes was less likely to be the largest CRISPR in the genome. A visual representa- tion for Class I can be seen in Fig. 5b. Therefore, it was concluded that CRISPRs tend to be larger when they are in closer proximity to a set of cas genes but as you increase the number of CRISPRs on a chromosome, then the probability of the largest CRISPRs being associated with those cas genes tends to for chromosomes with a single set of cas genes. Mechanistically, we hypothesize that this enables the host to control the expansion of one given locus, which would be advantageous transcriptionally, by having the ability to both initiate transcription from multiple loca- tions, and also limit the size of primary CRISPR ...
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Citations
... Quite contrary to this view, frequent deletion of spacers and renewal of spacer composition is essential for the effectiveness of the system [44] and could, in principle, have fostered the emergence of a dynamic deletion system similar to primed adaptation. Interestingly, deletion frequencies can be subject to selection indirectly when multiple CRISPR arrays with different lengths, and therefore due to the boundary effect also different per spacer deletion rates, coexist [34,54]. ...
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 to 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 374 times more frequently than mutations and are regularly deleted jointly, with an average of 2.7 spacers.
Surprisingly, we observed a decrease of 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.
... Recently, rank abundance curves of spacer types were constructed from a mathematical model of bacteria-phage interaction in a chemostat [18], and the model was seen to show a quick drop in the abundance of rare spacers. However, to the best of our knowledge, the quantitative aspect of spacer abundance in microbial population, i.e. the distribution of the total number of spacers in CRISPR arrays among individual bacteria, still remains largely unaddressed (but see [19] where Class I CRISPR-Cas systems were found to tend to follow a geometric distribution, although the sample size was rather small). From the previous studies, it is known that the number of spacers increases as a result of interaction with microbial viruses and decreases due to non-specific deletions during genome replication [20]. ...
Understanding CRISPR-Cas systems-the adaptive defence mechanism that about half of bacterial species and most of archaea use to neutralise viral attacks-is important for explaining the biodiversity observed in the microbial world as well as for editing animal and plant genomes effectively. The CRISPR-Cas system learns from previous viral infections and integrates small pieces from phage genomes called spacers into the microbial genome. The resulting library of spacers collected in CRISPR arrays is then compared with the DNA of potential invaders. One of the most intriguing and least well understood questions about CRISPR-Cas systems is the distribution of spacers across the microbial population. Here, using empirical data, we show that the global distribution of spacer numbers in CRISPR arrays across multiple biomes worldwide typically exhibits scale-invariant power law behaviour, and the standard deviation is greater than the sample mean. We develop a mathematical model of spacer loss and acquisition dynamics which fits observed data from almost four thousand metagenomes well. In analogy to the classical 'rich-get-richer' mechanism of power law emergence, the rate of spacer acquisition is proportional to the CRISPR array size, which allows a small proportion of CRISPRs within the population to possess a significant number of spacers. Our study provides an alternative explanation for the rarity of all-resistant super microbes in nature and why proliferation of phages can be highly successful despite the effectiveness of CRISPR-Cas systems.
... Interestingly, the Type I-C and I-E systems detected here contain longer CRISPR arrays than the Type II systems, averaging 60 spacers in Type I systems and 25 in Type II systems ( Figure 4A). The average CRISPR array size for the Type I system in Bifidobacterium is longer than the previously reported 40 spacers for a Type I system [40], reflecting the unusually large Type I CRISPR array in Bifidobacterium. Type I-E systems displayed the highest average number of spacers (66), representing the majority of longest arrays detected in Bifidobacterium genomes, although Type I-C was close behind with an average of 55 repeats. ...
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated cas) systems constitute the adaptive immune system in prokaryotes, which provides resistance against bacteriophages and invasive genetic elements. The landscape of applications in bacteria and eukaryotes relies on a few Cas effector proteins that have been characterized in detail. However, there is a lack of comprehensive studies on naturally occurring CRISPR-Cas systems in beneficial bacteria, such as human gut commensal Bifidobacterium species. In this study, we mined 954 publicly available Bifidobacterium genomes and identified CRIPSR-Cas systems in 57% of these strains. A total of five CRISPR-Cas subtypes were identified as follows: Type I-E, I-C, I-G, II-A, and II-C. Among the subtypes, Type I-C was the most abundant (23%). We further characterized the CRISPR RNA (crRNA), tracrRNA, and PAM sequences to provide a molecular basis for the development of new genome editing tools for a variety of applications. Moreover, we investigated the evolutionary history of certain Bifidobacterium strains through visualization of acquired spacer sequences and demonstrated how these hypervariable CRISPR regions can be used as genotyping markers. This extensive characterization will enable the repurposing of endogenous CRISPR-Cas systems in Bifidobacteria for genome engineering, transcriptional regulation, genotyping, and screening of rare variants.
... Type I, II, and III spacers grouped based on CRISPR-Cas systems. As expected, Type I systems encoded for a greater number of spacers than that of the Type II systems [18]. The spacers in L. fermentum as a whole were very diverse and we were unable to identify common ancestral spacers for the majority of the strains. ...
Background:
Lactobacillus fermentum, a member of the lactic acid bacteria complex, has recently garnered increased attention due to documented antagonistic properties and interest in assessing the probiotic potential of select strains that may provide human health benefits. Here, we genomically characterize L. fermentum using the type strain DSM 20052 as a canonical representative of this species.
Results:
We determined the polished whole genome sequence of this type strain and compared it to 37 available genome sequences within this species. Results reveal genetic diversity across nine clades, with variable content encompassing mobile genetic elements, CRISPR-Cas immune systems and genomic islands, as well as numerous genome rearrangements. Interestingly, we determined a high frequency of occurrence of diverse Type I, II, and III CRISPR-Cas systems in 72% of the genomes, with a high level of strain hypervariability.
Conclusions:
These findings provide a basis for the genetic characterization of L. fermentum strains of scientific and commercial interest. Furthermore, our study enables genomic-informed selection of strains with specific traits for commercial product formulation, and establishes a framework for the functional characterization of features of interest.
... The phage-encoded CRISPR arrays are often compact (median, six repeats per array) (Extended Data Fig. 10). This range is substantially smaller than typically found in prokaryotic genomes (mean of 41 repeats for class I systems) 29 . Some phage spacers target core structural and regulatory genes of other phages ( Fig. 4c and Supplementary Table 10). ...
Bacteriophages typically have small genomes and depend on their bacterial hosts for replication. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.
... The phage-encoded CRISPR arrays are often compact (median, six repeats per array) (Extended Data Fig. 10). This range is substantially smaller than typically found in prokaryotic genomes (mean of 41 repeats for class I systems) 29 . Some phage spacers target core structural and regulatory genes of other phages ( Fig. 4c and Supplementary Table 10). ...
Bacteriophages typically have small genomes¹ and depend on their bacterial hosts for replication². Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is—to our knowledge—the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR–Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR–Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR–Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth’s ecosystems.
... Many of the type I-F systems in L. pneumophila 387 have different array lengths, ranging from 8 spacers to 129 spacers, with an average length of 61 388 spacers (Table 2). A global analysis of class I CRISPR arrays found that the average array length 389 for type I-F systems was 33 spacers, with statistically significant differences between the array 390 lengths of different type I subtypes (Toms and Barrangou 2017). Accordingly, if spacer 391 acquisition is a driving force in array divergence, it is likely coupled to spacer loss. ...
In bacteria and archaea, several distinct types of CRISPR-Cas systems provide adaptive immunity through broadly similar mechanisms: short nucleic acid sequences derived from foreign DNA, known as spacers, engage in complementary base pairing with invasive genetic elements setting the stage for nucleases to degrade the target DNA. A hallmark of type I CRISPR-Cas systems is their ability to acquire spacers in response to both new and previously encountered invaders (naïve and primed acquisition, respectively). Our phylogenetic analyses of 43 L. pneumophila type I-F CRISPR-Cas systems and their resident genomes suggest that many of these systems have been horizontally acquired. These systems are frequently encoded on plasmids and can co-occur with nearly identical chromosomal loci. We show that two such co-occurring systems are highly protective and undergo efficient primed acquisition in the lab. Furthermore, we observe that targeting by one system's array can prime spacer acquisition in the other. Lastly, we provide experimental and genomic evidence for a model in which primed acquisition can efficiently replenish a depleted type I CRISPR array following a mass spacer deletion event.
... Orphan CRISPR arrays are smaller (eight repeats on average) than distant arrays (13), which are smaller than arrays within CRISPR-Cas loci (24, Figure 4A). The latter result, is consistent with previous findings (42). In consequence, the presence, proximity and subtype of cas clusters impact the number of repeats in CRISPRs. ...
Prokaryotes use CRISPR-Cas systems for adaptive immunity, but the reasons for the frequent existence of multiple CRISPRs and cas clusters remain poorly understood. Here, we analysed the joint distribution of CRISPR and cas genes in a large set of fully sequenced bacterial genomes and their mobile genetic elements. Our analysis suggests few negative and many positive epistatic interactions between Cas subtypes. The latter often result in complex genetic organizations, where a locus has a single adaptation module and diverse interference mechanisms that might provide more effective immunity. We typed CRISPRs that could not be unambiguously associated with a cas cluster and found that such complex loci tend to have unique type I repeats in multiple CRISPRs. Many chromosomal CRISPRs lack a neighboring Cas system and they often have repeats compatible with the Cas systems encoded in trans. Phages and 25 000 prophages were almost devoid of CRISPR-Cas systems, whereas 3% of plasmids had CRISPR-Cas systems or isolated CRISPRs. The latter were often compatible with the chromosomal cas clusters, suggesting that plasmids can co-opt the latter. These results highlight the importance of interactions between CRISPRs and cas present in multiple copies and in distinct genomic locations in the function and evolution of bacterial immunity.
... Many of the type I-F systems in L. 238 pneumophila have different array lengths, ranging from 8 spacers to 129 spacers, with an average 239 length of 61 spacers (Table 2). Toms and Barrangou have performed a global analysis of class I 240 CRISPR arrays and found that the average array length for type I-F systems was 33 spacers, with 241 statistically significant differences between the array lengths of different type I subtypes (Toms 242 and Barrangou 2017). Accordingly, if spacer acquisition is a driving force in array divergence, it 243 is likely coupled to spacer loss. ...
In bacteria and archaea, several distinct types of CRISPR-Cas systems provide adaptive immunity through broadly similar mechanisms: short nucleic acid sequences derived from foreign DNA, known as spacers, engage in complementary base pairing with invasive genetic elements setting the stage for nucleases to degrade the target DNA. A hallmark of type I CRISPR-Cas systems is their ability to acquire spacers in response to both new and previously encountered invaders (naïve and primed acquisition, respectively). Our phylogenetic analyses of 47 L. pneumophila type I-F CRISPR-Cas systems and their resident genomes suggest that many of these systems have been horizontally acquired. These systems are frequently encoded on plasmids and can co-occur with nearly identical chromosomal loci. We show that two such co-occurring systems are highly protective and undergo efficient primed acquisition in the lab. Furthermore, we observe that targeting by one system’s array can prime spacer acquisition in the other. Lastly, we provide experimental and genomic evidence for a model in which primed acquisition can efficiently replenish a depleted type I CRISPR array following a mass spacer deletion event.
... This region is likely to be a genomic hotspot for horizontally acquired genes and/or CRISPR-Cas systems. The tendency of CRISPRs to be distributed non-randomly on bacterial chromosomes, but occurring in specific chromosome regions, has already been reported [36][37][38]. For instance, within the Enterobacteriaceae family genome, hotspots involved in the acquisition of CRISPR-Cas systems have been described for Klebsiella spp. ...
The CRISPR-Cas adaptive immune system has been attracting increasing scientific interest for biological functions and biotechnological applications. Data on the Serratia marcescens system are scarce. Here, we report a comprehensive characterisation of CRISPR-Cas systems identified in S. marcescens strains isolated as secondary symbionts of Rhynchophorus ferrugineus, also known as Red Palm Weevil (RPW), one of the most invasive pests of major cultivated palms. Whole genome sequencing was performed on four strains (S1, S5, S8, and S13), which were isolated from the reproductive apparatus of RPWs. Subtypes IF and I-E were harboured by S5 and S8, respectively. No CRISPR-Cas system was detected in S1 or S13. Two CRISPR arrays (4 and 51 spacers) were detected in S5 and three arrays (11, 31, and 30 spacers) were detected in S8. The CRISPR-Cas systems were located in the genomic region spanning from ybhR to phnP, as if this were the only region where CRISPR-Cas loci were acquired. This was confirmed by analyzing the S. marcescens complete genomes available in the NCBI database. This region defines a genomic hotspot for horizontally acquired genes and/or CRISPR-Cas systems. This study also supplies the first identification of subtype I-E in S. marcescens.
