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Title: Diverse CRISPRs Evolving in Human Microbiomes}
Article:
Abstract:
CRISPR( Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with cas( CRISPR–associated) genes, form the CRISPR/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human Microbiome Project( HMP) enables us to explore the distribution and diversity of known CRISPRs in human-associated microbial communities and to discover new CRISPRs. We propose a targeted assembly strategy to reconstruct CRISPR arrays, which whole-metagenome assemblies fail to identify. For each known CRISPR type( identified from reference genomes), we use its direct repeat consensus sequence to recruit reads from each HMP dataset and then assemble the recruited reads into CRISPR loci; the unique spacer sequences can then be extracted for analysis. We also identified novel CRISPRs or new CRISPR variants in contigs from whole-metagenome assemblies and used targeted assembly to more comprehensively identify these CRISPRs across samples. We observed that the distributions of CRISPRs( including 64 known and 86 novel ones) are largely body-site specific. We provide detailed analysis of several CRISPR loci, including novel CRISPRs. For example, known streptococcal CRISPRs were identified in most oral microbiomes, totaling ∼8, 000 unique spacers: samples resampled from the same individual and oral site shared the most spacers; different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences on the evolution of CRISPR defenses. We further demonstrate potential applications of CRISPRs to the tracing of rare species and the virus exposure of individuals. This work indicates the importance of effective identification and characterization of CRISPR loci to the study of the dynamic ecology of microbiomes.
Introduction:
CRISPRs, together with cas genes( CRISPR-associated genes), provide acquired resistance against viruses and conjugative plasmids[1],[2], and are found in most archaeal( ∼90%) and bacterial( ∼40%) genomes[3],[4],[5]. CRISPR arrays consist of 24–47 bp direct repeats, separated by unique sequences( spacers) that are acquired from viral or plasmid genomes[6]. Even though some CRISPR arrays may contain hundreds of spacers( an extreme case is the CRISPR array in the Haliangium ochraceum DSM 14365 genome, which has 588 copies of its repeat), they tend to be much smaller, generally with dozens of spacers. The repeat sequences of some CRISPRs are partially palindromic, and have stable, highly conserved RNA secondary structures, while others lack detectable structures[7]. CRISPR arrays are usually adjacent to cas genes, which encode a large and heterogeneous family of proteins with functional domains typical of nucleases, helicases, polymerases, and polynucleotide-binding proteins. CRISPR/Cas systems commonly use repeat and spacer-derived short guide CRISPR RNAs( crRNAs) to silence foreign nucleic acids in a sequence-specific manner[8],. CRISPR/Cas defense pathways involve several steps, including integration of viral or plasmid DNA-derived spacers into the CRISPR array, expression of short crRNAs consisting of unique single repeat-spacer units, and interference with invading foreign genomes at both the DNA and RNA levels, by mechanisms that are not yet fully understood[8],[10]. The diversity of cas genes suggests that multiple pathways have arisen to use the basic information contained in the repeat-spacer units in diverse defense mechanisms. The CRISPR components are evolutionarily closely linked and potentially evolve simultaneously as an intact locus—sequence analysis reveals that the direct repeats in CRISPR locus and the linked cas genes co-evolve under analogous evolutionary pressure[11]. Previous studies have shown that CRISPR loci are very diverse and abundant in the genomes of bacteria and archaea. In addition, it has been shown that CRISPR loci with the same repeat sequence and cas gene set can be found in multiple bacterial species, implying horizontal gene transfer( HGT)[12]. Moreover, CRISPR loci can change their spacer content rapidly, as a result of interactions between viruses( or plasmids) and bacteria: several metagenomic studies investigating host-virus population dynamics have shown that CRISPR loci evolve in response to viral predation and that CRISPR spacer content and sequential order provide both historically and geographically insights[13],[14],[15],[16]—essentially, epidemiology. As a reflection of the infectious dynamics of microbial communities, the study of CRISPRs is an essential compliment to the study of the human microbiome, encompassing both disease ecology and ecological immunology[17]. Infectious disease works to maintain both species diversity[18],[19] and genotypic diversity[20] within a species, as has recently been shown for marine microbiomes[21],[22]. As such, infectious agents may be at least partially responsible for the amazing species diversity and turnover found throughout the human microbiome[23]. The ability of CRISPR loci to prevent plasmid spread is medically relevant, in that the exchange of conjugative elements is perhaps the dominant mechanism by which antibiotic resistance genes( notably multi-drug resistance) move within a biome, and by which pathogens acquire resistance[24]; CRISPR activities could be expected to retard this exchange( e. g.[25]). CRISPR composition in human microbial communities, the relative rate of CRISPR locus change, or how CRISPR loci vary between different body sites and between the microbiota of different individuals are less studied, as compared to other environments. A recent analysis of streptococcal CRISPRs from human saliva, in which CRISPR spacers and repeats were amplified from salivary DNA, using the conserved streptococcal CRISPR repeat sequence for priming, revealed substantial spacer sequence diversity within and between subjects over time[26], which is imagined to reflect the dynamics of phage and other infectious agents in the human mouth[2]. The availability of more than 700 shotgun metagenomic datasets from the Human Microbiome Project( HMP) enables us to explore the distribution and diversity of many more CRISPRs, and to discover new ones, across different body sites, in a systematic manner. We developed a targeted assembly strategy( see Figure 1) to better identify CRISPRs in shotgun metagenomic sequences, as whole-metagenomic assembly failed to reconstruct many CRISPRs that otherwise could be identified. All of the programs available to date[27],[28],[29],[30] are designed to find CRISPRS from assembled contigs that are sufficiently long to contain at least partial CRISPR loci; however, it is very difficult to assemble metagenome reads into contigs containing CRISPR loci, because of their repeated structures. We thus needed to collect sequencing reads associated with CRISPRs and assemble them specifically. For known CRISPRs( identified in reference genomes), we identified consensus sequences of CRISPR repeats, collected the reads containing these sequences, and assembled these reads into CRISPR contigs. We also identified CRISPRs from the whole-metagenome assemblies, and for the novel CRISPRs or new CRISPR variants( that are not seen in the reference genomes), applied the same assembly strategy to achieve a more comprehensive identification of the novel CRISPRs across the samples. This approach allows us to study the evolution of CRISPRs in human microbiomes.
Discussion:
We have applied a targeted assembly approach to CRISPR identification, to characterize CRISPRs across body sites in different individuals. Our studies show that a directed approach—such as our targeted assembly approach—is important for a comprehensive( thus less biased) estimation of the distribution of CRISPRs across body sites and individuals, and their dynamics. Note that in this study, we only focused on CRISPRs identified in eubacterial genomes, since archaea are rare in human microbiomes( we looked for, but did not find, archaeal CRISPRs in the HMP assemblies). Also for the sake of simplicity, we derived a non-redundant list of CRISPRs based on the similarity of the CRISPR repeats( see Methods), and detailed targeted assembly was only applied to these CRISPRs. Although many CRISPR arrays may be missed by whole-metagenome assembly, we show that whole-metagenome assemblies are useful for identifying novel CRISPRs( as de novo prediction of CRISPRs relies on sequence features of CRISPRs that do not exist in short reads). Once seeding CRISPRs are identified from whole-metagenome assemblies, we can go back to the original short read datasets, and pursue a comprehensive characterization of the CRISPRs, using the targeted assembly approach. Also, we did not fully utilize the presence of cas genes for identification of novel CRISPRs in our study, since in many cases we can identify arrays of repeats, but not their associated cas genes. A future direction is to combine targeted assembly of CRISPRs and whole-metagenome assembly, aiming to achieve even better assembly of the CRISPR loci with more complete structures, including cas genes and the arrays of repeats and spacers. Such an improvement is necessary to achieve a more comprehensive characterization of especially the novel CRISPRs discovered in metagenomes, and the temporal order of spacer addition to arrays. The immediate utility of this study is to provide more complete inventories of CRISPR loci in human microbiomes and their distributions in different human body sites, and the spacer content of these loci. The identification of CRISPR spacers opens up several potential applications, including tracing the viral exposure of the hosts, studying the sequence patterns of the regions adjoining the spacer precursors in viral genomes, and discovering viral contigs in metagenome assemblies. It has been shown that short sequence motifs found in the regions adjacent to the spacer precursors in the viral genomes determine the targets of the CRISPR defense system[38], and we were able to analyze the sequence patterns of regions adjacent to spacer precursors for several CRISPRs with the most spacers identified in the HMP datasets( including SmutaL36, LjensL36, and KoralL32; see sequence logos in Figure S7). When more metagenomic datasets become available, we will extend the analysis to more CRISPRs, which may provide insights into the mechanism of the CRISPR defense system( including the turnover patterns of the CRISPR spacers, and the target recognition of the CRISPR defense systems). Our preliminary exploration of viral contigs—by searching CRISPR spacers against whole-metagenome assemblies—suggests that we can identify new virus genomes in metagenome assemblies; further computational and experimental analysis will be needed to confirm these contigs. We look forward to being able to utilize CRISPR spacer sequences to understand human and human microbiome biology better, utilizing the metadata associated with the HMP datasets. This awaits a more complete sampling of individuals over time, and of known relationships; and a far better characterization of bacteriophage and other selfish genetic elements in the human biome( our inventory of spacers is a standard against which phage and plasmid collections can be judged).
Human bodies are complex ecological systems in which various microbial organisms and viruses interact with each other and with the human host . The Human Microbiome Project ( HMP ) has resulted in >700 datasets of shotgun metagenomic sequences , from which we can learn about the compositions and functions of human-associated microbial communities . CRISPR/Cas systems are a widespread class of adaptive immune systems in bacteria and archaea , providing acquired immunity against foreign nucleic acids: CRISPR/Cas defense pathways involve integration of viral- or plasmid-derived DNA segments into CRISPR arrays ( forming spacers between repeated structural sequences ) , and expression of short crRNAs from these single repeat-spacer units , to generate interference to future invading foreign genomes . Powered by an effective computational approach ( the targeted assembly approach for CRISPR ) , our analysis of CRISPR arrays in the HMP datasets provides the very first global view of bacterial immunity systems in human-associated microbial communities . The great diversity of CRISPR spacers we observed among different body sites , in different individuals , and in single individuals over time , indicates the impact of subtle niche differences on the evolution of CRISPR defenses and indicates the key role of bacteriophage ( and plasmids ) in shaping human microbial communities .