Spacer Acquisition Research Articles

Distinct Genomic Features Characterize Two Clades of Corynebacterium diphtheriae: Proposal of Corynebacterium diphtheriae Subsp. diphtheriae Subsp. nov. and Corynebacterium diphtheriae Subsp. lausannense Subsp. nov.

Corynebacterium diphtheriae is the etiological agent of diphtheria, a disease caused by the presence of the diphtheria toxin. However, an increasing number of records report non-toxigenic C. diphtheriae infections. Here, a C. diphtheriae strain was recovered from a patient with a past history of bronchiectasis who developed a severe tracheo-bronchitis with multiple whitish lesions of the distal trachea and the mainstem bronchi. Whole-genome sequencing (WGS), performed in parallel with PCR targeting the toxin gene and the Elek test, provided clinically relevant results in a short turnaround time, showing that the isolate was non-toxigenic. A comparative genomic analysis of the new strain (CHUV2995) with 56 other publicly available genomes of C. diphtheriae revealed that the strains CHUV2995, CCUG 5865 and CMCNS703 share a lower average nucleotide identity (ANI) (95.24 to 95.39%) with the C. diphtheriae NCTC 11397T reference genome than all other C. diphtheriae genomes (>98.15%). Core genome phylogeny confirmed the presence of two monophyletic clades. Based on these findings, we propose here two new C. diphtheriae subspecies to replace the lineage denomination used in previous multilocus sequence typing studies: C. diphtheriae subsp. lausannense subsp. nov. (instead of lineage-2), regrouping strains CHUV2995, CCUG 5865, and CMCNS703, and C. diphtheriae subsp. diphtheriae subsp. nov, regrouping all other C. diphtheriae in the dataset (instead of lineage-1). Interestingly, members of subspecies lausannense displayed a larger genome size than subspecies diphtheriae and were enriched in COG categories related to transport and metabolism of lipids (I) and inorganic ion (P). Conversely, they lacked all genes involved in the synthesis of pili (SpaA-type, SpaD-type and SpaH-type), molybdenum cofactor and of the nitrate reductase. Finally, the CHUV2995 genome is particularly enriched in mobility genes and harbors several prophages. The genome encodes a type II-C CRISPR-Cas locus with 2 spacers that lacks csn2 or cas4, which could hamper the acquisition of new spacers and render strain CHUV2995 more susceptible to bacteriophage infections and gene acquisition through various mechanisms of horizontal gene transfer.

Spacer acquisition mechanism in type II-A CRISPR system

Spacer acquisition mechanism in type II-A CRISPR system

Cas1 and Cas2 From the Type II-C CRISPR-Cas System of Riemerella anatipestifer Are Required for Spacer Acquisition.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide acquired genetic immunity against the entry of mobile genetic elements (MGEs). The immune defense provided by various subtypes of the CRISPR-Cas system has been confirmed and is closely associated with the formation of immunological memory in CRISPR arrays, called CRISPR adaptation or spacer acquisition. However, whether type II-C CRISPR-Cas systems are also involved in spacer acquisition remains largely unknown. This study explores and provides some definitive evidence regarding spacer acquisition of the type II-C CRISPR-Cas system from Riemerella anatipestifer (RA) CH-2 (RA-CH-2). Firstly, introducing an exogenous plasmid into RA-CH-2 triggered spacer acquisition of RA CRISPR-Cas system, and the acquisition of new spacers led to plasmid instability in RA-CH-2. Furthermore, deletion of cas1 or cas2 of RA-CH-2 abrogated spacer acquisition and subsequently stabilized the exogenous plasmid, suggesting that both Cas1 and Cas2 are required for spacer acquisition of RA-CH-2 CRISPR-Cas system, consistent with the reported role of Cas1 and Cas2 in type I-E and II-A systems. Finally, assays for studying Cas1 nuclease activity and the interaction of Cas1 with Cas2 contributed to a better understanding of the adaptation mechanism of RA CRISPR-Cas system. This is the first experimental identification of the naïve adaptation of type II-C CRISPR-Cas system.

DnaQ exonuclease-like domain of Cas2 promotes spacer integration in a type I-E CRISPR-Cas system.

CRISPR-Cas systems constitute an adaptive immune system that provides acquired resistance against phages and plasmids in prokaryotes. Upon invasion of foreign nucleic acids, some cells integrate short fragments of foreign DNA as spacers into the CRISPR locus to memorize the invaders and acquire resistance in the subsequent round of infection. This immunization step called adaptation is the least understood part of the CRISPR-Cas immunity. We have focused here on the adaptation stage of Streptococcus thermophilus DGCC7710 type I-E CRISPR4-Cas (St4) system. Cas1 and Cas2 proteins conserved in nearly all CRISPR-Cas systems are required for spacer acquisition. The St4 CRISPR-Cas system is unique because the Cas2 protein is fused to an additional DnaQ exonuclease domain. Here, we demonstrate that St4 Cas1 and Cas2-DnaQ form a multimeric complex, which is capable of integrating DNA duplexes with 3'-overhangs (protospacers) in vitro We further show that the DnaQ domain of Cas2 functions as a 3'-5'-exonuclease that processes 3'-overhangs of the protospacer to promote integration.

Cas4 Nucleases Define the PAM, Length, and Orientation of DNA Fragments Integrated at CRISPR Loci

To achieve adaptive and heritable immunity againstviruses and other mobile genetic elements, CRISPR-Cas systems must capture and store short DNA fragments (spacers) from these foreign elements into host genomic CRISPR arrays. This process is catalyzed by conserved Cas1/Cas2 integration complexes, but the specific roles of another highly conserved protein linked to spacer acquisition, the Cas4 nuclease, are just now emerging. Here, we show that two Cas4 nucleases (Cas4-1 and Cas4-2) play critical roles in CRISPR spacer acquisition in Pyrococcus furiosus. The nuclease activities of both Cas4 proteins are required to process protospacers to the correct size. Cas4-1 specifies the upstream PAM (protospacer adjacent motif), while Cas4-2 specifies the conserved downstream motif. Both Cas4 proteins ensure CRISPR spacer integration in a defined orientation leading to CRISPR immunity. Collectively, these findings provide invivo evidence for critical roles of Cas4 nucleases in protospacer generation and functional spacer integration at CRISPR arrays.

Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays

CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specificendonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration.

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.

Characterization of a novel lytic bacteriophage from an industrial Escherichia coli fermentation process and elimination of virulence using a heterologous CRISPR\u2013Cas9 system

Bacterial–bacteriophage interactions are a well-studied and ecologically-important aspect of microbiology. Many commercial fermentation processes are susceptible to bacteriophage infections due to the use of high-density, clonal cell populations. Lytic infections of bacterial cells in these fermentations are especially problematic due to their negative impacts on product quality, asset utilization, and fouling of downstream equipment. Here, we report the isolation and characterization of a novel lytic bacteriophage, referred to as bacteriophage DTL that is capable of rapid lytic infections of an Escherichia coli K12 strain used for commercial production of 1,3-propanediol (PDO). The bacteriophage genome was sequenced and annotated, which identified 67 potential open-reading frames (ORF). The tail fiber ORF, the largest in the genome, was most closely related to bacteriophage RTP, a T1-like bacteriophage reported from a commercial E. coli fermentation process in Germany. To eliminate virulence, both a fully functional Streptococcus thermophilus CRISPR3 plasmid and a customized S. thermophilus CRISPR3 plasmid with disabled spacer acquisition elements and seven spacers targeting the bacteriophage DTL genome were constructed. Both plasmids were separately integrated into a PDO production strain, which was subsequently infected with bacteriophage DTL. The native S. thermophilus CRISPR3 operon was shown to decrease phage susceptibility by approximately 96%, while the customized CRISPR3 operon provided complete resistance to bacteriophage DTL. The results indicate that the heterologous bacteriophage-resistance system described herein is useful in eliminating lytic infections of bacteriophage DTL, which was prevalent in environment surrounding the manufacturing facility.

Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration.

The adaptation phase of CRISPR-Cas immunity depends on the precise integration of short segments of foreign DNA (spacers) into a specific genomic location within the CRISPR locus by the Cas1-Cas2 integration complex. Although off-target spacer integration outside of canonical CRISPR arrays has been described in vitro, no evidence of non-specific integration activity has been found in vivo. Here, we show that non-canonical off-target integrations can occur within bacterial chromosomes at locations that resemble the native CRISPR locus by characterizing hundreds of off-target integration locations within Escherichia coli. Considering whether such promiscuous Cas1-Cas2 activity could have an evolutionary role through the genesis of neo-CRISPR loci, we combed existing CRISPR databases and available genomes for evidence of off-target integration activity. This search uncovered several putative instances of naturally occurring off-target spacer integration events within the genomes of Yersinia pestis and Sulfolobus islandicus. These results are important in understanding alternative routes to CRISPR array genesis and evolution, as well as in the use of spacer acquisition in technological applications.

Analysis of the CRISPR-Cas system in bacteriophages active on epidemic strains of Vibrio cholerae in Bangladesh

CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated proteins) are microbial nuclease systems involved in defense against phages. Bacteria also resist phages by hosting phage-inducible chromosomal islands (PICI) which prevent phage reproduction. Vibrio cholerae which causes cholera epidemics, interacts with numerous phages in the environment and in cholera patients. Although CRISPR-Cas systems are usually carried by bacteria and archea, recently V. cholerae specific ICP1 phages were found to host a CRISPR-Cas system that inactivates PICI-like elements (PLE) in V. cholerae. We analyzed a collection of phages and V. cholerae isolated during seasonal cholera epidemics in Bangladesh, to study the distribution, and recent evolution of the phage-encoded CRISPR-Cas system. Five distinct but related phages carrying the CRISPR-Cas system, and possible CRISPR-Cas negative progenitor phages were identified. Furthermore, CRISPR arrays in the phages were found to have evolved by acquisition of new spacers targeting diverse regions of PLEs carried by the V. cholerae strains, enabling the phages to efficiently grow on PLE positive strains. Our results demonstrate a continuing arms-race involving genetic determinants of phage-resistance in V. cholerae, and the phage-encoded CRISPR-Cas system in the co-evolution of V. cholerae and its phages, presumably fostered by their enhanced interactions during seasonal epidemics of cholera.

Methanosarcina Spherical Virus, a Novel Archaeal Lytic Virus Targeting Methanosarcina Strains.

A novel archaeal lytic virus targeting species of the genus Methanosarcina was isolated using Methanosarcina mazei strain Gö1 as the host. Due to its spherical morphology, the virus was designated Methanosarcina spherical virus (MetSV). Molecular analysis demonstrated that MetSV contains double-stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs), all oriented in the same direction. Functions were predicted for some of these ORFs, i.e., such as DNA polymerase, ATPase, and DNA-binding protein as well as envelope (structural) protein. MetSV-derived spacers in CRISPR loci were detected in several published Methanosarcina draft genomes using bioinformatic tools, revealing a potential protospacer-adjacent motif (PAM) motif (TTA/T). Transcription and expression of several predicted viral ORFs were validated by reverse transcription-PCR (RT-PCR), PAGE analysis, and liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Analysis of core lipids by atmospheric pressure chemical ionization (APCI) mass spectrometry showed that MetSV and Methanosarcina mazei both contain archaeol and glycerol dialkyl glycerol tetraether without a cyclopentane moiety (GDGT-0). The MetSV host range is limited to Methanosarcina strains growing as single cells (M. mazei, Methanosarcina barkeri and Methanosarcina soligelidi). In contrast, strains growing as sarcina-like aggregates were apparently protected from infection. Heterogeneity related to morphology phases in M. mazei cultures allowed acquisition of resistance to MetSV after challenge by growing cultures as sarcina-like aggregates. CRISPR/Cas-mediated resistance was excluded since neither of the two CRISPR arrays showed MetSV-derived spacer acquisition. Based on these findings, we propose that changing the morphology from single cells to sarcina-like aggregates upon rearrangement of the envelope structure prevents infection and subsequent lysis by MetSV.IMPORTANCE Methanoarchaea are among the most abundant organisms on the planet since they are present in high numbers in major anaerobic environments. They convert various carbon sources, e.g., acetate, methylamines, or methanol, to methane and carbon dioxide; thus, they have a significant impact on the emission of major greenhouse gases. Today, very little is known about viruses specifically infecting methanoarchaea that most probably impact the abundance of methanoarchaea in microbial consortia. Here, we characterize the first identified Methanosarcina-infecting virus (MetSV) and show a mechanism for acquiring resistance against MetSV. Based on our results, we propose that growth as sarcina-like aggregates prevents infection and subsequent lysis. These findings allow new insights into the virus-host relationship in methanogenic community structures, their dynamics, and their phase heterogeneity. Moreover, the availability of a specific virus provides new possibilities to deepen our knowledge of the defense mechanisms of potential hosts and offers tools for genetic manipulation.

CRISPR: A Useful Genetic Feature to Follow Vaginal Carriage of Group B Streptococcus.

Clustered regularly interspaced short palindromic repeats (CRISPR) and Cas (CRISPR-associated proteins) play a critical role in adaptive immunity against mobile genetic elements, especially phages, through their ability to acquire novel spacer sequences. Polarized spacer acquisition results in spacer polymorphism and temporal organization of CRISPR loci, making them attractive epidemiological markers. Group B Streptococcus (GBS), a genital commensal for 10 to 30% of healthy women and a major neonatal pathogen, possesses a ubiquitous and functional CRISPR1 locus. Our aim was to assess the CRISPR1 locus as an epidemiological marker to follow vaginal carriage of GBS in women. This study also allowed us to observe the evolution of the CRISPR1 locus in response to probable phage infection occurring in vivo. We followed carriage of GBS among 100 women over an 11-year period, with a median duration of approximately 2 years. The CRISPR1 locus was highly conserved over time. The isolates that show the same CRISPR1 genotype were collected from 83% of women. There was an agreement between CRISPR genotyping and other typing methods [MLVA (multilocus variable number of tandem repeat Analysis) and MLST (multilocus sequence typing)] for 94% of the cases. The CRISPR1 locus of the isolates from 18 women showed modifications, four of which acquired polarized spacer, highlighting the in vivo functionality of the system. The novel spacer of one isolate had sequence similarity with phage, suggesting that phage infection occurred during carriage. These findings improve our understanding of CRISPR-Cas evolution in GBS and provide a glimpse of host-phage dynamics in vivo.

Mobile Genetic Elements and Evolution of CRISPR-Cas Systems: All the Way There and Back.

The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) systems of bacterial and archaeal adaptive immunity show multifaceted evolutionary relationships with at least five classes of mobile genetic elements (MGE). First, the adaptation module of CRISPR-Cas that is responsible for the formation of the immune memory apparently evolved from a Casposon, a self-synthesizing transposon that employs the Cas1 protein as the integrase and might have brought additional cas genes to the emerging immunity loci. Second, a large subset of type III CRISPR-Cas systems recruited a reverse transcriptase from a Group II intron, providing for spacer acquisition from RNA. Third, effector nucleases of Class 2 CRISPR-Cas systems that are responsible for the recognition and cleavage of the target DNA were derived from transposon-encoded TnpB nucleases, most likely, on several independent occasions. Fourth, accessory nucleases in some variants of types I and III toxin and type VI effectors RNases appear to be ultimately derived from toxin nucleases of microbial toxin–antitoxin modules. Fifth, the opposite direction of evolution is manifested in the recruitment of CRISPR-Cas systems by a distinct family of Tn7-like transposons that probably exploit the capacity of CRISPR-Cas to recognize unique DNA sites to facilitate transposition as well as by bacteriophages that employ them to cope with host defense. Additionally, individual Cas proteins, such as the Cas4 nuclease, were recruited by bacteriophages and transposons. The two-sided evolutionary connection between CRISPR-Cas and MGE fits the “guns for hire” paradigm whereby homologous enzymatic machineries, in particular nucleases, are shuttled between MGE and defense systems and are used alternately as means of offense or defense.

Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition

CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

Broad Targeting Specificity during Bacterial Type III CRISPR-Cas Immunity Constrains Viral Escape

CRISPR loci are a cluster of repeats separated by short "spacer" sequences derived from prokaryotic viruses and plasmids that determine the targets of the host's CRISPR-Cas immune response against its invaders. For type I and II CRISPR-Cas systems, single-nucleotide mutations in the seed or protospacer adjacent motif (PAM) of the target sequence cause immune failure and allow viral escape. This is overcome by the acquisition of multiple spacers that target the same invader. Here we show that targeting by the Staphylococcus epidermidis type III-A CRISPR-Cas system does not require PAM or seed sequences, and thus prevents viral escape via single-nucleotide substitutions. Instead, viral escapers can only arise through complete target deletion. Our work shows that, as opposed to type I and II systems, the relaxed specificity of type III CRISPR-Cas targeting provides robust immune responses that can lead to viral extinction with a single spacer targeting an essential phage sequence.

Priming in a permissive type I-C CRISPR-Cas system reveals distinct dynamics of spacer acquisition and loss.

CRISPR–Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR–Cas system in Legionella pneumophila that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3′–5′ translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe increased interference efficiency, which, when coupled with forced maintenance of a targeted plasmid, provides a useful experimental system to study spacer loss. Based on these observations, we propose that the Legionella pneumophila type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation.

Coupling transcriptional activation of CRISPR-Cas system and DNA repair genes by Csa3a in Sulfolobus islandicus.

CRISPR–Cas system provides the adaptive immunity against invading genetic elements in prokaryotes. Recently, we demonstrated that Csa3a regulator mediates spacer acquisition in Sulfolobus islandicus by activating the expression of Type I-A adaptation cas genes. However, links between the activation of spacer adaptation and CRISPR transcription/processing, and the requirement for DNA repair genes during spacer acquisition remained poorly understood. Here, we demonstrated that de novo spacer acquisition required Csa1, Cas1, Cas2 and Cas4 proteins of the Sulfolobus Type I-A system. Disruption of genes implicated in crRNA maturation or DNA interference led to a significant accumulation of acquired spacers, mainly derived from host genomic DNA. Transcriptome and proteome analyses showed that Csa3a activated expression of adaptation cas genes, CRISPR RNAs, and DNA repair genes, including herA helicase, nurA nuclease and DNA polymerase II genes. Importantly, Csa3a specifically bound the promoters of the above DNA repair genes, suggesting that they were directly activated by Csa3a for adaptation. The Csa3a regulator also specifically bound to the leader sequence to activate CRISPR transcription in vivo. Our data indicated that the Csa3a regulator couples transcriptional activation of the CRISPR–Cas system and DNA repair genes for spacer adaptation and efficient interference of invading genetic elements.

On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.

ABSTRACTCas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium—Arthrospira platensis. Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown.

CRISPR-Cas encoding of a digital movie into the genomes of a population of living bacteria.

DNA is an excellent medium for data archival. Recent efforts have illustrated the potential for information storage in DNA using synthesized oligonucleotides assembled in vitro1–6. A relatively unexplored avenue of information storage in DNA is the ability to write information into the genome of a living cell by the addition of nucleotides over time. Using the Cas1-Cas2 integrase, the CRISPR-Cas microbial immune system stores the nucleotide content of invading viruses to confer adaptive immunity7. Harnessed, this system has the potential to write arbitrary information into the genome8. Here, we use the CRISPR-Cas system to encode images and a short movie into the genomes of a population of living bacteria. In doing so, we push the technical limits of this information storage system and optimize strategies to minimize those limitations. We additionally uncover underlying principles of the CRISPR-Cas adaptation system, including sequence determinants of spacer acquisition relevant for understanding both the basic biology of bacterial adaptation as well as its technological applications. This work demonstrates that this system can capture and stably store practical amounts of real data within the genomes of populations of living cells.

Structural and dynamic insights into the role of conformational switching in the nuclease activity of the Xanthomonas albilineans Cas2 in CRISPR-mediated adaptive immunity.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute a microbial, adaptive immune system countering invading nucleic acids. Cas2 is a universal Cas protein found in all types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition into CRISPR loci. In subtype I-C CRISPR-Cas systems, Cas2 proteins are metal-dependent double-stranded DNA (dsDNA) nucleases, and a pH-dependent conformational transition has been proposed as a prerequisite for catalytic action. Here, we report the crystal structure of Xanthomonas albilineans Cas2 (XaCas2) and provide experimental evidence of a pH-dependent conformational change during functional activation. XaCas2 crystallized at an acidic pH represented a catalytically inactive conformational state in which two Asp8 residues were too far apart to coordinate a single catalytic metal ion. Consistently, XaCas2 exhibited dsDNA nuclease activity only under neutral and basic conditions. Despite the overall structural similarity of the two protomers, significant conformational heterogeneity was evident in the putative hinge regions, suggesting that XaCas2 engages in hinge-bending conformational switching. The presence of a Trp residue in the hinge region enabled the investigation of hinge dynamics by fluorescence spectroscopy. The pH dependence of the fluorescence intensity overlapped precisely with that of nuclease activity. Mutational analyses further suggested that conformational activation proceeded via a rigid-body hinge-bending motion as both D8E and hinge mutations significantly reduced nuclease activity. Together, our results reveal strong correlations between the conformational states, catalytic activity, and hinge dynamics of XaCas2, and provide structural and dynamic insights into the conformational activation of the nuclease function of Cas2.