PAM-repeat associations and spacer selection preferences in single and co-occurring CRISPR-Cas systems

Background: The adaptive CRISPR-Cas immune system stores sequences from past invaders as spacers in CRISPR arrays and thereby provides direct evidence that links invaders to hosts. Mapping CRISPR spacers has revealed many aspects of CRISPR-Cas biology, including target requirements such as the protospacer adjacent motif (PAM). However, studies have so far been limited by a low number of mapped spacers in the database. Results: By using vast metagenomic sequence databases, we map approximately one-third of more than 200,000 unique CRISPR spacers from a variety of microbes and derive a catalog of more than two hundred unique PAM sequences associated with specific CRISPR-Cas subtypes. These PAMs are further used to correctly assign the orientation of CRISPR arrays, revealing conserved patterns between the last nucleotides of the CRISPR repeat and PAM. We could also deduce CRISPR-Cas subtype-specific preferences for targeting either template or coding strand of open reading frames. While some DNA-targeting systems (type I-E and type II systems) prefer the template strand and avoid mRNA, other DNA- and RNA-targeting systems (types I-A and I-B and type III systems) prefer the coding strand and mRNA. In addition, we find large-scale evidence that both CRISPR-Cas adaptation machinery and CRISPR arrays are shared between different CRISPR-Cas systems. This could lead to simultaneous DNA and RNA targeting of invaders, which may be effective at combating mobile genetic invaders. Conclusions: This study has broad implications for our understanding of how CRISPR-Cas systems work in a wide range of organisms for which only the genome sequence is known.BN/Stan Brouns La

Conserved motifs in the CRISPR leader sequence control spacer acquisition levels in Type I-D CRISPR-Cas systems

Integrating short DNA fragments at the correct leader-repeat junction is key to successful CRISPR-Cas memory formation. The Cas1-2 proteins are responsible to carry out this process. However, the CRISPR adaptation process additionally requires a DNA element adjacent to the CRISPR array, called leader, to facilitate efficient localization of the correct integration site. In this work, we introduced the core CRISPR adaptation genes cas1 and cas2 from the Type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and assessed spacer integration efficiency. Truncation of the leader resulted in a significant reduction of spacer acquisition levels and revealed the importance of different conserved regions for CRISPR adaptation rates. We found three conserved sequence motifs in the leader of I-D CRISPR arrays that each affected spacer acquisition rates, including an integrase anchoring site. Our findings support the model in which the leader sequence is an integral part of type I-D adaptation in Synechocystis sp. acting as a localization signal for the adaptation complex to drive CRISPR adaptation at the first repeat of the CRISPR array.BN/Stan Brouns La

Complete genome sequences of two T4-like Escherichia coli bacteriophages

Bacteriophages and their proteins have potential applications in biotechnology for the detection and control of bacterial diseases. Here, we describe the sequencing and genome annotations of two strictly virulent Escherichia coli bacteriophages that may be explored for biocontrol strategies and to expand the understanding of phage-host interactions.BN/Stan Brouns La

RNA-targeting CRISPR–Cas systems

CRISPR–Cas is a widespread adaptive immune system in bacteria and archaea that protects against viral infection by targeting specific invading nucleic acid sequences. Whereas some CRISPR–Cas systems sense and cleave viral DNA, type III and type VI CRISPR–Cas systems sense RNA that results from viral transcription and perhaps invasion by RNA viruses. The sequence-specific detection of viral RNA evokes a cell-wide response that typically involves global damage to halt the infection. How can one make sense of an immune strategy that encompasses broad, collateral effects rather than specific, targeted destruction? In this Review, we summarize the current understanding of RNA-targeting CRISPR–Cas systems. We detail the composition and properties of type III and type VI systems, outline the cellular defence processes that are instigated upon viral RNA sensing and describe the biological rationale behind the broad RNA-activated immune responses as an effective strategy to combat viral infection.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BN/Stan Brouns LabScience Centre & Programmerin

Prophages are associated with extensive CRISPR-Cas auto-immunity

CRISPR-Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing self-targeting spacers (STS), i.e. CRISPR spacers targeting protospacers on the same genome. STS has been suggested to reflect potential auto-immunity as an unwanted side effect of CRISPR-Cas defense, or a regulatory mechanism for gene expression. Here we investigated the incidence, distribution, and evasion of STS in over 100 000 bacterial genomes. We found STS in all CRISPR-Cas types and in one fifth of all CRISPR-carrying bacteria. Notably, up to 40% of I-B and I-F CRISPR-Cas systems contained STS. We observed that STS-containing genomes almost always carry a prophage and that STS map to prophage regions in more than half of the cases. Despite carrying STS, genetic deterioration of CRISPR-Cas systems appears to be rare, suggesting a level of escape from the potentially deleterious effects of STS by other mechanisms such as anti-CRISPR proteins and CRISPR target mutations. We propose a scenario where it is common to acquire an STS against a prophage, and this may trigger more extensive STS buildup by primed spacer acquisition in type I systems, without detrimental autoimmunity effects as mechanisms of auto-immunity evasion create tolerance to STS-targeted prophages.BN/Stan Brouns La

Complete genome sequence of the Escherichia coli phage Ayreon

We report the whole-genome sequence of a new Escherichia coli temperate phage, Ayreon, comprising a linear double-stranded DNA (dsDNA) genome of 44,708 bp.BN/Stan Brouns La

Approaches for bacteriophage genome engineering

In recent years, bacteriophage research has been boosted by a rising interest in using phage therapy to treat antibiotic-resistant bacterial infections. In addition, there is a desire to use phages and their unique proteins for specific biocontrol applications and diagnostics. However, the ability to manipulate phage genomes to understand and control gene functions, or alter phage properties such as host range, has remained challenging due to a lack of universal selectable markers. Here, we discuss the state-of-the-art techniques to engineer and select desired phage genomes using advances in cell-free methodologies and clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR-Cas) counter-selection approaches.BN/Stan Brouns La

Craspase Orthologs Cleave a Nonconserved Site in Target Protein Csx30

The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus “Jettenia caeni” (Jc-Craspase). We find that Jc-Craspase cleaves Jc-Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus “Scalindua brodae” and Desulfonema ishimotonii, respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.BN/Stan Brouns LabBT/Environmental Biotechnolog

Mining moon & mars with microbes: Biological approaches to extract iron from Lunar and Martian regolith

The logistical supply of terrestrial materials to space is costly and puts limitations on exploration mission scenarios. In-situ resource utilization (ISRU) can alleviate logistical requirements and thus enables sustainable exploration of space. In this paper, a novel approach to ISRU, utilizing microorganisms to extract iron from Lunar or Martian regolith, is presented. Process yields, and kinetics are used to verify the theoretical feasibility of applying four different microorganisms. Based on yields alone, three of the four organisms were not investigated further for use in biological ISRU. For the remaining organism, Shewanella oneidensis, the survivability impact of Martian regolith simulant JSC-MARS1 and Mars-abundant magnesium perchlorate were studied and found to be minimal. The payback time of the infrastructure installation needed for the process with S. oneidensis on Mars was analyzed and the sensitivity to various parameters was investigated. Water recycling efficiency and initial regolith concentration were found to be key to process performance. With a water recycling efficiency of 99.99% and initial regolith concentration of 300 ​g/L, leading to an iron concentration of approximately 44.7 ​g/L, a payback time of 3.3 years was found.Accepted Author ManuscriptBN/Stan Brouns La

Structural basis for broad anti-phage immunity by DISARM

In the evolutionary arms race against phage, bacteria have assembled a diverse arsenal of antiviral immune strategies. While the recently discovered DISARM (Defense Island System Associated with Restriction-Modification) systems can provide protection against a wide range of phage, the molecular mechanisms that underpin broad antiviral targeting but avoiding autoimmunity remain enigmatic. Here, we report cryo-EM structures of the core DISARM complex, DrmAB, both alone and in complex with an unmethylated phage DNA mimetic. These structures reveal that DrmAB core complex is autoinhibited by a trigger loop (TL) within DrmA and binding to DNA substrates containing a 5′ overhang dislodges the TL, initiating a long-range structural rearrangement for DrmAB activation. Together with structure-guided in vivo studies, our work provides insights into the mechanism of phage DNA recognition and specific activation of this widespread antiviral defense system.BN/Stan Brouns La