Regulatory sequence-based discovery of anti-defense genes in archaeal viruses.

In silico identification of viral anti-CRISPR proteins (Acrs) has relied largely on the guilt-by-association method using known Acrs or anti-CRISPR associated proteins (Acas) as the bait. However, the low number and limited spread of the characterized archaeal Acrs and Aca hinders our ability to identify Acrs using guilt-by-association. Here, based on the observation that the few characterized archaeal Acrs and Aca are transcribed immediately post viral infection, we hypothesize that these genes, and many other unidentified anti-defense genes (ADG), are under the control of conserved regulatory sequences including a strong promoter, which can be used to predict anti-defense genes in archaeal viruses. Using this consensus sequence based method, we identify 354 potential ADGs in 57 archaeal viruses and 6 metagenome-assembled genomes. Experimental validation identified a CRISPR subtype I-A inhibitor and the first virally encoded inhibitor of an archaeal toxin-antitoxin based immune system. We also identify regulatory proteins potentially akin to Acas that can facilitate further identification of ADGs combined with the guilt-by-association approach. These results demonstrate the potential of regulatory sequence analysis for extensive identification of ADGs in viruses of archaea and bacteria.

Fanzor is a eukaryotic programmable RNA-guided endonuclease.

RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR-Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors such as Cas9 and Cas12 perform guide-RNA-dependent DNA cleavage1. Although a few eukaryotic RNA-guided systems have been studied, including RNA interference2 and ribosomal RNA modification3, it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided systems (termed OMEGA) was reported4,5. The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity4,6. TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins4,7, raising the possibility that eukaryotes are also equipped with CRISPR-Cas or OMEGA-like programmable RNA-guided endonucleases. Here we report the biochemical characterization of Fz, showing that it is an RNA-guided DNA endonuclease. We also show that Fz can be reprogrammed for human genome engineering applications. Finally, we resolve the structure of Spizellomyces punctatus Fz at 2.7 Å using cryogenic electron microscopy, showing the conservation of core regions among Fz, TnpB and Cas12, despite diverse cognate RNA structures. Our results show that Fz is a eukaryotic OMEGA system, demonstrating that RNA-guided endonucleases are present in all three domains of life.

Prokaryotic innate immunity through pattern recognition of conserved viral proteins.

Many organisms have evolved specialized immune pattern-recognition receptors, including nucleotide-binding oligomerization domain-like receptors (NLRs) of the STAND superfamily that are ubiquitous in plants, animals, and fungi. Although the roles of NLRs in eukaryotic immunity are well established, it is unknown whether prokaryotes use similar defense mechanisms. Here, we show that antiviral STAND (Avs) homologs in bacteria and archaea detect hallmark viral proteins, triggering Avs tetramerization and the activation of diverse N-terminal effector domains, including DNA endonucleases, to abrogate infection. Cryo-electron microscopy reveals that Avs sensor domains recognize conserved folds, active-site residues, and enzyme ligands, allowing a single Avs receptor to detect a wide variety of viruses. These findings extend the paradigm of pattern recognition of pathogen-specific proteins across all three domains of life.

Evolutionary plasticity and functional versatility of CRISPR systems.

The principal biological function of bacterial and archaeal CRISPR systems is RNA-guided adaptive immunity against viruses and other mobile genetic elements (MGEs). These systems show remarkable evolutionary plasticity and functional versatility at multiple levels, including both the defense mechanisms that lead to direct, specific elimination of the target DNA or RNA and those that cause programmed cell death (PCD) or induction of dormancy. This flexibility is also evident in the recruitment of CRISPR systems for nondefense functions. Defective CRISPR systems or individual CRISPR components have been recruited by transposons for RNA-guided transposition, by plasmids for interplasmid competition, and by viruses for antidefense and interviral conflicts. Additionally, multiple highly derived CRISPR variants of yet unknown functions have been discovered. A major route of innovation in CRISPR evolution is the repurposing of diverged repeat variants encoded outside CRISPR arrays for various structural and regulatory functions. The evolutionary plasticity and functional versatility of CRISPR systems are striking manifestations of the ubiquitous interplay between defense and "normal" cellular functions.

Type II anti-CRISPR proteins as a new tool for synthetic biology.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) system represents, in prokaryotes, an adaptive and inheritable immune response against invading DNA. The discovery of anti-CRISPR proteins (Acrs), which are inhibitors of CRISPR-Cas, mainly encoded by phages and prophages, showed a co-evolution history between prokaryotes and phages. In the past decade, the CRISPR-Cas systems together with the corresponding Acrs have been turned into a genetic-engineering tool. Among the six types of CRISPR-Cas characterized so far, type II CRISPR-Cas system is the most popular in biotechnology. Here, we discuss about the discovery, the reported inhibitory mechanisms, and the applications in both gene editing and gene transcriptional regulation of type II Acrs. Moreover, we provide insights into future potential research and feasible applications.

Prokaryote autoimmunity in the context of self-targeting by CRISPR-Cas systems.

Prokaryote adaptive immunity (CRISPR-Cas systems) can be a threat to its carriers. We analyze the risks of autoimmune reactions related to adaptive immunity in prokaryotes by computational methods. We found important differences between bacteria and archaea with respect to autoimmunity potential. According to the results of our analysis, CRISPR-Cas systems in bacteria are more prone to self-targeting even though they possess fewer spacers per organism on average than archaea. The results of our study provide opportunities to use self-targeting in prokaryotes for biological and medical applications.

Anti-CRISPR Proteins in Archaea.

Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas immune systems. To date, Acrs inhibiting types I, II, III, V, and VI CRISPR-Cas systems have been characterized. While most known Acrs are derived from bacterial phages and prophages, very few have been characterized in the domain Archaea, despite the nearly ubiquitous presence of CRISPR-Cas in archaeal cells. Here we summarize the discovery and characterization of the archaeal Acrs with the representatives encoded by a model archaeal virus, Sulfolobus islandicus rod-shaped virus 2 (SIRV2). AcrID1 inhibits subtype I-D CRISPR-Cas immunity through direct interaction with the large subunit Cas10d of the effector complex, and AcrIIIB1 inhibits subtype III-B CRISPR-Cas immunity through a mechanism interfering with middle/late gene targeting. Future development of efficient screening methods will be key to uncovering the diversity of archaeal Acrs.

Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems.

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.

CRISPR/Cas: from adaptive immune system in prokaryotes to therapeutic weapon against immune-related diseases.

CRISPR/Cas evolved as an adaptive immune system in bacteria and archaea to inactivate foreign viral and plasmid DNA. However, the capacities of various CRISPR/Cas systems for precise genome editing based on sequence homology also allow their use as tools for genomic and epigenomic modification in eukaryotes. Indeed, these genetic characteristics have proven useful for disease modeling and testing the specific functions of target genes under pathological conditions. Moreover, recent studies provide compelling evidence that CRISPR/Cas systems could be useful therapeutic tools against human diseases, including cancer, monogenic disorders, and autoimmune disorders.HighlightsCRISPR/Cas evolved as an adaptive immune system in bacteria and archaea.CRISPR/Cas systems are nowadays used as tools for genomic modification.CRISPR/Cas systems could be useful therapeutic tools against human disease, including autoimmune conditions.

Approaches to study CRISPR RNA biogenesis and the key players involved.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide an inheritable and adaptive immune system against phages and foreign genetic elements in many bacteria and archaea. The three stages of CRISPR-Cas immunity comprise adaptation, CRISPR RNA (crRNA) biogenesis and interference. The maturation of the pre-crRNA into mature crRNAs, short guide RNAs that target invading nucleic acids, is crucial for the functionality of CRISPR-Cas defense systems. Mature crRNAs assemble with Cas proteins into the ribonucleoprotein (RNP) effector complex and guide the Cas nucleases to the cognate foreign DNA or RNA target. Experimental approaches to characterize these crRNAs, the specific steps toward their maturation and the involved factors, include RNA-seq analyses, enzyme assays, methods such as cryo-electron microscopy, the crystallization of proteins, or UV-induced protein-RNA crosslinking coupled to mass spectrometry analysis. Complex and multiple interactions exist between CRISPR-cas-encoded specific riboendonucleases such as Cas6, Cas5d and Csf5, endonucleases with dual functions in maturation and interference such as the enzymes of the Cas12 and Cas13 families, and nucleases belonging to the cell's degradosome such as RNase E, PNPase and RNase J, both in the maturation as well as in interference. The results of these studies have yielded a picture of unprecedented diversity of sequences, enzymes and biochemical mechanisms.

Anti-CRISPR proteins targeting the CRISPR-Cas system enrich the toolkit for genetic engineering.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune defense systems, which are widely distributed in bacteria and Archaea, can provide sequence-specific protection against foreign DNA or RNA in some cases. However, the evolution of defense systems in bacterial hosts did not lead to the elimination of phages, and some phages carry anti-CRISPR genes that encode products that bind to the components mediating the defense mechanism and thus antagonize CRISPR-Cas immune systems of bacteria. Given the extensive application of CRISPR-Cas9 technologies in gene editing, in this review, we focus on the anti-CRISPR proteins (Acrs) that inhibit CRISPR-Cas systems for gene editing. We describe the discovery of Acrs in immune systems involving type I, II, and V CRISPR-Cas immunity, discuss the potential function of Acrs in inactivating type II and V CRISPR-Cas systems for gene editing and gene modulation, and provide an outlook on the development of important biotechnology tools for genetic engineering using Acrs.

Methanogenic Archaea: Emerging Partners in the Field of Allergic Diseases.

Archaea, which form one of four domains of life alongside Eukarya, Bacteria, and giant viruses, have long been neglected as components of the human microbiota and potential opportunistic infectious pathogens. In this review, we focus on methanogenic Archaea, which rely on hydrogen for their metabolism and growth. On one hand, methanogenic Archaea in the gut are functional associates of the fermentative digestion of dietary fibers, favoring the production of beneficial short-chain fatty acids and likely contributing to the weaning reaction during the neonatal window of opportunity. On the other hand, methanogenic Archaea trigger the activation of innate and adaptive responses and the generation of specific T and B cells in animals and humans. In mouse models, lung hypersensitivity reactions can be induced by inhaled methanogenic Archaea mimicking human professional exposure to organic dust. Changes in methanogenic Archaea of the microbiota are detected in an array of dysimmune conditions comprising inflammatory bowel disease, obesity, malnutrition, anorexia, colorectal cancer, and diverticulosis. At the subcellular level, methanogenic Archaea are activators of the TLR8-dependent NLRP3 inflammasome, modulate the release of antimicrobial peptides and drive the production of proinflammatory, Th-1, Th-2, and Th-17 cytokines. Our objective was to introduce the most recent and major pieces of evidence supporting the involvement of Archaea in the balance between health and dysimmune diseases, with a particular focus on atopic and allergic conditions.

Archaeal glycolipid adjuvanted vaccines induce strong influenza-specific immune responses through direct immunization in young and aged mice or through passive maternal immunization.

Vaccine induced responses are often weaker in those individuals most susceptible to infection, namely the very young and the elderly, highlighting the need for safe and effective vaccine adjuvants. Herein we evaluated different archaeosome formulations as an adjuvant to the H1N1 influenza hemagglutinin protein and compared immune responses (anti-HA IgG and hemagglutination inhibition assay titers) as well as protection to an influenza A virus (strainA/PuertoRico/8/1934H1N1)homologous challenge to those generated using a squalene-based oil-in-water nano-emulsion, AddaVax™ in a murine model. The impact of age (young adult vs aged) on vaccine induced immune responses as well as the protection in pups due to the transfer of maternal antibodies was measured. Overall, we show that archaeal lipid based adjuvants can induce potent anti-HA responses in young and aged mice that can also be passed from vaccinated mothers to pups. Furthermore, young and aged mice immunized with archaeal lipid adjuvants as well as pups from immunized mothers were protected from challenge with influenza. In addition, we show that a simple admixed archaeosome formulation composed of a single sulfated glycolipid namely sulfated lactosylarchaeol (SLA; 6'-sulfate-ß-D-Galp-(1,4)-ß-D-Glcp-(1,1)-archaeol) can give equal or better protection compared to AddaVax™ or the traditional antigen-encapsulated archaeosome formulations.

Visualization and prediction of CRISPR incidence in microbial trait-space to identify drivers of antiviral immune strategy.

Bacteria and archaea are locked in a near-constant battle with their viral pathogens. Despite previous mechanistic characterization of numerous prokaryotic defense strategies, the underlying ecological drivers of different strategies remain largely unknown and predicting which species will take which strategies remains a challenge. Here, we focus on the CRISPR immune strategy and develop a phylogenetically-corrected machine learning approach to build a predictive model of CRISPR incidence using data on over 100 traits across over 2600 species. We discover a strong but hitherto-unknown negative interaction between CRISPR and aerobicity, which we hypothesize may result from interference between CRISPR-associated proteins and non-homologous end-joining DNA repair due to oxidative stress. Our predictive model also quantitatively confirms previous observations of an association between CRISPR and temperature. Finally, we contrast the environmental associations of different CRISPR system types (I, II, III) and restriction modification systems, all of which act as intracellular immune systems.

Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems.

The constant selective pressure exerted by phages, the viruses that infect bacteria, has led to the evolution of a wide range of anti-phage defenses. One of these defense mechanisms, CRISPR-Cas, provides an adaptive immune system to battle phage infection and inhibit horizontal gene transfer by plasmids, transposons, and other mobile genetic elements. Although CRISPR-Cas systems are widespread in bacteria and archaea, they appear to have minimal long-term evolutionary effects with respect to limiting horizontal gene transfer. One factor that may contribute to this may be the presence of potent inhibitors of CRISPR-Cas systems, known as anti-CRISPR proteins. Forty unique families of anti-CRISPR proteins have been described to date. These inhibitors, which are active against both Class 1 and 2 CRISPR-Cas systems, have a wide range of mechanisms of activity. Studies of these proteins have provided important insight into the evolutionary arms race between bacteria and phages, and have contributed to the development of biotechnological tools that can be harnessed for control of CRISPR-Cas genome editing.

Origins and evolution of CRISPR-Cas systems.

CRISPR-Cas, the bacterial and archaeal adaptive immunity systems, encompass a complex machinery that integrates fragments of foreign nucleic acids, mostly from mobile genetic elements (MGE), into CRISPR arrays embedded in microbial genomes. Transcripts of the inserted segments (spacers) are employed by CRISPR-Cas systems as guide (g)RNAs for recognition and inactivation of the cognate targets. The CRISPR-Cas systems consist of distinct adaptation and effector modules whose evolutionary trajectories appear to be at least partially independent. Comparative genome analysis reveals the origin of the adaptation module from casposons, a distinct type of transposons, which employ a homologue of Cas1 protein, the integrase responsible for the spacer incorporation into CRISPR arrays, as the transposase. The origin of the effector module(s) is far less clear. The CRISPR-Cas systems are partitioned into two classes, class 1 with multisubunit effectors, and class 2 in which the effector consists of a single, large protein. The class 2 effectors originate from nucleases encoded by different MGE, whereas the origin of the class 1 effector complexes remains murky. However, the recent discovery of a signalling pathway built into the type III systems of class 1 might offer a clue, suggesting that type III effector modules could have evolved from a signal transduction system involved in stress-induced programmed cell death. The subsequent evolution of the class 1 effector complexes through serial gene duplication and displacement, primarily of genes for proteins containing RNA recognition motif domains, can be hypothetically reconstructed. In addition to the multiple contributions of MGE to the evolution of CRISPR-Cas, the reverse flow of information is notable, namely, recruitment of minimalist variants of CRISPR-Cas systems by MGE for functions that remain to be elucidated. Here, we attempt a synthesis of the diverse threads that shed light on CRISPR-Cas origins and evolution. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

Exploration of Microbial Diversity to Discover Novel Molecular Technologies.

Many powerful molecular biology tools have their origin in nature. From restriction enzymes to CRISPR-Cas9, microbes utilize a diverse array of systems to get ahead evolutionarily. We are exploring this natural diversity through bioinformatics, biochemical, and molecular work to better understand the fundamental ways in which microbes and other living organisms sense and respond to their environment and as possible to develop these natural systems as molecular tools and to improve human health. Building on our demonstration that Cas9 can be repurposed for precision genome editing in mammalian cells, we look for novel CRISPR-Cas systems that are different and may have other useful properties. This led to the discovery of several new CRISPR systems, including the CRISPR-Cas13 family that target RNA, rather than DNA. We have developed a toolbox for RNA modulation based on Cas13, including methods for precision base editing, adding to our robust toolbox for DNA based on Cas9 and Cas12. We are expanding our biodiscovery efforts to search for new microbial proteins that may be adapted for applications beyond genome and transcriptome modulation, capitalizing on the growing volume of microbial genomic sequences. We are particularly interested in identifying new therapeutic modalities and vehicles for delivering them into patients. We hope that additional robust tools and delivery options will further accelerate research into human disease and open up new therapeutic possibilities.

A comparison of the immune responses induced by antigens in three different archaeosome-based vaccine formulations.

Archaeosomes are liposomes composed of natural or synthetic archaeal lipids that can be used as adjuvants to induce strong long-lasting humoral and cell-mediated immune responses against entrapped antigen. However, the entrapment efficiency of antigen within archaeosomes constituted using standard liposome forming methodology is often only 5-40%. In this study, we evaluated different formulation methods using a simple semi-synthetic archaeal lipid (SLA, sulfated lactosyl archaeol) and two different antigens, ovalbumin (OVA) and hepatitis B surface antigen (HBsAg). Antigen was entrapped within archaeosomes using the conventional thin film hydration-rehydration method with or without removal of non-entrapped antigen, or pre-formed empty archaeosomes were simply admixed with an antigen solution. Physicochemical characteristics were determined (size distribution, zeta potential, vesicle morphology and lamellarity), as well as location of antigen relative to bilayer using cryogenic transmission electron microscopy (TEM). We demonstrate that antigen (OVA or HBsAg) formulated with SLA lipid adjuvants using all the different methodologies resulted in a strong antigen-specific immune response. Nevertheless, the advantage of using a drug substance process that comprises of simply admixing antigen with pre-formed empty archaeosomes, represents a simple, efficient and antigenic dose-sparing formulation for adjuvanting and delivering vaccine antigens.

Open questions: CRISPR biology.

CRISPR-Cas systems, the purveyors of adaptive immunity in archaea and bacteria and sources of the new generation of genome engineering tools, have been studied in exquisite molecular detail. However, when it comes to biological functions, ecology, and evolution of CRISPR-Cas, many more intriguing questions remain than there are answers.