Diversity, classification 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’.

CRISPR-Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, we examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR-Cas immune systems and deployed for wide-ranging genome editing applications. We explore the adaptive and interference aspects of CRISPR-Cas function as well as open questions about the molecular mechanisms responsible for genome targeting. These structural insights reflect close evolutionary links between CRISPR-Cas systems and mobile genetic elements, including the origins and evolution of CRISPR-Cas systems from DNA transposons, retrotransposons and toxin-antitoxin modules. We discuss how the evolution and structural diversity of CRISPR-Cas systems explain their functional complexity and utility as genome editing tools.

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.

The rapid emergence of antibiotic-resistant bacteria and the comparatively limited development of new antibacterial molecules pose a major threat to modern medicine by jeopardizing our ability to treat and prevent infections [1]. The overuse of antibiotics in both medical and agricultural settings has facilitated the emergence of multidrug-resistant (MDR) bacteria [2]. Furthermore, many antibiotics lack specificity, indiscriminately killing pathogenic and nonpathogenic bacteria and contributing to antibiotic-associated infections [3,4]. This highlights the critical need for novel therapeutics that circumvent existing modes of drug resistance while adding specificity [5]. Toward this goal, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are being explored as possible antimicrobials to target bacteria and/or antibiotic resistance genes in a sequence-specific manner. These promising targeted therapies are the focus of synthetic biology companies such as Locus Biosciences, Intellia Therapeutics, and Eligo Bioscience, to name a few. Although these technologies are mostly in the preclinical phase, the main goal is to provide precision antimicrobials for infectious diseases. Here, we discuss the present state of CRISPR antimicrobial research, with a specific focus on the biological differences between targeting plasmids versus chromosomes, the efficacy of CRISPR antimicrobials in infection and colonization models, and the various delivery mechanisms for these potential therapeutic tools. Sequence-specific targeting by CRISPR/Cas CRISPR/Cas systems are adaptive defense mechanisms against mobile genetic elements (MGEs). A key hallmark of these systems is the CRISPR array, a genomic locus comprised of unique spacers alternated by identical repeats [6]. Effector proteins function as interference molecules to silence foreign genetic elements [6]. During adaptation, some CRISPR types integrate a short fragment of foreign DNA into the CRISPR array, thereby providing a genetic memory of MGE invasion, referred to as a spacer (Fig 1). Transcription of the CRISPR array yields precursor CRISPR RNAs (pre-crRNAs) that are enzymatically processed into mature crRNAs (Fig 1). Upon invasion of bacteria by MGEs with complementary sequence, crRNAs guide effector proteins to these targets for enzymatic cleavage, ultimately causing sequence-specific elimination of the invading molecule [6] (Fig 1). Open in a separate window Fig 1 Mechanisms of action of CRISPR/Cas immunity. Left: Class I CRISPR/Cas systems (modeled by Type I system) utilize a multi-subunit complex, termed “Cascade,” as the effector machinery. Right: Class II CRISPR/Cas systems (modeled by Type II system) use a single effector protein (such as Cas9) for interference. Both classes also consist of spacer (diamonds) and repeat (squares) arrays. Top: During adaptation, the Cas1–Cas2 complex takes a sequence of the invading DNA and integrates it into the CRISPR array as a novel spacer. Center: In the next stage, termed “expression,” the CRISPR array is transcribed into pre-crRNAs that are further processed into mature interfering crRNAs. Bottom: During the interference stage, the mature crRNAs guide the Cas proteins to their DNA target. Upon binding of the crRNA to their cognate DNA target, the Cas protein generates a double-stranded DNA break in the target. Created with BioRender.com. Cascade, CRISPR-associated complex for antiviral defense; CRISPR/Cas, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein.

Classified as the genospecies Clostridium novyi sensu lato and distributed into four lineages (I–IV), Clostridium botulinum (group III), Clostridium novyi, and Clostridium haemolyticum are clostridial pathogens that cause animal diseases. Clostridium novyi sensu lato contains a large mobilome consisting of plasmids and circular bacteriophages. Here, we explored clustered regularly interspaced short palindromic repeats (CRISPR) arrays and their associated proteins (Cas) to shed light on the link between evolution of CRISPR-Cas systems and the plasmid and phage composition in a study of 58 Clostridium novyi sensu lato genomes. In 55 of these genomes, types I-B (complete or partial), I-D, II-C, III-B, III-D, or V-U CRISPR-Cas systems were detected in chromosomes as well as in mobile genetic elements (MGEs). Type I-B predominated (67.2%) and was the only CRISPR type detected in the Ia, III, and IV genomic lineages. Putative type V-U CRISPR Cas14a genes were detected in two different cases: next to partial type-IB CRISPR loci on the phage encoding the botulinum neurotoxin (BoNT) in lineage Ia and in 12 lineage II genomes, as part of a putative integrative element related to a phage-inducible chromosomal island (PICI). In the putative PICI, Cas14a was associated with CRISPR arrays and restriction modification (RM) systems as part of an accessory locus. This is the first time a PICI containing such locus has been detected in C. botulinum. Mobilome composition and dynamics were also investigated based on the contents of the CRISPR arrays and the study of spacers. A large proportion of identified protospacers (20.2%) originated from Clostridium novyi sensu lato (p1_Cst, p4_BKT015925, p6_Cst, CWou-2020a, p1_BKT015925, and p2_BKT015925), confirming active exchanges within this genospecies and the key importance of specific MGEs in Clostridium novyi sensu lato.

Being frequently exposed to foreign nucleic acids, bacteria and archaea have developed an ingenious adaptive defense system, called CRISPR-Cas. The system is composed of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array, together with CRISPR (cas)-associated genes. This system consists of a complex machinery that integrates fragments of foreign nucleic acids from viruses and mobile genetic elements (MGEs), into CRISPR arrays. The inserted segments (spacers) are transcribed and then used by cas proteins as guide RNAs for recognition and inactivation of the targets. Different types and families of CRISPR-Cas systems consist of distinct adaptation and effector modules with evolutionary trajectories, partially independent. The origin of the effector modules and the mechanism of spacer integration/deletion is far less clear. A review of the most recent data regarding the structure, ecology, and evolution of CRISPR-Cas systems and their role in the modulation of accessory genomes in prokaryotes is proposed in this article. The CRISPR-Cas system's impact on the physiology and ecology of prokaryotes, modulation of horizontal gene transfer events, is also discussed here. This system gained popularity after it was proposed as a tool for plant and animal embryo editing, in cancer therapy, as antimicrobial against pathogenic bacteria, and even for combating the novel coronavirus – SARS-CoV-2; thus, the newest and promising applications are reviewed as well.

The CRISPR (clustered regularly interspaced short palindromic repeats) system is an adaptive immune system that targets viruses and other mobile genetic elements in bacteria and archaea. Cells store information of past infections in their genome in repeat-spacer arrays. After transcription, these arrays are processed into unit-length crRNA (CRISPR RNA) that is loaded into effector complexes encoded by Cas (CRISPR-associated) genes. CRISPR-Cas complexes target invading nucleic acid for degradation. CRISPR effector complexes have been classified into three main types (I-III). Type III effector complexes share the Cas10 subunit. In the present paper, we discuss the structures of the two Type III effector complexes from Sulfolobus solfataricus, SsoCSM (subtype III-A) and SsoCMR (subtype III-B), obtained by electron microscopy and single particle analysis. We also compare these structures with Cascade (CRISPR-associated complex for antiviral defence) and with the RecA nucleoprotein.

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports.

The Greater Virus World and its Evolution

In this study, we describe a rapid immunoaffinity purification procedure for gel-free tandem mass spectrometry-based analysis of endogenous protein complexes and apply it to the characterization of complexes containing the SRm160 (serine/arginine repeat-related nuclear matrix protein of 160 kDa) splicing coactivator. In addition to promoting splicing, SRm160 stimulates 3'-end processing via its N-terminal PWI nucleic acid-binding domain and is found in a post-splicing exon junction complex that has been implicated in coupling splicing with mRNA turnover, export, and translation. Consistent with these known functional associations, we found that the majority of proteins identified in SRm160-containing complexes are associated with pre-mRNA processing. Interestingly, SRm160 is also associated with factors involved in chromatin regulation and sister chromatid cohesion, specifically the cohesin subunits SMC1alpha, SMC3, RAD21, and SA2. Gradient fractionation suggested that there are two predominant SRm160-containing complexes, one enriched in splicing components and the other enriched in cohesin subunits. Co-immunoprecipitation and co-localization experiments, as well as combinatorial RNA interference in Caenorhabditis elegans, support the existence of conserved and functional interactions between SRm160 and cohesin.

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3′ end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes, utilising small CRISPR RNAs which direct effector complexes to degrade invading nucleic acids1–3. Type III effector complexes were recently demonstrated to synthesise a novel second messenger, cyclic oligoadenylate (cOA), on binding target RNA4,5. cOA in turn binds to and activates ribonucleases and other factors via a CARF (CRISPR associated Rossman Fold) domain, inducing an antiviral state in the cell that is important for immunity. The mechanism of the “off-switch” that resets the system is not understood. Here, we report the identification of the nuclease that degrades these cOA ring molecules. The “Ring nuclease” is itself a CARF family protein with a metal independent mechanism, which cleaves cA4 rings to generate linear di-adenylate species and switches off the antiviral state. The identification of Ring nucleases adds an important insight to the CRISPR system.

In type I CRISPR–Cas systems, primed adaptation of new spacers into CRISPR arrays occurs when the effector Cascade–crRNA complex recognizes imperfectly matched targets that are not subject to efficient CRISPR interference. Thus, primed adaptation allows cells to acquire additional protection against mobile genetic elements that managed to escape interference. Biochemical and biophysical studies suggested that Cascade–crRNA complexes formed on fully matching targets (subject to efficient interference) and on partially mismatched targets that promote primed adaption are structurally different. Here, we probed Escherichia coli Cascade–crRNA complexes bound to matched and mismatched DNA targets using a magnetic tweezers assay. Significant differences in complex stabilities were observed consistent with the presence of at least two distinct conformations. Surprisingly, in vivo analysis demonstrated that all mismatched targets stimulated robust primed adaptation irrespective of conformational states observed in vitro. Our results suggest that primed adaptation is a direct consequence of a reduced interference efficiency and/or rate and is not a consequence of distinct effector complex conformations on target DNA.

Background Bacteria and archaea have developed a number of strategies to keep the influx of mobile genetic elements (MGEs) such as viruses and plasmids in check. CRISPR-Cas systems provide adaptive immunity; all types of CRISPR-Cas systems have in common the genes for uptake of genetic information from the invading MGE but differ in the composition of the effector complex that directs and enforces the immune response. The genome of Mycobacterium tuberculosis encodes a Class 1, type III-A CRISPR-Cas system that has not been studied in detail. Methodology We used heterologous production of the mycobacterial CRISPR-Cas genes. Purification of the enzymes enabled in vitro biochemical characterisation of the adaptation, maturation and interference proteins. Reconstitution of the system in E. coli was used to demonstrate functionality in vivo. Results Maturation of crRNA through the action of Cas6 proceeded as expected, generating canonical CRISPR RNAs (crRNAs). The type III effector complex, consisting of Csm1-5, was shown to bind crRNA and cleave target RNA with the typical 6 nt spacing, display ssDNase activity and produce the cyclic oligoadenylate signalling molecule. The latter clearly activated the ribonuclease Csm6, an essential element in type III immunity. The M. tuberculosis type III CRISPR-Cas system was also reconstituted in E. coli where it provided plasmid immunity, demonstrating the functionality in vivo. Conclusions All elements of the M. tuberculosis type III CRISPR-Cas system are functional in vitro and in vivo. These studies lay the foundation for further investigations into the mechanism of adaptive immunity and possible applications in biotechnology.

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity have become a household name among biologists and even the general public thanks to the unprecedented success of the new generation of genome editing tools utilizing Cas proteins. However, the fundamental biological features of CRISPR-Cas are of no lesser interest and have major impacts on our understanding of the evolution of antivirus defense, host-parasite coevolution, self versus non-self discrimination and mechanisms of adaptation. CRISPR-Cas systems present the best known case in point for Lamarckian evolution, i.e. generation of heritable, adaptive genomic changes in response to encounters with external factors, in this case, foreign nucleic acids. CRISPR-Cas systems employ multiple mechanisms of self versus non-self discrimination but, as is the case with immune systems in general, are nevertheless costly because autoimmunity cannot be eliminated completely. In addition to the autoimmunity, the fitness cost of CRISPR-Cas systems appears to be determined by their inhibitory effect on horizontal gene transfer, curtailing evolutionary innovation. Hence the dynamic evolution of CRISPR-Cas loci that are frequently lost and (re)acquired by archaea and bacteria. Another fundamental biological feature of CRISPR-Cas is its intimate connection with programmed cell death and dormancy induction in microbes. In this and, possibly, other immune systems, active immune response appears to be coupled to a different form of defense, namely, “altruistic” shutdown of cellular functions resulting in protection of neighboring cells. Finally, analysis of the evolutionary connections of Cas proteins reveals multiple contributions of mobile genetic elements (MGE) to the origin of various components of CRISPR-Cas systems, furthermore, different biological systems that function by genome manipulation appear to have evolved convergently from unrelated MGE. The shared features of adaptive defense systems and MGE, namely the ability to recognize and cleave unique sites in genomes, make them ideal candidates for genome editing and engineering tools.