Genetic Invaders Research Articles

A B cell screen against endogenous retroviruses identifies glycan-reactive IgM that recognizes a broad array of enveloped viruses.

Endogenous retroviruses (ERVs), comprising a substantial portion of the vertebrate genome, are remnants of ancient genetic invaders. ERVs with near-intact coding potential reactivate in B cell-deficient mice. To study how B cells contribute to host anti-ERV immunity, we used an antigen-baiting strategy to enrich B cells reactive to ERV surface antigens. We identified ERV-reactive B-1 cells expressing germline-encoded natural IgM antibodies in naïve mice, the level of which further increases upon innate immune sensor stimulation. B cell receptor repertoire profiling of ERV-reactive B-1 cells revealed increased usage of the Igh VH gene that gives rise to glycan-specific antibodies targeting terminal N-acetylglucosamine moieties on ERV glycoproteins, which further engage the complement pathway to mediate anti-ERV responses. These same antibodies also recognize glycoproteins of other enveloped viruses but not self-proteins. These results reveal an innate antiviral mechanism of germline-encoded antibodies with broad reactivity to enveloped viruses, which constitutes a natural antibody repertoire capable of preventing the emergence of infectious ERVs.

The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity.

Type III CRISPR systems provide immunity against genetic invaders through the production of cyclic oligo-adenylate (cAn) molecules that activate effector proteins that contain CRISPR-associated Rossman fold (CARF) domains. Here, we characterized the function and structure of an effector in which the CARF domain is fused to an adenosine deaminase domain, CRISPR-associated adenosine deaminase 1 (Cad1). We show that upon binding of cA4 or cA6 to its CARF domain, Cad1 converts ATP to ITP, both invivo and invitro. Cryoelectron microscopy (cryo-EM) structural studies on full-length Cad1 reveal an hexameric assembly composed of a trimer of dimers, with bound ATP at inter-domain sites required for activity and ATP/ITP within deaminase active sites. Upon synthesis of cAn during phage infection, Cad1 activation leads to a growth arrest of the host that prevents viral propagation. Our findings reveal that CRISPR-Cas systems employ a wide range of molecular mechanisms beyond nucleic acid degradation to provide adaptive immunity in prokaryotes.

CRISPR-repressed toxin-antitoxin provides herd immunity against anti-CRISPR elements.

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are highly vulnerable to phage-encoded anti-CRISPR (Acr) factors. How CRISPR-Cas systems protect themselves remains unclear. Here we uncovered a broad-spectrum anti-anti-CRISPR strategy involving a phage-derived toxic protein. Transcription of this toxin is normally repressed by the CRISPR-Cas effector but is activated to halt cell division when the effector is inhibited by any anti-CRISPR proteins or RNAs. We showed that this abortive infection-like effect efficiently expels Acr elements from bacterial population. Furthermore, we exploited this anti-anti-CRISPR mechanism to develop a screening method for specific Acr candidates for a CRISPR-Cas system and successfully identified two distinct Acr proteins that enhance the binding of CRISPR effector to nontarget DNA. Our data highlight the broad-spectrum role of CRISPR-repressed toxins in counteracting various types of Acr factors. We propose that the regulatory function of CRISPR-Cas confers host cells herd immunity against Acr-encoding genetic invaders whether they are CRISPR targeted or not.

Widespread RNA-based cas regulation monitors crRNA abundance and anti-CRISPR proteins

CRISPR RNAs (crRNAs) and Cas proteins work together to provide prokaryotes with adaptive immunity against genetic invaders like bacteriophages and plasmids. However, the coordination of crRNA production and cas expression remains poorly understood. Here, we demonstrate that widespread modulatory mini-CRISPRs encode cas-regulating RNAs (CreRs) that mediate autorepression of type I-B, I-E, and V-A Cas proteins, based on their limited complementarity to cas promoters. This autorepression not only reduces autoimmune risks but also responds to changes in the abundance of canonical crRNAs that compete with CreR for Cas proteins. Furthermore, the CreR-guided autorepression of Cas proteins can be alleviated or even subverted by diverse bacteriophage anti-CRISPR (Acr) proteins that inhibit Cas effectors, which, in turn, promotes the generation of new Cas proteins. Our findings reveal a general RNA-guided autorepression paradigm for diverse Cas effectors, shedding light on the intricate self-coordination of CRISPR-Cas and its transcriptional counterstrategy against Acr proteins.

Investigating the Relationship between CRISPR-Cas Content and Growth Rate in Bacteria.

CRISPR-Cas systems provide adaptive immunity for prokaryotic cells by recognizing and eliminating the recurrent genetic invaders whose sequences had been captured in a prior infection and stored in the CRISPR arrays as spacers. However, the biological/environmental factors determining the efficiency of this immune system have yet to be fully characterized. Recent studies in cultured bacteria showed that slowing the growth rate of bacterial cells could promote their acquisition of novel spacers. This study examined the relationship between the CRISPR-Cas content and the minimal doubling time across the bacteria and the archaea domains. Every completely sequenced genome could be used to predict a minimal doubling time. With a large data set of 4,142 bacterial samples, we found that the predicted minimal doubling times are positively correlated with spacer number and other parameters of the CRISPR-Cas systems, like array number, Cas gene cluster number, and Cas gene number. Different data sets gave different results. Weak results were obtained in analyzing bacterial empirical minimal doubling times and the archaea domain. Still, the conclusion of more spacers in slowly grown prokaryotes was supported. In addition, we found that the minimal doubling times are negatively correlated with the occurrence of prophages, and the spacer numbers per array are negatively associated with the number of prophages. These observations support the existence of an evolutionary trade-off between bacterial growth and adaptive defense against virulent phages. IMPORTANCE Accumulating evidence indicates that slowing the growth of cultured bacteria could stimulate their CRISPR spacer acquisition. We observed a positive correlation between CRISPR-Cas content and cell cycle duration across the bacteria domain. This observation extends the physiological conclusion to an evolutionary one. In addition, the correlation provides evidence supporting the existence of a trade-off between bacterial growth/reproduction and antiviral resistance.

Glycan-specific B-1 cells mediate blockade of endogenous retroviruses emergence through recognition of conserved glycan epitopes

Abstract Endogenous retroviruses (ERVs), comprising a substantial portion of the vertebrate genome, are remnants of ancient genetic invaders. Although multiple layers of cell-intrinsic control are utilized to prevent retroviral reactivation, ERVs with intact coding potential were found to reactivate in immunodeficient mice. While previous studies have indicated that B cells are indispensable for preventing ERV reactivation, it is not yet clear which B cell population mediates the blockade of ERV emergence to prevent subsequent damage in the host. Here, we employed direct labeling of B cells reactive with emerged ERV particles to characterize the B cell population and clonal repertoire responsible for recognition of ERV, and to study the mechanism by which B cells provide protection against ERV emergence. We found that ERV-reactive B cells are enriched in innate-like B-1 cell compartment that predominantly reside in peritoneal and pleural cavities. We identified ERV-reactive antibodies in unimmunized mice, the level of which further increases upon innate sensor stimulation. B cell receptor repertoire profiling of ERV-reactive B-1 cells revealed increased usage of Igh VH genes that give rise to glycan-specific antibodies, which were further determined to target terminal N-Acetylglucosamine moieties exhibited by endogenous and exogenous viral antigens. We demonstrated that these glycan-specific natural antibodies engage complement pathway to facilitate clearance of reactivated ERV particles. In conclusion, we elucidated the role of glycan-specific B-1 cells and secreted natural antibodies in mediating blockade of ERV emergence through recognition of conserved glycan epitopes. Supported by grants from Howard Hughes Medical Institute

Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System.

The emergence and persistence of selfish genetic elements is an intrinsic feature of all living systems. Cellular organisms have evolved a plethora of elaborate defense systems that limit the spread of such genetic parasites. CRISPR-Cas are RNA-guided defense systems used by prokaryotes to recognize and destroy foreign nucleic acids. These systems acquire and store fragments of foreign nucleic acids and utilize the stored sequences as guides to recognize and destroy genetic invaders. CRISPR-Cas systems have been extensively studied, as some of them are used in various genome editing technologies. Although Type III CRISPR-Cas systems are among the most common CRISPR-Cas systems, they are also some of the least investigated ones, mostly due to the complexity of their action compared to other CRISPR-Cas system types. Type III effector complexes specifically recognize and cleave RNA molecules. The recognition of the target RNA activates the effector large subunit – the so-called CRISPR polymerase – which cleaves DNA and produces small cyclic oligonucleotides that act as signaling molecules to activate auxiliary effectors, notably non-specific RNases. In this review, we provide a historical overview of the sometimes meandering pathway of the Type III CRISPR research. We also review the current data on the structures and activities of Type III CRISPR-Cas systems components, their biological roles, and evolutionary history. Finally, using structural modeling with AlphaFold2, we show that the archaeal HRAMP signature protein, which heretofore has had no assigned function, is a degenerate relative of Type III CRISPR-Cas signature protein Cas10, suggesting that HRAMP systems have descended from Type III CRISPR-Cas systems or their ancestors.

Glycan-reactive natural antibodies mediate blockade of endogenous retroviruses emergence

Abstract Endogenous retroviruses (ERVs), comprising a substantial portion of the vertebrate genome, are remnants of ancient genetic invaders. Although multiple layers of cell-intrinsic control are utilized to prevent retroviral reactivation, ERVs with intact coding potential are able to reactivate in immunodeficient mice. While previous studies have indicated that B cells are indispensable for preventing ERV reactivation, it is not yet clear which B cell population mediates the blockade of ERV emergence to prevent subsequent damage in the host. Here, we employed direct labeling of B cells reactive with emerged ERV particles to characterize the B cell population and clonal repertoire responsible for recognition of ERV, and to study the mechanism by which B cells provide protection against ERV emergence. We found that ERV-reactive B cells are enriched in innate-like B-1 cell compartment that predominantly reside in peritoneal and pleural cavities. B-1 cells specificity is biased toward conserved epitopes shared by bacterial- and self-antigens due to distinct developmental pathways in fetal haematopoiesis. We identified ERV-reactive antibodies in unimmunized mice, the level of which further increases upon innate sensor stimulation. B cell receptor repertoire profiling of ERV-reactive B-1 cells revealed increased usage of Igh VH genes that give rise to glycan-specific antibodies targeting glycan structures exhibited by bacterial and viral antigens. We demonstrated that these glycan-specific natural antibodies engage complement pathway to facilitate clearance of reactivated ERV particles. In conclusion, we elucidated the role of glycan-specific B-1 cells and secreted natural antibodies in mediating blockade of ERV emergence.

Small RNAs guard CRISPR-Cas

CRISPR The microbial adaptive immunity system CRISPR-Cas benefits microbes by warding off genetic invaders, but it also inflicts a fitness cost because of occasional autoimmune reactions, rendering CRISPR loci evolutionarily unstable. Li et al. identified previously unnoticed toxin-antitoxin RNA pairs embedded within diverse CRISPR-Cas loci. The antitoxin RNA mimics a CRISPR RNA and repurposes the CRISPR immunity effector to transcriptionally repress a toxin RNA that would otherwise arrest cell growth by sequestering a rare transfer RNA. These small RNAs thus form a symbiosis with CRISPR, rendering CRISPR addictive to the host despite its fitness cost. These findings reveal how CRISPR-Cas can operate as a selfish genetic element. Science , this issue p. [eabe5601][1] [1]: /lookup/doi/10.1126/science.abe5601

Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems.

CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA-resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.

Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.

Detection of CRISPR adaptation.

Prokaryotic adaptive immunity is built when short DNA fragments called spacers are acquired into CRISPR (clustered regularly interspaced short palindromic repeats) arrays. CRISPR adaptation is a multistep process which comprises selection, generation, and incorporation of prespacers into arrays. Once adapted, spacers provide immunity through the recognition of complementary nucleic acid sequences, channeling them for destruction. To prevent deleterious autoimmunity, CRISPR adaptation must therefore be a highly regulated and infrequent process, at least in the absence of genetic invaders. Over the years, ingenious methods to study CRISPR adaptation have been developed. In this paper, we discuss and compare methods that detect CRISPR adaptation and its intermediates in vivo and propose suppressing PCR as a simple modification of a popular assay to monitor spacer acquisition with increased sensitivity.

Structure of Csx1-cOA4 complex reveals the basis of RNA decay in Type III-B CRISPR-Cas

Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA4 binding site and a ssRNA catalytic pocket. cOA4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5′-C-C-3′. The degradation of cOA4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.

Cas4-Cas1 fusions drive efficient PAM selection and control CRISPR adaptation.

Microbes have the unique ability to acquire immunological memories from mobile genetic invaders to protect themselves from predation. To confer CRISPR resistance, new spacers need to be compatible with a targeting requirement in the invader's DNA called the protospacer adjacent motif (PAM). Many CRISPR systems encode Cas4 proteins to ensure new spacers are integrated that meet this targeting prerequisite. Here we report that a gene fusion between cas4 and cas1 from the Geobacter sulfurreducens I-U CRISPR–Cas system is capable of introducing functional spacers carrying interference proficient TTN PAM sequences at much higher frequencies than unfused Cas4 adaptation modules. Mutations of Cas4-domain catalytic residues resulted in dramatically decreased naïve and primed spacer acquisition, and a loss of PAM selectivity showing that the Cas4 domain controls Cas1 activity. We propose the fusion gene evolved to drive the acquisition of only PAM-compatible spacers to optimize CRISPR interference.

Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers.

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against genetic invaders, such as bacteriophages. The systems integrate short sequences from the phage genome into the bacterial CRISPR array. These 'spacers' provide sequence-specific immunity but drive natural selection of evolved phage mutants that escape the CRISPR-Cas defence. Spacer acquisition occurs by either naive or primed adaptation. Naive adaptation typically results in the incorporation of a single spacer. By contrast, priming is a positive feedback loop that often results in acquisition of multiple spacers, which occurs when a pre-existing spacer matches the invading phage. We predicted that single and multiple spacers, representative of naive and primed adaptation, respectively, would cause differing outcomes after phage infection. We investigated the response of two phages, ϕTE and ϕM1, to the Pectobacterium atrosepticum type I-F CRISPR-Cas system and observed that escape from single spacers typically occurred via point mutations. Alternatively, phages escaped multiple spacers through deletions, which can occur in genes encoding structural proteins. Cryo-EM analysis of the ϕTE structure revealed shortened tails in escape mutants with tape measure protein deletions. We conclude that CRISPR-Cas systems can drive phage genetic diversity, altering morphology and fitness, through selective pressures arising from naive and primed acquisition events. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

Type III CRISPR-Cas systems can provide redundancy to counteract viral escape from type I systems.

CRISPR-Cas-mediated defense utilizes information stored as spacers in CRISPR arrays to defend against genetic invaders. We define the mode of target interference and role in antiviral defense for two CRISPR-Cas systems in Marinomonas mediterranea. One system (type I-F) targets DNA. A second system (type III-B) is broadly capable of acquiring spacers in either orientation from RNA and DNA, and exhibits transcription-dependent DNA interference. Examining resistance to phages isolated from Mediterranean seagrass meadows, we found that the type III-B machinery co-opts type I-F CRISPR-RNAs. Sequencing and infectivity assessments of related bacterial and phage strains suggests an 'arms race' in which phage escape from the type I-F system can be overcome through use of type I-F spacers by a horizontally-acquired type III-B system. We propose that the phage-host arms race can drive selection for horizontal uptake and maintenance of promiscuous type III interference modules that supplement existing host type I CRISPR-Cas systems.

KRAB zinc-finger proteins contribute to the evolution of gene regulatory networks.

The human genome encodes some 350 Krüppel-associated box (KRAB) domain-containing zinc-finger proteins (KZFPs), the products of a rapidly evolving gene family that has been traced back to early tetrapods. The function of most KZFPs is unknown, but a few have been demonstrated to repress transposable elements in embryonic stem (ES) cells by recruiting the transcriptional regulator TRIM28 and associated mediators of histone H3 Lys9 trimethylation (H3K9me3)-dependent heterochromatin formation and DNA methylation. Depletion of TRIM28 in human or mouse ES cells triggers the upregulation of a broad range of transposable elements, and recent data based on a few specific examples have pointed to an arms race between hosts and transposable elements as an important driver of KZFP gene selection. Here, to obtain a global view of this phenomenon, we combined phylogenetic and genomic studies to investigate the evolutionary emergence of KZFP genes in vertebrates and to identify their targets in the human genome. First, we unexpectedly reassigned the root of the family to a common ancestor of coelacanths and tetrapods. Second, although we confirmed that the majority of KZFPs bind transposable elements and pinpoint cases of ongoing co-evolution, we found that most of their transposable element targets have lost all transposition potential. Third, by examining the interplay between human KZFPs and other transcriptional modulators, we obtained evidence that KZFPs exploit evolutionarily conserved fragments of transposable elements as regulatory platforms long after the arms race against these genetic invaders has ended. Together, our results demonstrate that KZFPs partner with transposable elements to build a largely species-restricted layer of epigenetic regulation.

Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile.

ABSTRACTClostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites.

Drawing a fine line on endogenous retroelement activity.

Endogenous retroelements (EREs) are essential motors of evolution yet require careful control to prevent genomic catastrophes, notably during the vulnerable phases of epigenetic reprogramming that occur immediately after fertilization and in germ cells. Accordingly, a variety of mechanisms restrict these mobile genetic units. Previous studies have revealed the importance of KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor, KAP1, in the early embryonic silencing of endogenous retroviruses and so-called SVAs, but the implication of this transcriptional repression system in the control of LINE-1, the only known active autonomous retrotransposon in the human genome, was thought to be marginal. Two recent studies straighten the record by revealing that the KRAB/KAP system is key to the control of L1 in embryonic stem (ES) cells, and go further in demonstrating that DNA methylation and KRAB/KAP1-induced repression contribute to this process in an evolutionally dynamic fashion. These results shed light on the delicate equilibrium between higher vertebrates and endogenous retroelements, which are not just genetic invaders calling for strict control but rather a constantly renewed and nicely exploitable source of evolutionary potential.

Whole genome sequencing reveals a novel CRISPR system in industrial Clostridium acetobutylicum.

Clostridium acetobutylicum is an important organism for biobutanol production. Due to frequent exposure to bacteriophages during fermentation, industrial C. acetobutylicum strains require a strong immune response against foreign genetic invaders. In the present study, a novel CRISPR system was reported in a C. acetobutylicum GXAS18-1 strain by whole genome sequencing, and several specific characteristics of the CRISPR system were revealed as follows: (1) multiple CRISPR loci were confirmed within the whole bacterial genome, while only one cluster of CRISPR-associated genes (Cas) was found in the current strain; (2) similar leader sequences at the 5' end of the multiple CRISPR loci were identified as promoter elements by promoter prediction, suggesting that these CRISPR loci were under the control of the same transcriptional factor; (3) homology analysis indicated that the present Cas genes shared only low sequence similarity with the published Cas families; and (4) concerning gene similarity and gene cluster order, these Cas genes belonged to the csm family and originated from the euryarchaeota by horizontal gene transfer.