An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts. Significance Statement: The phage nucleus is an enclosed replication compartment built by Chimalliviridae phages that, similar to the eukaryotic nucleus, separates transcription from translation and selectively imports certain proteins. This allows the phage to concentrate proteins required for DNA replication and transcription while excluding DNA-targeting host defense proteins. However, the mechanism of selective trafficking into the phage nucleus is currently unknown. Here we determine the region of a phage nuclear protein that targets it for nuclear import and identify a conserved, essential nuclear shell-associated protein that plays a key role in this process. This work provides the first mechanistic model of selective import into the phage nucleus.

Hachiman is a genome integrity sensor.

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman comprises a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact dsDNA. When the HamAB complex detects DNA damage, HamB helicase activity liberates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating 'phantom' cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and eukaryotic enzymes suggest this bacterial immune system has been repurposed for diverse functions across all domains of life.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes.

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the Chimalliviridae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.

Sequential membrane- and protein-bound organelles compartmentalize genomes during phage infection.

Eukaryotic viruses assemble compartments required for genome replication, but no such organelles are known to be essential for prokaryotic viruses. Bacteriophages of the family Chimalliviridae sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of viral protein ChmA. Using the dRfxCas13d-based knockdown system CRISPRi-ART, we show that ChmA is essential for the E. coli phage Goslar life cycle. Without ChmA, infections are arrested at an early stage in which the injected phage genome is enclosed in a membrane-bound vesicle capable of gene expression but not DNA replication. Not only do we demonstrate that the phage nucleus is essential for genome replication, but we also show that the Chimalliviridae early phage infection (EPI) vesicle is a transcriptionally active, phage-generated organelle.

Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes.

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12. In eukaryotes and their viruses, TnpB homologs occur as two distinct types, Fanzor1 and Fanzor2. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, revealing that a clade of IS607 TnpBs with unusual active site arrangement found primarily in Cyanobacteriota likely gave rise to both types of Fanzors. The wide-spread nature of Fanzors imply that the properties of this particular group of IS607 TnpBs were particularly suited to adaptation and evolution in eukaryotes and their viruses. Experimental characterization of a prokaryotic IS607 TnpB and virally encoded Fanzor1s uncovered features that may have fostered coevolution between TnpBs/Fanzors and their cognate transposases. Our results provide insight into the evolutionary origins of a ubiquitous family of RNA-guided proteins that shows remarkable conservation across domains of life.

Multicopy suppressor screens reveal convergent evolution of single-gene lysis proteins.

Single-strand RNA (ssRNA) Fiersviridae phages cause host lysis with a product of single gene (sgl for single-gene lysis; product Sgl) that induces autolysis. Many different Sgls have been discovered, but the molecular targets of only a few have been identified. In this study, we used a high-throughput genetic screen to uncover genome-wide host suppressors of diverse Sgls. In addition to validating known molecular mechanisms, we discovered that the Sgl of PP7, an ssRNA phage of Pseudomonas aeruginosa, targets MurJ, the flippase responsible for lipid II export, previously shown to be the target of the Sgl of coliphage M. These two Sgls, which are unrelated and predicted to have opposite membrane topology, thus represent a case of convergent evolution. We extended the genetic screens to other uncharacterized Sgls and uncovered a common set of multicopy suppressors, suggesting that these Sgls act by the same or similar mechanism.

Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing.

CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.

A naturally DNase-free CRISPR-Cas12c enzyme silences gene expression.

Used widely for genome editing, CRISPR-Cas enzymes provide RNA-guided immunity to microbes by targeting foreign nucleic acids for cleavage. We show here that the native activity of CRISPR-Cas12c protects bacteria from phage infection by binding to DNA targets without cleaving them, revealing that antiviral interference can be accomplished without chemical attack on the invader or general metabolic disruption in the host. Biochemical experiments demonstrate that Cas12c is a site-specific ribonuclease capable of generating mature CRISPR RNAs (crRNAs) from precursor transcripts. Furthermore, we find that crRNA maturation is essential for Cas12c-mediated DNA targeting. These crRNAs direct double-stranded DNA binding by Cas12c using a mechanism that precludes DNA cutting. Nevertheless, Cas12c represses transcription and can defend bacteria against lytic bacteriophage infection when targeting an essential phage gene. Together, these results show that Cas12c employs targeted DNA binding to provide antiviral immunity in bacteria, providing a native DNase-free pathway for transient antiviral immunity.

The genetic basis of phage susceptibility, cross-resistance and host-range in Salmonella.

Though bacteriophages (phages) are known to play a crucial role in bacterial fitness and virulence, our knowledge about the genetic basis of their interaction, cross-resistance and host-range is sparse. Here, we employed genome-wide screens in Salmonella enterica serovar Typhimurium to discover host determinants involved in resistance to eleven diverse lytic phages including four new phages isolated from a therapeutic phage cocktail. We uncovered 301 diverse host factors essential in phage infection, many of which are shared between multiple phages demonstrating potential cross-resistance mechanisms. We validate many of these novel findings and uncover the intricate interplay between RpoS, the virulence-associated general stress response sigma factor and RpoN, the nitrogen starvation sigma factor in phage cross-resistance. Finally, the infectivity pattern of eleven phages across a panel of 23 genome sequenced Salmonella strains indicates that additional constraints and interactions beyond the host factors uncovered here define the phage host range.

High-throughput mapping of the phage resistance landscape in E. coli.

Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.

Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a.

CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.

A versatile platform strain for high-fidelity multiplex genome editing.

Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (i) a refactored λ-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (ii) genetic modifications that boost recombination efficiency, and (iii) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a 7-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context--neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.

Drug-refractory aggression, self-injurious behavior, and severe tantrums in autism spectrum disorders: a chart review study.

Aggression, self-injurious behavior, and severe tantrums are impairing symptoms frequently experienced by individuals with autism spectrum disorders. Despite US Food and Drug Administration approval of two atypical antipsychotics targeting these symptoms in youth with autistic disorder, they remain frequently drug refractory. We define drug-refractory aggression, self-injurious behavior, and severe tantrums in people with autism spectrum disorders as behavioral symptoms requiring medication adjustment despite previous trials of risperidone and aripiprazole or previous trials of three psychotropic drugs targeting the symptom cluster, one of which was risperidone or aripiprazole. We reviewed the medical records of individuals of all ages referred to our clinic for autism spectrum disorder diagnostic evaluation, as well as pharmacotherapy follow-up notes for all people meeting autism spectrum disorder criteria, for drug-refractory symptoms. Among 250 consecutively referred individuals, 135 met autism spectrum disorder and enrollment criteria, and 53 of these individuals met drug-refractory symptom criteria. Factors associated with drug-refractory symptoms included age 12 years or older (p < 0.0001), diagnosis of autistic disorder (p = 0.0139), and presence of intellectual disability (p = 0.0273). This pilot report underscores the significance of drug-refractory aggression, self-injurious behavior, and severe tantrums; suggests the need for future study clarifying factors related to symptom development; and identifies the need for focused treatment study of this impairing symptom domain.

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.