CRISPR-Cas Genome Editing in Ex Vivo Human Lungs to Rewire the Translational Path of Genome-Targeting Therapeutics.

The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both in vivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (IL-10). We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an in vivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.

Anti-phage defence through inhibition of virion assembly.

Bacteria have evolved diverse antiviral defence mechanisms to protect themselves against phage infection. Phages integrated into bacterial chromosomes, known as prophages, also encode defences that protect the bacterial hosts in which they reside. Here, we identify a type of anti-phage defence that interferes with the virion assembly pathway of invading phages. The protein that mediates this defence, which we call Tab (for 'Tail assembly blocker'), is constitutively expressed from a Pseudomonas aeruginosa prophage. Tab allows the invading phage replication cycle to proceed, but blocks assembly of the phage tail, thus preventing formation of infectious virions. While the infected cell dies through the activity of the replicating phage lysis proteins, there is no release of infectious phage progeny, and the bacterial community is thereby protected from a phage epidemic. Prophages expressing Tab are not inhibited during their own lytic cycle because they express a counter-defence protein that interferes with Tab function. Thus, our work reveals an anti-phage defence that operates by blocking virion assembly, thereby both preventing formation of phage progeny and allowing destruction of the infected cell due to expression of phage lysis genes.

F-Type Pyocins Are Diverse Noncontractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa.

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or noncontractile phage tails known as R- and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3' end of the F-type pyocin cluster are divergent in sequence and likely determine bactericidal specificity. We use sequence similarities among these proteins to define eleven distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly reassorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins may have emerged earlier. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as antibacterial therapeutics. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes antibiotic-resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great need for the development of alternative therapeutics. In this study, we investigate one such potential therapeutic: F-type pyocins, which are bacteriocins naturally produced by P. aeruginosa that resemble noncontractile phage tails. We show that they are potent killers of P. aeruginosa and identify their probable bactericidal specificity determinants, which opens up the possibility of engineering them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well-characterized phage tails will greatly facilitate their development into effective antibacterials.

Immunomodulation of the donor lung with CRISPR-mediated activation of IL-10 expression.

BACKGROUND: Inflammatory injury in the donor lung remains a persistent challenge in lung transplantation that limits donor organ usage and post-transplant outcomes. Inducing immunomodulatory capacity in donor organs could address this unsolved clinical problem. We sought to apply clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) technologies to the donor lung to fine-tune immunomodulatory gene expression, exploring for the first time the therapeutic use of CRISPR-mediated transcriptional activation in the whole donor lung. METHODS: We explored the feasibility of CRISPR-mediated transcriptional upregulation of interleukin 10 (IL-10), a key immunomodulatory cytokine, in vitro and in vivo. We first evaluated the potency, titratability, and multiplexibility of the gene activation in rat and human cell lines. Next, in vivo CRISPR-mediated IL-10 activation was characterized in rat lungs. Finally, the IL-10-activated donor lungs were transplanted into recipient rats to assess the feasibility in a transplant setting. RESULTS: The targeted transcriptional activation induced robust and titrable IL-10 upregulation in vitro. The combination of guide RNAs also facilitated multiplex gene modulation, that is, simultaneous activation of IL-10 and IL1 receptor antagonist. In vivo profiling demonstrated that adenoviral delivery of Cas9-based activators to the lung was feasible with the use of immunosuppression, which is routinely applied to organ transplant recipients. The transcriptionally modulated donor lungs retained IL-10 upregulation in isogeneic and allogeneic recipients. CONCLUSIONS: Our findings highlight the potential of CRISPR epigenome editing to improve lung transplant outcomes by creating a more favorable immunomodulatory environment in the donor organ, a paradigm that may be extendable to other organ transplants.

Complete genomes and comparative analyses of Streptomyces phages that influence secondary metabolism and sporulation.

Bacteria in the genus Streptomyces are found ubiquitously in nature and are known for the number and diversity of specialized metabolites they produce, as well as their complex developmental lifecycle. Studies of the viruses that prey on Streptomyces, known as phages, have aided the development of tools for genetic manipulation of these bacteria, as well as contributing to a deeper understanding of Streptomyces and their behaviours in the environment. Here, we present the genomic and biological characterization of twelve Streptomyces phages. Genome analyses reveal that these phages are closely related genetically, while experimental approaches show that they have broad overlapping host ranges, infect early in the Streptomyces lifecycle, and induce secondary metabolite production and sporulation in some Streptomyces species. This work expands the group of characterized Streptomyces phages and improves our understanding of Streptomyces phage-host dynamics.

Diverse yeast antiviral systems prevent lethal pathogenesis caused by the L-A mycovirus.

Recent studies show that antiviral systems are remarkably conserved from bacteria to mammals, demonstrating that unique insights into these systems can be gained by studying microbial organisms. Unlike in bacteria, however, where phage infection can be lethal, no cytotoxic viral consequence is known in the budding yeast Saccharomyces cerevisiae even though it is chronically infected with a double-stranded RNA mycovirus called L-A. This remains the case despite the previous identification of conserved antiviral systems that limit L-A replication. Here, we show that these systems collaborate to prevent rampant L-A replication, which causes lethality in cells grown at high temperature. Exploiting this discovery, we use an overexpression screen to identify antiviral functions for the yeast homologs of polyA-binding protein (PABPC1) and the La-domain containing protein Larp1, which are both involved in viral innate immunity in humans. Using a complementary loss of function approach, we identify new antiviral functions for the conserved RNA exonucleases REX2 and MYG1; the SAGA and PAF1 chromatin regulatory complexes; and HSF1, the master transcriptional regulator of the proteostatic stress response. Through investigation of these antiviral systems, we show that L-A pathogenesis is associated with an activated proteostatic stress response and the accumulation of cytotoxic protein aggregates. These findings identify proteotoxic stress as an underlying cause of L-A pathogenesis and further advance yeast as a powerful model system for the discovery and characterization of conserved antiviral systems.

A contractile injection system is required for developmentally regulated cell death in Streptomyces coelicolor.

Diverse bacterial species produce extracellular contractile injection systems (eCISs). Although closely related to contractile phage tails, eCISs can inject toxic proteins into eukaryotic cells. Thus, these systems are commonly viewed as cytotoxic defense mechanisms that are not central to other aspects of bacterial biology. Here, we provide evidence that eCISs appear to participate in the complex developmental process of the bacterium Streptomyces coelicolor. In particular, we show that S. coelicolor produces eCIS particles during its normal growth cycle, and that strains lacking functional eCIS particles exhibit pronounced alterations in their developmental program. Furthermore, eCIS-deficient mutants display reduced levels of cell death and altered morphology during growth in liquid media. Our results suggest that the main role of eCISs in S. coelicolor is to modulate the developmental switch that leads to aerial hyphae formation and sporulation, rather than to attack other species.

Anti-CRISPR Protein AcrIIC5 Inhibits CRISPR-Cas9 by Occupying the Target DNA Binding Pocket.

Anti-CRISPR proteins inhibit CRISPR-Cas immune systems through diverse mechanisms. Previously, the anti-CRISPR protein AcrIIC5Smu was shown to potently inhibit a type II-C Cas9 from Neisseria meningitidis (Nme1Cas9). In this work, we explore the mechanism of activity of the AcrIIC5 homologue from Neisseria chenwenguii (AcrIIC5Nch) and show that it prevents Cas9 binding to target DNA. We show that AcrIIC5Nch targets the PAM-interacting domain (PID) of Nme1Cas9 for inhibition, agreeing with previous findings for AcrIIC5Smu, and newly establish that strong binding of the anti-CRISPR requires guide RNA be pre-loaded on Cas9. We determined the crystal structure of AcrIIC5Nch using X-ray crystallography and identified amino acid residues that are critical for its function. Using a protein docking algorithm we show that AcrIIC5Nch likely occupies the Cas9 DNA binding pocket, thereby inhibiting target DNA binding through a mechanism similar to that previously described for AcrIIA2 and AcrIIA4.

The small genome, virulent, non-contractile tailed bacteriophages that infect Enterobacteriales hosts.

Tailed bacteriophages are abundant and extremely diverse. Understanding this diversity is a challenge, and here we examine a small slice of that diversity in some detail. We contrast and compare the small genome, virulent, non-contractile tailed phages that infect the bacterial order Enterobacteriales. These phages, with genomes in the 35-60 kbp range, have very similar virions that are often difficult to distinguish by negative stain electron microscopy. There are currently 651 genome sequences of such phages in the public database. We show that these can be robustly parsed into fifteen well-defined clusters that have very different nucleotide sequences. We examine the similarities and differences among these clusters, as well as genetic exchange among clusters and the relationships between host species and phage clusters.

Identification of the tail assembly chaperone genes of T4-Like phages suggests a mechanism other than translational frameshifting for biogenesis of their encoded proteins.

Tape measure (TM) proteins are essential for the formation of long-tailed phages. TM protein assembly into tails requires the action of tail assembly chaperones (TACs). TACs (e.g. gpG and gpT of E. coli phage lambda) are usually produced in a short (TAC-N) and long form (TAC-NC) with the latter comprised of TAC-N with an additional C-terminal domain (TAC-C). TAC-NC is generally synthesized through a ribosomal frameshifting mechanism. TAC encoding genes have never been identified in the intensively studied Escherichia coli phage T4, or any related phages. Here, we have bioinformatically identified putative TAC encoding genes in diverse T4-like phage genomes. The frameshifting mechanism for producing TAC-NC appears to be conserved in several T4-like phage groups. However, the group including phage T4 itself likely employs a different strategy whereby TAC-N and TAC-NC are encoded by separate genes (26 and 51 in phage T4).

Structural and Mechanistic Insight into CRISPR-Cas9 Inhibition by Anti-CRISPR Protein AcrIIC4Hpa.

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPR proteins have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal structure of AcrIIC4Hpa and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4Hpa likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

Anti-CRISPR AcrIF9 functions by inducing the CRISPR-Cas complex to bind DNA non-specifically.

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complementarity to the CRISPR RNA. Induction of non-specific DNA-binding is a shared property of diverse AcrIF9 homologues. Substitution of a conserved positively charged surface on AcrIF9 abrogated non-specific dsDNA-binding of the Csy:AcrIF9 complex, but specific dsDNA binding was maintained. AcrIF9 mutants with impaired non-specific dsDNA binding activity in vitro displayed a reduced ability to inhibit CRISPR-Cas activity in vivo. We conclude that misdirecting the CRISPR-Cas complex to bind non-specific DNA is a key component of the inhibitory mechanism of AcrIF9. This inhibitory mechanism is distinct from a previously characterized anti-CRISPR, AcrIF1, that sterically blocks DNA-binding, even though AcrIF1and AcrIF9 bind to the same site on the Csy complex.

A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa.

The arms race between bacteria and phages has led to the evolution of diverse anti-phage defenses, several of which are controlled by quorum-sensing pathways. In this work, we characterize a quorum-sensing anti-activator protein, Aqs1, found in Pseudomonas phage DMS3. We show that Aqs1 inhibits LasR, the master regulator of quorum sensing, and present the crystal structure of the Aqs1-LasR complex. The 69-residue Aqs1 protein also inhibits PilB, the type IV pilus assembly ATPase protein, which blocks superinfection by phages that require the pilus for infection. This study highlights the remarkable ability of small phage proteins to bind multiple host proteins and disrupt key biological pathways. As quorum sensing influences various anti-phage defenses, Aqs1 provides a mechanism by which infecting phages might simultaneously dampen multiple defenses. Because quorum-sensing systems are broadly distributed across bacteria, this mechanism of phage counter-defense may play an important role in phage-host evolutionary dynamics.

Anti-CRISPR AcrIE2 Binds the Type I-E CRISPR-Cas Complex But Does Not Block DNA Binding.

Anti-CRISPRs are protein inhibitors of CRISPR-Cas systems. They are produced by phages and other mobile genetic elements to evade CRISPR-Cas-mediated destruction. Anti-CRISPRs are remarkably diverse in sequence, structure, and functional mechanism; thus, structural and mechanistic investigations of anti-CRISPRs continue to yield exciting new insights. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AcrIE2, an anti-CRISPR that inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa. Guided by the structure, we used site-directed mutagenesis to identify key residues that are required for AcrIE2 function. Using affinity purification experiments, we found that AcrIE2 binds the type I-E CRISPR-Cas complex (Cascade). In vivo transcriptional assays, in which Cascade was targeted to promoter regions, demonstrated that Cascade still binds to DNA in the presence of AcrIE2. This is the first instance of a type I anti-CRISPR that binds to a CRISPR-Cas complex but does not prevent DNA-binding. Another unusual property of AcrIE2 is that the effect of Cascade:AcrIE2 complex binding to promoter regions varied depending on the position of the binding site. Most surprisingly, Cascade:AcrIE2 binding led to transcriptional activation in some cases rather than repression, which did not occur when Cascade alone bound to the same sites. We conclude that AcrIE2 operates through a distinct mechanism compared to other type I anti-CRISPRs. While AcrIE2 does not prevent Cascade from binding DNA, it likely blocks subsequent recruitment of the Cas3 nuclease to Cascade thereby preventing DNA cleavage.

Phage Proteins Required for Tail Fiber Assembly Also Bind Specifically to the Surface of Host Bacterial Strains.

To initiate their life cycle, phages must specifically bind to the surface of their bacterial hosts. Long-tailed phages often interact with the cell surface using fibers, which are elongated intertwined trimeric structures. The folding and assembly of these complex structures generally requires the activity of an intra- or intermolecular chaperone protein. Tail fiber assembly (Tfa) proteins are a very large family of proteins that serve as chaperones for fiber folding in a wide variety of phages that infect diverse species. A recent structural study showed that the Tfa protein from Escherichia coli phage Mu (TfaMu) mediates fiber folding and stays bound to the distal tip of the fiber, becoming a component of the mature phage particle. This finding revealed the potential for TfaMu to also play a role in cell surface binding. To address this issue, we have here shown that TfaMu binds to lipopolysaccharide (LPS), the cell surface receptor of phage Mu, with a similar strength as to the fiber itself. Furthermore, we have found that TfaMu and the Tfa protein from E. coli phage P2 bind LPS with distinct specificities that mirror the host specificity of these two phages. By comparing the sequences of these two proteins, which are 93% identical, we identified a single residue that is responsible for their distinct LPS-binding behaviors. Although we have not yet found conditions under which Tfa proteins influence host range, the potential for such a role is now evident, as we have demonstrated their ability to bind LPS in a strain-specific manner.IMPORTANCE With the growing interest in using phages to combat antibiotic-resistant infections or manipulate the human microbiome, establishing approaches for the modification of phage host range has become an important research topic. Tfa proteins are a large family of proteins known previously to function as chaperones for the folding of phage fibers, which are crucial determinants of host range for long-tailed phages. Here, we reveal that some Tfa proteins are bi-functional, with the additional activity of binding to LPS, the surface binding receptor for many phages. This discovery opens up new potential avenues for altering phage host range through engineering of the surface binding specificity of Tfa proteins.

AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided surveillance complex.

Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.

Listeria Phages Induce Cas9 Degradation to Protect Lysogenic Genomes.

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy phage (viral) DNA. Phages, in turn, have evolved diverse "anti-CRISPR" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode two to three distinct anti-Cas9 proteins, with acrIIA1 always present. However, the significance of AcrIIA1's pervasiveness and its mechanism are unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain. During lysogeny in Listeria, AcrIIA1 triggers Cas9 degradation. During lytic infection, however, AcrIIA1 fails to block Cas9 due to its multi-step inactivation mechanism. Thus, phages encode an additional Acr that rapidly binds and inactivates Cas9. AcrIIA1 also uniquely inhibits a highly diverged Cas9 found in Listeria (similar to SauCas9) and Type II-C Cas9s, likely due to Cas9 HNH domain conservation. In summary, Listeria phages inactivate Cas9 in lytic growth using variable, narrow-spectrum inhibitors, while the broad-spectrum AcrIIA1 stimulates Cas9 degradation for protection of the lysogenic genome.

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.

Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing.

CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction.