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Cell Aug 2023Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size...
Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxΔRNaseH, the truncated editor used in dual-AAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed in vivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.
Topics: Humans; Animals; Mice; Protein Engineering; Bacteriophages; Brain; Cerebral Cortex; DNA-Directed RNA Polymerases
PubMed: 37657419
DOI: 10.1016/j.cell.2023.07.039 -
Nature Nov 2023Many bacteria use CRISPR-Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids. In turn, these invasive elements have evolved anti-CRISPR...
Many bacteria use CRISPR-Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids. In turn, these invasive elements have evolved anti-CRISPR proteins to block host immunity. Here we unveil a distinct type of CRISPR-Cas Inhibition strategy that is based on small non-coding RNA anti-CRISPRs (Racrs). Racrs mimic the repeats found in CRISPR arrays and are encoded in viral genomes as solitary repeat units. We show that a prophage-encoded Racr strongly inhibits the type I-F CRISPR-Cas system by interacting specifically with Cas6f and Cas7f, resulting in the formation of an aberrant Cas subcomplex. We identified Racr candidates for almost all CRISPR-Cas types encoded by a diverse range of viruses and plasmids, often in the genetic context of other anti-CRISPR genes. Functional testing of nine candidates spanning the two CRISPR-Cas classes confirmed their strong immune inhibitory function. Our results demonstrate that molecular mimicry of CRISPR repeats is a widespread anti-CRISPR strategy, which opens the door to potential biotechnological applications.
Topics: Bacteria; Bacteriophages; Biotechnology; CRISPR-Associated Proteins; CRISPR-Cas Systems; Molecular Mimicry; Plasmids; Prophages; RNA, Viral
PubMed: 37853129
DOI: 10.1038/s41586-023-06612-5 -
MBio Oct 2023Invasions by eukaryotes dependent on environmentally acquired bacterial mutualists are often limited by the ability of bacterial partners to survive and establish... (Review)
Review
Invasions by eukaryotes dependent on environmentally acquired bacterial mutualists are often limited by the ability of bacterial partners to survive and establish free-living populations. Focusing on the model legume-rhizobium mutualism, we apply invasion biology hypotheses to explain how bacteriophages can impact the competitiveness of introduced bacterial mutualists. Predicting how phage-bacteria interactions affect invading eukaryotic hosts requires knowing the eco-evolutionary constraints of introduced and native microbial communities, as well as their differences in abundance and diversity. By synthesizing research from invasion biology, as well as bacterial, viral, and community ecology, we create a conceptual framework for understanding and predicting how phages can affect biological invasions through their effects on bacterial mutualists.
Topics: Bacteriophages; Bacteria; Rhizobium; Ecology; Microbiota
PubMed: 37812005
DOI: 10.1128/mbio.01886-23 -
Nature Jan 2024Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation. Gabija is one of the most prevalent anti-phage...
Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.
Topics: Bacteria; Bacterial Proteins; Bacteriophages; Cryoelectron Microscopy; Crystallography, X-Ray; Deoxyribonucleases; DNA, Viral; Immune Evasion; Protein Multimerization
PubMed: 37992757
DOI: 10.1038/s41586-023-06855-2 -
Annual Review of Virology Sep 2023Two decades of metagenomic analyses have revealed that in many environments, small (∼5 kb), single-stranded DNA phages of the family dominate the virome. Although the... (Review)
Review
Two decades of metagenomic analyses have revealed that in many environments, small (∼5 kb), single-stranded DNA phages of the family dominate the virome. Although the emblematic microvirus X174 is ubiquitous in the laboratory, most other microviruses, particularly those of the gokushovirus and amoyvirus lineages, have proven to be much more elusive. This puzzling lack of representative isolates has hindered insights into microviral biology. Furthermore, the idiosyncratic size and nature of their genomes have resulted in considerable misjudgments of their actual abundance in nature. Fortunately, recent successes in microvirus isolation and improved metagenomic methodologies can now provide us with more accurate appraisals of their abundance, their hosts, and their interactions. The emerging picture is that X174 and its relatives are rather rare and atypical microviruses, and that a tremendous diversity of other microviruses is ready for exploration.
Topics: Microvirus; Microviridae; Bacteriophages; Phylogeny; Metagenomics
PubMed: 37774127
DOI: 10.1146/annurev-virology-100120-011239 -
Cell Host & Microbe Jul 2023Glycosylation of eukaryotic virus particles is common and influences their uptake, trafficking, and immune recognition. In contrast, glycosylation of bacteriophage...
Glycosylation of eukaryotic virus particles is common and influences their uptake, trafficking, and immune recognition. In contrast, glycosylation of bacteriophage particles has not been reported; phage virions typically do not enter the cytoplasm upon infection, and they do not generally inhabit eukaryotic systems. We show here that several genomically distinct phages of Mycobacteria are modified with glycans attached to the C terminus of capsid and tail tube protein subunits. These O-linked glycans influence antibody production and recognition, shielding viral particles from antibody binding and reducing production of neutralizing antibodies. Glycosylation is mediated by phage-encoded glycosyltransferases, and genomic analysis suggests that they are relatively common among mycobacteriophages. Putative glycosyltransferases are also encoded by some Gordonia and Streptomyces phages, but there is little evidence of glycosylation among the broader phage population. The immune response to glycosylated phage virions in mice suggests that glycosylation may be an advantageous property for phage therapy of Mycobacterium infections.
Topics: Animals; Mice; Mycobacteriophages; Glycosylation; Bacteriophages; Virion; Glycosyltransferases; Polysaccharides
PubMed: 37329881
DOI: 10.1016/j.chom.2023.05.028 -
Ugeskrift For Laeger Jan 2024We present a case report detailing therapeutic application of two lytic antipseudomonal bacteriophages to treat a chronic relapsing Pseudomonas aeruginosa infection of a...
We present a case report detailing therapeutic application of two lytic antipseudomonal bacteriophages to treat a chronic relapsing Pseudomonas aeruginosa infection of a prosthetic aortic graft. As there are currently no Danish laboratories offering phages for clinical therapy, and this case, to our knowledge represents the first applied phage therapy in Denmark, the practical and regulatory aspects of offering this treatment option in Denmark is briefly reviewed along with the clinical case.
Topics: Humans; Bacteriophages; Pseudomonas; Pseudomonas Phages; Blood Vessel Prosthesis; Pseudomonas aeruginosa
PubMed: 38305316
DOI: 10.61409/V09230617 -
Annual Review of Microbiology Sep 2023The gut microbiome is a dense and metabolically active consortium of microorganisms and viruses located in the lower gastrointestinal tract of the human body. Bacteria... (Review)
Review
The gut microbiome is a dense and metabolically active consortium of microorganisms and viruses located in the lower gastrointestinal tract of the human body. Bacteria and their viruses (phages) are the most abundant members of the gut microbiome. Investigating their biology and the interplay between the two is important if we are to understand their roles in human health and disease. In this review, we summarize recent advances in resolving the taxonomic structure and ecological functions of the complex community of phages in the human gut-the gut phageome. We discuss how age, diet, and geography can all have a significant impact on phageome composition. We note that alterations to the gut phageome have been observed in several diseases such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer, and we evaluate whether these phageome changes can directly or indirectly contribute to disease etiology and pathogenesis. We also highlight how lack of standardization in studying the gut phageome has contributed to variation in reported results.
Topics: Humans; Virome; Bacteriophages; Gastrointestinal Microbiome
PubMed: 37307857
DOI: 10.1146/annurev-micro-032421-105754 -
Microbial Biotechnology Jan 2024Mobile genetic elements (MGEs) are crucial for horizontal gene transfer (HGT) in bacteria and facilitate their rapid evolution and adaptation. MGEs include plasmids,... (Review)
Review
Mobile genetic elements (MGEs) are crucial for horizontal gene transfer (HGT) in bacteria and facilitate their rapid evolution and adaptation. MGEs include plasmids, integrative and conjugative elements, transposons, insertion sequences and bacteriophages. Notably, the spread of antimicrobial resistance genes (ARGs), which poses a serious threat to public health, is primarily attributable to HGT through MGEs. This mini-review aims to provide an overview of the mechanisms by which MGEs mediate HGT in microbes. Specifically, the behaviour of conjugative plasmids in different environments and conditions was discussed, and recent methodologies for tracing the dynamics of MGEs were summarised. A comprehensive understanding of the mechanisms underlying HGT and the role of MGEs in bacterial evolution and adaptation is important to develop strategies to combat the spread of ARGs.
Topics: Interspersed Repetitive Sequences; Gene Transfer, Horizontal; Plasmids; Bacteria; Bacteriophages; Anti-Bacterial Agents
PubMed: 38226780
DOI: 10.1111/1751-7915.14408 -
Cell Reports Jul 2023Prokaryotic adaptation is strongly influenced by the horizontal acquisition of beneficial traits via mobile genetic elements (MGEs), such as viruses/bacteriophages and... (Review)
Review
Prokaryotic adaptation is strongly influenced by the horizontal acquisition of beneficial traits via mobile genetic elements (MGEs), such as viruses/bacteriophages and plasmids. However, MGEs can also impose a fitness cost due to their often parasitic nature and differing evolutionary trajectories. In response, prokaryotes have evolved diverse immune mechanisms against MGEs. Recently, our understanding of the abundance and diversity of prokaryotic immune systems has greatly expanded. These defense systems can degrade the invading genetic material, inhibit genome replication, or trigger abortive infection, leading to population protection. In this review, we highlight these strategies, focusing on the most recent discoveries. The study of prokaryotic defenses not only sheds light on microbial evolution but also uncovers novel enzymatic activities with promising biotechnological applications.
Topics: Prokaryotic Cells; Plasmids; Bacteriophages; Genome; Interspersed Repetitive Sequences
PubMed: 37347666
DOI: 10.1016/j.celrep.2023.112672