Biology subjects

Castelle, C. J.

Publications and source records attributed to Castelle, C. J..

13 recordsLinked to original sources

bioRxiv2026

Compact adenine base editors to enable therapeutic rescue of Duchenne muscular dystrophy

Adenine base editors (ABEs) have emerged as a powerful gene-editing technology enabling precise and programmable adenine-to-guanine substitutions across the genome. However, their translation into in vivo therapeutics is limited by delivery challenges, as their size exceeds the packaging capacity of adeno-associated virus (AAV). Here, we report the discovery, structural characterization, and engineering of two compact, highly active ABEs built on novel deaminases and Cas9d nucleases, enabling all-in-one, single-vector AAV delivery. Applying these compact ABEs to Duchenne muscular dystrophy (DMD), we demonstrate efficient disruption of conserved splice-acceptor sites at dystrophin exons 45 and 51 in human skeletal muscle cells, enabling therapeutically relevant exon skipping. Together, these ABEs help expand the therapeutic reach of base editing towards diverse tissue types and disease targets.

bioengineering↗

bioRxiv2025

Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency

Miniature CRISPR-Cas12f nucleases are attractive candidates for therapeutic genome editing owing to their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems such as Cas12a and SpCas9. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here, we characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Cas12f-MG119-28, which supports robust genome editing in human cells. Through structural, biochemical, and kinetic analyses, we compare Cas12f-MG119-28 with two recently described orthologs, Oscillibacter sp. Cas12f (OsCas12f) and Ruminiclostridium herbifermentans Cas12f (RhCas12f). These orthologs present divergent architectures and regulatory features governing PAM recognition, gRNA binding, dimerization, and DNA cleavage. Notably, Cas12f-MG119-28 achieves efficient R-loop formation via a stable dimer interface and a naturally optimized guide RNA. These discoveries elucidate key mechanistic determinants of Cas12f activity and may offer a framework for engineering compact genome editors with therapeutic potential.

biochemistry↗

bioRxiv2022

Variable impact of geochemical gradients on the functional potential of bacteria, archaea, and phages from the permanently stratified Lac Pavin

Permanently stratified lakes contain diverse microbial communities that vary with depth, and so serve as useful models for studying the relationships between microbial community structure and geochemistry. Recent work has shown that these lakes can also harbor numerous bacteria and archaea from novel lineages, including those from the Candidate Phyla Radiation (CPR). However, the extent to which geochemical stratification differentially impacts carbon metabolism and overall genetic potential in CPR bacteria compared to other organisms is not well defined. Here, we determine the distribution of microbial lineages along an oxygen gradient in Lac Pavin, a deep, stratified lake in central France, and examine the influence of this gradient on their metabolism. Genome-based analyses revealed an enrichment of distinct C1 and CO2 fixation pathways in the oxic lake interface and anoxic zone/sediments, suggesting that oxygen likely plays a role in structuring metabolic strategies in non-CPR bacteria and archaea. Notably, we find that oxidation of methane and its byproducts is largely spatially separated from methane production, which is mediated by diverse communities of sediment methanogens that vary on the centimeter scale. In contrast, we detected evidence for RuBisCO throughout the water column and sediments, including form II/III and form III-related enzymes encoded by CPR bacteria in the water column and DPANN archaea in the sediments. Overall, CPR bacteria and phages did not show strong signals of gene content differentiation by depth, despite the fact that distinct species groups populate different lake and sediment compartments. Thus, environmental gradients in Lac Pavin probably select for the capacities of CPR bacteria and phages to a lesser extent than other bacteria and archaea, possibly because selection on the former groups is indirect and depends on host characteristics.

microbiology↗

bioRxiv2021

Protein family content uncovers lineage relationships and bacterial pathway maintenance mechanisms in DPANN archaea

DPANN are small-celled archaea that are generally predicted to be symbionts, and in some cases are known episymbionts of other archaea. As the monophyly of the DPANN remains uncertain, we hypothesized that proteome content could reveal relationships among DPANN lineages, constrain genetic overlap with bacteria, and illustrate how organisms with hybrid bacterial and archaeal protein sets might function. We tested this hypothesis using protein family content that was defined in part using 569 newly reconstructed genomes. Protein family content clearly separates DPANN from other archaea, paralleling the separation of Candidate Phyla Radiation (CPR) bacteria from all other bacteria. This separation is partly driven by hypothetical proteins, some of which may be symbiosis-related. Pacearchaeota with the most limited predicted metabolic capacities have Form II/III and III-like Rubisco, suggesting metabolisms based on scavenged nucleotides. Intriguingly, the Pacearchaeota and Woesearchaeota with the smallest genomes also tend to encode large extracellular murein-like lytic transglycosylase domain proteins that may bind and degrade components of bacterial cell walls, indicating that some might be episymbionts of bacteria. The pathway for biosynthesis of bacterial isoprenoids is widespread in Woesearchaeota genomes and is encoded in proximity to genes involved in bacterial fatty acids synthesis. Surprisingly, in some DPANN genomes we identified a pathway for synthesis of queuosine, an unusual nucleotide in tRNAs of bacteria. Other bacterial systems are predicted to be involved in protein refolding. For example, many DPANN have the complete bacterial DnaK-DnaJ-GrpE system and many Woesearchaeota and Pacearchaeota possess bacterial group I chaperones. Thus, many DPANN appear to have mechanisms to ensure efficient protein folding of both archaeal and laterally acquired bacterial proteins.

microbiology↗

bioRxiv2020

Early acquisition of conserved, lineage-specific proteins currently lacking functional predictions were central to the rise and diversification of archaea

Recent genomic analyses of Archaea have profoundly reshaped our understanding of their distribution, functionalities and roles in eukaryotic evolution. Within the domain, major supergroups are Euryarchaeota, which includes many methanogens, the TACK, which includes Thaumarchaeaota that impact ammonia oxidation in soils and the ocean, the Asgard, which includes lineages inferred to be ancestral to eukaryotes, and the DPANN, a group of mostly symbiotic small-celled archaea. Here, we investigated the extent to which clustering based on protein family content recapitulates archaeal phylogeny and identified the proteins that distinguish the major subdivisions. We also defined 10,866 archaeal protein families that will serve as a community resource. Clustering based on these families broadly recovers the archaeal phylogenetic tree. Interestingly, all major groups are distinguished primarily by the presence of families of conserved hypothetical proteins that are either novel or so highly diverged that their functions are obscured. Given that these hypothetical proteins are near ubiquitous within phyla, we conclude that they were important in the origin of most of the major archaeal lineages.

microbiology↗

bioRxiv2019

The rise of diversity in metabolic platforms across the Candidate Phyla Radiation

A unifying feature of the bacterial Candidate Phyla Radiation (CPR) is a limited and highly variable repertoire of biosynthetic capabilities. However, the distribution of metabolic traits across the CPR and the evolutionary processes underlying them are incompletely resolved. Here, we selected [~]1,000 genomes of CPR bacteria from diverse environments to construct a robust internal phylogeny that was consistent across two unlinked marker sets. Mapping of glycolysis, the pentose phosphate pathway, and pyruvate metabolism onto the tree showed that some components of these pathways are sparsely distributed and that similarity between metabolic platforms is only partially predicted by phylogenetic relationships. To evaluate the extent to which gene loss and lateral gene transfer have shaped trait distribution, we analyzed the patchiness of gene presence in a phylogenetic context, examined the phylogenetic depth of clades with shared traits, and compared the reference tree topology with those of specific metabolic proteins. While the central glycolytic pathway in CPR is widely conserved and has likely been shaped primarily by vertical transmission, there is evidence for both gene loss and transfer especially in steps that convert glucose into fructose 1,6-bisphosphate and glycerate 3P into pyruvate. Additionally, the distribution of Group 3 and Group 4-related NiFe hydrogenases is patchy and suggests multiple events of ancient gene transfer. Overall, patterns of gene gain and loss, including acquisition of accessory traits in independent transfer events, may have been driven by shifts in host-derived resources and led to sparse but varied genetic inventories.

microbiology↗

bioRxiv2019

Aquatic Elusimicrobia are metabolically diverse compared to gut microbiome Elusimicrobia and some have novel nitrogenase-like gene clusters

Currently described members of Elusimicrobia, a relatively recently defined phylum, are animal-associated and rely on fermentation. However, free-living Elusimicrobia have been detected in sediments, soils and groundwater, raising questions regarding their metabolic capacities and evolutionary relationship to animal-associated species. Here, we analyzed 94 draft-quality, non-redundant genomes, including 30 newly reconstructed genomes, from diverse animal-associated and natural environments. Genomes group into 12 clades, 10 of which previously lacked reference genomes. Groundwater-associated Elusimicrobia are predicted to be capable of heterotrophic or autotrophic lifestyles, reliant on oxygen or nitrate/nitrite-dependent respiration, or a variety of organic compounds and Rhodobacter nitrogen fixation-dependent (Rnf-dependent) acetogenesis with hydrogen and carbon dioxide as the substrates. Genomes from two clades of groundwater-associated Elusimicrobia often encode a new group of nitrogenase paralogs that co-occur with an extensive suite of radical S-Adenosylmethionine (SAM) proteins. We identified similar genomic loci in genomes of bacteria from the Gracilibacteria phylum and the Myxococcales order and predict that the gene clusters reduce a tetrapyrrole, possibly to form a novel cofactor. The animal-associated Elusimicrobia clades nest phylogenetically within two free-living-associated clades. Thus, we propose an evolutionary trajectory in which some Elusimicrobia adapted to animal-associated lifestyles from free-living species via genome reduction.

microbiology↗

bioRxiv2019

Unusual metabolism and hypervariation in the genome of a Gracilibacteria (BD1-5) from an oil degrading community

The Candidate Phyla Radiation (CPR) comprises a large monophyletic group of bacterial lineages known almost exclusively based on genomes obtained using cultivation-independent methods. Within the CPR, Gracilibacteria (BD1-5) are particularly poorly understood due to undersampling and the inherent fragmented nature of available genomes. Here, we report the first closed, curated genome of a Gracilibacteria from an enrichment experiment inoculated from the Gulf of Mexico and designed to investigate hydrocarbon degradation. The gracilibacterium rose in abundance after the community switched to dominance by Colwellia. Notably, we predict that this gracilibacterium completely lacks glycolysis, the pentose phosphate and Entner-Doudoroff pathways. It appears to acquire pyruvate, acetyl-CoA and oxaloacetate via degradation of externally derived citrate, malate and amino acids and may use compound interconversion and oxidoreductases to generate and recycle reductive power. The initial genome assembly was fragmented in an unusual gene that is hypervariable within a repeat region. Such extreme local variation is rare, but characteristic of genes that confer traits under pressure to diversify within a population. Notably, the four major repeated 9-mer nucleotide sequences all generate a proline-threonine-aspartic acid (PTD) repeat. The genome of an abundant Colwellia psychrerythraea population has a large extracellular protein that also contains the repeated PTD motif. Although we do not know the host for the BD1-5 cell, the high relative abundance of the C. psychrerythraea population and the shared surface protein repeat may indicate an association between these bacteria.\n\nImportanceCPR bacteria are generally predicted to be symbionts due to their extensive biosynthetic deficits. Although monophyletic, they are not monolithic in terms of their lifestyles. The organism described here appears to have evolved an unusual metabolic platform not reliant on glucose or pentose sugars. Its biology appears to be centered around bacterial host-derived compounds and/or cell detritus. Amino acids likely provide building blocks for nucleic acids, peptidoglycan and protein synthesis. We resolved an unusual repeat region that would be invisible without genome curation. The nucleotide sequence is apparently under strong diversifying selection but the amino acid sequence is under stabilizing selection. The amino acid repeat also occurs in a surface protein of a coexisting bacterium, suggesting co-location and possibly interdependence.

microbiology↗

bioRxiv2019

Clades of huge phage from across Earth’s ecosystems

Phage typically have small genomes and depend on their bacterial hosts for replication. DNA sequenced from many diverse ecosystems revealed hundreds of huge phage genomes, between 200 kbp and 716 kbp in length. Thirty-four genomes were manually curated to completion, including the largest phage genomes yet reported. Expanded genetic repertoires include diverse and new CRISPR-Cas systems, tRNAs, tRNA synthetases, tRNA modification enzymes, translation initiation and elongation factors, and ribosomal proteins. Phage CRISPR-Cas systems have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phage may repurpose bacterial CRISPR-Cas systems to eliminate competing phage. We phylogenetically define major clades of huge phage from human and other animal microbiomes, oceans, lakes, sediments, soils and the built environment. We conclude that their large gene inventories reflect a conserved biological strategy, observed over a broad bacterial host range and across Earths ecosystems.

microbiology↗

bioRxiv2018

Lipid analysis of CO2-rich subsurface aquifers suggests an autotrophy-based deep biosphere with lysolipids enriched in CPR bacteria

Sediment-hosted CO2-rich aquifers deep below the Colorado Plateau (USA) contain a remarkable diversity of uncultivated microorganisms, including Candidate Phyla Radiation (CPR) bacteria that are putative symbionts unable to synthesize membrane lipids. The origin of organic carbon in these ecosystems is unknown and the source of CPR membrane lipids remains elusive. We collected cells from deep groundwater brought to the surface by eruptions of Crystal Geyser, sequenced the community, and analyzed the whole community lipidome over time. Characteristic stable carbon isotopic compositions of microbial lipids suggest that bacterial and archaeal CO2 fixation ongoing in the deep subsurface provides organic carbon for the complex communities that reside there. Coupled lipidomic-metagenomic analysis indicates that CPR bacteria lack complete lipid biosynthesis pathways but still possess regular lipid membranes. These lipids may therefore originate from other community members, which also adapt to high in situ pressure by increasing fatty acid unsaturation. An unusually high abundance of lysolipids attributed to CPR bacteria may represent an adaptation to membrane curvature stress induced by their small cell sizes. Our findings provide new insights into the carbon cycle in the deep subsurface and suggest the redistribution of lipids into putative symbionts within this community.

microbiology↗

bioRxiv2018

Lateral gene transfer shapes the distribution of RuBisCO among Candidate Phyla Radiation bacteria and DPANN archaea

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is considered to be the most abundant enzyme on Earth. Despite this, its full diversity and distribution across the domains of life remain to be determined. Here, we leverage a large set of bacterial, archaeal, and viral genomes recovered from the environment to expand our understanding of existing RuBisCO diversity and the evolutionary processes responsible for its distribution. Specifically, we report a new type of RuBisCO present in Candidate Phyla Radiation (CPR) bacteria that is related to the archaeal Form III enzyme and contains the amino acid residues necessary for catalytic activity. Genome-level metabolic analyses supported the inference that these RuBisCO function in a nucleotide-based, CO2-incorporating pathway. Importantly, some Gottesmanbacteria (CPR) also encode a phosphoribulokinase that may augment carbon metabolism through a partial Calvin-Benson-Bassham Cycle. Based on the scattered distribution of RuBisCO and its discordant evolutionary history, we conclude that this enzyme has been extensively laterally transferred across the CPR bacteria and DPANN archaea. We also report RuBisCO-like proteins in phage genomes from diverse environments. These sequences cluster with proteins in the Beckwithbacteria (CPR), implicating phage as a possible mechanism of RuBisCO transfer. Finally, we synthesize our metabolic and evolutionary analyses to suggest that lateral gene transfer of RuBisCO may have facilitated major shifts in carbon metabolism in several important bacterial and archaeal lineages.

microbiology↗

bioRxiv2018

Biological capacities clearly define a major subdivision in Domain Bacteria

ResumePhylogenetic analyses separate candidate phyla radiation (CPR) bacteria from other bacteria, but the degree to which their proteomes are distinct remains unclear. Here, we leveraged a proteome database that includes sequences from thousands of uncultivated organisms to identify protein families and examine their organismal distributions. We focused on widely distributed protein families that co-occur in genomes, as they likely foundational for metabolism. Clustering of genomes using the protein family presence/absence patterns broadly recapitulates the phylogenetic structure of the tree, suggesting persistence of core sets of protein families after lineage divergence. CPR bacteria group together and away from all other bacteria and archaea, in part due to novel proteins, some of which may be involved in cell-cell interactions. The diversity of combinations of protein families in CPR may exceed that of all other bacteria. Overall, the results extend the phylogeny-based suggestion that the CPR represent a major subdivision within Bacteria.

microbiology↗

bioRxiv2018

Hydrogen-based metabolism - An ancestral trait in lineages sibling to the Cyanobacteria

The metabolic machinery from which microbial aerobic respiration evolved is tightly linked to the origins of oxygenic Cyanobacteria (Oxyphotobacteria). Even though the majority of Oxyphotobacteria are photoautotrophs and can use carbohydrates with oxygen (O2) as the electron acceptor, all are fermenters under dark anoxic conditions. Studies suggest that the ancestor of Oxyphotobacteria may have used hydrogen (H2) as an electron donor and that two types of NiFe hydrogenases are essential for its oxidation. Melainabacteria and Sericytochromatia, close phylogenetic neighbors to Oxyphotobacteria comprise fermentative and aerobic representatives, or organisms capable of both. Margulisbacteria (candidate divisions RBX-1 and ZB3) and Saganbacteria (candidate division WOR-1), a novel cluster of bacteria phylogenetically related to Melainabacteria, Sericytochromatia and Oxyphotobacteria may further constrain the metabolic platform in which oxygenic photosynthesis and aerobic respiration arose. Here, we predict the metabolisms of Margulisbacteria and Saganbacteria from new and published metagenome-assembled genomes (MAGs) and single amplified genomes (SAGs), and compare them to their phylogenetic neighbors. Sediment-associated Margulisbacteria are predicted to have a fermentation-based metabolism featuring a variety of hydrogenases, a nitrogenase for nitrogen (N2) fixation, and electron bifurcating complexes involved in cycling of ferredoxin and NAD(P)H. Overall, the genomic features suggest the capacity for metabolic fine-tuning under strictly anoxic conditions. In contrast, the genomes of Margulisbacteria from the ocean ecosystem encode an electron transport chain that supports aerobic growth. Similarly, some Saganbacteria genomes encode various hydrogenases, and others may have the ability to use O2 under certain conditions via a putative novel type of heme copper O2 reductase. Like Melainabacteria and Sericytochromatia, Margulisbacteria and Saganbacteria have diverse energy metabolisms capable of fermentation, and aerobic or anaerobic respiration. In summary, our findings support the hypothesis that the ancestor of these groups was an anaerobe in which fermentation and H2 metabolism were central metabolic features. Our genomic data also suggests that contemporary lineages sibling to the Oxyphotobacteria may have acquired the ability to use O2 as a terminal electron acceptor under certain environmental conditions.

microbiology↗