Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.

Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of ß2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.

Photobiological Effects at Earth's Surface Following a 50 pc Supernova.

We investigated the potential biological impacts at Earth's surface of stratospheric O3 depletion caused by nearby supernovae known to have occurred about 2.5 and 8 million years ago at about 50 pc distance. New and previously published atmospheric chemistry modeling results were combined with radiative transfer modeling to determine changes in surface-level solar irradiance and biological responses. We find that UVB irradiance is increased by a factor of 1.1 to 2.8, with large variation in latitude, and seasonally at high-latitude regions. Changes in UVA and PAR (visible light) are much smaller. DNA damage (in vitro) is increased by factors similar to UVB, while other biological impacts (erythema, skin cancer, cataracts, marine phytoplankton photosynthesis inhibition, and plant damage) are increased by smaller amounts. We conclude that biological impacts due to increased UV irradiance in this SN case are not mass-extinction level but might be expected to contribute to changes in species abundances; this result fits well with species turnover observed around the Pliocene-Pleistocene boundary. Key Words: UV radiation-Supernovae-Ozone-Radiative transfer. Astrobiology 18, 481-490.

Unusual respiratory capacity and nitrogen metabolism in a Parcubacterium (OD1) of the Candidate Phyla Radiation.

The Candidate Phyla Radiation (CPR) is a large group of bacteria, the scale of which approaches that of all other bacteria. CPR organisms are inferred to depend on other community members for many basic cellular building blocks and all appear to be obligate anaerobes. To date, there has been no evidence for any significant respiratory capacity in an organism from this radiation. Here we report a curated draft genome for 'Candidatus Parcunitrobacter nitroensis' a member of the Parcubacteria (OD1) superphylum of the CPR. The genome encodes versatile energy pathways, including fermentative and respiratory capacities, nitrogen and fatty acid metabolism, as well as the first complete electron transport chain described for a member of the CPR. The sequences of all of these enzymes are highly divergent from sequences found in other organisms, suggesting that these capacities were not recently acquired from non-CPR organisms. Although the wide respiration-based repertoire points to a different lifestyle compared to other CPR bacteria, we predict similar obligate dependence on other organisms or the microbial community. The results substantially expand the known metabolic potential of CPR bacteria, although sequence comparisons indicate that these capacities are very rare in members of this radiation.

Inhibition by ultraviolet and photosynthetically available radiation lowers model estimates of depth-integrated picophytoplankton photosynthesis: global predictions for Prochlorococcus and Synechococcus.

Phytoplankton photosynthesis is often inhibited by ultraviolet (UV) and intense photosynthetically available radiation (PAR), but the effects on ocean productivity have received little consideration aside from polar areas subject to periodic enhanced UV-B due to depletion of stratospheric ozone. A more comprehensive assessment is important for understanding the contribution of phytoplankton production to the global carbon budget, present and future. Here, we consider responses in the temperate and tropical mid-ocean regions typically dominated by picophytoplankton including the prokaryotic lineages, Prochlorococcus and Synechococcus. Spectral models of photosynthetic response for each lineage were constructed using model strains cultured at different growth irradiances and temperatures. In the model, inhibition becomes more severe once exposure exceeds a threshold (Emax ) related to repair capacity. Model parameters are presented for Prochlorococcus adding to those previously presented for Synechococcus. The models were applied to estimate midday, water column photosynthesis based on an atmospheric model of spectral radiation, satellite-derived spectral water transparency and temperature. Based on a global survey of inhibitory exposure severity, a full-latitude section of the mid-Pacific and near-equatorial region of the east Pacific were identified as representative regions for prediction of responses over the entire water column. Comparing predictions integrated over the water column including versus excluding inhibition, production was 7-28% lower due to inhibition depending on strain and site conditions. Inhibition was consistently greater for Prochlorococcus compared to two strains of Synechococcus. Considering only the surface mixed layer, production was inhibited 7-73%. On average, including inhibition lowered estimates of midday productivity around 20% for the modeled region of the Pacific with UV accounting for two-thirds of the reduction. In contrast, most other productivity models either ignore inhibition or only include PAR inhibition. Incorporation of Emax model responses into an existing spectral model of depth-integrated, daily production will enable efficient global predictions of picophytoplankton productivity including inhibition.

Solar Irradiance Changes and Phytoplankton Productivity in Earth's Ocean Following Astrophysical Ionizing Radiation Events.

Two atmospheric responses to simulated astrophysical ionizing radiation events significant to life on Earth are production of odd-nitrogen species, especially NO2, and subsequent depletion of stratospheric ozone. Ozone depletion increases incident short-wavelength ultraviolet radiation (UVB, 280-315 nm) and longer (>600 nm) wavelengths of photosynthetically available radiation (PAR, 400-700 nm). On the other hand, the NO2 haze decreases atmospheric transmission in the long-wavelength UVA (315-400 nm) and short-wavelength PAR. Here, we use the results of previous simulations of incident spectral irradiance following an ionizing radiation event to predict changes in terran productivity focusing on photosynthesis of marine phytoplankton. The prediction is based on a spectral model of photosynthetic response, which was developed for the dominant genera in central regions of the ocean (Synechococcus and Prochlorococcus), and on remote-sensing-based observations of spectral water transparency, temperature, wind speed, and mixed layer depth. Predicted productivity declined after a simulated ionizing event, but the effect integrated over the water column was small. For integrations taking into account the full depth range of PAR transmission (down to 0.1% of utilizable PAR), the decrease was at most 2-3% (depending on strain), with larger effects (5-7%) for integrations just to the depth of the surface mixed layer. The deeper integrations were most affected by the decreased utilizable PAR at depth due to the NO2 haze, whereas shallower integrations were most affected by the increased surface UV. Several factors tended to dampen the magnitude of productivity responses relative to increases in surface-damaging radiation, for example, most inhibition in the modeled strains is caused by UVA and PAR, and the greatest relative increase in damaging exposure is predicted to occur in the winter when UV and productivity are low.

Analysis of five complete genome sequences for members of the class Peribacteria in the recently recognized Peregrinibacteria bacterial phylum.

Five closely related populations of bacteria from the Candidate Phylum (CP) Peregrinibacteria, part of the bacterial Candidate Phyla Radiation (CPR), were sampled from filtered groundwater obtained from an aquifer adjacent to the Colorado River near the town of Rifle, CO, USA. Here, we present the first complete genome sequences for organisms from this phylum. These bacteria have small genomes and, unlike most organisms from other lineages in the CPR, have the capacity for nucleotide synthesis. They invest significantly in biosynthesis of cell wall and cell envelope components, including peptidoglycan, isoprenoids via the mevalonate pathway, and a variety of amino sugars including perosamine and rhamnose. The genomes encode an intriguing set of large extracellular proteins, some of which are very cysteine-rich and may function in attachment, possibly to other cells. Strain variation in these proteins is an important source of genotypic variety. Overall, the cell envelope features, combined with the lack of biosynthesis capacities for many required cofactors, fatty acids, and most amino acids point to a symbiotic lifestyle. Phylogenetic analyses indicate that these bacteria likely represent a new class within the Peregrinibacteria phylum, although they ultimately may be recognized as members of a separate phylum. We propose the provisional taxonomic assignment as 'Candidatus Peribacter riflensis', Genus Peribacter, Family Peribacteraceae, Order Peribacterales, Class Peribacteria in the phylum Peregrinibacteria.

Ground-Level Ozone Following Astrophysical Ionizing Radiation Events: An Additional Biological Hazard?

Astrophysical ionizing radiation events such as supernovae, gamma-ray bursts, and solar proton events have been recognized as a potential threat to life on Earth, primarily through depletion of stratospheric ozone and subsequent increase in solar UV radiation at Earth's surface and in the upper levels of the ocean. Other work has also considered the potential impact of nitric acid rainout, concluding that no significant threat is likely. Not yet studied to date is the potential impact of ozone produced in the lower atmosphere following an ionizing radiation event. Ozone is a known irritant to organisms on land and in water and therefore may be a significant additional hazard. Using previously completed atmospheric chemistry modeling, we examined the amount of ozone produced in the lower atmosphere for the case of a gamma-ray burst and found that the values are too small to pose a significant additional threat to the biosphere. These results may be extended to other ionizing radiation events, including supernovae and extreme solar proton events.

Genome-Resolved Metagenomic Analysis Reveals Roles for Candidate Phyla and Other Microbial Community Members in Biogeochemical Transformations in Oil Reservoirs.

UNLABELLED: Oil reservoirs are major sites of methane production and carbon turnover, processes with significant impacts on energy resources and global biogeochemical cycles. We applied a cultivation-independent genomic approach to define microbial community membership and predict roles for specific organisms in biogeochemical transformations in Alaska North Slope oil fields. Produced water samples were collected from six locations between 1,128 m (24 to 27°C) and 2,743 m (80 to 83°C) below the surface. Microbial community complexity decreased with increasing temperature, and the potential to degrade hydrocarbon compounds was most prevalent in the lower-temperature reservoirs. Sulfate availability, rather than sulfate reduction potential, seems to be the limiting factor for sulfide production in some of the reservoirs under investigation. Most microorganisms in the intermediate- and higher-temperature samples were related to previously studied methanogenic and nonmethanogenic archaea and thermophilic bacteria, but one candidate phylum bacterium, a member of the Acetothermia (OP1), was present in Kuparuk sample K3. The greatest numbers of candidate phyla were recovered from the mesothermic reservoir samples SB1 and SB2. We reconstructed a nearly complete genome for an organism from the candidate phylum Parcubacteria (OD1) that was abundant in sample SB1. Consistent with prior findings for members of this lineage, the OD1 genome is small, and metabolic predictions support an obligately anaerobic, fermentation-based lifestyle. At moderate abundance in samples SB1 and SB2 were members of bacteria from other candidate phyla, including Microgenomates (OP11), Atribacteria (OP9), candidate phyla TA06 and WS6, and Marinimicrobia (SAR406). The results presented here elucidate potential roles of organisms in oil reservoir biological processes. IMPORTANCE: The activities of microorganisms in oil reservoirs impact petroleum resource quality and the global carbon cycle. We show that bacteria belonging to candidate phyla are present in some oil reservoirs and provide the first insights into their potential roles in biogeochemical processes based on several nearly complete genomes.

Angioimmunoblastic t-cell lymphoma presenting as purpura fulminans.

Purpura fulminans is a nonspecific hematologic emergency with high initial mortality, representing a thrombotic occlusion of blood vessels leading to skin necrosis and disseminated intravascular coagulation, and often reported in the setting of sepsis. We report a case of nonfatal purpura fulminans in the context of angioimmunoblastic T-cell lymphoma (AITL).

Solar irradiance changes and photobiological effects at earth's surface following astrophysical ionizing radiation events.

Astrophysical ionizing radiation events have been recognized as a potential threat to life on Earth, primarily through depletion of stratospheric ozone and subsequent increase in surface-level solar ultraviolet radiation. Simulations of the atmospheric effects of a variety of events (such as supernovae, gamma-ray bursts, and solar proton events) have been previously published, along with estimates of biological damage at Earth's surface. In this work, we employed the Tropospheric Ultraviolet and Visible (TUV) radiative transfer model to expand and improve calculations of surface-level irradiance and biological impacts following an ionizing radiation event. We considered changes in surface-level UVB, UVA, and photosynthetically active radiation (visible light) for clear-sky conditions and fixed aerosol parameter values. We also considered a wide range of biological effects on organisms ranging from humans to phytoplankton. We found that past work overestimated UVB irradiance but that relative estimates for increase in exposure to DNA-damaging radiation are still similar to our improved calculations. We also found that the intensity of biologically damaging radiation varies widely with organism and specific impact considered; these results have implications for biosphere-level damage following astrophysical ionizing radiation events. When considering changes in surface-level visible light irradiance, we found that, contrary to previous assumptions, a decrease in irradiance is only present for a short time in very limited geographical areas; instead we found a net increase for most of the modeled time-space region. This result has implications for proposed climate changes associated with ionizing radiation events.

Fermentation, hydrogen, and sulfur metabolism in multiple uncultivated bacterial phyla.

BD1-5, OP11, and OD1 bacteria have been widely detected in anaerobic environments, but their metabolisms remain unclear owing to lack of cultivated representatives and minimal genomic sampling. We uncovered metabolic characteristics for members of these phyla, and a new lineage, PER, via cultivation-independent recovery of 49 partial to near-complete genomes from an acetate-amended aquifer. All organisms were nonrespiring anaerobes predicted to ferment. Three augment fermentation with archaeal-like hybrid type II/III ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) that couples adenosine monophosphate salvage with CO(2) fixation, a pathway not previously described in Bacteria. Members of OD1 reduce sulfur and may pump protons using archaeal-type hydrogenases. For six organisms, the UGA stop codon is translated as tryptophan. All bacteria studied here may play previously unrecognized roles in hydrogen production, sulfur cycling, and fermentation of refractory sedimentary carbon.

Astrophysical ionizing radiation and Earth: a brief review and census of intermittent intense sources.

Cosmic radiation backgrounds are a constraint on life, and their distribution will affect the Galactic Habitable Zone. Life on Earth has developed in the context of these backgrounds, and characterizing event rates will elaborate the important influences. This in turn can be a base for comparison with other potential life-bearing planets. In this review, we estimate the intensities and rates of occurrence of many kinds of strong radiation bursts by astrophysical entities, ranging from gamma-ray bursts at cosmological distances to the Sun itself. Many of these present potential hazards to the biosphere; on timescales long compared with human history, the probability of an event intense enough to disrupt life on the land surface or in the oceans becomes large. Both photons (e.g., X-rays) and high-energy protons and other nuclei (often called "cosmic rays") constitute hazards. For either species, one of the mechanisms that comes into play even at moderate intensities is the ionization of Earth's atmosphere, which leads through chemical changes (specifically, depletion of stratospheric ozone) to increased ultraviolet B flux from the Sun reaching the surface. UVB is extremely hazardous to most life due to its strong absorption by the genetic material DNA and subsequent breaking of chemical bonds. This often leads to mutation or cell death. It is easily lethal to the microorganisms that lie at the base of the food chain in the ocean. We enumerate the known sources of radiation and characterize their intensities at Earth and rates or upper limits on these quantities. When possible, we estimate a "lethal interval," our best estimate of how often a major extinction-level event is probable given the current state of knowledge; we base these estimates on computed or expected depletion of stratospheric ozone. In general, moderate-level events are dominated by the Sun, but the far more severe infrequent events are probably dominated by gamma-ray bursts and supernovae. We note for the first time that so-called "short-hard" gamma-ray bursts are a substantial threat, comparable in magnitude to supernovae and greater than that of the higher-luminosity long bursts considered in most past work. Given their precursors, short bursts may come with little or no warning.

Amphibian nitrate stress as an additional terrestrial threat from astrophysical ionizing radiation events?

Abstract Various astrophysical events have been suggested as sources of ionizing radiation that, by way of destruction of the ozone layer and the subsequent increase in UVB and deposition of nitrate, could pose a threat to life on Earth. We have investigated whether the nitrate deposition that follows an ionizing event is sufficient to cause an additional stress beyond that of the heightened UVB previously considered. Our results show that, subsequent to the most intense ionization event likely to have occurred in the last billion years, the increase in nitrate concentration in bodies of water would not be sufficient to cause serious additional stress on amphibian populations and may actually provide some benefit by acting as fertilizer.

Arabidopsis intragenomic conserved noncoding sequence.

After the most recent tetraploidy in the Arabidopsis lineage, most gene pairs lost one, but not both, of their duplicates. We manually inspected the 3,179 retained gene pairs and their surrounding gene space still present in the genome using a custom-made viewer application. The display of these pairs allowed us to define intragenic conserved noncoding sequences (CNSs), identify exon annotation errors, and discover potentially new genes. Using a strict algorithm to sort high-scoring pair sequences from the bl2seq data, we created a database of 14,944 intragenomic Arabidopsis CNSs. The mean CNS length is 31 bp, ranging from 15 to 285 bp. There are approximately 1.7 CNSs associated with a typical gene, and Arabidopsis CNSs are found in all areas around exons, most frequently in the 5' upstream region. Gene ontology classifications related to transcription, regulation, or "response to ..." external or endogenous stimuli, especially hormones, tend to be significantly overrepresented among genes containing a large number of CNSs, whereas protein localization, transport, and metabolism are common among genes with no CNSs. There is a 1.5% overlap between these CNSs and the 218,982 putative RNAs in the Arabidopsis Small RNA Project database, allowing for two mismatches. These CNSs provide a unique set of noncoding sequences enriched for function. CNS function is implied by evolutionary conservation and independently supported because CNS-richness predicts regulatory gene ontology categories.

Gene-balanced duplications, like tetraploidy, provide predictable drive to increase morphological complexity.

Controversy surrounds the apparent rising maximums of morphological complexity during eukaryotic evolution, with organisms increasing the number and nestedness of developmental areas as evidenced by morphological elaborations reflecting area boundaries. No "predictable drive" to increase this sort of complexity has been reported. Recent genetic data and theory in the general area of gene dosage effects has engendered a robust "gene balance hypothesis," with a theoretical base that makes specific predictions as to gene content changes following different types of gene duplication. Genomic data from both chordate and angiosperm genomes fit these predictions: Each type of duplication provides a one-way injection of a biased set of genes into the gene pool. Tetraploidies and balanced segments inject bias for those genes whose products are the subunits of the most complex biological machines or cascades, like transcription factors (TFs) and proteasome core proteins. Most duplicate genes are removed after tetraploidy. Genic balance is maintained by not removing those genes that are dose-sensitive, which tends to leave duplicate "functional modules" as the indirect products (spandrels) of purifying selection. Functional modules are the likely precursors of coadapted gene complexes, a unit of natural selection. The result is a predictable drive mechanism where "drive" is used rigorously, as in "meiotic drive." Rising morphological gain is expected given a supply of duplicate functional modules. All flowering plants have survived at least three large-scale duplications/diploidizations over the last 300 million years (Myr). An equivalent period of tetraploidy and body plan evolution may have ended for animals 500 million years ago (Mya). We argue that "balanced gene drive" is a sufficient explanation for the trend that the maximums of morphological complexity have gone up, and not down, in both plant and animal eukaryotic lineages.

Following tetraploidy in an Arabidopsis ancestor, genes were removed preferentially from one homeolog leaving clusters enriched in dose-sensitive genes.

Approximately 90% of Arabidopsis' unique gene content is found in syntenic blocks that were formed during the most recent whole-genome duplication. Within these blocks, 28.6% of the genes have a retained pair; the remaining genes have been lost from one of the homeologs. We create a minimized genome by condensing local duplications to one gene, removing transposons, and including only genes within blocks defined by retained pairs. We use a moving average of retained and non-retained genes to find clusters of retention and then identify the types of genes that appear in clusters at frequencies above expectations. Significant clusters of retention exist for almost all chromosomal segments. Detailed alignments show that, for 85% of the genome, one homeolog was preferentially (1.6x) targeted for fractionation. This homeolog fractionation bias suggests an epigenetic mechanism. We find that islands of retention contain "connected genes," those genes predicted-by the gene balance hypothesis-to be resistant to removal because the products they encode interact with other products in a dose-sensitive manner, creating a web of dependency. Gene families that are overrepresented in clusters include those encoding components of the proteasome/protein modification complexes, signal transduction machinery, ribosomes, and transcription factor complexes. Gene pair fractionation following polyploidy or segmental duplication leaves a genome enriched for "connected" genes. These clusters of duplicate genes may help explain the evolutionary origin of coregulated chromosomal regions and new functional modules.

Gene structure and minimal promoter of mouse rdh1.

Mouse rdh1 encodes retinol dehydrogenase type 1 (RDH1), a short-chain dehydrogenase, which recognizes as substrates all-trans-retinol, 9-cis-retinol, 5alpha-androstan-3,17-diol and 5alpha-androstan-3-ol-17-one. RDH1 is the most efficient known mouse short-chain dehydrogenase that catalyzes dehydrogenation of all-trans-retinol, and contributes to a reconstituted path of all-trans-retinoic acid biosynthesis, when coexpressed in reporter cells with any one of three retinal dehydrogenases. Rdh1 shows widespread, if not ubiquitous, mRNA expression in the mouse beginning no later than embryo day 7. Here we report genomic organization, chromosomal localization and analysis of a minimum promoter of mouse rdh1. Rdh1 consists of four exons and three introns and spans approximately 14412 bp. Rdh1 is a single copy gene that maps to chromosome 10D3 with rdh5-9, but no known disorder maps precisely to rdh1. Rdh1 has three transcription start sites in kidney and one start site in liver. The rdh1 5'-region between -424 and +43 induces transcription maximally in COS7, mouse kidney RAG, and mouse liver NMu3Li cells. This section has no TATA box, but has a CCAAT box beginning 65 bp upstream of the major transcription start site, which is required for transcription of transfected reporter constructs. An AP1 binding site at -119 also activates transfected reporter constructs, and mediates 2-O-tetradecanoylphorbol-13-acetate (TPA) induced transcription. All-trans-retinoic acid antagonizes the TPA affect; however, no RARE or RXRE was found in the proximal promoter region, consistent with indirect regulation by all-trans-retinoic acid.