Structure-guided discovery of ancestral CRISPR-Cas13 ribonucleases.

The RNA-guided ribonuclease CRISPR-Cas13 enables adaptive immunity in bacteria and programmable RNA manipulation in heterologous systems. Cas13s share limited sequence similarity, hindering discovery of related or ancestral systems. To address this, we developed an automated structural-search pipeline to identify an ancestral clade of Cas13 (Cas13an) and further trace Cas13 origins to defense-associated ribonucleases. Despite being one-third the size of other Cas13s, Cas13an mediates robust programmable RNA depletion and defense against diverse bacteriophages. However, unlike its larger counterparts, Cas13an uses a single active site for both CRISPR RNA processing and RNA-guided cleavage, revealing that the ancestral nuclease domain has two modes of activity. Discovery of Cas13an deepens our understanding of CRISPR-Cas evolution and expands opportunities for precision RNA editing, showcasing the promise of structure-guided genome mining.

The extracellular matrix integrates mitochondrial homeostasis.

Cellular homeostasis is intricately influenced by stimuli from the microenvironment, including signaling molecules, metabolites, and pathogens. Functioning as a signaling hub within the cell, mitochondria integrate information from various intracellular compartments to regulate cellular signaling and metabolism. Multiple studies have shown that mitochondria may respond to various extracellular signaling events. However, it is less clear how changes in the extracellular matrix (ECM) can impact mitochondrial homeostasis to regulate animal physiology. We find that ECM remodeling alters mitochondrial homeostasis in an evolutionarily conserved manner. Mechanistically, ECM remodeling triggers a TGF-ß response to induce mitochondrial fission and the unfolded protein response of the mitochondria (UPRMT). At the organismal level, ECM remodeling promotes defense of animals against pathogens through enhanced mitochondrial stress responses. We postulate that this ECM-mitochondria crosstalk represents an ancient immune pathway, which detects infection- or mechanical-stress-induced ECM damage, thereby initiating adaptive mitochondria-based immune and metabolic responses.

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

RNA language models predict mutations that improve RNA function.

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. While advances in deep learning enable the prediction of accurate protein structural models, RNA structure prediction is not possible at present due to a lack of abundant high-quality reference data1. Furthermore, available sequence data are generally not associated with organismal phenotypes that could inform RNA function2-4. We created GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB)5. GARNET links RNA sequences derived from GTDB genomes to experimental and predicted optimal growth temperatures of GTDB reference organisms. This enables construction of deep and diverse RNA sequence alignments to be used for machine learning. Using GARNET, we define the minimal requirements for a sequence- and structure-aware RNA generative model. We also develop a GPT-like language model for RNA in which overlapping triplet tokenization provides optimal encoding. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identified mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB-derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

Non-Thermal Solar Wind Electron Velocity Distribution Function.

The quiet-time solar wind electrons feature non-thermal characteristics when viewed from the perspective of their velocity distribution functions. They typically have an appearance of being composed of a denser thermal "core" population plus a tenuous energetic "halo" population. At first, such a feature was empirically fitted with the kappa velocity space distribution function, but ever since the ground-breaking work by Tsallis, the space physics community has embraced the potential implication of the kappa distribution as reflecting the non-extensive nature of the space plasma. From the viewpoint of microscopic plasma theory, the formation of the non-thermal electron velocity distribution function can be interpreted in terms of the plasma being in a state of turbulent quasi-equilibrium. Such a finding brings forth the possible existence of a profound inter-relationship between the non-extensive statistical state and the turbulent quasi-equilibrium state. The present paper further develops the idea of solar wind electrons being in the turbulent equilibrium, but, unlike the previous model, which involves the electrostatic turbulence near the plasma oscillation frequency (i.e., Langmuir turbulence), the present paper considers the impact of transverse electromagnetic turbulence, particularly, the turbulence in the whistler-mode frequency range. It is found that the coupling of spontaneously emitted thermal fluctuations and the background turbulence leads to the formation of a non-thermal electron velocity distribution function of the type observed in the solar wind during quiet times. This demonstrates that the whistler-range turbulence represents an alternative mechanism for producing the kappa-like non-thermal distribution, especially close to the Sun and in the near-Earth space environment.

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.

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.

The hypothalamus predates the origin of vertebrates.

The hypothalamus coordinates neuroendocrine functions in vertebrates. To explore its evolutionary origin, we describe integrated transcriptome/connectome brain maps for swimming tadpoles of Ciona, which serves as an approximation of the ancestral proto-vertebrate. This map features several cell types related to different regions of the vertebrate hypothalamus, including the mammillary nucleus, the arcuate nucleus, and magnocellular neurons. Coronet cells express melanopsin and share additional properties with the saccus vasculosus, a specialized region of the hypothalamus that mediates photoperiodism in nontropical fishes. Comparative transcriptome analyses identified orthologous cell types for mechanosensory switch neurons, and VP+ and VPR+ relay neurons in different regions of the mouse hypothalamus. These observations provide evidence that the hypothalamus predates the evolution of the vertebrate brain. We discuss the possibility that switch neurons, coronet cells, and FoxP+ /VPR+ relay neurons comprise a behavioral circuit that helps trigger metamorphosis of Ciona larvae in response to twilight.

The Effects of Upper-Hybrid Waves on Energy Dissipation in the Electron Diffusion Region.

Using a two-dimensional particle-in-cell simulation, we investigate the effects and roles of upper-hybrid waves (UHW) near the electron diffusion region (EDR). The energy dissipation via the wave-particle interaction in our simulation agrees with J · E ' measured by magnetospheric multiscale (MMS) spacecraft. It means that UHW contributes to the local energy dissipation. As a result of wave-particle interactions, plasma parameters which determine the larger-scale energy dissipation in the EDR are changed. The y-directional current decreases while the pressure tensor P y z increases/decreases when the agyrotropic beam density is low/high, where (x, y, z)-coordinates correspond the (L, M, N)-boundary coordinates. Because the reconnection electric field comes from -∂ P y z /∂ z, our result implies that UHW plays an additional role in affecting larger-scale energy dissipation in the EDR by changing plasma parameters. We provide a simple diagram that shows how the UHW activities change the profiles of plasma parameters near the EDR comparing cases with and without UHW.

High-Frequency Waves Driven by Agyrotropic Electrons Near the Electron Diffusion Region.

National Aeronautics and Space Administration's Magnetosphere Multiscale mission reveals that agyrotropic electrons and intense waves are prevalently present in the electron diffusion region. Prompted by two distinct Magnetosphere Multiscale observations, this letter investigates by theoretical means and the properties of agyrotropic electron beam-plasma instability and explains the origin of different structures in the wave spectra. The difference is owing to the fact that in one instance, a continuous beam mode is excited, while in the other, discrete Bernstein modes are excited, and the excitation of one mode versus the other depends on physical input parameters, which are consistent with observations. Analyses of dispersion relations show that the growing mode becomes discrete when the maximum growth rate is lower than the electron cyclotron frequency. Making use of particle-in-cell simulations, we found that the broadening angle Δ in the gyroangle space is also an important factor controlling the growth rate. Ramifications of the present finding are also discussed.

High-Frequency Wave Generation in Magnetotail Reconnection: Nonlinear Harmonics of Upper Hybrid Waves.

MMS3 spacecraft passed the vicinity of the electron diffusion region of magnetotail reconnection on 3 July 2017, observing discrepancies between perpendicular electron bulk velocities and E → × B → drift, and agyrotropic electron crescent distributions. Analyzing linear wave dispersions, Burch et al. (2019, https://doi.org/10.1029/2019GL082471) showed the electron crescent generates high-frequency waves. We investigate harmonics of upper-hybrid (UH) waves using both observation and particle-in-cell (PIC) simulation, and the generation of electromagnetic radiation from PIC simulation. Harmonics of UH are linearly polarized and propagate along the perpendicular direction to the ambient magnetic field. Compared with two-dimensional PIC simulation and nonlinear kinetic theory, we show that the nonlinear beam-plasma interaction between the agyrotropic electrons and the core electrons generates harmonics of UH. Moreover, PIC simulation shows that agyrotropic electron beam can lead to electromagnetic (EM) radiation at the plasma frequency and harmonics.

Comprehensive single-cell transcriptome lineages of a proto-vertebrate.

Ascidian embryos highlight the importance of cell lineages in animal development. As simple proto-vertebrates, they also provide insights into the evolutionary origins of cell types such as cranial placodes and neural crest cells. Here we have determined single-cell transcriptomes for more than 90,000 cells that span the entirety of development-from the onset of gastrulation to swimming tadpoles-in Ciona intestinalis. Owing to the small numbers of cells in ascidian embryos, this represents an average of over 12-fold coverage for every cell at every stage of development. We used single-cell transcriptome trajectories to construct virtual cell-lineage maps and provisional gene networks for 41 neural subtypes that comprise the larval nervous system. We summarize several applications of these datasets, including annotating the synaptome of swimming tadpoles and tracing the evolutionary origin of cell types such as the vertebrate telencephalon.

Solar-wind proton anisotropy versus beta relation.

The present Letter puts forth a possible explanation for the outstanding problem of measured proton temperature anisotropy in the solar wind at 1 AU apparently being regulated by the mirror and oblique fire-hose instabilities. Making use of the fact that the local magnetic field intensity near 1 AU undergoes intermediate-scale temporal variations, the present Letter carries out the quasilinear analysis of the temperature anisotropy-driven instabilities with a time-varying local B field, assuming arbitrary initial temperature ratios and parallel betas. It is found that the saturated states in (ß(∥), T(⊥)/T(∥)) space are bounded by the mirror and oblique fire-hose instabilities, which is superficially similar to the observation.

Self-consistent generation of superthermal electrons by beam-plasma interaction.

It has been known since the early days of plasma physics research that superthermal electrons are generated during beam-plasma laboratory experiments. Superthermal electrons (the kappa distribution) are also ubiquitously observed in space. To explain such a feature, various particle acceleration mechanisms have been proposed. However, self-consistent acceleration of electrons in the context of plasma kinetic theory has not been demonstrated to date. This Letter reports such a demonstration. It is shown that the collisionality, defined via the "plasma parameter" g=1/n(lambda(D)(3), plays a pivotal role. It is found that a small but moderately finite value of is necessary for the superthermal tail to be generated, implying that purely collisionless (g=0) Vlasov theory cannot produce a superthermal population.

Exact energy principle in magnetic reconnection for current-sheet models.

On the basis of an exact nonlinear energy principle, it is shown that the change in magnetic topology (i.e., reconnection) in a finite-domain system leads to the conversion of magnetic field energy to particle energy. However, it is also shown that the conversion efficiency gradually disappears as the system size increases. This principle is demonstrated with model current-sheet equilibria including Harris and Fadeev solutions, as well as a current-sheet equilibrium which contains a singular current layer. The finding that energy conversion in reconnection is highly dependent on the system size may have an important implication for numerical simulations performed under finite geometry.

Unified formulation for inhomogeneity-driven instabilities in the lower-hybrid range.

A local dispersion relation that describes inhomogeneity-driven instabilities in the lower-hybrid range is derived following a procedure that correctly describes energy exchange between waves and particles in inhomogeneous media, correcting some inherent ambiguities associated with the standard formalism found in the literature. Numerical solutions of this improved dispersion relation show that it constitutes a unified formulation for the instabilities in the lower-hybrid range, describing the so-called modified two-stream instability, excited by the ion cross-field drift, including the ion Weibel instability, and also describing the lower-hybrid drift instability, which is due to inhomogeneity effects on the electron population.