Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.

Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.

Fast acrobatic maneuvers enable arboreal spiders to hunt dangerous prey.

Spiders, the most specious taxon of predators, have evolved an astounding range of predatory strategies, including group hunting, specialized silk traps, pheromone-loaded bolas, and aggressive mimicry. Spiders that hunt prey defended with behavioral, mechanical, or chemical means are under additional selection pressure to avoid injury and death. Ants are considered dangerous because they can harm or kill their predators, but some groups of spiders, such as the Theridiidae, have a very high diversification of ant-hunting species and strategies [J. Liu et al., Mol. Phylogenet. Evol. 94, 658-675 (2016)]. Here, we provide detailed behavioral analyses of the highly acrobatic Australian ant-slayer spider, Euryopis umbilicata (Theridiidae), that captures much larger and defended Camponotus ants on vertical tree trunks. The hunting sequence consists of ritualized steps performed within split seconds, resulting in an exceptionally high prey capture success rate.

Facilitating taxonomy and phylogenetics: An informative and cost-effective protocol integrating long amplicon PCRs and third-generation sequencing.

Phylogenetic inference has become a standard technique in integrative taxonomy and systematics, as well as in biogeography and ecology. DNA barcodes are often used for phylogenetic inference, despite being strongly limited due to their low number of informative sites. Also, because current DNA barcodes are based on a fraction of a single, fast-evolving gene, they are highly unsuitable for resolving deeper phylogenetic relationships due to saturation. In recent years, methods that analyse hundreds and thousands of loci at once have improved the resolution of the Tree of Life, but these methods require resources, experience and molecular laboratories that most taxonomists do not have. This paper introduces a PCR-based protocol that produces long amplicons of both slow- and fast-evolving unlinked mitochondrial and nuclear gene regions, which can be sequenced by the affordable and portable ONT MinION platform with low infrastructure or funding requirements. As a proof of concept, we inferred a phylogeny of a sample of 63 spider species from 20 families using our proposed protocol. The results were overall consistent with the results from approaches based on hundreds and thousands of loci, while requiring just a fraction of the cost and labour of such approaches, making our protocol accessible to taxonomists worldwide.

Building behavior does not drive rates of phenotypic evolution in spiders.

Do animals set the course for the evolution of their lineage when manipulating their environment? This heavily disputed question is empirically unexplored but critical to interpret phenotypic diversity. Here, we tested whether the macroevolutionary rates of body morphology correlate with the use of built artifacts in a megadiverse clade comprising builders and nonbuilders-spiders. By separating the inferred building-dependent rates from background effects, we found that variation in the evolution of morphology is poorly explained by artifact use. Thus natural selection acting directly on body morphology rather than indirectly via construction behavior is the dominant driver of phenotypic diversity.

Dynamic evolution of locomotor performance independent of changes in extended phenotype use in spiders.

Many animals use self-built structures (extended phenotypes) to enhance body functions, such as thermoregulation, prey capture or defence. Yet, it is unclear whether the evolution of animal constructions supplements or substitutes body functions-with disparate feedbacks on trait evolution. Here, using brown spiders (Araneae: marronoid clade), we explored if the evolutionary loss and gain of silken webs as extended prey capture devices correlates with alterations in traits known to play an important role in predatory strikes-locomotor performance (sprint speed) and leg spination (expression of capture spines on front legs). We found that in this group high locomotor performance, with running speeds of over 100 body lengths per second, evolved repeatedly-both in web-building and cursorial spiders. There was no correlation with running speed, and leg spination only poorly correlated, relative to the use of extended phenotypes, indicating that web use does not reduce selective pressures on body functions involved in prey capture and defence per se. Consequently, extended prey capture devices serve as supplements rather than substitutions to body traits and may only be beneficial in conjunction with certain life-history traits, possibly explaining the rare evolution and repeated loss of trapping strategies in predatory animals.

Stabilized Morphological Evolution of Spiders Despite Mosaic Changes in Foraging Ecology.

A prominent question in animal research is how the evolution of morphology and ecology interacts in the generation of phenotypic diversity. Spiders are some of the most abundant arthropod predators in terrestrial ecosystems and exhibit a diversity of foraging styles. It remains unclear how spider body size and proportions relate to foraging style, and if the use of webs as prey capture devices correlates with changes in body characteristics. Here, we present the most extensive data set to date of morphometric and ecological traits in spiders. We used this data set to estimate the change in spider body sizes and shapes over deep time and to test if and how spider phenotypes are correlated with their behavioral ecology. We found that phylogenetic variation of most traits best fitted an Ornstein-Uhlenbeck model, which is a model of stabilizing selection. A prominent exception was body length, whose evolutionary dynamics were best explained with a Brownian Motion (free trait diffusion) model. This was most expressed in the araneoid clade (ecribellate orb-weaving spiders and allies) that showed bimodal trends toward either miniaturization or gigantism. Only few traits differed significantly between ecological guilds, most prominently leg length and thickness, and although a multivariate framework found general differences in traits among ecological guilds, it was not possible to unequivocally associate a set of morphometric traits with the relative ecological mode. Long, thin legs have often evolved with aerial webs and a hanging (suspended) locomotion style, but this trend is not general. Eye size and fang length did not differ between ecological guilds, rejecting the hypothesis that webs reduce the need for visual cue recognition and prey immobilization. For the inference of the ecology of species with unknown behaviors, we propose not to use morphometric traits, but rather consult (micro-)morphological characters, such as the presence of certain podal structures. These results suggest that, in contrast to insects, the evolution of body proportions in spiders is unusually stabilized and ecological adaptations are dominantly realized by behavioral traits and extended phenotypes in this group of predators. This work demonstrates the power of combining recent advances in phylogenomics with trait-based approaches to better understand global functional diversity patterns through space and time. [Animal architecture; Arachnida; Araneae; extended phenotype; functional traits; macroevolution; stabilizing selection.].

pegIT - a web-based design tool for prime editing.

Prime editing (PE) is a novel CRISPR-derived genome editing technique facilitating precision editing without double-stranded DNA breaks. PE, mediated by a Cas9-reverse transcriptase fusion protein, is based on dual-functioning prime editing guide RNAs (pegRNAs), serving both as guide molecules and as templates carrying the desired edits. Due to such diverse functions, manual pegRNA design is a subject to error and not suited for large-scale setups. Here, we present pegIT, a user-friendly web tool for rapid pegRNA design for numerous user-defined edits, including large-scale setups. pegIT is freely available at https://pegit.giehmlab.dk.

Delivering genes with human immunodeficiency virus-derived vehicles: still state-of-the-art after 25 years.

Viruses are naturally endowed with the capacity to transfer genetic material between cells. Following early skepticism, engineered viruses have been used to transfer genetic information into thousands of patients, and genetic therapies are currently attracting large investments. Despite challenges and severe adverse effects along the way, optimized technologies and improved manufacturing processes are driving gene therapy toward clinical translation. Fueled by the outbreak of AIDS in the 1980s and the accompanying focus on human immunodeficiency virus (HIV), lentiviral vectors derived from HIV have grown to become one of the most successful and widely used vector technologies. In 2022, this vector technology has been around for more than 25 years. Here, we celebrate the anniversary by portraying the vector system and its intriguing properties. We dive into the technology itself and recapitulate the use of lentiviral vectors for ex vivo gene transfer to hematopoietic stem cells and for production of CAR T-cells. Furthermore, we describe the adaptation of lentiviral vectors for in vivo gene delivery and cover the important contribution of lentiviral vectors to basic molecular research including their role as carriers of CRISPR genome editing technologies. Last, we dwell on the emerging capacity of lentiviral particles to package and transfer foreign proteins.

Locomotion and kinematics of arachnids.

A basic feature of animals is the capability to move and disperse. Arachnids are one of the oldest lineages of terrestrial animals and characterized by an octopodal locomotor apparatus with hydraulic limb extension. Their locomotion repertoire includes running, climbing, jumping, but also swimming, diving, abseiling, rolling, gliding and -passively- even flying. Studying the unique locomotor functions and movement ecology of arachnids is important for an integrative understanding of the ecology and evolution of this diverse and ubiquitous animal group. Beyond biology, arachnid locomotion is inspiring robotic engineers. The aim of this special issue is to display the state of the interdisciplinary research on arachnid locomotion, linking physiology and biomechanics with ecology, ethology and evolutionary biology. It comprises five reviews and ten original research reports covering diverse topics, ranging from the neurophysiology of arachnid movement, the allometry and sexual dimorphism of running kinematics, the effect of autotomy or heavy body parts on locomotor efficiency, and the evolution of silk-spinning choreography, to the biophysics of ballooning and ballistic webs. This closes a significant gap in the literature on animal biomechanics.

Evolutionary kinematics of spinneret movements for rapid silk thread anchorage in spiders.

Many organisms secrete structural materials from their bodies to enhance protection, foraging or signalling. The function of such secretion products can be further extended by their assembly into complex structures, so-called extended phenotypes, such as shells, nests and biofilms. Understanding the variation in the efficacy of such assembly processes could help to explain why extended phenotypes are common on some lineages and rare in others. Here, I comparatively studied the assembly of sticky silk fibres into thread anchorages by the innate 'printing' behaviour in 92 species of spiders from 45 families, representing the so-far largest comparative study of construction-related motion patterns. I found a global evolutionary trend towards a faster production of silk thread anchorages, in both web builders and hunting spiders. The slowest producers of silk anchors belong to a clade with an ancestral configuration of respiratory organs, suggesting that a major constraint to the evolution of spinning speed is the efficiency of oxygen uptake. Motion patterns were found to contain a high phylogenetic signal, but did not correlate with spinning speeds. These results help to explain the variation in diversity and ecological success among the spider fauna and showcase the value of comparative kinematics in biodiversity studies.

Robust substrate anchorages of silk lines with extensible nano-fibres.

Living systems are built of multiscale-composites: materials formed of components with different properties that are assembled in complex micro- and nano-structures. Such biological multiscale-composites often show outstanding physical properties that are unachieved by artificial materials. A major scientific goal is thus to understand the assembly processes and the relationship between structure and function in order to reproduce them in a new generation of biomimetic high-performance materials. Here, we tested how the assembly of spider silk nano-fibres (i.e. glue coated 0.5 µm thick fibres produced by so-called piriform glands) into different micro-structures correlates with mechanical performance by empirically and numerically exploring the mechanical behaviour of line anchors in an orb weaver, a hunting spider and two ancient web builders. We demonstrate that the anchors of orb weavers exhibit outstanding mechanical robustness with minimal material use by the indirect attachment of the silk line to the substrate through a soft domain ('bridge'). This principle can be used to design new artificial high-performance attachment systems.

Limits of piriform silk adhesion-similar effects of substrate surface polarity on silk anchor performance in two spider species with disparate microhabitat use.

It has been suggested that physical interactions between biological and environmental surfaces may constrain ecological niche spaces. However, the mechanistic understanding of niche formation is frequently limited by the lack of information on the function and variation of these interactions. Here, we hypothesised that two closely related species of orb-web spiders have evolved different adhesion performance of web attachment (i.e. piriform silk) facilitating the occupation of contrasting microhabitats: plants versus rocks. Contrary to our prediction, we found that piriform silk adhesion was equally affected by surface chemistry in both species, with maximal adhesion on surfaces with high surface polarity and an average adhesion loss of 70-75% on low polar surfaces. Spiders did not respond to adhesion losses by increasing the anchor size, despite the repeated failure to attach their web to low polar surfaces. In a natural setting, poor adhesion on low polar surfaces may be mitigated by behavioural means, like the preference to place anchors on corrugated surface features such as leaf edges, or the spinning of multiple anchorages and formation of a bundled anchor line. Thus, microhabitat choice for web-building spiders may be governed by structural properties rather than surface chemistry. These results suggest that the repeatedly demonstrated effects of surface chemistry on bio-adhesion may be ecologically less important than assumed and that the role of behaviour in the evolution of bio-adhesion performance has been underestimated.

Traction reinforcement in prehensile feet of harvestmen (Arachnida, Opiliones).

Prehensile and gripping organs are recurring structures in different organisms that enhance friction by the reinforcement and redirection of normal forces. The relationship between organ structure and biomechanical performance is poorly understood, despite a broad relevance for microhabitat choice, movement ecology and biomimetics. Here, we present the first study of the biomechanics of prehensile feet in long-legged harvestmen. These arachnids exhibit the strongest sub-division of legs among arthropods, permitting extreme hyperflexion (i.e. curling up the foot tip). We found that despite the lack of adhesive foot pads, these moderately sized arthropods are able to scale vertical smooth surfaces, if the surface is curved. Comparison of three species of harvestmen differing in leg morphology shows that traction reinforcement by foot wrapping depends on the degree of leg sub-division, not leg length. Differences are explained by adaptation to different microhabitats on trees. The exponential increase of foot section length from distal to proximal introduces a gradient of flexibility that permits adaptation to a wide range of surface curvature while maintaining integrity at strong flexion. A pulley system of the claw depressor tendon ensures the controlled flexion of the high number of adesmatic joints in the harvestman foot. These results contribute to the general understanding of foot function in arthropods and showcase an interesting model for the biomimetic engineering of novel transportation systems and surgical probes.

The assassination of a hypothesis by non-critical interpretation of molecular data: A comment on.

In a recent paper, Sharma et al. (2017) tested the hypothesis that eggs attached to males' legs in podoctid harvestmen are laid by conspecifics. Using molecular methods, they falsify the "paternal care hypothesis" and suggest that the eggs belong to spiders. Here we raise several criticisms to the authenticity of this finding and present arguments supporting the hypothesis that eggs belong to harvestmen and are not accidentally attached to the males. We argue that the falsification of the paternal care hypothesis in podoctids is premature and based on non-critical interpretation of molecular data.

Distinct spinning patterns gain differentiated loading tolerance of silk thread anchorages in spiders with different ecology.

Building behaviour in animals extends biological functions beyond bodies. Many studies have emphasized the role of behavioural programmes, physiology and extrinsic factors for the structure and function of buildings. Structure attachments associated with animal constructions offer yet unrealized research opportunities. Spiders build a variety of one- to three-dimensional structures from silk fibres. The evolution of economic web shapes as a key for ecological success in spiders has been related to the emergence of high performance silks and thread coating glues. However, the role of thread anchorages has been widely neglected in those models. Here, we show that orb-web (Araneidae) and hunting spiders (Sparassidae) use different silk application patterns that determine the structure and robustness of the joint in silk thread anchorages. Silk anchorages of orb-web spiders show a greater robustness against different loading situations, whereas the silk anchorages of hunting spiders have their highest pull-off resistance when loaded parallel to the substrate along the direction of dragline spinning. This suggests that the behavioural 'printing' of silk into attachment discs along with spinneret morphology was a prerequisite for the evolution of extended silk use in a three-dimensional space. This highlights the ecological role of attachments in the evolution of animal architectures.

Hunting with sticky tape: functional shift in silk glands of araneophagous ground spiders (Gnaphosidae).

Foraging is one of the main evolutionary driving forces shaping the phenotype of organisms. In predators, a significant, though understudied, cost of foraging is the risk of being injured by struggling prey. Hunting spiders that feed on dangerous prey like ants or other spiders are an extreme example of dangerous feeding, risking their own life over a meal. Here, we describe an intriguing example of the use of attachment silk (piriform silk) for prey immobilization that comes with the costs of reduced silk anchorage function, increased piriform silk production and additional modifications of the extrusion structures (spigots) to prevent their clogging. We show that the piriform silk of gnaphosids is very stretchy and tough, which is an outstanding feat for a functional glue. This is gained by the combination of an elastic central fibre and a bi-layered glue coat consisting of aligned nanofibrils. This represents the first tensile test data on the ubiquitous piriform gland silk, adding an important puzzle piece to the mechanical catalogue of silken products in spiders.

Numerical simulation of colloidal self-assembly of super-hydrophobic arachnid cerotegument structures.

Certain arachnids exhibit complex coatings of their exoskeleton, consisting of globular structures with complex surface features. This, so-called, cerotegument is formed by a multi-component colloidal secretion that self-assembles and cures on the body surface, and leads to high water repellency. Previous ultrastructural studies revealed the involvement of different glandular cells that contribute different components to the secretion mixture, but the overall process of self-assembly into the complex regular structures observed remained highly unclear. Here we study this process from a theoretical point of view, starting from the so-called Tammes-problem. We show that slight changes of simple parameters lead to a variety of morphologies that are highly similar to the ones observed in the species specific cerotegument structures of whip-spiders. These results are not only important for our understanding of the formation of globular hierarchical structures in nature, but also for the fabrication of novel surface coatings by colloidal lithography.

Adhesive pad differentiation in Drosophila melanogaster depends on the Polycomb group gene Su(z)2.

The ability of many insects to walk on vertical smooth surfaces such as glass or even on the ceiling has fascinated biologists for a long time, and has led to the discovery of highly specialized adhesive organs located at the distal end of the animals' legs. So far, research has primarily focused on structural and ultrastructural investigations leading to a deeper understanding of adhesive organ functionality and to the development of new bioinspired materials. Genetic approaches, e.g. the analysis of mutants, to achieve a better understanding of adhesive organ differentiation have not been used so far. Here, we describe the first Drosophila melanogaster mutant that develops malformed adhesive organs, resulting in a complete loss of climbing ability on vertical smooth surfaces. Interestingly, these mutants fail to make close contact between the setal tips and the smooth surface, a crucial condition for wet adhesion mediated by capillary forces. Instead, these flies walk solely on their claws. Moreover, we were able to show that the mutation is caused by a P-element insertion into the Su(z)2 gene locus. Remobilization of the P-element restores climbing ability. Furthermore, we provide evidence that the P-element insertion results in an artificial Su(z)2 transcript, which most likely causes a gain-of-function mutation. We presume that this transcript causes deregulation of yet unknown target genes involved in pulvilli differentiation. Our results nicely demonstrate that the genetically treatable model organism Drosophila is highly suitable for future investigations on adhesive organ differentiation.

Spider's super-glue: thread anchors are composite adhesives with synergistic hierarchical organization.

Silk is a key innovation in spiders, fascinating both biologists and material scientists. However, to fulfil their biological function silken threads must be strongly fastened to substrates or other threads. The majority of modern spiders produce a unique and rather unexplored bio-adhesive: the two-compound pyriform secretion, which is spun into elaborate patterns (so called attachment discs) and used to anchor silken threads to substrates. Strong adhesion is achieved on a high variety of surfaces with a minimum of material consumption. Pyriform threads polymerize under ambient conditions, become functional within less than a second and can remain stable for years. They are biodegradable, biocompatible and highly versatile - the adhesion and the overall toughness of the attachment disc can be controlled by spinneret movements on a macroscopic level (ref. 1: V. Sahni et al., Nat. Commun., 2012, 3, 1106, DOI: 10.1038/ncomms2099). We found that the pyriform thread is a silk fibre that is coated with glue-like cement consisting of aligned nanofibrils, lipid enclosures and a dense, isotropic boundary layer. The threads are spun in a meshwork pattern that promotes stress distribution and crack arresting. Our results demonstrate, that hierarchical organization and fibre embedding may explain the high adhesive strength and flaw tolerance of a structure made by the same, rather simple type of silk glands.

The whole is more than the sum of all its parts: collective effect of spider attachment organs.

Dynamic attachment is the key to moving safely and fast in a three-dimensional environment. Among lizards, hexapods and arachnids, several lineages have evolved hairy foot pads that can generate strong friction and adhesion on both smooth and rough surfaces. A strongly expressed directionality of attachment structures results in an anisotropy of frictional properties, which might be crucial for attachment control. In a natural situation, more than one leg is usually in contact with the substrate. In order to understand the collective effect of hairy foot pads in the hunting spider Cupiennius salei (Arachnida, Ctenidae), we performed vertical pulling experiments combined with stepwise disabling of the pads. We found the attachment force of the spider to be not simply the sum of single leg forces because with leg pair deactivation a much greater decrease in attachment forces was found than was predicted by just the loss of available adhesive pad area. This indicates that overall adhesion ability of the spider is strongly dependent on the antagonistic work of opposing legs, and the apparent contact area plays only a minor role. It is concluded that the coordinated action of the legs is crucial for adhesion control and for fast and easy detachment. The cumulative effect of anisotropic fibrillar adhesive structures could be potentially interesting for biomimetic applications, such as novel gripping devices.