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Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

The evolution of fishes to tetrapods (four-limbed vertebrates) was one of the most important transformations in vertebrate evolution. Hypotheses of tetrapod origins rely heavily on the anatomy of a few tetrapod-like fish fossils from the Middle and Late Devonian period (393-359 million years ago).sup.1. These taxa--known as elpistostegalians--include Panderichthys.sup.2, Elpistostege.sup.3,4 and Tiktaalik.sup.1,5, none of which has yet revealed the complete skeletal anatomy of the pectoral fin. Here we report a 1.57-metre-long articulated specimen of Elpistostege watsoni from the Upper Devonian period of Canada, which represents--to our knowledge--the most complete elpistostegalian yet found. High-energy computed tomography reveals that the skeleton of the pectoral fin has four proximodistal rows of radials (two of which include branched carpals) as well as two distal rows that are organized as digits and putative digits. Despite this skeletal pattern (which represents the most tetrapod-like arrangement of bones found in a pectoral fin to date), the fin retains lepidotrichia (fin rays) distal to the radials. We suggest that the vertebrate hand arose primarily from a skeletal pattern buried within the fairly typical aquatic pectoral fin of elpistostegalians. Elpistostege is potentially the sister taxon of all other tetrapods, and its appendages further blur the line between fish and land vertebrates. The pectoral fin of an Elpistostege watsoni specimen from the Upper Devonian period of Canada combines digits and fin rays, blurring the line between the appendages of fish and land vertebrates., Author(s): Richard Cloutier [sup.1] [sup.2] , Alice M. Clement [sup.2] , Michael S. Y. Lee [sup.2] [sup.3] , Roxanne Noël [sup.1] , Isabelle Béchard [sup.1] , Vincent Roy [sup.1] , [...]

Reproduction in jawed vertebrates (gnathostomes) involves either external or internal fertilization (1). It is commonly argued that internal fertilization can evolve from external, but not the reverse. Male copulatory claspers are present in certain placoderms (2-4), fossil jawed vertebrates retrieved as a paraphyletic segment of the gnathostome stem group in recent studies (5-8). This suggests that internal fertilization could be primitive for gnathostomes, but such a conclusion depends on demonstrating that copulation was not just a specialized feature of certain placoderm subgroups. The reproductive biology of antiarchs, consistently identified as the least crownward placoderms (5-8) and thus of great interest in this context, has until now remained unknown. Here we show that certain antiarchs possessed dermal claspers in the males, while females bore paired dermal plates inferred to have facilitated copulation. These structures are not associated with pelvic fins. The clasper morphology resembles that of ptyctodonts, a more crownward placoderm group (7 8), suggesting that all placoderm claspers are homologous and that internal fertilization characterized all placoderms. This implies that external fertilization and spawning, which characterize most extant aquatic gnathostomes, must be derived from internal fertilization, even though this transformation has been thought implausible. Alternatively, the substantial morphological evidence for placoderm paraphyly must be rejected., Among living fish (non-tetrapod vertebrates) internal fertilization is mostly effected by copulatory structures representing modifications of the pelvic fins (chondrichthyans (9)) or anal fins (the gonopodium of some teleost fishes [...]

DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death (1). Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response (2). Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage., Detection and repair of damaged DNA is integral for cell survival and accurate transmission of genetic information to progeny. Defects in the DNA damage response (DDR) contribute to oncogenesis and [...]

New fossils from Australia reveal that the Cambrian apex predator Anomalocaris possessed compound eyes more powerful than those of most living arthropods. A sharp-eyed Cambrian predator The metre-long swimming invertebrate Anomalocaris was the top predator in the Cambrian ocean more than 500 million years ago. Recent discoveries of fly-like compound eyes attributable to this creature confirm suggestions that it is related to the arthropods -- jointed-limbed creatures such as insects, crustaceans and trilobites -- and show that compound eyes evolved before hardened exoskeletons. The superbly preserved fossils from South Australia show that Anomalocaris had exceptional vision. Its compound eyes are among the largest and most acute to have ever existed; each eye is up to 3 centimetres long and contains more than 16,000 lenses. The existence of highly visual hunters during the Cambrian would have accelerated the pace of the predator-prey 'arms race' then under way. Until recently.sup.1, intricate details of the optical design of non-biomineralized arthropod eyes remained elusive in Cambrian Burgess-Shale-type deposits, despite exceptional preservation of soft-part anatomy in such Konservat-Lagerstätten.sup.2,3. The structure and development of ommatidia in arthropod compound eyes support a single origin some time before the latest common ancestor of crown-group arthropods.sup.4, but the appearance of compound eyes in the arthropod stem group has been poorly constrained in the absence of adequate fossils. Here we report 2-3-cm paired eyes from the early Cambrian (approximately 515 million years old) Emu Bay Shale of South Australia, assigned to the Cambrian apex predator Anomalocaris. Their preserved visual surfaces are composed of at least 16,000 hexagonally packed ommatidial lenses (in a single eye), rivalling the most acute compound eyes in modern arthropods. The specimens show two distinct taphonomic modes, preserved as iron oxide (after pyrite) and calcium phosphate, demonstrating that disparate styles of early diagenetic mineralization can replicate the same type of extracellular tissue (that is, cuticle) within a single Burgess-Shale-type deposit. These fossils also provide compelling evidence for the arthropod affinities of anomalocaridids, push the origin of compound eyes deeper down the arthropod stem lineage, and indicate that the compound eye evolved before such features as a hardened exoskeleton. The inferred acuity of the anomalocaridid eye is consistent with other evidence that these animals were highly mobile visual predators in the water column.sup.5,6. The existence of large, macrophagous nektonic predators possessing sharp vision--such as Anomalocaris--within the early Cambrian ecosystem probably helped to accelerate the escalatory 'arms race' that began over half a billion years ago.sup.7,8., Author(s): John R. Paterson [sup.1] , Diego C. García-Bellido [sup.2] , Michael S. Y. Lee [sup.3] [sup.4] , Glenn A. Brock [sup.5] , James B. Jago [sup.3] [sup.6] , Gregory [...]

Despite the status of the eye as an 'organ of extreme perfection' (1), theory suggests that complex eyes can evolve very rapidly (2). The fossil record has, until now, been inadequate in providing insight into the early evolution of eyes during the initial radiation of many animal groups known as the Cambrian explosion. This is surprising because Cambrian Burgess-Shale-type deposits are replete with exquisitely preserved animals, especially arthropods, that possess eyes (3-5). However, with the exception of biomineralized trilobite eyes, virtually nothing is known about the details of their optical design. Here we report exceptionally preserved fossil eyes from the Early Cambrian (~515 million years ago) Emu Bay Shale of South Australia, revealing that some of the earliest arthropods possessed highly advanced compound eyes, each with over 3,000 large ommatidial lenses and a specialized 'bright zone'. These are the oldest nonbiomineralized eyes known in such detail, with preservation quality exceeding that found in the Burgess Shale and Chengjiang deposits. Non-biomineralized eyes of similar complexity are otherwise unknown until about 85 million years later (6,7). The arrangement and size of the lenses indicate that these eyes belonged to an active predator that was capable of seeing in low light. The eyes are more complex than those known from contemporaneous trilobites and are as advanced as those of many living forms. They provide further evidence that the Cambrian explosion involved rapid innovation in fine-scale anatomy as well as gross morphology, and are consistent with the concept that the development of advanced vision helped to drive this great evolutionary event (8)., The anatomy of many Early and Middle Cambrian (-520-500 million years (Myr) ago) organisms is known in considerable detail from numerous Burgess-Shale-type deposits worldwide (3-5), but the fine structure of [...]

Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C-C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosyl-L-methionine (SAM). However, further details of the biosynthesis remain unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron-sulphur-cluster enzyme, Dph2. Dph2 is a homodimer and each of its monomers can bind a [4Fe-4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5'-deoxyadenosyl radical. Instead, it breaks the [C.sub.γ,Met]-S bond of SAM and generates a 3-amino-3-carboxypropyl radical. Our results suggest that P. horikoshii Dph2 represents a previously unknown, SAM-dependent, [4Fe-4S]-containing enzyme that catalyses unprecedented chemistry., Corynebacterium diphtheriae is a pathogenic bacterium that causes the infectious disease diphtheria in humans (1). This bacterium kills host cells by secreting a protein factor, diphtheria toxin (2), that catalyses [...]

An amendment to this paper has been published and can be accessed via a link at the top of the paper., Author(s): Richard Cloutier [sup.1] [sup.2] , Alice M. Clement [sup.2] , Michael S. Y. Lee [sup.2] [sup.3] , Roxanne Noël [sup.1] , Isabelle Béchard [sup.1] , Vincent Roy [sup.1] , [...]

Author(s): Gary N. Parkinson; Michael P. H. Lee; Stephen Neidle (corresponding author) Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, are fundamental in protecting the cell [...]

Author(s): John D. Scanlon (corresponding author) [1, 2]; Michael S. Y. Lee [1] The Madtsoiidae were medium sized to gigantic snakes with a fossil record extending from the mid-Cretaceous to [...]

Snakes can engulf extremely large prey, but the evolutionary transition from the largely inflexible lizard skull to the mobile snake skull is not well understood. There is evidence that mosasaurs, marine lizards related to snakes, may be an important intermediate stage. It is likely that the highly flexible lower jaw evolved before the highly flexible upper jaw.

Analysis of the fossilized specimens of Pachyrachis problematicus found in the limestone quarries in Jerusalem, Israel establishes an evidence that the squamate is a primitive snake with a well developed hindlimb. The skull reveals the most semblance to the modern snake together with the long and slender body. The presence of separate astralagus and and calcaneum however supports its close relation with most squamates.

Turtles have undergone more radical postcranial changes compared to other vertebrates. Their dorsal vertebrae and ribs have fused wirth dermal armor to form a strong trunk region. Such a chelonian morphology approaches certain pareiasaurs more closely than previously believed. Evolutionary trends observed within pareiasaurs indicated that the armored body of turtles evolved slowly through correlated progression.

Amyloid precursor protein (APP) gives rise to the beta-amyloid protein (A-beta) present in the senile plaques associated with Alzheimer's disease. Analysis of human brain cultures and cerebrospinal fluid shows that much of APP consists of a newly identified form separated at the alpha terminus of A-beta. Hence a secretory pathway may be implicated in producing A-beta, a finding that could lead to a better understanding of familial Alzheimer's.

Beta-amyloid peptide (A-beta), which forms deposits in the brains of people with Alzheimer's disease, has been detected in cerebrospinal fluid and in conditioned medium of mixed-brain cell cultures. This detection was made with antibodies unique for an epitope within A-beta that covers the site of normal constitutive cleavage. The isolation of A-beta in these two fluids may lead to new methods of diagnosing Alzheimer's disease and to new treatments based on removing cerebral deposits of A-beta.

The race to describe and archive the planet's dwindling biodiversity (see K.-D. B. Dijkstra Nature 533, 172-174; 2016) becomes even more urgent with the realization that the task's scale may [...]

Vanishing profit margins in dairy farming are intensifying pressure on the ubiquitous, genetically elite, high-yielding Holstein milk cow. A radical change in strategy is needed--less-intensive agriculture and a reduction in [...]

A correction to the article "Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme" that was published in the 2010 issue is presented.