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RNA-guided nucleases of the CRISPR/Cas type can be repurposed as programmable nucleotide deaminases to mediate targeted nucleotide substitutions. Such base editors have enormous potential in genome editing, gene therapy and precision breeding. However, current editors suffer from limited specificity in that they edit different and/or multiple bases within a larger sequence window. Using cytidine deaminase base editors that elicit C-to-T mutations, we show here that high editing precision can be achieved by engineering the connection between the deaminase domain and the Cas domain of the editor. By systematically testing different linker sequences and removing non-essential sequences from the deaminase, we obtain high-precision base editors with narrow activity windows that can selectively edit a single cytidine at a specific position with high accuracy and efficiency. These base editors will enable the use of genome editing in applications where single-nucleotide changes are required and off-target editing of adjacent nucleotides is not tolerable. Base editors can target multiple bases within a window around the target site, reducing their specificity. Here the authors engineer the connection between the deaminase and Cas domain to narrow the window of activity.

Base editors (BEs) are RNA-guided CRISPR-Cas-derived genome editing tools that induce single-nucleotide changes. The limitations of current BEs lie in their low precision (especially when multiple target nucleotides of the deaminase are present within the activity window) and their restriction to targets that are in proper distance from the PAM sequence. We have recently developed high-precision cytidine BEs by engineering CDA1 truncations and nCas9 fusions that predominantly edit nucleotide C −18 relative to the PAM sequence NGG. Here, by testing fusions with Cas9 variants that recognize alternative PAMs, we provide a series of high-precision BEs that greatly expand the versatility of base editing. In addition, we obtained BEs that selectively edit C −15 or C −16 . We also show that our high-precision BEs can substantially reduce off-target effect. These improved base editing tools will be widely applicable in basic research, biotechnology and gene therapy. Base editors can be limited by precision and the size of the target window. Here the authors test Cas9s that recognise alternative PAMs to obtain a series of high-precision editors.

The formation of a new species can occur by an asexual mechanism by transfer of entire nuclear genomes between plant cells as shown by the creation of a new allopolyploid plant from parental herbaceous and woody plant species, this mechanism is a potential new tool for crop improvement. Speciation by growing together Grafting is a common occurrence in nature and is familiar as a means of manipulating plants and trees for use in agriculture and horticulture. Here Ralph Bock and colleagues demonstrate that entire nuclear genomes can be transferred across the graft junction from plant to plant. In grafting experiments between the tree tobacco Nicotiana glauca and the cigarette tobacco N. tabacum (woody and herbaceous species, respectively), horizontal transfer of nuclear genomes can result in the formation of a new polyploid species — Nicotiana tabauca . This is an example of allopolyploidization, the combination of the genomes from two different species that has contributed to evolutionary innovation, adaptation, speciation and domestication. It is thought to occur through hybridization events between species, accompanied or followed by genome duplication, but this work shows that it can also occur through an asexual mechanism that is readily available as a potential tool for crop improvement. Allopolyploidization, the combination of the genomes from two different species, has been a major source of evolutionary innovation and a driver of speciation and environmental adaptation 1 , 2 , 3 , 4 . In plants, it has also contributed greatly to crop domestication, as the superior properties of many modern crop plants were conferred by ancient allopolyploidization events 5 , 6 . It is generally thought that allopolyploidization occurred through hybridization events between species, accompanied or followed by genome duplication 6 , 7 . Although many allopolyploids arose from closely related species (congeners), there are also allopolyploid species that were formed from more distantly related progenitor species belonging to different genera or even different tribes 8 . Here we have examined the possibility that allopolyploidization can also occur by asexual mechanisms. We show that upon grafting—a mechanism of plant–plant interaction that is widespread in nature—entire nuclear genomes can be transferred between plant cells. We provide direct evidence for this process resulting in speciation by creating a new allopolyploid plant species from a herbaceous species and a woody species in the nightshade family. The new species is fertile and produces fertile progeny. Our data highlight natural grafting as a potential asexual mechanism of speciation and also provide a method for the generation of novel allopolyploid crop species.

The engineering of complex metabolic pathways requires the concerted expression of multiple genes. In plastids (chloroplasts) of plant cells, genes are organized in operons that are coexpressed as polycistronic transcripts and then often are processed further into monocistronic mRNAs. Here we have used the tocochromanol pathway (providing tocopherols and tocotrienols, collectively also referred to as “vitamin E”) as an example to establish principles of successful multigene engineering by stable transformation of the chloroplast genome, a technology not afflicted with epigenetic variation and/or instability of transgene expression. Testing a series of single-gene constructs (encoding homogentisate phytyltransferase, tocopherol cyclase, and γ-tocopherol methyltransferase) and rationally designed synthetic operons in tobacco and tomato plants, we (i) confirmed previous results suggesting homogentisate phytyltransferase as the limiting enzymatic step in the pathway, (ii) comparatively characterized the bottlenecks in tocopherol biosynthesis in transplastomic leaves and tomato fruits, and (iii) achieved an up to tenfold increase in total tocochromanol accumulation. In addition, our results uncovered an unexpected light-dependent regulatory link between tocochromanol metabolism and the pathways of photosynthetic pigment biosynthesis. The synthetic operon design developed here will facilitate future synthetic biology applications in plastids, especially the design of artificial operons that introduce novel biochemical pathways into plants.

Artemisinin-based therapies are the only effective treatment for malaria, the most devastating disease in human history. To meet the growing demand for artemisinin and make it accessible to the poorest, an inexpensive and rapidly scalable production platform is urgently needed. Here we have developed a new synthetic biology approach, combinatorial supertransformation of transplastomic recipient lines (COSTREL), and applied it to introduce the complete pathway for artemisinic acid, the precursor of artemisinin, into the high-biomass crop tobacco. We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output. Malaria is by far the most devastating tropical disease in the world. It affects hundreds of millions of people – mainly in Africa and Asia – with almost half a million deaths every year. The most effective therapies against malaria all include the drug artemisinin, which is naturally found in an Asian plant called Artemisia annua. Unfortunately, the artemisinin content of A. annua plants is relatively low and the demand for this drug outstrips the supply of the plant. The costly production process makes artemisinin-based treatments inaccessible to many of the people in the most badly affected regions, and so researchers have been trying to find new ways to produce this drug. Genetically modifying crop plants, such as tobacco, to produce artemisinin or related compounds could potentially provide a more sustainable and cheaper source of the drug. Inside plant cells, a structure called the nucleus contains DNA that encodes most of a plant’s genes, but compartments called mitochondria and chloroplasts also contain some DNA. Existing methods to genetically modify plants are able to insert a few genes into either the nucleus or the chloroplasts at a time. However, the production of artemisinin in A. annua involves many different genes that act at different stages of the process, and the precise roles played by many of them remain unclear. Fuentes et al. developed a new approach to insert many of the A. annua genes involved in artemisinin production into tobacco plants at the same time, instead of one-by-one. The new method, referred to as COSTREL, takes advantage of the researchers’ ability to insert new genes into both the nucleus and the chloroplast of the tobacco plants. In the first step, Fuentes et al. inserted a core set of genes that are essential to make artemisinin into the chloroplast. This enabled the plants to produce a molecule called artemisinic acid, which the researchers can extract from the plants and convert into artemisinin by simple chemical reactions. After testing different arrangements of the genes in the chloroplast, the plant line that had the highest levels of artemisinic acid was used to introduce a set of “accessory” genes into the nuclear DNA. These accessory genes are not strictly required to make the drug, but they help to regulate the process in a largely unknown manner. The experiments generated hundreds of genetically modified plant lines that each have different combinations of the accessory genes. Fuentes et al. examined these lines and were able to identify plants that could produce large amounts of artemisinic acid. Therefore, these findings lay the foundations for a cheap way to produce this life-saving drug in tobacco. In the future, the COSTREL method developed by Fuentes et al. could also be used to genetically engineer other complex biochemical processes into plants.

Antimicrobial peptides (AMPs) kill microbes or inhibit their growth and are promising next-generation antibiotics. Harnessing their full potential as antimicrobial agents will require methods for cost-effective large-scale production and purification. Here, we explore the possibility to exploit the high protein synthesis capacity of the chloroplast to produce AMPs in plants. Generating a large series of 29 sets of transplastomic tobacco plants expressing nine different AMPs as fusion proteins, we show that high-level constitutive AMP expression results in deleterious plant phenotypes. However, by utilizing inducible expression and fusions to the cleavable carrier protein SUMO, the cytotoxic effects of AMPs and fused AMPs are alleviated and plants with wild-type-like phenotypes are obtained. Importantly, purified AMP fusion proteins display antimicrobial activity independently of proteolytic removal of the carrier. Our work provides expression strategies for the synthesis of toxic polypeptides in chloroplasts, and establishes transplastomic plants as efficient production platform for antimicrobial peptides. Antimicrobial peptides (AMPs) are promising next-generation antibiotics, but are difficult to produce due to the toxicity to bacterial hosts. Here, the authors report the utilization of transplastomic tobacco plants for AMPs production without cytotoxic effects via inducible expression systems and fusions to cleavable carrier protein.

Riboswitches are natural RNA sensors that regulate gene expression in response to ligand binding. Riboswitches have been identified in prokaryotes and eukaryotes but are unknown in organelles (mitochondria and plastids). Here we have tested the possibility to engineer riboswitches for plastids (chloroplasts), a genetic system that largely relies on translational control of gene expression. To this end, we have used bacterial riboswitches and modified them in silico to meet the requirements of translational regulation in plastids. These engineered switches were then tested for functionality in vivo by stable transformation of the tobacco chloroplast genome. We report the identification of a synthetic riboswitch that functions as an efficient translational regulator of gene expression in plastids in response to its exogenously applied ligand theophylline. This riboswitch provides a novel tool for plastid genome engineering that facilitates the tightly regulated inducible expression of chloroplast genes and transgenes and thus has wide applications in functional genomics and biotechnology.

Plastids (chloroplasts) are maternally inherited in most crops. Maternal inheritance excludes plastid genes and transgenes from pollen transmission. Therefore, plastid transformation is considered a superb tool for ensuring transgene containment and improving the biosafety of transgenic plants. Here, we have assessed the strictness of maternal inheritance and the extent to which plastid transformation technology confers an increase in transgene confinement. We describe an experimental system facilitating stringent selection for occasional paternal plastid transmission. In a large screen, we detected low-level paternal inheritance of transgenic plastids in tobacco. Whereas the frequency of transmission into the cotyledons of F₁ seedlings was [almost equal to]1.58 x 10⁻⁵ (on 100% cross-fertilization), transmission into the shoot apical meristem was significantly lower (2.86 x 10⁻⁶). Our data demonstrate that plastid transformation provides an effective tool to increase the biosafety of transgenic plants. However, in cases where pollen transmission must be prevented altogether, stacking with other containment methods will be necessary to eliminate the residual outcrossing risk.

To avoid photodamage plants regulate the amount of excitation energy in the membrane at the level of the light-harvesting complexes (LHCs). It has been proposed that the energy absorbed in excess is dissipated via protein conformational changes of individual LHCs. However, the exact quenching mechanism remains unclear. Here we study the mechanism of quenching in LHCs that bind a single carotenoid species and are constitutively in a dissipative conformation. Via femtosecond spectroscopy we resolve a number of carotenoid dark states, demonstrating that the carotenoid is bound to the complex in different conformations. Some of those states act as excitation energy donors for the chlorophylls, whereas others act as quenchers. Via in silico analysis we show that structural changes of carotenoids are expected in the LHC protein domains exposed to the chloroplast lumen, where acidification triggers photoprotection in vivo. We propose that structural changes of LHCs control the conformation of the carotenoids, thus permitting access to different dark states responsible for either light harvesting or photoprotection. Carotenoids can dissipate excess energy captured by photosynthetic light-harvesting complexes to prevent photodamage. Here, via spectroscopic and in silico approaches, Liguori et al. resolve different carotenoid dark states and propose conformational changes that permit them to act as either energy donors or quenchers.

Carotenoids are essential in oxygenic photosynthesis: they stabilize the pigment–protein complexes, are active in harvesting sunlight and in photoprotection. In plants, they are present as carotenes and their oxygenated derivatives, xanthophylls. While mutant plants lacking xanthophylls are capable of photoautotrophic growth, no plants without carotenes in their photosystems have been reported so far, which has led to the common opinion that carotenes are essential for photosynthesis. Here, we report the first plant that grows photoautotrophically in the absence of carotenes: a tobacco plant containing only the xanthophyll astaxanthin. Surprisingly, both photosystems are fully functional despite their carotenoid-binding sites being occupied by astaxanthin instead of β-carotene or remaining empty (i.e. are not occupied by carotenoids). These plants display non-photochemical quenching, despite the absence of both zeaxanthin and lutein and show that tobacco can regulate the ratio between the two photosystems in a very large dynamic range to optimize electron transport. Most life on Earth depends on photosynthesis, the process used by plants and many other organisms to store energy from sunlight and produce oxygen. The first steps of photosynthesis, the capture and conversion of sunlight into chemical energy, happen in large assemblies of proteins containing many pigment molecules called photosystems. In plants, the pigments involved in photosynthesis are green chlorophylls and carotenoids. In addition to harvesting light, carotenoids have an important role in preventing damage caused by overexposure to sunlight There are over one thousand different carotenoids in living beings, but only one, β-carotene, is present in every organism that performs the type of photosynthesis in which oxygen is released, and is thought to be essential for the process. However, this could never be proved because it is impossible to remove β-carotene from cells using typical genetic approaches without affecting all other carotenoids. Xu et al. used genetic engineering to create tobacco plants that produced a pigment called astaxanthin in place of β-carotene. Astaxanthin is a carotenoid from salmon and shrimp, not normally found in plants. These plants are the first living things known to perform photosynthesis without β-carotene and demonstrate that this pigment is not essential for photosynthesis as long as other carotenoids are present. Xu et al. also show that the photosystems can adapt to using different carotenoids, and can even operate with a reduced number of them. Xu et al’s findings show the high flexibility of photosynthesis in plants, which are able to incorporate non-native elements to the process. These results are also important in the context of increasing the photosynthetic efficiency, and thus the productivity of crops, since they show that a radical redesign of the photosynthetic machinery is feasible.