Clustered regularly interspaced short palindromic repeat (CRISPR)-associated systems have revolutionized genome engineering by facilitating a wide range of targeted DNA perturbations. These systems ...have resulted in the development of powerful new screens to test gene functions at the genomic scale. While there is tremendous potential to map and interrogate gene regulatory networks at unprecedented speed and scale using CRISPR screens, their implementation in plants remains in its infancy. Here we discuss the general concepts, tools, and workflows for establishing CRISPR screens in plants and analyze the handful of recent reports describing the use of this strategy to generate mutant knockout collections or to diversify DNA sequences. In addition, we provide insight into how to design CRISPR knockout screens in plants given the current challenges and limitations and examine multiple design options. Finally, we discuss the unique multiplexing capabilities of CRISPR screens to investigate redundant gene functions in highly duplicated plant genomes. Combinatorial mutant screens have the potential to routinely generate higher-order mutant collections and facilitate the characterization of gene networks. By integrating this approach with the numerous genomic profiles that have been generated over the past two decades, the implementation of CRISPR screens offers new opportunities to analyze plant genomes at deeper resolution and will lead to great advances in functional and synthetic biology.
The ability to selectively alter genomic DNA sequences in vivo is a powerful tool for basic and applied research. The CRISPR/Cas9 system precisely mutates DNA sequences in a number of organisms. ...Here, the CRISPR/Cas9 system is shown to be effective in soybean by knocking-out a green fluorescent protein (GFP) transgene and modifying nine endogenous loci.
Targeted DNA mutations were detected in 95% of 88 hairy-root transgenic events analyzed. Bi-allelic mutations were detected in events transformed with eight of the nine targeting vectors. Small deletions were the most common type of mutation produced, although SNPs and short insertions were also observed. Homoeologous genes were successfully targeted singly and together, demonstrating that CRISPR/Cas9 can both selectively, and generally, target members of gene families. Somatic embryo cultures were also modified to enable the production of plants with heritable mutations, with the frequency of DNA modifications increasing with culture time. A novel cloning strategy and vector system based on In-Fusion® cloning was developed to simplify the production of CRISPR/Cas9 targeting vectors, which should be applicable for targeting any gene in any organism.
The CRISPR/Cas9 is a simple, efficient, and highly specific genome editing tool in soybean. Although some vectors are more efficient than others, it is possible to edit duplicated genes relatively easily. The vectors and methods developed here will be useful for the application of CRISPR/Cas9 to soybean and other plant species.
Quantitative text analysis tools have become increasingly popular methods for the operationalization of various types of discourse analysis. However, their application usually remains fairly simple ...and superficial, and fails to exploit the resources which the digital era holds for discourse analysis to their full extent. This paper discusses the discourse-analytic potential of a more complex and advanced text analysis tool, which is already frequently employed in other approaches to textual analysis, notably topic modelling. We argue that topic modelling promises advances in areas where discourse analysis has traditionally struggled, such as scaling, repetition, and systematization, which go beyond the contributions of simpler frequency and collocation counts. At the same time, it does not violate the epistemological premises and methodological ethos of even the more radical theories of discourse, we will demonstrate. Finally, we present two small case studies to show how topic modelling - when used with appropriate parameters - can straightforwardly enhance our ability to systematically investigate and interpret discourses in large collections of text. Abbreviations: CDA: Critical Discourse Analysis; LDA: Latent Dirichlet Allocation
The high efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mutagenesis in plants enables the development of high-throughput mutagenesis strategies. By ...transforming pooled CRISPR libraries into tomato (Solanum lycopersicum), collections of mutant lines were generated with minimal transformation attempts and in a relatively short period of time. Identification of the targeted gene(s) was easily determined by sequencing the incorporated guide RNA(s) in the primary transgenic events. From a single transformation with a CRISPR library targeting the immunity-associated leucine-rich repeat subfamily XII genes, heritable mutations were recovered in 15 of the 54 genes targeted. To increase throughput, a second CRISPR library was made containing three guide RNAs per construct to target 18 putative transporter genes. This resulted in stable mutations in 15 of the 18 targeted genes, with some primary transgenic plants having as many as five mutated genes. Furthermore, the redundancy in this collection of plants allowed for the association of aberrant T0 phenotypes with the underlying targeted genes. Plants with mutations in a homolog of an Arabidopsis (Arabidopsis thaliana) boron efflux transporter displayed boron deficiency phenotypes. The strategy described here provides a technically simple yet high-throughput approach for generating a collection of lines with targeted mutations and should be applicable to any plant transformation system.
Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In ...particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (
), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function.
ABSTRACT Studying the properties of young planetary systems can shed light on how the dynamics and structure of planets evolve during their most formative years. Recent K2 observations of nearby ...young clusters (10-800 Myr) have facilitated the discovery of such planetary systems. Here we report the discovery of a Neptune-sized planet transiting an M4.5 dwarf (K2-25) in the Hyades cluster (650-800 Myr). The light curve shows a strong periodic signal at 1.88 days, which we attribute to spot coverage and rotation. We confirm that the planet host is a member of the Hyades by measuring the radial velocity of the system with the high-resolution near-infrared spectrograph Immersion Grating Infrared Spectrometer. This enables us to calculate a distance based on K2-25's kinematics and membership to the Hyades, which in turn provides a stellar radius and mass to 5%-10%, better than what is currently possible for most Kepler M dwarfs (12%-20%). We use the derived stellar density as a prior on fitting the K2 transit photometry, which provides weak constraints on eccentricity. Utilizing a combination of adaptive optics imaging and high-resolution spectra, we rule out the possibility that the signal is due to a bound or background eclipsing binary, confirming the transits' planetary origin. K2-25b has a radius ( R⊕) much larger than older Kepler planets with similar orbital periods (3.485 days) and host-star masses (0.29 M ). This suggests that close-in planets lose some of their atmospheres past the first few hundred million years. Additional transiting planets around the Hyades, Pleiades, and Praesepe clusters from K2 will help confirm whether this planet is atypical or representative of other close-in planets of similar age.
Exoplanets can evolve significantly between birth and maturity, as their atmospheres, orbits, and structures are shaped by their environment. Young planets (<1 Gyr) offer an opportunity to probe the ...critical early stages of this evolution, where planets evolve the fastest. However, most of the known young planets orbit prohibitively faint stars. We present the discovery of two planets transiting HD 63433 (TOI 1726, TIC 130181866), a young Sun-like ( ) star. Through kinematics, lithium abundance, and rotation, we confirm that HD 63433 is a member of the Ursa Major moving group (τ = 414 23 Myr). Based on the TESS light curve and updated stellar parameters, we estimate that the planet radii are 2.15 0.10 R⊕ and 2.67 0.12 R⊕, the orbital periods are 7.11 and 20.55 days, and the orbital eccentricities are lower than about 0.2. Using High Accuracy Radial velocity Planet Searcher for the Northern hemisphere velocities, we measure the Rossiter-McLaughlin signal of the inner planet, demonstrating that the orbit is prograde. Since the host star is bright (V = 6.9), both planets are amenable to transmission spectroscopy, radial velocity measurements of their masses, and more precise determination of the stellar obliquity. This system is therefore poised to play an important role in our understanding of planetary system evolution in the first billion years after formation.
The green seaweed Ulva mutabilis is an ecologically important marine primary producer as well as a promising cash crop cultivated for multiple uses. Despite its importance, several molecular tools ...are still needed to better understand seaweed biology. Here, we report the development of a flexible and modular molecular cloning toolkit for the green seaweed U. mutabilis based on a Golden Gate cloning system. The toolkit presently contains 125 entry vectors, 26 destination vectors, and 107 functionally validated expression vectors. We demonstrate the importance of endogenous regulatory sequences for transgene expression and characterize three endogenous promoters suitable to drive transgene expression. We describe two vector architectures to express transgenes via two expression cassettes or a bicistronic approach. The majority of selected transformants (50%-80%) consistently give clear visual transgene expression. Furthermore, we made different marker lines for intracellular compartments after evaluating 13 transit peptides and 11 tagged endogenous Ulva genes. Our molecular toolkit enables the study of Ulva gain-of-function lines and paves the way for gene characterization and large-scale functional genomics studies in a green seaweed.
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