Conformational landscape of the type V-K CRISPR-associated transposon integration assembly.

CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.

Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon.

CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opted CRISPR-Cas systems for RNA-guided transposition. Here we present the 2.4 Å cryo-EM structure of the Scytonema hofmannii (sh) TnsB transposase from Type V-K CAST, bound to the strand transfer DNA. The strand transfer complex displays an intertwined pseudo-symmetrical architecture. Two protomers involved in strand transfer display a catalytically competent active site composed by DDE residues, while other two, which play a key structural role, show active sites where the catalytic residues are not properly positioned for phosphodiester hydrolysis. Transposon end recognition is accomplished by the NTD1/2 helical domains. A singular in trans association of NTD1 domains of the catalytically competent subunits with the inactive DDE domains reinforces the assembly. Collectively, the structural features suggest that catalysis is coupled to protein-DNA assembly to secure proper DNA integration. DNA binding residue mutants reveal that lack of specificity decreases activity, but it could increase transposition in some cases. Our structure sheds light on the strand transfer reaction of DDE transposases and offers new insights into CAST transposition.

Transposons and CRISPR: Rewiring Gene Editing.

CRISPR-Cas is driving a gene editing revolution because of its simple reprogramming. However, off-target effects and dependence on the double-strand break repair pathways impose important limitations. Because homology-directed repair acts primarily in actively dividing cells, many of the current gene correction/replacement approaches are restricted to a minority of cell types. Furthermore, current approaches display low efficiency upon insertion of large DNA cargos (e.g., sequences containing multiple gene circuits with tunable functionalities). Recent research has revealed new links between CRISPR-Cas systems and transposons providing new scaffolds that might overcome some of these limitations. Here, we comment on two new transposon-associated RNA-guided mechanisms considering their potential as new gene editing solutions. Initially, we focus on a group of small RNA-guided endonucleases of the IS200/IS605 family of transposons, which likely evolved into class 2 CRISPR effector nucleases (Cas9s and Cas12s). We explore the diversity of these nucleases (named OMEGA, obligate mobile element-guided activity) and analyze their similarities with class 2 gene editors. OMEGA nucleases can perform gene editing in human cells and constitute promising candidates for the design of new compact RNA-guided platforms. Then, we address the co-option of the RNA-guided activity of different CRISPR effector nucleases by a specialized group of Tn7-like transposons to target transposon integration. We describe the various mechanisms used by these RNA-guided transposons for target site selection and integration. Finally, we assess the potential of these new systems to circumvent some of the current gene editing challenges.

Nematode Signaling Molecules Are Extensively Metabolized by Animals, Plants, and Microorganisms.

Many bacterivorous and parasitic nematodes secrete signaling molecules called ascarosides that play a central role regulating their behavior and development. Combining stable-isotope labeling and mass spectrometry-based comparative metabolomics, here we show that ascarosides are taken up from the environment and metabolized by a wide range of phyla, including plants, fungi, bacteria, and mammals, as well as nematodes. In most tested eukaryotes and some bacteria, ascarosides are metabolized into derivatives with shortened fatty acid side chains, analogous to ascaroside biosynthesis in nematodes. In plants and C. elegans, labeled ascarosides were additionally integrated into larger, modular metabolites, and use of different ascaroside stereoisomers revealed the stereospecificity of their biosynthesis. The finding that nematodes extensively metabolize ascarosides taken up from the environment suggests that pheromone editing may play a role in conspecific and interspecific interactions. Moreover, our results indicate that plants, animals, and microorganisms may interact with associated nematodes via manipulation of ascaroside signaling.

Plant metabolism of nematode pheromones mediates plant-nematode interactions.

Microorganisms and nematodes in the rhizosphere profoundly impact plant health, and small-molecule signaling is presumed to play a central role in plant rhizosphere interactions. However, the nature of the signals and underlying mechanisms are poorly understood. Here we show that the ascaroside ascr#18, a pheromone secreted by plant-parasitic nematodes, is metabolized by plants to generate chemical signals that repel nematodes and reduce infection. Comparative metabolomics of plant tissues and excretions revealed that ascr#18 is converted into shorter side-chained ascarosides that confer repellency. An Arabidopsis mutant defective in two peroxisomal acyl-CoA oxidases does not metabolize ascr#18 and does not repel nematodes, indicating that plants, like nematodes, employ conserved peroxisomal ß-oxidation to edit ascarosides and change their message. Our results suggest that plant-editing of nematode pheromones serves as a defense mechanism that acts in parallel to conventional pattern-triggered immunity, demonstrating that plants may actively manipulate chemical signaling of soil organisms.