Advanced Bioscience Laboratories (United States)

CRISPR-Cas9 is a widely used platform for plant genome editing, but its outcomes are typically dominated by small insertions and deletions (indels). Such limited mutation profiles restrict its utility in functional studies of non-coding RNAs and regulatory elements, such as microRNAs (miRNAs), untranslated regions (UTRs), and promoter sequences, where larger sequence disruptions are often required. Here, we developed enhanced exonuclease-Cas9 platforms, termed multiple nucleotide deletion Cas9 (MND-Cas9) systems, for efficient generation of large deletions in rice. By screening four exonucleases (RecJ, T5, TREX2, and SbcB), we established MND-Cas9v1 systems based on TREX2 or SbcB that produced substantially larger deletions without reducing editing efficiency. Further optimization with an inserted DNA-binding domain (DBD) between Cas9 and exonuclease yielded MND-Cas9v2, which simultaneously enhanced efficiency and deletion size. To expand PAM compatibility, we introduced PAM-relaxed Cas9-NG and SpG variants, generating MND-Cas9-NG/SpGv2 systems with broader targeting scope and superior performance compared to their parental nucleases. Finally, we demonstrated the utility of these systems in two applications: MND-Cas9v2 efficiently knocked out the miRNA gene OsMIR530, producing larger seeds, and generated extended deletions in the 3'UTR of OsGhd2, which upregulated its expression and increased grain size. These results demonstrate that MND-Cas9 systems enable high-efficiency generation of extended deletions and facilitate functional analyses of non-coding RNAs and regulatory sequences. Overall, this work establishes a versatile and expandable exonuclease-Cas9 platform that substantially broadens the mutational spectrum and application potential of CRISPR-Cas9 for plant genome engineering.

Key messageBiolistic particle bombardment was used to deliver CRISPR-Cas9 ribonucleoprotein complexes (RNP) into the shoot apical meristem tissue of citrus and axillary meristem tissue of poplar, generating directed mutations in target genes.The use of meristematic tissues offers a strategic approach to genome editing in woody species, especially those that are recalcitrant to conventional tissue culture, as these regions contain totipotent, highly regenerative cells capable of giving rise to whole plants. Here, we employed biolistic delivery of genome-editing reagents into theshoot apical meristem (SAM) of citrus and the axillary meristems (AXM) of poplar. The system was first validated using a GFP expression construct and subsequently applied for targeted genome editing. In citrus, edited plants were obtained at the CsNPR3 locus exclusively through the delivery of CRISPR/Cas9 ribonucleoproteins (RNPs), whereas plasmid-based vectors were unsuccessful. Similarly, genome editing in poplar was achieved using RNPs targeting the Pt4CL1 gene. Although chimeric events were detected, this approach provides a feasible and innovative framework for producing transgene-free edited perennial plants.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00299-026-03741-9.

Proteolysis targeting chimeras, or PROTACs, are an emerging class of drugs that offer the potential to develop therapeutics targeting "undruggable" proteins by stabilizing protein-protein interactions (PPI). This involves leveraging the physiological protein degradation mechanism based on ubiquitination through stabilization of target protein-E3 ubiquitin ligase PPI mediated by the PROTAC. Existing computational methods for ligand design are not typically designed for the ternary complex problem and may have limited accuracy or efficiency due to the use of either rigid docking or full molecular dynamics (MD) simulations. Here we present a method which uses SILCS (site identification by ligand competitive saturation) to address the challenge of designing ligands which stabilize PPI by using precomputed ensembles of (1) functional group affinity patterns, termed FragMaps, for efficient and accurate ligand docking and of (2) a collection of putative PPI dimer 3D structures as docking targets. SILCS simulations involving aqueous, multisolute grand canonical Monte Carlo (GCMC)/MD calculations generate the FragMaps for both the target and ligase proteins. An ensemble of PPI dimer conformations is generated using the FragMaps and then dimer FragMaps are generated by merging the two sets of FragMaps. PROTAC molecules are docked into the ensemble of dimer FragMaps, and the final scoring metrics are extracted from the most favorable ternary complex. The scoring metrics, including energetics, binding site geometry, and physicochemical terms, are weighted together to construct an activity score. The method is benchmarked on a diverse set of ternary crystal structures of different proteins and PROTACs, and the derived activity score shows modest correlation with DC50 values in cells for a wide variety of systems. The SILCS-xTAC method is a powerful tool to facilitate PROTAC optimization by predicting the binding geometries and energetics of ternary complexes.

Accurate biomolecular structure prediction enables the prediction of mutational effects, the speculation of function based on predicted structural homology, the analysis of ligand binding modes, experimental model building and many other applications. Such algorithms to predict essential functional and structural features remain out of reach for biomolecular complexes containing nucleic acids. Here, we report quantitative and qualitative evaluation of nucleic acid structures for the CASP16 blind prediction challenge by 12 of the experimental groups who provided nucleic acid targets. Blind predictions accurately model secondary structure and some aspects of tertiary structure, including reasonable global folds for some complex RNAs, however, predictions often lack accuracy in the regions of highest functional importance. All models have inaccuracies in non-canonical regions where, e.g., the nucleic-acid backbone bends deviating from an A-form helix geometry or a base forms a non-standard hydrogen bond (not a Watson-Crick base pair). These bends and non-canonical interactions are integral to form functionally important regions such as RNA enzymatic active sites. Additionally, the modeling of conserved and functional interfaces between nucleic acids and ligands, proteins, or other nucleic acids remains poor. For some targets, the experimental structures may not represent the only structure the biomolecular complex occupies in solution or in its functional life-cycle, posing a future challenge for the community.

Acute exposure to a seizure-inducing dose of an organophosphorus nerve agent inhibits acetylcholinesterase, leading to pharmacoresistance if benzodiazepine treatment is delayed. Following soman-induced status epilepticus (SE) in rats, prolonged seizure is associated with severe and widespread neurodegeneration. We evaluated the aminothiol cystamine, the oxidized form of cysteamine, for neuroprotective potential against soman-induced SE and associated neurodegeneration. Cystamine has a myriad of effects including antioxidant properties, neuroprotective effects, and immunomodulation, among others, which is of interest in evaluating neuroprotective efficacy against cholinergic-induced neurodegeneration. Adult male rats implanted with telemetry transmitters for continuous EEG recording were exposed to soman and treated with the muscarinic antagonist atropine sulfate and the oxime asoxime dimethanesulfonate 1min after exposure to increase survival. Midazolam was administered 30 min after seizure onset. Cystamine (10 or 50mg/kg) or vehicle was administered 30min after seizure onset and again 4h after soman exposure. The initial seizure duration, the EEG power integral at 6h after exposure, and the percentage of rats that developed spontaneous recurrent seizure were reduced in rats treated with cystamine, compared to those that received only midazolam. In addition, cystamine reduced neurodegeneration in seizure-sensitive brain regions following soman exposure, compared to midazolam. Our findings highlight the potential for aminothiols to serve as adjunctive therapy to midazolam in treating cholinergic-induced toxicity and suggest broader applications of aminothiols in neuroprotection and neurological disorders.

The host machinery in the secretory organelles, the endoplasmic reticulum and the Golgi network, is pivotal in directing the biogenesis of enveloped virus proteins during natural infection and vaccination. Secretory trafficking of the viral proteins in these organelles is accompanied by post-translational modifications (PTMs), which modify interactions with host receptors and antibodies. Hence, elucidating the fundamental basis of PTM modulation by secretory trafficking is essential for designing genetic vaccines that encode stable and immunogenic viral proteins. For instance, the spike protein in COVID-19 mRNA vaccines undergoes bidirectional secretory trafficking after export from the endoplasmic reticulum to the cis-Golgi. This supplies the spike from the cis-Golgi to the coronavirus assembly site in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) by retrograde trafficking and to the plasma membrane by anterograde trafficking for coronavirus transmission and for immune display. However, surprisingly, little is known about the modulation of spike PTMs by this secretory recycling and retention in the endoplasmic reticulum-ERGIC-Golgi pathway before export to the plasma membrane. Since PTMs such as N-glycans modulate the conformations of immunogenic epitopes in the spike protein, addressing this knowledge gap in secretory routing is critical for understanding spike-immune system interactions. Furthermore, the bidirectional trafficking of the spike protein engages and diverts the secretory machinery, which can cause secretory stress, inflammation, and immune dysfunction. Hence, elucidating the fundamental interactions that govern spike biogenesis and trafficking will expedite the design of next- generation genetic vaccines that interfere minimally with the secretory trafficking machinery while undergoing proper PTMs for stability and efficient delivery to the plasma membrane. Here, we report atomic-level insights into the PTM and structural biology of novel spike protein constructs arrested in various stages of secretory trafficking. Using a combination of mass spectrometry, single particle cryoEM, and MD simulations on these differently arrested spike constructs, we identified clusters of N-glycans whose remodeling is intimately linked to spike recycling and secretory routing. These spike constructs demonstrate robust binding to a panel of neutralizing antibodies despite differences in their N-glycans and secretory routing to the plasma membrane. Finally, building on these structure-function insights, we engineered a novel spike vaccine candidate with enhanced secretion to the cell surface. We determined its structures in various conformational states by single particle cryoEM, demonstrating the presentation of immunogenic epitopes. Thus, our investigation provides novel strategies for designing a new generation of genetic vaccines with enhanced secretion and immune display at the host cell plasma membrane. Bottom of Form

Key messageTransgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing the ALS gene via transient transgene expression of an efficient cytosine base editor.CRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation when Agrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit the ALS gene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00299-025-03627-2.