Genetius

The study engineered Tobacco rattle virus vectors incorporating distinct RNA secondary structures as mobility factors to improve guide RNA delivery to plant meristems. Using Nicotiana benthamiana plants expressing Cas9, optimal virus constructs were identified that generated both somatic and heritable edits, and these constructs were successfully applied to edit the emerging oilseed crop pennycress (Thlaspi arvense).

The authors introduce the ENABLE(R) Gene Editing in planta toolkit, a streamlined two‑step cloning system for creating CRISPR/Cas9 knockout vectors suitable for transient or stable transformation. Validation was performed in Oryza sativa protoplasts and Arabidopsis thaliana plants, and the toolkit includes low‑cost protocols aimed at facilitating adoption in the Global South.

The authors created a fast‑cycling, isogenic barley line (GP‑rapid) by introgressing the wild‑type Ppd‑H1 allele from Igri into the Golden Promise cultivar and performing two backcrosses to limit the donor genome, achieving a 25% reduction in generation time under speed‑breeding conditions while retaining high transformation efficiency. CRISPR/Cas9‑mediated editing of Ppd‑H1 showed regeneration and transformation rates comparable to the original Golden Promise, establishing GP‑rapid as a rapid platform for transgenic and gene‑edited barley research.

The study applied CRISPR/Cas9 gene editing to Physalis peruviana to modify plant‑architecture genes and create a compact growth ideotype. This compact phenotype is intended to increase per‑plot yield and support future breeding efforts for this nutritionally valuable minor crop.

The study created transgenic Arabidopsis lines enabling inducible plasmodesmal closure via an overactive CALLOSE SYNTHASE3 allele (icals3m) and the C‑terminal domain of PDLP1, independent of pathogen signals. Induced closure triggered stress‑responsive gene expression, elevated salicylic acid levels, and enhanced resistance to Pseudomonas syringae, while also causing starch accumulation, reduced growth, and increased susceptibility to Botrytis cinerea, indicating that plasmodesmal closure itself can activate immune signaling.

The study identified twenty survival of SA-induced death (ssd) mutants that are defective in starch, glucose, nitrate metabolism, and circadian regulation, leading to excessive carbohydrate accumulation and susceptibility to salicylic acid (SA)-induced death in prolonged darkness. Glucose application rescues SA‑treated plants by antagonizing oxidative stress and restoring metabolic balance, as revealed by transcriptomic analyses that link SA‑induced cell death to effector‑triggered immunity pathways.

The study used CRISPR/Cas9 to edit the downstream region of the Arabidopsis thaliana FLOWERING LOCUS T (FT) gene, identifying a 2.3‑kb segment containing the Block E enhancer as crucial for normal FT expression and flowering. Fine‑scale deletions pinpointed a 63‑bp core module with CCAAT‑ and G‑boxes, and revealed a cryptic CCAAT‑box that becomes active when repositioned, highlighting the importance of local chromatin context and motif arrangement for enhancer function.

The study optimized three wheat transformation methods—immature embryo, callus, and in planta injection—by systematically adjusting Agrobacterium strain, bacterial density, acetosyringone concentration, and incubation conditions, achieving transformation efficiencies up to 66.84%. Using these protocols, CRISPR/Cas9 knockout of the negative regulator TaARE1-D produced mutants with increased grain number, spike length, grain size, and a stay‑green phenotype, demonstrating the platform’s potential to accelerate yield and stress‑tolerance improvements in wheat.

The study reveals that the methyl‑CpG‑binding domain protein MBD8 interacts with the histone demethylase LDL2 to facilitate removal of H3K4me1 and transcriptional repression downstream of H3K9me2 in Arabidopsis. MBD8 binds GC‑poor DNA independently of cytosine methylation and stabilizes LDL2 protein levels, indicating a broader role for MBD proteins beyond methyl‑DNA recognition.

The authors introduced a polycistronic tRNA‑gRNA array for CRISPR/Cas9 editing in Physcomitrium patens that doubled the frequency of large, targeted deletions compared with conventional single‑gRNA constructs. Using dual‑gRNA targeting, they achieved simultaneous deletion of two to four genes (katanin and TPX2 families) in a single transformation, reaching up to 42% efficiency per gene, though efficiency depended on gRNA pair design.