CRISPR/Cas12a-mediated allele engineering of SmAPRR2 and SmGLK2 reveals complementary control of fruit peel and flesh chlorophyll pigmentation in eggplant

MAD7-mediated genome editing via Agrobacterium enables recovery of hexa-allelic edited lines carrying distinct mutations in all six gene copies in wheat (Triticum aestivum L.) T0 plants and stable inheritance of edits into the following generation

The expansion of genome-editing tools for bread wheat ( Triticum aestivum L.) is essential to accelerate trait improvement while addressing intellectual property constraints associated with widely used CRISPR systems. MAD7 (ErCas12a), a royalty-accessible CRISPR nuclease, represents a potential alternative to CRISPR-Cas9; however, its performance in complex polyploid crops remains insufficiently characterized. In this study, the in planta genome-editing efficiency of MAD7 in hexaploid wheat was evaluated using Agrobacterium -mediated transformation. Conserved coding regions of the TaLCYε ( LYCOPENE EPSILON CYCLASE ) gene, a key regulator of carotenoid flux, were targeted across all three wheat subgenomes (A, B, and D). MAD7-mediated editing showed strong dependence on protospacer adjacent motif (PAM) composition, with detectable activity only at a T-rich PAM (TTTG), resulting in targeted mutations in 26% of transgenic T 0 plants. Notably, MAD7 enabled recovery of hexa-allelic edited lines carrying distinct mutations in all six gene copies already in the T 0 generation, with edits stably inherited in the T 1 progeny and evidence of continued nuclease activity across generations. In contrast, CRISPR-Cas9 achieved higher editing efficiencies under experimental conditions for two of three guide RNAs tested (up to 86%) and produced a broader spectrum of mutations, predominantly small insertions and deletions. MAD7-induced edits were characterized mainly by medium-sized deletions (6–15 bp), consistent with Cas12a-type staggered cleavage. This is the first study that demonstrated recovery of hexa-allelic MAD7-edited mutants in hexaploid wheat T 0 plants via Agrobacterium -mediated transformation, with stable inheritance into T 1 .

CRISPR/Cas9-mediated transformation enables functional characterization of the effector Avr4 in the banana pathogen Pseudocercospora fijiensis

Pseudocercospora fijiensis is the causal agent of Black Leaf Streak Disease (BLSD), also known as black Sigatoka, in banana. The disease affects many banana varieties, including the highly susceptible Cavendish banana that dominates global production and the export trade, and several cooking bananas that are a staple food for hundreds of millions of people worldwide. Currently, the disease is controlled using preventative fungicide treatments with up to 70 applications per year in Cavendish plantations, which accounts for approximately 30% of the production costs. Resistant cultivars are required for more sustainable production, but no resistance gene to BLSD has been identified. This is partly due to the poor genetic amenability of P. fijiensis and the lack of methods for functional gene analysis. To address these limitations, we developed a CRISPR/Cas9-mediated transformation system specifically optimized for P. fijiensis. We established a protocol to produce protoplasts, evaluated their capacity to regenerate into new colonies, and assessed antibiotic sensitivity. Subsequently, we confirmed the integration of foreign DNA, including resistance markers, using PEG-mediated transformation. We demonstrated targeted transformation using CRISPR-Cas9 to knockout the polyketide synthase gene PKS10-1, which is responsible for the production of the pigment melanin, and the mitogen-activated protein kinase (MAPK) gene Fus3. Following the successful generation of knockout mutants for these genes, achieving gene targeting efficiencies of respectively 96% and 58%, we subsequently generated knockout mutants of the renowned effector Avr4 in P. fijiensis. The resulting mutants exhibited no reduction in virulence on the susceptible banana cultivar Cavendish. In addition, we used the wild-type isolate and Avr4 knockout strains to test the resistant banana accession Calcutta 4. Contrary to a previous study, we demonstrate that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors. The established CRISPR/Cas9-mediated disruption system is highly efficient and enables routine functional gene characterization, which will help to elucidate genes involved in banana-P. fijiensis interaction, thereby supporting the discovery of resistance genes against BLSD.

A minimal two-amino-acid deletion in SmMYB1 converts an activator into a dominant repressor, reshaping global eggplant anthocyanin pigmentation during domestication.

Eggplant fruits exhibit remarkable natural variation in both the intensity and spatial distribution of anthocyanin pigmentation, yet the genetic bases underlying the dominant anthocyaninless fruit (ALF) phenotype in many white- and green-fruited accessions remain unclear. Using bulked segregant analysis, we identified a 6-bp deletion within the coding sequence of SmMYB1 as the causal mutation underlying the ALF trait. Functional characterization revealed that this deletion converts the core fruit coloration regulator SmMYB1 from a transcriptional activator into a dominant-negative repressor, designated SmMYB1alf-D. While SmMYB1alf-D loses its ability to bind target gene promoters, it retains the complete protein-interaction network of the wild-type SmMYB1, thereby sequestering essential partners and effectively suppressing the expression of anthocyanin biosynthetic genes. This strong suppression of anthocyanin structural genes by SmMYB1alf-D enables reliable prediction of fruit color and provides a breeding strategy to precisely manipulate anthocyanin metabolism. Notably, unlike previously reported dominant-negative mutants that often involve large protein truncations, this study uncovers a rare case in which a deletion of just two amino acids is sufficient to generate a potent dominant-negative regulator. Phylogenetic, geographic, haplotypic and historical evidence strongly suggests that alf-D originated as a spontaneous mutation from a purple eggplant cultivar in North China approximately 1200 years ago. Moreover, its emergence and subsequent introgression represent a major mechanism underlying the origin of green/white-fruited eggplant varieties globally. Collectively, these findings highlight how a single, minimally altered natural allele originated, spread, and reshaped eggplant fruit pigmentation and offer a potential tool for precise phenotype engineering in molecular breeding.

SAM-Targeted CRISPR-Cas9 RNP Delivery Combined with Leaf Regeneration Enables DNA-Free, Non-Chimeric Genome Editing in ‘Fuji’ Apple

Molecular Mapping and CRISPR/Cas9 Validation Reveal That SlVQ10 Regulates Seed Size in Tomato

Seed size affects seed vigor, seedling establishment, and seed utilization in tomato. Yet the genetic basis of this trait remains poorly defined in tomato itself. Here, we describe a stable small–seed mutant, T31, isolated from an ethyl methanesulfonate (EMS)–mutagenized population of the elite inbred line DL5. Relative to the wild type, T31 showed a 24% reduction in seed width, whereas vegetative growth and major fruit traits were largely unchanged. Throughout this study, ‘seed size’ refers to seed width, which was used as the principal index of seed size because tomato seeds are oblate. Genetic analysis of six populations (P1, P2, F1, F2, BC1, and BC2) indicated that the phenotype is controlled by a single recessive locus, designated ssm. Bulked segregant analysis sequencing (BSA–seq) placed ssm within a 1.77 –Mb interval on chromosome 4. KASP–based fine mapping reduced this interval to 390 kb and identified six EMS–type SNPs. Only one of these SNPs was located in an exon of Solyc04g073950.2, where it caused a Pro337Ser substitution. This gene encodes a VQ motif–containing protein and was designated SlVQ10. To test gene function, we generated CRISPR/Cas9 knockout lines in the DL5 background. Two independent homozygous knockout lines reproduced the small–seed phenotype. Seed size was reduced by 31–33%, and thousand–seed weight decreased by 33–35%. Histological analysis further showed reduced seed–coat cell expansion in the mutants. Together, the genetic and genome–editing data support SlVQ10 as the gene underlying ssm and indicate that it promotes seed size in tomato.

CRISPR/Cas9-Mediated Mutagenesis of OsERF94 Enhances Pre-Harvest Sprouting in Rice.

Pre-harvest sprouting (PHS), where seeds germinate on panicles before harvest under humid conditions, is a serious global issue in cereal crop production, including rice. Fine-mapping of the previously reported chromosome 4 locus identified OsERF94 as a strong candidate gene for functional validation. In this study, we investigated the role of OsERF94 in PHS using CRISPR/Cas9 gene editing. The CRISPR/Cas9-mediated mutagenesis of OsERF94 induced frameshift mutations, resulting in a loss-of-function of OsERF94 in the 1-I-ET and 2-D-ET lines. The 1-I-ET and 2-D-ET lines exhibited significantly higher germination rates under PHS conditions compared to the wild type, indicating increased susceptibility to PHS. Whole-genome re-sequencing confirmed that few or no mutations could be detected at off-target candidate sites in both edited lines, ensuring the precision of the CRISPR/Cas9 gene editing. A transcriptome analysis revealed altered expression patterns of several GA-related genes, including OsLOL1, OsKO3, OsGA3ox2, and OsGA2ox5 in the OsERF94 mutant lines. The up-regulation of GA biosynthetic genes and the down-regulation of GA deactivation genes observed in both the OsERF94 mutant lines suggest possible alterations in GA metabolism during the early stages of PHS. Transient luciferase reporter assays using a single-luciferase system suggested that OsERF94 may be associated with changes in the promoter activities of several GA- and ethylene-related genes. These findings suggest that OsERF94 may contribute to the regulation of PHS, potentially through moderation of GA- and ethylene-related pathways. Overall, this study improves our understanding of the molecular role of OsERF94 in PHS and highlights its potential as a target for the genetic improvement of PHS resistance in rice-breeding programs.