AgroGem: A Rapid and Scalable Transient Transformation System for Functional Genetics in Multiple Plant Species

Establishment of an efficient Agrobacterium-mediated transformation system and CRISPR/Cas9-mediated genome editing of the OvPDS1 gene in Oxalis vulcanicola ‘Sunset Velvet’

CRISPR–Cas technologies for precision genome editing in plants: advances, applications, and future perspectives

This review provides a comprehensive synthesis of a recent advances in CRISPR–Cas technologies and their strategic applications in crop genetics and hybrid breeding, and showcases how these technologies accelerate hybrid breeding by engineering male sterility systems, fixing heterosis, and generating high-throughput mutant libraries for trait discovery.

Virus-induced Genome Editing in Plants: Viral Vector Biology, Germline Access and the Evidence Base for Heritable, Transgene-Free Crop Improvement

Delivery, rather than nuclease chemistry, now sets the practical limit on plant genome editing. Conventional delivery depends on tissue culture and stable transformation, which remain slow, genotype-dependent and unavailable for most cultivated germplasm. Plant viruses offer an alternative because they replicate to high copy number, move systemically and can carry heterologous sequences into cells that no transformation protocol reaches. Virus-induced genome editing exploits this behaviour, and the field has moved within a decade from transient somatic mutagenesis in a model tobacco to heritable, transgene-free mutations in hexaploid bread wheat. This critical review evaluates the strength and the limits of that evidence. Vector chassis are compared on the properties that actually determine outcome, namely cargo capacity, insert stability, host range, movement behaviour and access to reproductive tissue, rather than on reported somatic editing percentages, which are shown to be poor predictors of germline transmission. The central unresolved problem is identified as the disjunction between efficient editing in infected somatic tissue and rare, stochastic entry of editing reagents into cells that give rise to gametes. Three engineering responses to that problem, namely fusion of guide RNAs to mobile RNA motifs, control of nuclease expression through meristem-competent promoters, and exploitation of axillary or adventitious growth points, are assessed against the evidence for each. Reagent miniaturisation, guide array design and virus-delivered precision editing are examined as partial solutions to the cargo constraint. Recurrent weaknesses in the literature are documented, including reliance on visible reporter loci, small progeny samples, inconsistent definitions of editing efficiency, near-absence of independent replication and the complete absence of field evaluation. The claim that virus-derived products are transgene-free is examined against the regulatory frameworks that will govern them, and the biosafety implications of releasing engineered, potentially insect-transmissible vectors are considered. Prioritised research directions are proposed, emphasising standardised reporting of progeny-level outcomes, biocontained vector design, and genotype-spanning validation in elite crop backgrounds.

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

iPB-REG is established as a practical strategy for producing uniform genome-edited fruit trees and provide a valuable platform for DNA-free genetic improvement and functional genomics in clonally propagated perennial crops.

Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement

An inducer-independent, single-plasmid CRISPR-Cas9 system for genome editing in Bacillus species

Advances in molecular biology tools are essential for streamlining and accelerating genetic engineering of cells across industrial and academic applications. While CRISPR-Cas improves genome editing efficiency, current systems have limitations and are often host specific, which restricts their versatility. This study describes a versatile CRISPR-Cas9 system for genome editing in industrially relevant Bacillus species. By adapting the well-established pJOE8999 vector-based CRISPR-Cas9 genome editing system, we constructed an inducer-independent, broad-host-range genome editing system. It maintains the benefits of low toxicity to the target cell and the cloning host as well as the ease to use of a single-plasmid CRISPR-Cas9 system. We utilized the constitutive Sigma70-type promoter from the conserved veg gene of Bacillus, to develop and test the suitability of promoter variants of different strengths for Cas9 expression. Successful gene deletions in three different Bacillus species demonstrated the versatility of the modified system for this industrially important genus. This was further confirmed by the integration of a reporter gene fusion and the introduction of a single point mutation in the genome of Bacillus licheniformis. This one-step CRISPR-based transformation protocol developed in this study enables fast genome editing workflows with minimal hands-on time. • Editing and screening of promoter variants for balanced Cas9 expression in Bacillus. • Development of a versatile inducer-independent, single-plasmid CRISPR-Cas-based system. • Verification of the modified CRISPR-based system for genome editing in different Bacilli.