Establishment of a CRISPR/Cas9-mediated system for targeted editing of the MFS gene in mint

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

A Novel, Simple, and Highly Efficient Dual-sgRNA CRISPR/Cas9-Based Editing System in the Filamentous Fungus Fusarium oxysporum

Fusarium oxysporum, as one of the most common filamentous fungi, possesses great biosynthetic potential for natural products. However, the lack of efficient genetic tools has hindered functional genome mining and metabolic engineering in this fungus. In this study, a novel highly efficient CRISPR/Cas9-based dual-sgRNA expression editing system for F. oxysporum was successfully developed through construction of a robust plasmid platform pFRCas9-G418 using incorporation of an endogenous histone H2B nuclear localization signal and a 5S rRNA promoter-driven polycistronic tRNA−sgRNA cassette. This system is suitable not only for single-gene editing but also for large-fragment deletion and multiplex gene editing, although the editing efficiency is somewhat lower. First, this new CRISPR/Cas9 system exhibited a high efficiency of 93.75% ± 6.25% for deletion of the Fusarium cyclin C1 (fcc1) gene (∼1 kb), which was usually selected as the target gene responsible for yellow pigment accumulation. Then, knockout of the core NRPS gene sanB (∼19 kb) and knock-in of the strong promoter gpdA in the N-methylsansalvamide (SA) biosynthetic gene cluster (BGC) in strain F. oxysporum R1 using this system, respectively, led to no SA yield and an increase of 26.4% SA titer, confirming its capacity for large gene deletion and gene knock-in. Furthermore, one-step dual-gene knockout of hat1 (histone acetyltransferase gene, ∼1.5 kb) and pacC (pH-responsive transcription factor, ∼2 kb) was first achieved in Fusarium species. This versatile platform provides a powerful tool for editing gene(s) of various sizes in F. oxysporum.

Establishment of an Efficient CRISPR-Cas9-Mediated Gene Disruption System in the Lichen-Forming Fungus Umbilicaria muhlenbergii

Compared with conventional homologous recombination, the CRISPR-Cas9 system substantially improved gene disruption efficiency, thereby overcoming a major limitation in the genetic manipulation of lichen-forming fungi.

Application of Genome editing-CRISPR/Cas9 for the Crop Improvement

The application of genome editing, CRISPR/Cas9 has revolutionized plant breeding by enabling precise, efficient, and targeted modification of native genes, significantly accelerating the development of improved agronomic traits of crops. Therefore, CRISPR/Cas9 technology currently the most extensively used genome editing technique worldwide because of its simple design, cost-effectiveness, high efficiency, good reproducibility, high engineering feasibility, ability to create gene knockout, RNA editing, and quick cycle. It is used to knock in or knock out genes of interest and for generating models for genetic studies. The main components of the CRISPR/Cas9 system are an RNA-guided Cas9 endonuclease and a single-guide RNA (sgRNA). The workflow of CRISPR/Cas9 gene editing comprises selecting target sites, designing and synthesizing sgRNA, introducing transformation constructs or ribonucleoprotein (RNP) in plant cells, followed by transformation and identification of edited lines. This approach bypasses the formal regulations on GMOs, thus encouraging the widespread adoption RNA-guided gene editing in agricultural sciences and biotechnology. The system is now being utilized in the biofortification of cereal crops such as rice, wheat, barley, and maize, including vegetable crops such as potato and tomato. The world's first genome-edited rice varieties are DRR Dhan 100 (Kamala) and Pusa DST Rice 1 developed by the Indian Council of Agricultural Research (ICAR), New Delhi, India in 2025 with the objective of bringing about revolutionary changes in terms of higher production, climate adaptability, and water conservation. The CRISPR/Cas9-based crop genome editing has been utilized in imparting/producing qualitative enhancement in aroma, shelf life, sweetness, and quantitative improvement in starch, protein, gamma-aminobutyric acid (GABA), oleic acid, anthocyanin, phytic acid, gluten, and steroidal glycoalkaloid contents. Some varieties have even been modified to become disease and stress-resistant. Therefore, CRISPR/Cas9 is aiding in developing climate-ready crops and improving crop quality parameters such as appearance, palatability, nutritional components, and other preferred traits. Gene editing tools are used to generate changes to the native genetic material. Unlike GMOs, which introduce novel configurations of genetic materials typically derived from other organisms, gene editing methods modify existing genetic material in ways that can yield beneficial outcomes.

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.

Targeted Gene Expression Modulation Using CRISPR/dCas9 to Investigate Pathogenic Outcomes in Tomato.