A novel plant genome editing system is developed by delivering Cas9, Cas12f, and Cas12j nucleases along with their guide RNAs using a broad-host-range geminivirus, Wheat dwarf India virus (WDIV), in combination with Ageratum yellow leaf curl betasatellite (AYLCB).
Abstract
The use of viral vectors offers a promising alternative to traditional transformation methods for creating gene-edited plants. In this study, we developed a novel plant genome editing system by delivering Cas9, Cas12f, and Cas12j nucleases along with their guide RNAs using a broad-host-range geminivirus, Wheat dwarf India virus (WDIV), in combination with Ageratum yellow leaf curl betasatellite (AYLCB). Cas9, Cas12f, and Cas12j nucleases were efficiently expressed along with corresponding guide RNAs under viral promoters. By leveraging tRNA spacers in place of external promoters and terminators, we significantly reduced the overall cargo size, streamlining vector design. Additionally, we compared the traditional AtU6-driven gRNA delivery with a novel spacer:gRNA:spacer format in Cas9-expressing lines and observed comparable editing efficiencies. The broad host range of WDIV and AYLCB, combined with the novel genome-editing platform, opens possibilities for editing across a wide range of plant species.
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.
C. Nishitani, Nozomi Tsujino, Misa Kuroki et al.· bioRxiv· 0 citations
A novel highly efficient CRISPR/Cas9-based dual-sgRNA expression editing system for F. oxysporum suitable not only for single-gene editing but also for large-fragment deletion and multiplex gene editing, although the editing efficiency is somewhat lower.
Wangjie Zhu, Jiao Liao, Yuanyuan Liu et al.· ACS Synthetic Biology· 0 citations
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.
Ravindra B. Malabadi, Raju K. Chalannavar· World Journal of Advanced Re...· 0 citations
