Three endogenous promoters with high expression levels in the hypocotyls and callus are identified, to replace the 35S promoter of Cas9, and enhanced sgRNA (esgRNA) enhanced CRISPR/Cas9 editing efficiency by 52.1% compared to native sgRNA.
Abstract
CRISPR/Cas9 is currently the most powerful genome editing tool for crop improvement and gene function exploration. However, the mutation efficiency of CRISPR/Cas9 in Brassica napus, a globally important allotetraploid oil crop, so far achieved remains significant potential for further improvement compared to that observed in model species. In this study, we systematically evaluated three key factors influencing editing efficiency: sgRNA promoters, sgRNA structural modifications, and Cas9-driven promoters. First, we compared all sgRNA promoters currently used in B. napus side by side. The average editing efficiency of AtU6–26 was the highest (44.74%), followed by AtU3b (36.18%), AtU6–29 (28.49%), AtU3d (17.73%) and AtU6–1 (0.53%). Second, we demonstrated that enhanced sgRNA (esgRNA) enhanced CRISPR/Cas9 editing efficiency by 52.1% compared to native sgRNA. The combination of AtU6–26 with esgRNA achieved the highest editing efficiency (average 61.4%). Third, we identified three endogenous promoters (pCAB1, pLTP2, and pTCTP) with high expression levels in the hypocotyls and callus, to replace the 35S promoter of Cas9. The CAB1 promoter was the most efficient and significantly enhanced the mutation efficiency by 31.46% over the commonly used 35S promoter. Thus, these optimizations provide valuable strategies for improving CRISPR/Cas9 efficiency in Brassica crops.
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
A substantial decrease in menthofuran content in the essential oil of the edited line #10 compared to the wild-type control is revealed, thereby demonstrating a viable strategy for improving mint essential oil quality through genome-editing.
Rice (Oryza sativa L.) is a staple food crop worldwide, and improving disease resistance is a core target in rice breeding. In this study, we employed CRISPR/Cas9 genome editing to modify the coding sequence (CDS) of two susceptibility genes, Bsr-d1 and Pi21, in the elite maintainer line Gengxiang B to enhance its blast resistance. We generated Bsr-d1/Pi21 double homozygous mutants via Agrobacterium-mediated genetic transformation. Quantitative RT-PCR revealed significantly suppressed transcript accumulation of both target genes in the edited lines compared with the wild type Gengxiang B. Upon inoculation with Magnaporthe oryzae, multiple defense-related marker genes were markedly upregulated in the double mutants. Phenotypic assays demonstrated significantly reduced disease severity for both leaf and panicle blast in the edited lines compared with the wild type. Importantly, no statistically detectable differences were found between the double mutants and wild-type plants for key agronomic or grain quality traits. Collectively, these results demonstrate that CRISPR/Cas9-mediated editing of susceptibility loci generates genetically stable blast-resistant rice germplasm without compromising agronomic traits or grain quality, providing valuable genetic resources for future rice varietal improvement.
Ke Lan, Lin Yuan, Da-Cheng Zhao et al.· Plants· 0 citations
The results demonstrate the successful deployment of CRISPR/Cas9 for targeted genome engineering in sugarbeet and establish a reliable platform for future gene-editing efforts aimed at enhancing resistance to a wide range of pathogens and diseases affecting the crop.
Z. Khan, Tinley Hathaway, C. Chu et al.· Frontiers in Genome Editing· 0 citations
The first successful application of CRISPR/Cas9 genome editing in G. boninense is reported, establishing a robust platform for functional genetic analysis and dissecting pathogenicity in G. boninense, ultimately advancing strategies to mitigate basal stem rot disease in oil palm.
Anis Farhan Fatimi Ab Wahab, Mohd Azinuddin Ahmad Mokhtar, S. Vetaryan et al.· Journal of Fungi· 0 citations
CRISPR/Cas-based genome editing has emerged as a powerful and precise tool for crop improvement, enabling targeted modification of genes associated with agriculturally important traits. In horticultural crops, CRISPR technologies have accelerated the improvement of disease resistance, abiotic stress tolerance, yield, nutritional quality, shelf life, flowering behavior, and ornamental characteristics. Among available genome-editing platforms, CRISPR/Cas9 is the most widely utilized because of its simplicity, efficiency, and versatility. The technology enables precise genome modification through targeted DNA cleavage followed by endogenous repair mechanisms, facilitating gene knockout, insertion, or sequence alteration. Recent advances in genome editing have significantly expanded its applications in vegetable, fruit, and ornamental crops. Successful modifications targeting genes associated with stress tolerance, fruit ripening, pigment biosynthesis, flowering regulation, and pathogen resistance demonstrate the enormous potential of CRISPR-mediated breeding for horticultural improvement. However, several challenges, including low transformation efficiency, genotype-dependent regeneration, prolonged juvenile phases, polyploidy, and regulatory concerns, continue to limit its broader application in many horticultural species. This review summarizes recent progress in CRISPR/Cas-mediated genome editing in horticultural crops, including strategies for guide RNA design, transformation, regeneration, development of transgene-free plants, and regulatory considerations. Furthermore, emerging advances such as precision editing technologies and improved delivery systems are discussed as promising approaches for enhancing editing efficiency and expanding future applications. Overall, CRISPR/Cas technologies hold substantial potential for accelerating the development of climate-resilient, high-quality, and nutritionally improved horticultural crops.
Prerna Srivastava, D. Singh, Rima Kumari et al.· Discover Plants· 0 citations