Rapeseed (Brassica napus L.) supplies a major share of global vegetable oil, yet breeders must improve oil accumulation, yield, plant architecture, and resilience within a complex polyploid genome. Conventional mutagenesis can create useful variation, but mutations are random and causal genes often require large populations and lengthy screening. Targeted clustered regularly interspaced short palindromic repeats-associated nuclease 9 (CRISPR-Cas9) libraries can connect a designed mutation with an observed trait, making gene discovery faster and more traceable. However, elite cultivars can be difficult to transform and regenerate, limiting their use as editing platforms. Based on these challenges, there is a need to develop cultivar-specific CRISPR resources and conduct in-depth research on their value for rapeseed trait improvement.
A research team from Xianghu Laboratory, Huazhong University of Science and Technology, New York University, and the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences published (DOI: 10.1093/hr/uhag087) the study on March 5, 2026, in Horticulture Research. The article reports a targeted knockout collection in Zhongshuang 11 (ZS11) and evaluates its ability to reveal genes linked to plant height, seed oil accumulation, and fatty acid composition in an elite rapeseed background.
The team first mapped a pooled CRISPR library to the ZS11 genome. Among 18,414 single guide RNAs (sgRNAs), 15,426 matched perfectly and together targeted 10,078 genes, including genes near quantitative trait loci (QTLs) for SOC, thousand seed weight (TSW), and silique length (SL). They then improved transformation and regeneration by fine-tuning hygromycin B (HyB) selection pressure and optimizing the rooting medium. This produced 326 independent first-generation transformed (T0) lines. In the first 139 tested plants, polymerase chain reaction (PCR) analysis showed a 94.2% positive rate, and sequencing of 114 decoded plants found a 68.4% mutagenesis frequency. The edited loci were stably inherited, and no off-target editing was detected at 10 predicted sites tested in five randomly selected lines. Field and seed analyses revealed multiple trait changes. A dwarf, compact plant was associated with knockout of BnUUAT1.C09, while one line with reduced seed oil content (SOC) carried an edited BnER-ANT1.C09 allele. Most importantly, the team validated two seed-oil genes across generations: loss of BnFAB1B increased SOC, whereas knockout of BnEDA32 reduced SOC. Complementation restored SOC to wild-type (WT) levels, and BnEDA32 overexpression raised SOC, supporting its role as a positive regulator of oil accumulation.
The authors said the library is valuable because it brings genome editing into a commercial rapeseed cultivar rather than only a laboratory-friendly background. They said BnFAB1B is particularly promising because reducing its function consistently promoted oil accumulation, suggesting a possible target for high-oil breeding. They also said BnEDA32 opens a different window into seed lipid metabolism, because it appears to support oil accumulation from the nucleus and may connect seed development with broader regulatory networks that remain to be explored through multi-omics studies.
The findings provide a usable gene-discovery platform for both functional genomics and breeding. For researchers, the ZS11 collection offers edited lines that can be linked to visible architecture, SOC, and fatty acid traits. For breeders, validated genes such as BnFAB1B and BnEDA32 offer new entry points for improving oil yield, while candidates such as BnUUAT1.C09 and BnER-ANT1.C09 can be tested in later generations. Because many sgRNAs from the pooled library also match other rapeseed genomes, the strategy could support collaborative screening across cultivars, help prioritize edits with agronomic value, and accelerate the development of superior oilseed germplasm.
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References
DOI
10.1093/hr/uhag087
Original Source URL
https://doi.org/10.1093/hr/uhag087
Funding information
This work was supported by the National Key Research and Development Program of China (2024YFD1200402, 2023YFD1201401), the National Natural Science Foundation of China (32272067), Zhejiang Provincial Science and Technology Key R&D Program (GG03208-1) and Scientific Research Start-up Funding for Xianghu Laboratory (2025C2STD00100).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.