Rapeseed (Brassica napus L.) is one of the most important oil crops worldwide. In our previous work, we generated a high-throughput CRISPR library whereby a knockout collection was established for rapeseed breeding and functional genomics. However, the collection remains small and several promising candidate genes still await functional validation. Here, we report an update of this collection by constructing a small-scale CRISPR mutant library based on the elite commercial cultivar Zhongshuang 11 (ZS11). We first generated 326 independent T₀ lines using an optimized protocol for ZS11 transformation and regeneration with a high positive rate of 94.2%. Analysis of the editing outcomes revealed a mutagenesis frequency of 68.4%. We then phenotyped this new collection and unraveled possible key genes underlying the variations in seed oil content (SOC) and plant height. Finally, we functionally validated BnFAB1B and BnEDA32, two candidate genes identified from our knockout collection. The results confirmed that loss of function of BnFAB1B significantly increases SOC, indicating its great agronomic potential, whereas knockout of the nuclear-localized BnEDA32 severely disrupts seed oil accumulation. This study provides a valuable knockout collection of the elite cultivar ZS11 and new genes for creating superior rapeseed germplasm.

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