A small-scale CRISPR mutant library in rapeseed of commercial cultivar Zhongshuang 11

Abstract

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.

References

[1] Mohamed IAA, Shalby N, el-Badri AM, et al. Multipurpose uses of rapeseed (Brassica napus L.) crop (food, feed, industrial, medicinal, and environmental conservation uses) and improvement strategies in China . J Agric Food Res. 2025; 20: 101794.

[2] Chen K, Wang Y, Zhang R, et al. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu Rev Plant Biol. 2019; 70: 667-97.

[3] Chen L, Duan L, Sun M, et al. Current trends and insights on EMS mutagenesis application to studies on plant abiotic stress tolerance and development. Front Plant Sci. 2023; 13: 1052569.

[4] Jan S, Parween T, Siddiqi TO, et al. Effect of gamma radiation on morphological, biochemical, and physiological aspects of plants and plant products. Environ Rev. 2012; 20: 17-39.

[5] Tang S, Liu DX, Lu S, et al. Development and screening of EMS mutants with altered seed oil content or fatty acid composition in Brassica napus . Plant J. 2020; 104: 1410-22.

[6] Rao G, Huang S, Ashraf U, et al. Ultrasonic seed treatment improved cadmium (Cd) tolerance in Brassica napus L. Ecotoxicol Environ Saf. 2019; 185: 109659.

[7] Ma C, Zhu C, Zheng M, et al. CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNA-processing system . Hortic Res. 2019; 6: 20.

[8] Wan DY, Guo Y, Cheng Y, et al. CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera) . Hortic Res. 2020; 7: 116.

[9] Lu Y, Ye X, Guo R, et al. Genome-wide targeted mutagenesis in rice using the CRISPR/Cas9 system. Mol Plant. 2017; 10: 1242-5.

[10] Meng X, Yu H, Zhang Y, et al. Construction of a genome-wide mutant library in rice using CRISPR/Cas9. Mol Plant. 2017; 10: 1238-41.

[11] Bai M, Yuan J, Kuang H, et al. Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean. Plant Biotechnol J. 2020; 18: 721-31.

[12] Sun L, Alariqi M, Wang Y, et al. Construction of host plant insect-resistance mutant library by high-throughput CRISPR/Cas9 system and identification of a broad-spectrum insect resistance gene. Adv Sci. 2024; 11: 2306157.

[13] He J, Zhang K, Yan S, et al. Genome-scale targeted mutagenesis in Brassica napus using a pooled CRISPR library. Genome Res. 2023; 33: 798-809.

[14] Li H, Wu M, Chao H, et al. A rare dominant allele DYSOC1 determines seed coat color and improves seed oil content in Brassica napus . Sci Adv. 2025; 11: eads7620.

[15] Sun F, Fan G, Hu Q, et al. The high-quality genome of Brassica napus cultivar ZS11 reveals the introgression history in semi-winter morphotype. Plant J. 2017; 92: 452-68.

[16] Zhou X, Dai L, Wang P, et al. Mining favorable alleles for five agronomic traits from the elite rapeseed cultivar Zhongshuang 11 by QTL mapping and integration. Crop J. 2021; 9: 1449-59.

[17] Zhang K, Nie L, Cheng Q, et al. Effective editing for lysophosphatidic acid acyltransferase 2/5 in allotetraploid rapeseed (Brassica napus L.) using CRISPR-Cas9 system . Biotechnol Biofuels. 2019; 12: 225.

[18] Dai C, Li Y, Li L, et al. An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus . Mol Breed. 2020; 40: 96.

[19] Zhang K, He J, Liu L, et al. A convenient, rapid and efficient method for establishing transgenic lines of Brassica napus . Plant Methods. 2020; 16: 43.

[20] Nomani M, Tohidfar M . Plant regeneration and transformation of Trachyspermum ammi using Agrobacterium tumefaciens and zygotic embryos . J Genet Eng Biotechnol. 2021; 19: 68.

[21] Ma X, Zhang Q, Zhu Q, et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015; 8: 1274-84.

[22] Chen Z, Debernardi JM, Dubcovsky J, et al. Recent advances in crop transformation technologies. Nat Plants. 2022; 8: 1343-51.

[23] Bae S, Park J, Kim JS . Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014; 30: 1473-5.

[24] Leroch M, Neuhaus HE, Kirchberger S, et al. Identification of a novel adenine nucleotide transporter in the endoplasmic reticulum of Arabidopsis . Plant Cell. 2008; 20: 438-51.

[25] Shalem O, Sanjana NE, Zhang F . High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet. 2015; 16: 299-311.

[26] Zhong Z, Liu G, Tang Z, et al. Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system. Nat Commun. 2023; 14: 6102.

[27] Zhang X, Wang Z, Zhou X, et al. QTL mapping and candidate gene analysis for sclerotinia stem rot resistance in rapeseed cultivar Zhongshuang 11 by linkage, bulk segregant, and transcriptome analysis. Ind Crop Prod. 2025; 223: 120192.

[28] Song JM, Guan Z, Hu J, et al. Eight high-quality genomes reveal pan-genome architecture and ecotype differentiation of Brassica napus . Nat Plants. 2020; 6: 34-45.

[29] Li B, Yang Q, Yang L, et al. A gap-free reference genome reveals structural variations associated with flowering time in rapeseed (Brassica napus) . Hortic Res. 2023; 10: uhad171.

[30] Yu W, Wang X, Wang H, et al. Optimization and application of genome prediction model in rapeseed: flowering time, yield components, and oil content as examples. Hortic Res. 2025; 12: uhaf115.

[31] Liu Y, du Z, Lin S, et al. CRISPR/Cas9-targeted mutagenesis of BnaFAE1 genes confers low-erucic acid in Brassica napus . Front Plant Sci. 2022; 13: 848723.

[32] Hwang HH, Yu M, Lai EM . Agrobacterium-mediated plant transformation: biology and applications . Arabidopsis Book. 2017; 15: e0186.

[33] Li S, Liu Q, Zhong Z, et al. Exploration of hygromycin B biosynthesis utilizing CRISPR-Cas9-associated base editing. ACS Chem Biol. 2020; 15: 1417-23.

[34] Oung HM, Lin KC, Wu TM, et al. Hygromycin B-induced cell death is partly mediated by reactive oxygen species in rice (Oryza sativa L.) . Plant Mol Biol. 2015; 89: 577-88.

[35] Du JX, Pang SF, Xu Y, et al. Study on sensitivity of cotyledon and hypocotyl of Brassica napus L. to hygromycin. J Anhui Agric Sci. 2009; 37: 2384-5.

[36] Mueller-Roeber B, Pical C . Inositol phospholipid metabolism in Arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C. Plant Physiol. 2002; 130: 22-46.

[37] Laxalt AM, Van Hooren M, Munnik T . Plant PI-PLC signaling in stress and development. Plant Physiol. 2025; 197: kiae534.

[38] Bhandari S, Bates PD . Triacylglycerol remodeling in Physaria fendleri indicates oil accumulation is dynamic and not a metabolic endpoint. Plant Physiol. 2021; 187: 799-815.

[39] Hirano T, Matsuzawa T, Takegawa K, et al. Loss-of-function and gain-of-function mutations in FAB1A/ B impair endomembrane homeostasis, conferring pleiotropic developmental abnormalities in Arabidopsis . Plant Physiol. 2011; 155: 797-807.

[40] Pagnussat GC, Yu HJ, Ngo QA, et al. Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis . Development. 2005; 132: 603-14.

[41] Lei Y, Lu L, Liu HY, et al. CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant. 2014; 7: 1494-6.

[42] Brinkman EK, Van Steensel B . Rapid quantitative evaluation of CRISPR genome editing by TIDE and TIDER. In: Luo Y, ed. CRISPR Gene Editing.Vol. 1961. Springer New York: New York, NY, 2019; 29-44.

[43] Conant D, Hsiau T, Rossi N, et al. Inference of CRISPR edits from Sanger trace data. CRISPR J. 2022; 5: 123-30.

[44] Wittkop B, Snowdon RJ, Friedt W . New NIRS calibrations for fiber fractions reveal broad genetic variation in Brassica napus seed quality . J Agric Food Chem. 2012; 60: 2248-56.

[45] Tang S, Zhao H, Lu S, et al. Genome- and transcriptome-wide association studies provide insights into the genetic basis of natural variation of seed oil content in Brassica napus . Mol Plant. 2021; 14: 470-87.

[46] Yin Y, Raboanatahiry N, Chen K, et al. Class A lysophosphatidic acid acyltransferase 2 from Camelina sativa promotes very long-chain fatty acids accumulation in phospholipid and triacylglycerol. Plant J. 2022; 112: 1141-58.

[47] Wang J, Li M, Zhuo S, et al. Mitogen-activated protein kinase 4 is obligatory for late pollen and early fruit development in tomato. Hortic Res. 2022; 9: uhac048.

[48] Xie Z, Yu G, Lei S, et al. CCCH protein-PvCCCH69 acted as a repressor for leaf senescence through suppressing ABA-signaling pathway. Hortic Res. 2021; 8: 165.

[49] Garcia-Hernandez M, Berardini T, Chen G, et al. TAIR: a resource for integrated Arabidopsis data. Funct Integr Genomics. 2002; 2: 239-53.

[50] Yang Z, Wang S, Wei L, et al. BnIR: a multi-omics database with various tools for Brassica napus research and breeding. Mol Plant. 2023; 16: 775-89.