Dongxin Huai, Xiaomeng Xue, Jie Wu, Manish K. Pandey, Nian Liu, Li Huang, Liying Yan, Yuning Chen, Xin Wang, Qianqian Wang, Yanping Kang, Zhihui Wang, Huifang Jiang

Plant Biotechnology Journal; 30 June 2024; https://doi.org/10.1111/pbi.14423

Peanut (Arachis hypogaea L.) is a globally staple oilseed crop, extensively cultivated in tropical and subtropical regions. Due to its substantial oil (approximately 46%–58%) and protein (around 22%–32%) content, the peanut plays a pivotal role in addressing malnutrition and ensuring food security in many regions. The fatty acid profiles of vegetable oil and foods have recently garnered increased attention due to the potential impact on human health. Very long chain fatty acids (VLCFAs) are defined as fatty acids with a carbon chain length exceeding 18 atoms (Guyomarc'h et al., 2021). Peanut kernels contain various VLCFAs, such as arachidic acid (C20:0), eicosenoic acid (C20:1), behenic acid (C22:0) and lignoceric acid (C24:0), but most of them are saturated fatty acids (SFAs). It is well understood that high levels of very long chain saturated fatty acid (VLCSFA) are associated with prevalence of atherosclerosis and cardiovascular disease (Bloise et al., 2022). Therefore, reducing the VLCFA content in peanuts has gained more importance realizing its positive impact for improving the nutritional quality and health value.

The biosynthesis of VLCFAs in plants is known to be regulated by a key enzyme, β-ketoacyl-CoA synthase (KCS) (Wang et al., 2017). In our previous study, a total of 30 AhKCS genes were identified in peanut genomes. After gene expression profiling and functional analysis, a pair of homologous gene AhKCS1 and AhKCS28 were identified as putative regulators of VLCFA contents in peanut kernels. The VLCFA content in available peanut germplasm accessions ranges from 4.3% to 9.8%, but no sequence variation was observed within or surrounding the AhKCS1 and AhKCS28 genes, suggesting the only possibility of further reduction of VLCFA content through gene editing (Huai et al., 2020). Therefore, in this study, AhKCS1 and AhKCS28 were genetically disrupted using the CRISPR/Cas9 system to generate novel peanut mutants exhibiting significantly reduced levels of VLCFA content in kernels.

A CRISPR/Cas9 construct was designed to incorporate two single-guide RNAs (sgRNAs) that specifically target the homologous exon regions of AhKCS1 and AhKCS28 genes (Figure 1a,b). Firstly, this construct was introduced into normal oleate peanut cultivar Zhonghua 12 (ZH12) through Agrobacterium tumefaciens-mediated transformation (Huai et al., 2023). A total of 66 independent positive T₀ transgenic ZH12 plants were successfully obtained. Among them, 61 exhibited mutations in both target genes, while two showed mutations in only one gene (Table S1). Three homozygous T₁ lines (A-2, A-3 and A-9) with mutations at both target sites for sgRNA1 and sgRNA2 in AhKCS1 and AhKCS28 genes, which caused translational frameshifts and premature stop codons, were selected for further study (Figures 1b and S1). None of the AhKCS1/AhKCS28 double mutants exhibited any growth anomalies, and no apparent alteration in morphological and yield-related traits under both greenhouse and field conditions. Furthermore, resequencing of the three double mutants revealed no evidence of off-target mutations (Table S2).

In summary, we demonstrated that AhKCS1 and AhKCS28 genes with no natural variation are the key genes for controlling the seed VLCFA content in peanut, and developed novel germplasm lines with low seed VLCFA content using genome-editing system. Furthermore, we also provided an efficient CRISPR/Cas9 genome editing platform for peanut, with great potential for expediting breeding programmes aimed at improving traits such as yield, quality and stress resistance.

See https://onlinelibrary.wiley.com/doi/10.1111/pbi.14423