Enhancing peanut nutritional quality by editing AhKCS genes lacking natural variation

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). The fatty acid composition of the harvested seeds from ZH12 and each double mutant was determined by gas chromatography (Figure 1c). The VLCFAs contents in the double mutants have been significantly decreased by 70.6%–100.0%. The VLCFA profiles of ZH12 showed four distinct peaks corresponding to C20:0, C20:1, C22:0 and C24:0. However, the peak of C20:1 and C24:0 was absent in all the three double mutants (Figure 1c). Although the peak of C20:0 was observed in both ZH12 and the double mutants, its content significantly decreased from 1.7% to 0.4%–0.5% in the double mutants. Similarly, while the content of C22:0 amounted to 2.8% in ZH12, it dramatically reduced to 0.3% in A-2 and was absent altogether in A-3 and A-9. Consequently, there was a substantial reduction from total VLCFA content of 6.9% observed within ZH12 down to merely 0.9%, 0.5% and 0.4% in A-2, A-3 and A-9, respectively, which were considerably lower than the value (4.3%) in naturally evolved germplasm materials (Figure 1d). The CRISPR/Cas9 construct was also introduced into a high oleate peanut breeding line JC30. In total, 63 independent positive T0 transgenic JC30 plants were generated, out of which 60 exhibited mutations in both target genes (Table S1). Similarly, three homozygous T1 lines (B-37, B-38 and B-59) harbouring truncated proteins of AhKCS1 and AhKCS28 were chosen to analyse the seed fatty acid composition (Figures 1b and S1). The double mutants of JC 30 exhibited only three peaks representing to C20:0, C20:1 and C22:0, while the peak of C24:0 was not detected (Figure 1c). The contents of C20:0 and C20:1 in double mutants of JC30 were reduced from 1.0% to 0.4%, while the C22:0 content was decreased from 1.4% to 0.2%. The VLCFA content in the double mutants of JC30 was reduced from 4.1% to 1.0%, which was slightly higher than that of double mutants of ZH12 (0.4%–0.9%). This relatively higher content can be attributed to the higher C20:1 content in the double mutants of JC30, which was absent in the double mutants of ZH12 (Figure 1d). The increase of C20:1 in double mutants of JC30 can be explained by an augment availability of substrate C18:1 in kernels. Interestingly, there was no significant difference in total VLCSFA content between the double mutants derived from JC30 and ZH12 (0.6%–0.7% vs 0.4%–0.9%). Additionally, the levels of C16:0, C18:0 and C18:2 were found to be elevated, while the content of C18:1 was observed to be slightly reduced in both double mutants derived from JC30 and ZH12 (Figure 1c,d). 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. This work was supported by the Key Research and Development Program of China (2023YFD1202800), the Knowledge Innovation Program of Wuhan-Basic Research (2022020801010291), the Project of the Development for High-quality Seed Industry of Hubei province (HBZY2023B003) and Innovation Program of the Chinese Academy of Agricultural Sciences (2023-2060299-089-031). DH, RKV, BL and YL conceived and designed the experiments; HJ and LH supplied the peanut cultivars; XX, JW, NL, LY, YC, XW, QW, YK and ZW performed the experiments; DH, XX and MKP analysed the data; DH wrote the manuscript; DH, MKP, RKV, BL and YL contributed in data interpretation and revision of the manuscript. All authors have read and approved the final version of the manuscript. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Figure S1. The Sanger sequencing chromatograms of each target site in the homozygous T1 lines. Table S1. Summary of mutations at each target site in the T0 generation. Table S2. Detection of off-target mutation in A-2, A-3 and A-9 using genome resequencing. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.