Development of novel transgene‐free high‐oleic peanuts through <scp>CRISPR</scp>‐Cas9‐mediated gene editing of two <i>AhFAD2</i> homologues

Cultivated peanut (Arachis hypogaea L.), an allotetraploid crop (2n = 4X = 40, AABB), is a vital oilseed crop cultivated across temperate and tropical zones in over 100 countries (Norden et al., 1987). Commodity peanut seeds typically comprise 45%–56% oil, with two major fatty acids, oleic acid (C18:1) at approximately 42% and linoleic acid (C18:2) at about 37%. Oleic acid is notably more oxidatively stable than linoleic acid, offering an extended shelf life (Martín et al., 2018), preventing the development of off-flavours (Barkley et al., 2013) and reducing the formation of nutritionally undesirable trans fatty acids (Chu et al., 2007). Fatty acid desaturase 2 (FAD2) catalyses the conversion of oleic acid to linoleic acid by introducing a second double bond to oleic acid within the endoplasmic reticulum (ER) (Okuley et al., 1994). Genetic studies have shown that the simultaneous loss of function in two homeologous genes, AhFAD2A and AhFAD2B, located on chromosome 9 of the A subgenome and chromosome 19 of the B subgenome, respectively, in A. hypogaea, results in a high-oleic acid trait in peanut, characterized by approximately 80% oleic acid and just 2% linoleic acid in peanut oil. China is the world's largest peanut producer and consumer of peanuts, with peanut production ranking first among oilseed crops in the country. Despite the dominance of the upright peanut varieties in China, no high-oleic mutant or germplasm has been identified originally in the country to date. In this study, we leveraged CRISPR-Cas9 technology to knockout AhFAD2A and AhFAD2B genes, resulting in the generation of transgene-free high-oleic peanut plants. In our experiment, the expression of Cas9 was driven by the 2× CaMV35S promoter, while the Arabidopsis AtU6 promoter directed sgRNA expression (Figure 1a). The sgRNAs targeted regions adjacent to and downstream of the start ATG codon of AhFAD2A and AhFAD2B (Figure 1b). The CRISPR construct was introduced into the elite peanut cultivars Yuhua9326 via microprojectile bombardment of embryogenic tissue cultures. Following three consecutive cultures of 3 weeks each on MS medium containing hygromycin (Hyg), 13 independent T0 Hyg-resistant embryogenic tissue lines were obtained. DNA from the Hyg-resistant calli was extracted and amplified using gene-specific primers (Table S1) targeting AhFAD2A, AhFAD2B and a pseudogene. Sanger sequencing of the amplicons revealed that 11 out of 13 lines (84.6%) exhibited mutations in two or all targeted sequences (Figure 1c). Within the editing window, seven out of 39 target sites displayed a single peak on the chromatogram, indicating homozygous mutations, while 25 displayed overlapping peaks, signifying heterozygous mutations with two distinct alleles. Seven target sites retained a single peak corresponding to the unedited wild-type (WT) allele. Notably, one line was homozygous for mutations in both AhFAD2A and AhFAD2B. Overall, the editing efficiency for AhFAD2A, AhFAD2B and the pseudogene was approximately 82.0% (32/39). Diverse mutation types were observed at on-target sites, including deletions and insertions of varying lengths. These included single-nucleotide changes (e.g. T), short sequences (e.g. TT, TC and TGA), as well as longer insertions or deletions (InDels) ranging from 61 to 597 bp (Figure S1). All observed non-multiple-of-3-bp mutations resulted in the effective knockout of the AhFAD2A/B genes, demonstrating the precision and versatility of the CRISPR-Cas9 system in peanut gene editing. Out of the 11 gene-edited T0 lines, eight successfully produced seeds in varying quantities following regeneration and grafting. While the T0 plants carried heterozygous mutations at the targeted sites, their T1, T2 and T3 progeny exhibited both homozygous and heterozygous mutations. This observation suggests that the heteroduplexes resulted from the hybridization of distinct CRISPR-induced mutations. In the edited plants analysed, the T2 progeny derived from different T1 plants retained consistent mutation profiles. For instance, the mutation types observed in line #13-1 remained stable across its T1 and T2 offsprings (Figure 1d). This stability demonstrates that edited alleles introduced in the T0 plants can be reliably transmitted to subsequent generations, highlighting the heritability and precision of the CRISPR-Cas9 gene-editing system in peanuts. A total of 15 T3 plants, derived from six CRISPR-Cas9-edited AhFAD2A/B mutant lines (lines #1-3, #2-1, #3-1, #10-1, #13-1 and #20-1), were analysed using MGI2000 whole-genome sequencing at a 20× sequencing depth (Michno et al., 2020). To evaluate potential off-target effects of CRISPR-Cas9 in peanut plants, single nucleotide polymorphisms (SNPs) and InDels were detected in the edited plants and the WT Yuhua9326 plant using the reference genome of cultivar Yuanza9102 (reference genome link: https://doi.org/10.6084/m9.figshare.26551309.v1). No significant difference in the number of variations was observed between the edited lines and Yuhua9326. However, considerable genetic variation was noted between Yuhua9326 and reference genome sequences (Table S2). Across the 15 edited plants, 427–5836 unique SNPs and 529–3125 InDels were identified when compared to Yuhua9326 and the reference genome. Importantly, most variations lacked protospacer-adjacent motifs (PAMs), suggesting that these genetic changes arose from somaclonal variation or inherent variation from maternal plants, rather than off-target effects of CRISPR-Cas9 editing (Table S2). To comprehensively evaluate off-target mutations, the sgRNA and their PAM sequences were aligned with the reference genome using CRISPR-P and Cas-OFFinder software (Li et al., 2019). With ≤5 mismatches in the sgRNA and PAM sequences, 2128 (PAM: NGG), 1315 (PAM: NAG) and 1785 (PAM: NGA) potential off-target sites were predicted. Whole-genome sequencing (WGS) detected a very low off-target mutation frequency, identifying only 18 InDels (PAM: NGG) across different CRISPR/Cas9 transgenic lines, none of which were located within the 10 most likely off-target sites (Table S3). This indicates that no off-target mutations were present in the sequenced edited lines, effectively ruling out off-target effects as a concern. Among the 15 T3 offspring, six offspring derived from lines #1-3, #13-1 and #20-1 were confirmed to have lost the transgenes, as verified by next-generation sequencing (Table S4). Additionally, four T4 offspring derived from the non-transgenic T3 plants of lines #1-3, #13-1 and #20-1 were further analysed using WGS, and no transgenic elements were detected. No morphological abnormalities were observed in the vegetative or reproductive growth of FAD2A/B knockout lines compared to the WT (Figure 1e). The fatty acid composition, oil content and protein content of seeds harvested from homozygous T3 plants with AhFAD2A/B double mutations were analysed using Nile red staining, gas chromatography, the Kjeldahl method and Soxhlet extraction. The results revealed a substantial increase in oleic acid content, rising from 36.92% in the WT to 83.75%, 81.85% and 80.59% in the edited lines, accompanied by a drastic reduction in linoleic acid content from 39.75% to 2.84%, 3.84% and 3.23%. Additionally, palmitic acid content was significantly reduced from 13.69% in the WT to 6.77%, 6.79% and 7.27% in the edited lines (Figure 1g). Notably, no significant differences were observed in protein or oil content between the knockout edited lines and the WT (Figure 1f,h). In conclusion, we successfully generated high-oleic peanut lines in the elite commercial cultivar Yuhua9326 by specifically disrupting the AhFAD2A/B genes. Moreover, we demonstrated that CRISPR-Cas9 is a promising and efficient tool for precise and rapid improvement of elite peanut cultivars without negatively affecting growth or yield. This study was supported by grants from the National Key Research and Development Program of China (2022YFD1200400), the Key Project of Science and Technology of Henan Province (201300111000), the Earmarked Fund (CARS-13) and the Henan Province Agriculture Research System (S2012-5). The authors declare no conflict of interest. L.S. and X.Z. conceived the study; L.S., H.Z., H.L., X.W., X.L., P.Q., L.X., B.H., F.Q., X.D., J.X. and W.D. performed the experiments. L.S. wrote the manuscript and X.Z. revised the manuscript. All authors read and approved the final manuscript. The raw sequencing data of FAD2 gene-edited plants generated in this study are available at Genome Sequence Archive (GSA, https://ngdc.cncb.ac.cn/gsa/) of the National Genomics Data Center, under submission accession number subCRA039668 associated with BioProject PRJCA038148. Figure S1. Table S1–S4. 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.

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