Plant material for genome editing and its cultivation
Huayu 23, a popular peanut normal-oleic cultivar of runner market type widely accepted by growers and food processors in China, was used in this study. As expected, Sanger sequencing of its FAD2A and FAD2B and subsequent sequence alignment revealed that this cultivar had a mutated FAD2A (448 G > A) and a wild type FAD2B. Peanut for genome editing was sown under polyethylene film mulching in an isolated region in SPRI Laixi Experimental Station on May 5, 2021. Agronomic practices were followed as routine.
Vector construction and transformation of Escherichia coli
Target site of sgRNA, 501–520 position of the coding sequence (5’-catgaacaatccaccaggga-3’) of wild type FAD2B, was selected using CRISPR-GE (http://skl.scau.edu.cn/) (Fig. 1). To facilitate ligation in genome editing vector construction, two oligos which would produce overhangs after mixing, denaturation and annealing were generated with online tool (http://biogle.cn/index/excrispr) and synthesized (Tsingke, Qingdao) (FAD2B-1F: 5’-gggttgcatgaacaatccaccaggga-3’, FAD2B-1R: 5’-aaactccctggtggattgttcatgca-3’). CRISPR/Cas9 editing vector with target site incorporated was made with BGK41-Cas9 (Biogle Biotechnology Co. Ltd, Hangzhou) following manufacturer’s instructions. Briefly, BGK41-Cas9, oligo dimers and enzyme mix (Biogle CRISPR/Cas vector construction kit) were mixed on ice bath, and then incubated at 20 ℃ for 1 h. The ligation products were transformed into competent cells of E. coli strain DH5α. Positive clones identified using colony-PCR with Cas9-F/Cas9-R primer pair (5’-tcgtgctgaccctgacactgtttga-3’, 5’- cttggcggtagccttgccgatttcc-3’) were sequenced with primer CXYW1 (5’- cccagtcacgacgttgtaaa-3’) (Tsingke, Qingdao) to confirm the inclusion of the target site in the vector. The newly constructed plasmid was named as BGK41-Cas9 recombinant vector FAD2B-1 (Fig. 1)
Transformation of Agrobacterium and node injection transformation of peanut
Genome editing construct was transformed into Agrobacterium tumefaciens strain GV3101 chemically competent cells (Veidi Biotech, Shanghai) according to the attached user’s guide. Preparation of Agrobacterium for injection was based on Pan et al. (2020) with some modifications. Positive single clones were verified by bacterial suspension PCR using Cas9-F/Cas9-R primer pair. 100 µl of freshly prepared bacterial suspension were cultured in 10 ml of YEB liquid medium at 28 ℃ with agitation (250 rpm) until OD600 reached 0.6–0.8 (about 12 h). The cultures were centrifuged at 6 000 rpm for 1 min to collect the bacterial cells. Equal volume of infection solution containing 100 µmol/L acetosyringone (BBI Lifesciences, Hongkong), 10 mmol/L MES (Sangon Biotech, Shanghai) and 10 mmol/L MgCl2·6H2O was added to the pellets. Resuspended bacterial pellets were used for injection. Node injection procedure was essentially the same as that in our previous report except for the plant age (Wang et al. 2013) (Fig. 2). Injection was done between 6:00–8:00 a.m. on July 17, 2021, and the positions injected were marked with threads (Fig. 2). Inverted U-shaped mental wires were used to facilitate the entry of pegs into the soil from higher nodes (Fig. 2).
Fatty Acid Analysis Of Resultant T0 Seeds
Pods were harvested when matured (Sept. 17, 2021). These pods were sun-dried and hand shelled. Oleic and linoleic acid contents of the individual single seeds were predicted with NIRS (Wang et al. 2014). Seeds with at least 74% oleic acid along with the untreated control Huayu 23 were further analyzed for fatty acids by gas-chromatography using cotyledonary slices follow the protocol of Yang et al. (2012).
Comparison of FAD2 sequences between the high-oleic T0 seeds and Huayu 23
FAD2A and FAD2B sequences of the high-oleic T0 seeds and Huayu 23 were amplified by PCR using primers aF19 (5’-gattactgattattgactt-3’)/R1 (5’- ctctgactatgcatcag-3’) and bF19 (5’-cagaaccattagctttg-3’)/R1, respectively (Patel et al. 2004), and DNA templates prepared from cotyledonary slices (Yu et al. 2010). PCR products were directly sequenced by Tsingke, Qingdao. Sequence comparison was done with the DNAStar Lasergene version 7.1.0.
Amplification of bar gene
PCR amplification of bar gene were performed using DNA templates prepared from Cas9 empty vector (positive control), Huayu 23 (negative control) and 2 high-oleic T0 seeds respectively, with Bar-7F and Bar-6 primer (Bar-7F: 5’-caccatcgtcaaccactaca-3’,bar-6r༚5’-acttcagcaggtgggtgta-3’). The PCR mixture (10 µ1) consisted of 6.25 µ1 of 2×Taq Plus Master Mix Ⅱ (Vazyme, Najing), 1 µ1 of DNA template, 0.5 µ1of primers (10 µM) each, and 4.25 µ1 of double distilled water. PCR thermal profile was 95 ℃ 5 min, followed by 30 cycles of 95 ℃ for 40 s, 61 ℃ for 40 s, and 72 ℃ for 30 s, and a final extension of 72 ℃ for 10 min.
Cultivation Of T1 Plants
To obtain descendants as soon as possible, one T0 seed was sown in a pot in the winter of 2021, but unfortunately it died and did not set any pods. The remaining T0 seed along with a Huayu 23 seed was sown under film mulch on May 23, 2022. Both grew to maturity and were harvested on September 20, 2022.
Fatty Acid Profiling Of The T2 Seeds Harvested From T1 Plant
Oleic and linoleic acid contents of the T2 seeds from the T1 plant both as bulk seed sample and as individual single seeds were determined by NIRS (Wang et al. 2021, Wang et al. 2014). Huayu 23 CK was also analyzed for main fatty acid content.