Efficiency of potato genome editing: Targeted mutation on the genes involved in starch biosynthesis using the CRISPR/dMac3-Cas9 system

Potato (Solanum tuberosum L.) has a tetraploid genome. To make a mutant lacking a specific gene function, it is necessary to introduce mutations into all four gene alleles. To achieve this goal, we developed a powerful genome editing tool, CRISPR/dMac3-Cas9, which installed the translation enhancer dMac3 that greatly increased the translation of the downstream open reading frame. The CRISPR/dMac3-Cas9 system employing three guide RNAs (gRNAs) greatly elevated the frequency of the generation rate of mutation. This system enabled to create the 4-allele mutants of granule-bound starch synthase (GBSS) and starch branching enzyme (SBE). These mutants indicated functionally defective features, suggesting that we succeeded in efficient genome editing of the potato tetraploid genome. Here, we show the effect of the number of gRNAs for efficient mutagenesis of the target gene using the mutants of the GBSS1 gene. CRISPR/dMac3-Cas9 employing three gRNA genes achieved a higher mutation efficiency than the CRISPR/dMac3-Cas9 with two gRNAs, suggesting being influenced by the dose effect of the number of gRNAs at the target region. The alleles of the SBE3 gene contained SNPs that caused sequence differences in the gRNAs but these gRNAs functioned efficiently. However, many rearrangement events and large deletions were induced. These results support the importance of accurate binding of gRNA to the target sequence, which may lead to a hint to avoid the unexpected mutation on the off-target sites.

. A. tumefaciens was cultured with shaking at 28 °C for 24 hours in 2xYT liquid medium (Bacto trypton 1.6%, Bacto Yeast extract 0.1%, NaCl 0.5%) supplemented with 50 mg/L spectinomycin (Fuji Film Wako).Cells harvested at the late-stationary phase were resuspended in 2xYT liquid medium to adjust the cell concentration to OD600 = 0.1.Then, 2 μL of 10 mg/mL acetosyringone and 1 mL of 3C5ZR medium (Sheerman et al., 1988) were added to 150 μL of the suspended A. tumefaciens.The resultant suspension was used for the potato transformation.
Stem internodes were isolated from germ-free potato plants grown in a clean environment for 3 weeks and cut into approximately 0.7-cm pieces.These pieces were infected with A. tumefaciens harboring an appropriate plasmid.After treatment with Agrobacterium infection, the stem internodes were rinsed with sterilized water, placed onto 3C5ZR plates containing 0.3% Gelrites, and cultured in a growth chamber at 23 °C under a long-day conditions with a light period of 16 hours (200 μmol m -2 s -1 ) and a dark period of 8 hours for 4-6 days.Then, they were washed with sterile water.
After this treatment, callus induction and plant regeneration were performed by culturing for 2 months on a plate of 3C5ZR medium (Sheerman et al., 1988) containing 0.3% Gelrite supplemented with 5 mg L -1 hygromycin B (Fuji Film Wako).
Regenerated shoots were transplanted onto MS plates supplemented with 5 mg/L hygromycin B to generate regenerated plants.The regenerated plants were cultured on MS plates under long-day conditions.Introduction of the transgene into the regenerated plants was confirmed by polymerase chain reaction (PCR) using the primers, 5'-GGCGTAAGAATAGAATCTGTTAT-3' and 5'-GACAGCGCTATCAGATTTCCAA-3', which amplified a part of the Cas9 gene sequence.

Determination of the properties of tuber starch
Starch granules were prepared from potato tubers according to Noda et al. (2004).Sliced sections of potato tuber were dipped into 0.4% potassium iodide-0.12%iodide solution.Photographs were taken after washing with water.The amylose content in the potato tubers was analyzed according to a previous paper (Noda et al., 2004).The data were statistically analyzed using Dunnett's multiple comparison test.
Sectioned tissues of potato tuber (150 mg) were frozen in liquid nitrogen and ground into a fine powder using an SK mill (Tokken Inc., Kashiwa, Japan).Then, 400 μL of the protein extraction buffer (50 mM Tris-HCl, pH 6.8, 8 M urea, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 1 mM dithiothreitol (DTT), 0.01% bromophenol blue) was added to the powdered tuber.The mixture was well combined and centrifuged at 20,000 x g for 15 min at room temperature.The supernatant solution was collected as a crude protein fraction.This fraction (10 µL) was used for 7.5% SDS-polyacrylamide gel electrophoresis and subjected to Western blot analysis using antiserum raised against rice BEI (Kawasaki et al., 1996).SDS-PAGE and Western blot analysis were performed according to Wakasa et al. (2020).For this analysis, a 5,000-fold dilution of the antiserum was used.Protein interaction with the antibody was detected using the anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HPR)-linked secondary antibody (1:10,000 dilution) (GE Healthcare, Chalfont Saint Giles, UK).Signals were detected with Pierce TM ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA).
model target gene to test new technology Desirée Veillet et al. 2020 Int J Mol Sci 20: 402 StGBSS used as a model target gene to test new technology Desirée Zong et al. 2018 Nat Biotechnol 36:950-953 GBSS used as a model target gene to test new technology Sayaka Kusano et al. 2016 Sci Rep 6: 30234 SBE3 apparently amylose-rich starch, more sensitive to iodine staining Sayaka Takeuchi et al. 2021 Plant Biotechnol 38: 345-353 SBE1, SBE2 amylopectin-free starch in double full-allelic mutation line Desirée Zhao et al. 2021 Sci Rep 11: 4311 SBE1, SBE2 range of starch proparties, longer chain length, fewer blanch, smaller and many granules Desirée Tuncel et al. 2019 Plant Biotechnol J 17: 2259-2271 SS6 unknown isoform of starch synthase, discovered by genome analysis Desirée Sevestre et sl.2020 Sci Rep 10: 2045 GWD1 key regulatory enzyme in starch metabolism, hypothetical Saturna Carlsen et al. 2022 Front Genome Ed 3: 795644 VInv reduced CIS, lipid oxidation and H2O2 level in tuber; reduced plant wilting in dry environment Desirée, Brooke Teper-Bamnolker et al. 2022 Plant J 113: 327-341 VInv reduced CIS by partial knockdown/silencing AGB Purple Yasmeen et al. 2022 Planta 256: 107 StvacINV1, StBAM1 postharvest-related potential target genes Yagana-INIA Acha et al. 2021 Plants 10: 1882 VInv revealing molecular system consist of VInv, cytokinin and sugar, involved in lateral bud growth Desirée Salam et al. 2021 Plant Physiol 185: 1708α-solanine biosynthesis gene; α-solanine-free hairy root Mayqueen Nakayasu et al. 2018 Plant Physiol Biochem 131: 70-.Genome editing studies of genes involved in crop trait of potato Starch composition change Reducing acrylamide-generation, cold-induced sweetwening (CIS) and bud outgrowth Secondary metabolism, reducing toxic compounds and enzymatic browning Disease resistance DMR6-1 used as a model target gene to test new technology Desirée Veillet et al. 2020 Plant Cell Rep 38: 1065-1080 DMR6-1 used as a model target gene to test new technology Desirée Veillet et al. 2020 Int J Mol Sci 20: 402 Coilin used as a model target gene to test new technology Chicago Khromov et al. 2018 Dokl Biochem Biophys 479: 90-94 Coilin potato virus Y resistance, salt and osmotic stress tolerance Chicago Makhotenko et al. 2019 Dokl Biochem Biophys 484: 88-Nucleotide sequence of the rearrangement region in the allele #105d of mutant line #105.The nucleotide sequence of the insertion in the GBSS1 gene is indicated by blue letters.This mutant allele contains a 250-nt deletion along with a 1,484-nt insertion which starts from the position -56 in the target region.The inserted sequence shows similarity to an unidentified sequence of Solanum tuberosum genome.Traits of the GBSS1 and SBE3 mutants.(A) Amylose content of starch in tubers of GBSS1 and SBE3 mutants.Error bars indicate standard deviation (n=3).Asterisks indicate significant differences in the values of transformants compared to that of wild type at P < 0.05.(B) Iodine staining of tubers of GBSS1 mutants.Bar = 2 cm.(C) Iodine staining of tubers of SBE3 mutants.Bar = 2 cm.(D) Western blot analysis of the proteins in tubers of SBE3 mutants.A rabbit antiserum against the rice BEI was used for the detection of SBE3.The arrow corresponds to SBE3.The lower panel shows the corresponding SDS-PAGE results.M: molecular weight marker.WT: wild type.#105, #115 and #156 indicate the GBSS mutant lines, and #19, #31 and #86 are the SBE3 mutant lines.Detailed procedures used in this figure is described in Supplementary Methods.