Method for obtaining potato with low sugars content by site-directed nucleotide substitution

Abstract

The present invention relates to a method for obtaining potato with lower sugars content by site-directed nucleotide substitution, and also relates to a method for generating site- directed nucleotide substitution and fragment substitution.

Description

METHOD FOR OBTAINING POTATO WITH LOW SUGARS CONTENT BY SITE-DIRECTED NUCLEOTIDE SUBSTITUTIONTECHNICAL FIELD

[0001] The present invention belongs to the field of biotechnological breeding, relates to a method for obtaining potato with lower sugars content by site-directed nucleotide substitution, and also relates to a method for generating site-directed nucleotide substitution and fragment substitution.BACKGROUND

[0002] During several decades, the compound Chlorprofam (CIPC) was used as an efficient anti-sprouting agent for long term storage of potato. However, the recent prohibition of CIPC in the European Union is prompting the potato processing industry to search for alternative and safer anti-sprouting approaches. In this context, storage at cold temperature (i.e., 4°C) has emerged as a valuable option for long term storage of potato without the use of CIPC. However, most commercial potato varieties processed by the industry accumulate high levels of reducing sugars during cold storage, a phenomenon called cold-induced sweetening (CIS). During high temperature processing of potatoes into products such as crisps and French fries, the reducing sugars react with some free amino acids and peptides to produce the neurotoxin acrylamide, whose presence is evidenced by a brown-to-black coloration of the processed products. Therefore, it is key to prevent CIS in potato in order to unlock the potential of long-term storage at cold temperature in the processed potato value chain.

[0003] The potato processing industries currently rely on a few varieties which have been selected for their agronomical, technological and organoleptic properties (ref 4). Given the difficulty to breed CIS-resistant potato varieties to replace the ones that are CIS-susceptible, New Genomic Techniques (NGTs) are emerging as suitable approachesto rapidly introgress the CIS-resistant trait in the commercial varieties currently used for processing into crisps and French fries.

[0004] In the present invention, the Inventors have demonstrated that a specific edition of the 5’ UTR region of 50% of the alleles of Vacuolar invertase (VInv) is sufficient to significantly alter the CIS phenotype of stored potatoes.SUMMARY OF THE INVENTION

[0005] The present invention discloses a method for obtaining a plant with low sugars content.

[0006] The method for obtaining the plants with low sugars content provided by the present invention comprises the following steps: at least inserting an adenine in the 5' UTR sequence of the gene coding for Vacuolar invertase (VInv), after position -34, preferably between positions -34 and -35, to obtain a plant with reduced activity of the vacuolar invertase at 4°C, leading to low sugars content. The DNA sequence of the 5' UTR region of the gene coding for VInv of the target plant is set forth in SEQ ID NO: 1.

[0007] The numbering referred to herein, unless indicated otherwise, refers to the unmodified DNA sequence of the 5' UTR region of the gene coding for VInv. In one embodiment, the numbering referred to herein, unless indicated otherwise, refers to the sequence as set forth in SEQ ID NO: 1.

[0008] The amino acid sequence of the 5' UTR region obtained after the insertion is set forth in SEQ ID NO: 2.

[0009] According to the method, the steps of “inserting an adenine (A) after position - 34, preferably between positions -34 and -35, of the DNA sequence of the 5 ’UTR region of the gene coding for Vacuolar invertase (VInv)” are realized by introducing the following a), b), c), d), e) or f) into a cell or tissue of the target plant, and then culturing the cell or tissue as obtained into complete plants:a) a genetic material 1 (or “first genetic material”), a genetic material 2 (or “second genetic material”) and a donor vector: the genetic material 1 is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a sequence specific nuclease 1 (or “first sequence specific nuclease”); the genetic material 2 is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a sequence specific nuclease 2 (or “second sequence specific nuclease”); b) a genetic material 12 (or “third genetic material) and a donor vector: the genetic material 12 is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing the sequence specific nuclease 1 and also expressing the sequence specific nuclease 2; c) a non-genetic material 1, a non-genetic material 2 and a donor vector: the non- genetic material 1 is an mRNA capable of expressing the sequence specific nuclease 1; the non-genetic material 2 is an mRNA capable of expressing the sequence specific nuclease 2; d) a non-genetic material 1, a non-genetic material 2 and a donor vector: the non- genetic material 1 is the protein of the sequence specific nuclease 1 expressed in vitro; the non-genetic material 2 is the protein of the sequence specific nuclease 2 expressed in vitro; e) a donor vector; f) a donor vector capable of expressing the sequence specific nuclease 1 and also capable of expressing the sequence specific nuclease 2; the donor vector is a vector carrying a mutation target sequence; the nucleotide mutation is an insertion at position - of the 5’UTR region of the gene coding for VInv.

[0010] The mutation target sequence may be a DNA fragment sequence corresponding to a sequence in the genome of the target plant from the 5' end of a target fragment 1 tothe 3 ' end of a target fragment 2, which contains the desired nucleotide mutation; and may further contains an upstream and / or downstream homologous sequence, wherein the upstream homologous sequence is a segment of sequence positioned in the upstream of the target fragment 1 in the genome of the target plant, the downstream homologous sequence is a segment of sequence positioned in the downstream of the target fragment 2 in the genome of the target plant.

[0011] The sequence specific nuclease 1 is able to specifically cleave the target fragments 1 in the genome of the target plant and the donor vector; the sequence specific nuclease 2 is able to specifically cleave the target fragments 2 in the genome of the target plant and the donor vector; when the sequence specific nuclease 1 and the sequence specific nuclease 2 cleave the target fragments 1 and the target fragments 2 in the genome of the target plant and the donor vector at the same time, the fragment containing the inserted nucleotide between two target sites of the donor vector can be inserted between the two target sites of the genome of the target plant, thus obtaining a genome sequence with a site-directed nucleotide insertion.

[0012] Hence, in particular, the method for obtaining the plants with low sugars content provided by the present invention may be characterized in that it comprises introducing the following a), b), c), d), e) or f) into a cell or tissue of the target plant, and then culturing the cell or tissue as obtained into a complete plant: a) a first genetic material, a second genetic material and a donor vector, wherein the first genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a first sequence specific nuclease, and wherein the second genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a second sequence specific nuclease; b) a third genetic material and a donor vector, wherein the third genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro, and wherein the third genetic material is capable of expressing the first sequence specific nuclease and the second sequence specific nuclease;c) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is an mRNA capable of expressing the first sequence specific nuclease, and wherein the second non-genetic material is an mRNA capable of expressing the second sequence specific nuclease; d) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is a protein of the first sequence specific nuclease expressed in vitro, and wherein the second non-genetic material is a protein of the second sequence specific nuclease expressed in vitro; e) a donor vector; or f) a donor vector capable of expressing the first sequence specific nuclease and also capable of expressing the second sequence specific nuclease; wherein the donor vector is a vector carrying a mutation target sequence comprising a DNA fragment sequence corresponding to a sequence in the genome of the target plant from the 5' end of a first target fragment to the 3' end of a second target fragment which contains the desired nucleotide mutation, wherein the nucleotide mutation is at least an insertion of an adenine (A) after position -34, preferably between position -34 and -35, of the DNA sequence of the 5’UTR region of endogenous VInv protein of a target plant, and wherein the first sequence specific nuclease is able to specifically cleave the first target fragment in the genome of the target plant and the donor vector; and wherein the second sequence specific nuclease is able to specifically cleave the second target fragment in the genome of the target plant and the donor vector.

[0013] Both the sequence specific nuclease 1 (or “first sequence specific nuclease”) and the sequence specific nuclease 2 (or “second sequence specific nuclease”) may be a CRISPR / Cas9 nuclease, a TALEN nuclease, a zinc finger nuclease or any sequence specific nuclease capable of realizing genome editing; the sequence specific nuclease 1 and the sequence specific nuclease 2 may be of the same type or may be of different type.

[0014] According to the method, the plant may be a monocotyledon or dicotyledon. In one embodiment, the plant is a dicotyledon. In one embodiment, the dicotyledon is a Solanaceae. Specifically, the Solanaceae plant may be Solanum tuberosum, i.e., potato.

[0015] In an embodiment of the present invention, specifically, the plant is potato.

[0016] In one embodiment, the genetic material 1 (or “first genetic material”) is a recombinant plasmid.

[0017] According to the method, the cell may be any cell that can be used as an introduction recipient and can be regenerated into a complete plant by tissue culture; the tissue may be any cell that can be used as an introduction recipient and can be regenerated into a complete plant by tissue culture. Specifically, for example, the cell may be a protoplast cell or suspension cell; for example, the tissue may be callus, immature embryo or mature embryo.

[0018] According to the method, the approach for introducing a), b), c), d) or e) into the cell or tissue of the target plant may be a gene gun approach, an Agrobacterium infection approach, a PEG-mediated protoplast transformation approach or any other introduction approach.

[0019] The present invention further relates to a biological material selected from the group consisting of:(1) a protein formed by at least inserting an adenine (A) after position -34, preferably between position -34 and -35, of the DNA sequence of the 5’UTR region of potato endogenous VInv protein; wherein the amino acid sequence of the 5’UTR region of potato endogenous VInv protein is set forth in SEQ ID NO: 1;(2) a coding gene of said protein; and(3) an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line containing said coding gene.

[0020] Hence, any one of the following biological materials also falls within the protection scope of the present invention:(1) a protein formed by at least inserting at least an adenine (A) after position -34, preferably between positions -34 and -35, of the DNA sequence of the 5’ UTR region ofpotato endogenous VInv protein; the amino acid sequence of the 5’ UTR region of the potato endogenous VInv protein is SEQ ID NO: 1 in the sequence listing;(2) a coding gene of said protein;(3) an expression cassette recombinant vector, recombinant bacterium or transgenic cell line containing said coding gene.

[0021] Said transgenic cell line is a non-propagating material.

[0022] The present invention also provides a method for site-directed insertion of a target nucleotide in a target gene.

[0023] Hence, the present invention also provides a method for inserting a target nucleotide in a target gene of a target organism, comprising the step of introducing one of the following a), b), c), d) or e) into a cell or tissue of the target organism: a) a first genetic material, a second genetic material and a donor vector: the first genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a first sequence specific nuclease; the second genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a second sequence specific nuclease; b) a third genetic material and a donor vector, wherein the third genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing the first sequence specific nuclease and also capable of expressing the second sequence specific nuclease; c) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is an mRNA capable of expressing the sequence specific nuclease 1; and wherein the second non-genetic material is an mRNA capable of expressing the second sequence specific nuclease; d) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is a protein of the first sequence specificnuclease expressed in vitro; and wherein the second non-genetic material is a protein of the second sequence specific nuclease expressed in vitro; e) a donor vector capable of expressing the first sequence specific nuclease and also capable of expressing the second sequence specific nuclease; the donor vector is a vector carrying a mutation target sequence; the mutation target sequence contains a DNA fragment sequence corresponding to a sequence in the genome of the target organism from the 5' end of a first target fragment to the 3' end of a second target fragment, which contains the desired nucleotide substitution; wherein the first sequence specific nuclease is able to specifically cleave the first target fragment in the genome of the target organism and the donor vector; and wherein the second sequence specific nuclease is able to specifically cleave the second target fragment in the genome of the target organism and the donor vector

[0024] The invention has the advantages that one or more upstream open reading frames (uORF) are added in the 5' untranslated region (5' UTR) of the target gene promoter by using the single base editor, so that the translation of the major open reading frame (mORF) is greatly weakened, and the target gene is inactivated.

[0025] Compared with traditional gene inactivation means such as CRISPR / Cas9 gene editing, EMS mutagenesis or T-DNA random insertion, the method has the advantages of no initiation of DNA double-strand break, high accuracy and efficiency, small influence on target gene mRNA level and the like. In addition, experimental evidence shows that the phenotypes of the gene mutant obtained by the method, such as growth and development, protein function and the like, are consistent with the published phenotype of the mutant obtained by the traditional T-DNA insertion.

[0026] Hence, the present invention further relates to a specific sgRNA for editing a 5’ UTR of potato VInv gene based on CRISPR / Cas9 characterized in that the coding sequence of the sgRNA is shown as SEQ ID NO: 3.

[0027] Hence, the present invention further relates to a CRISPR / Cas9 vector for 5 ’ UTR of potato VInv gene, characterized by containing coding gene sequence for the specific sgRNA according to the invention.

[0028] Hence, the present invention further relates to a use of the specific sgRNA according to the invention to construct potato with low sugars content.

[0029] Hence, the present invention further relates to a use of the CRISPR / Cas9 vector according to the invention in the construction of potato with low sugars content.

[0030] Hence, the present invention further relates to a method for reducing sugars content of potato by gene editing, comprising the steps of: introducing the coding gene of the specific sgRNA according to the invention and the coding gene of the Cas9 protein into starting potato to obtain potato with 5’ UTR of VInv gene mutated; compared with the starting potato, the sugars content of the transgenic potato is reduced; the cDNA sequence of the 5’ UTR of VInv gene is shown as SEQ ID NO: 1.

[0031] Hence, the present invention further relates to a method for reducing sugars content of potato by gene editing characterized in that CRISPR / Cas9 vector according to the invention is introduced into starting potato, and potato VInv gene is subjected to gene editing, so that insertion is generated, a mutant of the VInv gene is formed, and transgenic potato is obtained; compared with the starting potato, the transgenic potato has lower sugars content, and the cDNA sequence of the 5’ UTR of VInv gene is shown in SEQ ID NO: 1.

[0032] According to particular embodiments, the starting potato may be Lady Rosetta or Verdi.

[0033] According to particular embodiments, the mutant nucleotide sequence of 5’ UTR of VInv gene may be as shown as SEQ ID NO: 2.

[0034] Hence, the present invention further relates to a mutant 5’ UTR of VInv characterized in that the nucleotide sequence is shown as SEQ ID NO: 2.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a representation of the target region from Illumina sequenced Lady Rosetta (LaRo) edited lines showing In-dels and the percentages of reads corresponding to the percentage of edited alleles. Wild-type Lady Rosetta and wild-type Verdi are nontransformed CIS-Susceptible and CIS-Resistant controls respectively.

[0036] Figures 2A-2C is a set of histograms. Figure 2A shows contents of sucrose and reducing sugars from tubers of transformed non-edited, non-transformed control and edited lines after 1 month storage at 4°C. Figure 2B shows corresponding protein activity of Vacuolar Invertase (VInv) quantified from potato tubers of the edited and control lines after 1 month of storage at 4°C. Figure 2C shows tuber from edited lines shown to have contrasting levels of reducing sugars content after cold storage, processed into crisps. Figure 2D shows acrylamide content of the chips and indication of the EU benchmark level for acrylamide in chips. Three technical replicates were performed. n=3 biological replicates for quantification of sugars and VInv protein activity, al, a2, a3 and a4 indicate statistically significant differences in reducing sugars and VInv protein activity between the different lines and controls, and bl, b2, b3, b4, b5 represent statistically significant difference in sucrose content between the different lines and controls, at P<0.05 computed by Scott knot test.

[0037] Figure 3A-3E is a set of histograms. Figure 3A shows sucrose and reducing sugars content quantified from CIS-contrasting potato lines and controls after storage for 1 month at room temperature. Figure 3B shows reducing sugars quantified from CIS- contrasting lines multiplied after 3 generations (T2) and stored at 4°C for 1 month. Figure 3C shows fold change of VInv gene expression in tubers stored for 1 month at 4°C relative to tubers stored at room temperature for same duration. Figure 3D shows fold change of VInv gene expression in leaves stored for 7 days at 4°C relative to leaves stored at room temperature. Figure 3E shows mean diameter of four largest tubers from selected T1 generation lines and varieties, al, a2, a3. . . a7 indicate statistically significant differences in reducing sugars and VInv gene expression between the different lines and controls, and bl, b2 represent statistically significant difference in sucrose content between the different lines and controls, at P<0.05 computed by Scott knot test.EXAMPLES

[0038] The present invention is further illustrated by the following examples.Example 1 :Materials and Methods1. sgRNA design and efficiency assay

[0039] The nucleotide sequence 5’ -T AT ATATAAAGC AGT AGAC-3 ’ (SEQ ID NO: 3) located in the 5’ UTR region of the potato Vinv gene was selected as the sgRNA target sequence, using the CRISPR RGEN Cas-Designer Tool. The sequence was checked for off targets using the Cas-OFFinder tool and by blasting against the potato genome on phytozome. The sgRNA cassette (T7promoter-target sequence-scaffold-terminator) was transcribed as described in the EnGen® sgRNA Synthesis Kit, S. pyogenes Protocol (NEB, USA).

[0040] The efficiency of the sgRNA was assessed in an in vitro cleavage assay, as described in the protocol for in vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (NEB, USA). A 1.2 bk PCR amplicon spanning the sgRNA target sequence was used as the template in the cleavage reaction. The control reaction was performed following the same procedure without adding the S. pyogenes Cas9 nuclease.2. Editing and characterization of potato lines2.1. Generation of the transformation vector (PC2300-pcoCas9-AtU6-sgRNA)

[0041] The plant codon optimized Cas9 sequence (pco-Cas9) was obtained from addgene, in the PYPQ152 plasmid (Addgene Plasmid #69303). The CaMV-35s promoter was amplified from pCambia2300 plasmid using the primer pair ApaI-EcoRI-35s-F and NcoI-35s-R and cloned in the Apal / Ncol sites of the pYPQ152 plasmid, to generate the pYPQ152_35s plasmid. The 35s-dCas9 fragment was then digested out from thepYPQ152_35s plasmid using EcoRI restriction enzyme and cloned into the EcoRI site of pCambia2300 to form pC2300-35s-dCas9 plasmid. The sgRNA cassette was assembled in a PCR reaction, using the primer pair BspEI-AtU6-gRNA-F and BspEI-Common-R and a synthesized sgRNA cassette as template. The purified sgRNA cassette was cloned into the Pm el site of the pC2300-35s-dCas9 plasmid, producing the pC2300-35s-dCas9- AtU6-sgRNA vector.2.2. Transformation of potato

[0042] Agrobacterium harboring the PC2300-pcoCas9-AtU6-sgRNA plasmid was grown in 20 mL LB liquid medium contain 20 mg / L of rifampicin, to OD600 of 0.9. The agro-suspension was centrifuged and resuspended in lOmL of MS-liquid supplemented with acetosyringone to a final concentration of 100 pM. All steps in the transformation were performed under sterile conditions. Inter-nodal cuttings obtained from four weeks old in vitro LaRo potato plantlets were incubated with the agrobacterium harbouring the PC2300-pcoCas9-AtU6-sgRNA plasmid for 30 minutes at room temperature, after which the stem cuttings were blotted dry with sterile filter paper and placed on MS-solid medium (autoclaved 4.4 g / L MS with vitamin + 20 g / L sucrose + 3 g / L gelrite). After 2 days of co-cultivation at 28°C in the dark, excess agrobacterium on the stem cuttings were removed by washing twice with sterile distilled water and once with sterile distilled water supplemented with cefotaxime (300 mg / L). The cuttings were again blotted to dry and place on Callus Induction Medium (CIM3 : MS-solid medium supplemented with 3 mg / L BAP + 2.5 mg / L NAA + 300 mg / L Cefotaxime +100 mg / L Kanamycin). Once callus development was initiated (after about 10 days on CIM3), the cuttings were transferred to Shoot Induction Medium (SIM1 : MS-solid medium supplemented with 2 mg / L BAP + 3 mg / L GA3 + 300 mg / L Cefotaxime +100 mg / L Kanamycin) for shoot production, shoots approximately 3 cm were cut from distant points on a callus and from different calli and placed on MS-solid medium for root development. Once the roots had established, each plantlet was screened for the presence of the transgene, by PCR using the Phire Plant Direct PCR kit as described by the manufacturer (ThermoFisher Scientific, France) and primers targeting the Kanamycin resistance gene (Kan500-F / R) and the Cas9 gene (Cas9-F / R). The plantlets positive for both Kanamycin and Cas9 were grown andmultiplied in vitro in MS-solid medium to generate at least four plantlets per line. Two plantlets per line were subsequently planted in soil to generate mini tubers (TO tubers). The mini-tubers were stored at room temperature until dormancy was broken, then they were again planted in soil to produce tubers of caliber >30mm in diameter (T1 tubers). Tubers from selected lines were grown for a further generation to produce T2 tubers.2.3. Potato Phenotyping and yield

[0043] The potato shoots were visually observed for any differences in growth between the transgenic lines and the control varieties in all generations. The shape of the T1 generation tubers were also visually examined, and the yield per was quantified by measuring the diameter of the four biggest tubers of each line and variety.2.4. Pre-screening for in-dels

[0044] DNA was extracted from the TO tubers of all the lines generated, using the CTAB extraction method. The primer pair vinvl.2-F / R was used to PCR amplify a 1.2kb fragment spanning the target region of the gene. Restriction enzyme digestion with Accl was performed using lOOmg of each amplicon according to the manufacture’s protocol (NEB, USA). Lines showing a range of digestion profiles (non-digested, partial digestion, full digestion) were selected for further analyses.2.5. Screening for in-dels

[0045] DNA was extracted from a portion of the T1 tubers for each line using the CTAB extraction method. A 402 bp fragment was amplified from the DNA extracted from each line using the primer pair Gl-adapt-F / R (containing the illumina adapter sequences at the 5’end of each primer) and Q5 polymerase (Bioke, Belgium). Each amplicon was sequenced using the 2*300 bp paired end MiSeq system (GIGA platform, University of Liege, Belgium), generating approximately 300,000 reads per sample. The sequencing data was analysed using the CRISPresso2 software, to identify in-dels in the different potato lines3. Sugar measurements

[0046] D- Sucrose and total reducing sugars (D-Glucose and D-Fructose) were assayed from 200 mg of each freeze-dried, ground tuber, making use of the K-SUFRG kit (Megazyme, UK), as described in the assay procedure (Megazyme, K-SUFRG 04 / 17) with some modifications: 1 mL of distilled water was added to 200 mg of each freeze- dried powder sample in a 2 mL tube and vortexed until a homogenous suspension was obtained. The suspension was centrifuged at 15000 rpm for 10 minutes at 4°C, and the supernatant was harvested for sugar quantification. D-sucrose, D-Glucose and D-Fructose content was quantified as described in the microplate procedure of the Megazyme K- SURFG assay procedure 04 / 17, using the Microplate 96 wells F-Bottom (Greiner bio- one, Germany). Each absorbance value was divided by the value 0.625 (a function of the diameter of the well and the total reaction volume), to adjust the path length of the microplate to 1 cm. Computation of content of the sugars in each sample was performed as described in the assay procedure.4. VInv activity assay

[0047] VINV activity was assessed using the freeze-dried, ground, 4°C-stored potato samples of the T1 generation. Each sample was composed of 300 mg of pooled 5 biological replicates per line (60 mg per biological replicate). Four pooled replicates were per line were assessed. Total protein extraction, desalting and enzyme assay was performed as previously described (Bhaskar et al., Plant physiology 154, 939-948, 2010). The glucose content of samples and controls was quantified using the K-SUFRG kit, as described in the assay procedure (Megazyme, K-SUFRG 04 / 17), and the amount of glucose produced was determined by calculating the difference in glucose content between samples and controls. Total protein content for each sample was quantified using the Bradford method and vacuolar invertase activity was calculated as glucose concentration produced per hour per microgram of total protein.5. Acrylamide content assay

[0048] A portion of the cold stored potato tubers from each line were processed into chips by frying in oil at 280°C for 3 minutes. The chips obtained from each line were later sealed in a plastic bag and manually ground into fine powder using a rolling pin.Acrylamide was extracted from the samples and quantified by GC-MS on a Trace GC / Trace DSQ instrument (Thermo Scientific, Dreieich, Germany) in chemical ionization mode exactly as described previously (Haase et al., Food Additives & Contaminants: Part A 29, 1230-1238, 2012). Extraction and quantification were performed in duplicates (technical replicates).6. Gene expression

[0049] Vinv gene expression was assayed from leaves and tubers by qRT-PCR. RNA extraction from tuber samples were performed as previously described (Kumar et al., Journal of Agricultural and Food Chemistry 55, 1674-1678, 2007), with some modifications: Approximately 100 mg of lyophilized potato powder per sample was used and the volumes of the reagents were reduced five folds. RNA was extracted from fresh leaves using a protocol further modified from the protocol described for RNA extraction from tuber sample. Lysis was performed using RLT buffer (Qiagen, France). After centrifugation, equal volumes of chloroform: isoamyl alcohol (24: 1) was added to the supernatant. No Tris-saturated phenol was used. The quality of the RNA from both tissues was assessed on a 1% agarose gel, after which DNAsel (Bioke, Netherlands) treatment was performed according to the manufacturer’ s instructions. cDNA was synthesized from 500 ng of each DNA-free RNA sample using GoTaq Reverse Transcription, Oligo dT kit (Promega, USA) according to the manufacturers’ instructions. The cDNA was diluted 5 folds with distilled water and used in a reaction mix composed of lx GoTaq qRT-PCR master mix (Promega, USA) and 10 nM primers for RT-qPCR. Each sample was analyzed in triplicates on a CFX96 Real-Time System (Bio-Rad, USA) using the following program: Initial denaturation at 95°C for 3 mins, then 40 cycles of 95°C for 10 sec (denaturation), 60°C for 30 sec (Annealing) and 72°C for 30 sec (elongation), followed by a plate read. 18S rRNA was used as the house keeping gene. The primer sequences for Vinv gene (StVinv-F / R) and the house keeping gene (rRNA-F / R) are listed in Table 1.7. Statistical analyses

[0050] The students t-test and the Tukey test were used to compute the statistically significant difference between lines, varieties and between storage temperatures in this work.Primer sequences

[0051] Table 1 : List of primers used in this work and their corresponding nucleotide sequencesResults

[0052] We designed a guide RNA targeting the 5’ UTR region of the VInv gene and verified its activity in an in vitro cleavage assay using an amplicon from potato DNA as template.

[0053] We next transformed potato (var. Lady Rosetta (LaRo)) with the construct PC2300-sgRNAl-Cas9-eGFP which contains the plant codon optimized Cas9 sequence and the Arabidopsis thaliana U6 promoter driving transcription of the sgRNA. LaRo is a variety used in the crisp industry and previously shown to be CIS-susceptible. The selected sgRNA was designed to target 50% of the alleles in LaRo based on sequence of the protospacer adjacent motif (PAM) of the sgRNA target sequence (NGG) (Fig. 1). Thirty-two independent potato lines were generated and the tubers were molecularly characterized by PCR-RE assay. Based on the profile of the PCR-RE assay, eleven potato lines were selected and vegetatively propagated over two generations (T1 and T2 respectively).

[0054] After storage at 4°C for 1 month, Illumina sequencing was performed on amplicons from the T1 generation of those lines. Wild-type potatoes from LaRo and Verdi varieties were used as controls in the cold storage assessment of the selected edited lines. Verdi is a reference CIS-resistant potato variety. We identified four lines carrying one adenine insertion in the two editable alleles (P4, P6, P24 and P26), one transformed line without edits in the targeted 5 ’UTR sequence (Pl), and six lines with different types and percentages of deletions in the two editable alleles (P5, P15, P21, P28, P30, P32) (Fig. 1).

[0055] Assessment of the levels of reducing sugars (glucose + fructose) in the selected 11 transgenic lines after storage at 4°C for 1 month, revealed that lines with the single A- insertion (P4, P6, P24 and P26) contained significantly lower levels of reducing sugars compared with the non-edited line (Pl) and the other lines displaying various editing profiles of the 5’UTR (P5, P15, P21, P28, P30, P32) (Fig. 2A). Noticeably, lines P6 and P26 showed significantly lower levels of reducing sugars relative to the reference CIS- resistant potato variety Verdi. There were no significant differences in the levels of reducing sugars between the wild-type LaRo potatoes, the non-edited line (Pl) and the edited lines P5, P15, P21, P28, P30, P32. In line with previous observations, the levels of sucrose in all samples inversely correlated with the levels of reducing sugars (Fig. 2A).

[0056] We assayed the levels of VInv protein activity in ten out of the eleven transgenic lines, as well as in the wild type LaRo and Verdi varieties after storage at 4°C for 1 month. We observed significantly lower VInv activities in the lines with A-insertions and in the Verdi variety compared with the wild-type LaRo variety, the non-edited line and the lines with various editing profiles (Fig. 2B). The measured VInv activities were in line with the observed trends in reducing sugars (Fig. 2A). These results indicate that a single A- insertion in 50% of the alleles at the 5’ UTR region cause a consistent and significative reduction in the levels of VInv activity.

[0057] We also assayed the levels of reducing sugars and sucrose in selected CIS- contrasting lines and varieties (Laro, Verdi, Pl, P4, P24, P26, P30 and P32) stored at room temperature for 1 month (Fig. 3A). Noticeably there was no significant difference in the levels of reducing sugars between the CIS-susceptible lines (Laro, Pl, P30 and P32) and the CIS-resistant lines (Verdi, P4, P24 and P26). The sucrose levels in the CIS- resistant edited lines were similar to the ones detected in the CIS-resistant variety Verdi, suggesting that the single A-insertion in the 5’UTR alters sugar metabolism and the accumulation of sucrose in potato tubers.

[0058] We assessed the effect of multi-generation vegetative propagation on the reducing sugars of the selected CIS-contrasting line and varieties (Laro, Verdi, Pl, P4, P24, P26, P30 and P32). The trends in reducing sugars for those lines after two generations of growth (T2 tubers) and after 1 month storage at 4°C were observed to bestable. However, among the lines with A-insertion, the reducing sugars for lines P4 and P24 showed more stability at the T2 generation compared to line P26 (Fig. 3B).

[0059] The tubers of the CIS-resistant edited lines displayed a significantly reduced induction of the VInv gene expression under cold treatment as compared to the controls (Fig. 3C) and were consistent with the trends in reducing sugars for the tubers of those lines stored at 4° C (Fig. 2A). The alteration of the VInv gene expression in the leaves of the selected CIS-resistant lines (Fig. 3D) did not follow the trends observed in the tubers, suggesting that the effect of the A-insertion in the 5’UTR could be tuber-specific.

[0060] In order to assess the potential of CIS-resistant edited lines for acrylamide production upon high temperature processing, crisps from potato tubers of the selected edited lines and their respective controls were fried in oil heated to 280°C for 3 minutes and subsequently evaluated qualitatively by observing the differences in crisp color and quantitatively by measuring their acrylamide content. The fried crisps from CIS- susceptible lines Pl, P21 and P28 and the non-transformed control LaRo variety displayed a brown-to-black color while the ones from the CIS-resistant edited lines (i.e. P4, P6 and P24) and the CIS-resistant variety Verdi consistently appeared pale yellow in color (Fig. 2C). Acrylamide content was measured by HPLC-MS and the CIS-resistant edited lines showed acrylamide levels below the benchmark value of 750ug / kg set by the EU for potato crispsl l. Conversely the fried crisps from the CIS-susceptible transgenic lines and the wild-type variety LaRo had acrylamide contents above the EU benchmark value (Fig. 2D). These results also corroborate previous reports of a linear correlation between reducing sugars content, potato crisps color and acrylamide content.

[0061] Taken together, our findings demonstrate that a single adenine insertion in the 5’UTR region of 50% of the VInv alleles from the Lady Rosetta potato variety, is sufficient to significantly alter the CIS phenotype of stored potatoes, consequently reducing the acrylamide content in the processed products to an acceptable level in the EU. VInv is known to regulate many aspects of growth and development in plants, however, no significant phenotypic differences were observed between the CIS- contrasting transgenic lines and the control variety (data not shown), nor any significantdifferences in the sizes (mean diameter) of the four largest tubers per line and variety (Fig. 3E).

[0062] Classical applications of CRISPR-Cas9 genome editing techniques for trait improvement in crop plants, have mostly relied on targeting the coding sequence for double strand breaks and introduction of frame shift via the error prone Non-Homologous End Joining (NHEJ) repair mechanism. The 5’ UTR region has been shown to play a role in the regulation of mRNA stability as it harbours the transcription initiation site in many eukaryotes. Therefore the 5 ’UTR region offers an interesting alternative to the traditional exon targeting for genome editing. VInv is known to play an important role in regulating sugar levels in potato. However, it may also play a key role in the regulation of growth and development as shown for crop species such as rice and cotton. This suggests that knockout of the VInv gene might interfere with other developmental processes. Our data indicate that, by targeting 50% of the alleles in the 5 ’UTR of VInv, we quantitatively regulate the gene expression while retaining the phenotype of the tubers produced. While trait engineering usually relies on targeting all allelic sequences of the 5 ’UTR or coding sequences, our study demonstrates that a stable CIS-resistance trait can be achieved by differential allele targeting of the 5 ’UTR. In some specific cases, differential allele targeting might also represent a more suitable approach to reduce the possible pleiotropic effects associated with targeting all four alleles as previously demonstrated for the VInv gene. The present results provide a timely potential solution to convert commercial CIS- susceptible potato varieties widely used by the European potato industry into CIS- resistant varieties for the subsequent production of safe and healthy crisps and French fries.

Claims

CLAIMS1. A method for obtaining a plant with low sugars content, which comprises inserting adenine after position -34 of the 5’UTR of the gene coding for the vacuolar invertase (VInv) of a target plant to obtain a plant with lower sugars content, wherein the 5’UTR of the gene coding for VInv of the target plant comprises the sequence of SEQ ID NO: 1.

2. The method according to claim 1, wherein the inserting steps comprise introducing the following a), b), c), d), e) or f) into a cell or tissue of the target plant, and then culturing the cell or tissue as obtained into a complete plant: a) a first genetic material, a second genetic material and a donor vector, wherein the first genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a first sequence specific nuclease, and wherein the second genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a second sequence specific nuclease; b) a third genetic material and a donor vector, wherein the third genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro, and wherein the third genetic material is capable of expressing the first sequence specific nuclease and the second sequence specific nuclease; c) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is an mRNA capable of expressing the first sequence specific nuclease, and wherein the second non-genetic material is an mRNA capable of expressing the second sequence specific nuclease; d) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is a protein of the first sequence specific nuclease expressed in vitro, and wherein the second non-genetic material is a protein of the second sequence specific nuclease expressed in vitro; e) a donor vector; orf) a donor vector capable of expressing the first sequence specific nuclease and also capable of expressing the second sequence specific nuclease; wherein the donor vector is a vector carrying a mutation target sequence comprising a DNA fragment sequence corresponding to a sequence in the genome of the target plant from the 5' end of a first target fragment to the 3' end of a second target fragment which contains the desired nucleotide mutation, wherein the nucleotide mutation is at least an insertion of an adenine (A) after position -34, preferably between position -34 and -35, of the DNA sequence of the 5’UTR region of endogenous VInv protein of a target plant, and wherein the first sequence specific nuclease is able to specifically cleave the first target fragment in the genome of the target plant and the donor vector; and wherein the second sequence specific nuclease is able to specifically cleave the second target fragment in the genome of the target plant and the donor vector.

3. The method according to claim 2, wherein the first sequence specific nuclease is a CRISPR / Cas9 nuclease, a TALEN nuclease, a zinc finger nuclease or any nuclease capable of realizing genome editing, and wherein the second sequence specific nuclease is a CRISPR / Cas9 nuclease, a TALEN nuclease, a zinc finger nuclease or any nuclease capable of realizing genome editing.

4. The method according to claim 2, wherein the plant is a monocotyledon or dicotyledon.

5. The method according to claim 2, wherein the cell is any cell that can be used as an introduction recipient and can be regenerated into a complete plant by tissue culture, and wherein the tissue is any tissue that can be used as an introduction recipient and can be regenerated into a complete plant by tissue culture, and wherein introducing step is optionally performed with a gene gun, an agrobacterium infection, or a PEG- mediated protoplast transformation.

6. A biological material selected from the group consisting of:(1) a protein formed by at least inserting an adenine (A) after position -34, preferably between position -34 and -35, of the DNA sequence of the 5’UTR region of potato endogenous VInv protein; wherein the amino acid sequence of the 5’UTR region of potato endogenous VInv protein is set forth in SEQ ID NO: 1;(2) a coding gene of said protein; and(3) an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line containing said coding gene.

7. A method for inserting a target nucleotide in a target gene of a target organism, comprising the step of introducing one of the following a), b), c), d) or e) into a cell or tissue of the target organism: a) a first genetic material, a second genetic material and a donor vector: the first genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a first sequence specific nuclease; the second genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a second sequence specific nuclease; b) a third genetic material and a donor vector, wherein the third genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing the first sequence specific nuclease and also capable of expressing the second sequence specific nuclease; c) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is an mRNA capable of expressing the sequence specific nuclease 1; and wherein the second non-genetic material is an mRNA capable of expressing the second sequence specific nuclease; d) a first non-genetic material, a second non-genetic material and a donor vector, wherein the first non-genetic material is a protein of the first sequence specific nuclease expressed in vitro; and wherein the second non-genetic material is a protein of the second sequence specific nuclease expressed in vitro; e) a donor vector capable of expressing the first sequence specific nuclease and also capable of expressing the second sequence specific nuclease;the donor vector is a vector carrying a mutation target sequence; the mutation target sequence contains a DNA fragment sequence corresponding to a sequence in the genome of the target organism from the 5' end of a first target fragment to the 3' end of a second target fragment, which contains the desired nucleotide substitution; wherein the first sequence specific nuclease is able to specifically cleave the first target fragment in the genome of the target organism and the donor vector; and wherein the second sequence specific nuclease is able to specifically cleave the second target fragment in the genome of the target organism and the donor vector.

8. The method according to claim 7, wherein the first sequence specific nuclease is a CRISPR / Cas9 nuclease, a TALEN nuclease, a zinc finger nuclease or any nuclease capable of realizing genome editing; and wherein the second sequence specific nuclease is a CRISPR / Cas9 nuclease, a TALEN nuclease, a zinc finger nuclease or any nuclease capable of realizing genome editing.

9. The method according to claim 7, wherein the cell is any cell that can be used as an introduction recipient and can be regenerated into a complete plant by tissue culture; the tissue is any cell that can be used as an introduction recipient and can be regenerated into a complete plant by tissue culture; wherein introducing step is optionally performed with a gene gun, an agrobacterium infection, or a PEG-mediated protoplast transformation.

10. A specific sgRNA for editing a 5’ UTR of potato VInv gene based on CRISPR / Cas9 characterized in that the coding sequence of the sgRNA is shown as SEQ ID NO: 3.

11. A CRISPR / Cas9 vector for 5’ UTR of potato VInv gene, characterized by containing coding gene sequence for the specific sgRNA of claim 10.

12. The use of the specific sgRNA of claim 10 to construct potato with low sugars content.

13. Use of the CRISPR / Cas9 vector according to claim 11 in the construction of potato with low sugars content.

14. A method for reducing sugars content of potato by gene editing, comprising the steps of: introducing the coding gene of the specific sgRNA of claim 10 and the coding gene of the Cas9 protein into starting potato to obtain potato with 5’ UTR of VInv gene mutated; compared with the starting potato, the sugars content of the transgenic potato is reduced; the cDNA sequence of the 5’ UTR of VInv gene is shown as SEQ ID NO: 1.

15. A method for reducing sugars content of potato by gene editing characterized in that CRISPR / Cas9 vector of claim 11 is introduced into starting potato, and potato VInv gene is subjected to gene editing, so that insertion is generated, a mutant of the VInv gene is formed, and transgenic potato is obtained; compared with the starting potato, the transgenic potato has lower sugars content, and the cDNA sequence of the 5’ UTR of VInv gene is shown in SEQ ID NO: 1.

16. The method according to claim 14 or 15, wherein the starting potato is Lady Rosetta or Verdi.

17. The method as claimed in claim 14 or 15, wherein the mutant nucleotide sequence of 5’ UTR of VInv gene is shown as SEQ ID NO: 2.

18. A mutant 5’ UTR of VInv characterized in that the nucleotide sequence is shown as SEQ ID NO: 2.

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