An alternative pathway to plant cold tolerance in the absence of vacuolar invertase activity.

To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. In potato (Solanum tuberosum) tubers, which are swollen stems, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Désirée' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4°C, sugar analysis indicated that the knockout lines showed low levels of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H2 O2 levels. Furthermore, whole plants of vinv lines exposed to cold stress without irrigation showed normal vigor, in contrast to WT plants, which wilted. Transcriptome analysis of vinv lines revealed upregulation of an osmoprotectant pathway and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugar measurements showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold tolerance response.

Amylose starch with no detectable branching developed through DNA-free CRISPR-Cas9 mediated mutagenesis of two starch branching enzymes in potato.

DNA-free genome editing was used to induce mutations in one or two branching enzyme genes (Sbe) in tetraploid potato to develop starch with an increased amylose ratio and elongated amylopectin chains. By using ribonucleoprotein (RNP) transfection of potato protoplasts, a mutation frequency up to 72% was achieved. The large variation of mutations was grouped as follows: Group 1 lines with all alleles of Sbe1 mutated, Group 2 lines with all alleles of Sbe1 as well as two to three alleles of Sbe2 mutated and Group 3 lines having all alleles of both genes mutated. Starch from lines in Group 3 was found to be essentially free of amylopectin with no detectable branching and a chain length (CL) distribution where not only the major amylopectin fraction but also the shortest amylose chains were lost. Surprisingly, the starch still formed granules in a low-ordered crystalline structure. Starch from lines of Group 2 had an increased CL with a higher proportion of intermediate-sized chains, an altered granule phenotype but a crystalline structure in the granules similar to wild-type starch. Minor changes in CL could also be detected for the Group 1 starches when studied at a higher resolution.

Protoplast-Based Method for Genome Editing in Tetraploid Potato.

The cultivated potato is tetraploid with four probably equivalent loci for each gene. A potato variety is furthermore commonly genetically heterogeneous and selected based on a beneficial genetic context which is maintained by clonal propagation. When introducing genetic changes by genome editing it is then desirable to achieve edits in all four loci for a certain gene target. This is in order to avoid crosses to achieve homozygosity for edited gene loci and at the same time reduce risk of inbreeding depression. In such a context transient transfection of protoplasts for the introduction of mutations, avoiding stable insertion of foreign DNA, would be very attractive. The protocol of this chapter has been shown to be applicable for the introduction of mutations by DNA vectors containing expression cassettes of TALEN, Cas9, and Cas9 deaminase fusions together with sgRNA expression cassettes on either single or separate vectors. Furthermore, the protoplast-based system has been shown to work very efficiently for mutations introduced by in vitro-produced and transfected RNP (ribonucleoprotein) complexes.

Reduced Enzymatic Browning in Potato Tubers by Specific Editing of a Polyphenol Oxidase Gene via Ribonucleoprotein Complexes Delivery of the CRISPR/Cas9 System.

Polyphenol Oxidases (PPOs) catalyze the conversion of phenolic substrates to quinones, leading to the formation of dark-colored precipitates in fruits and vegetables. This process, known as enzymatic browning, is the cause of undesirable changes in organoleptic properties and the loss of nutritional quality in plant-derived products. In potato (Solanum tubersoum L.), PPOs are encoded by a multi-gene family with different expression patterns. Here, we have studied the application of the CRISPR/Cas9 system to induce mutations in the StPPO2 gene in the tetraploid cultivar Desiree. We hypothesized that the specific editing of this target gene would result in a lower PPO activity in the tuber with the consequent reduction of the enzymatic browning. Ribonucleoprotein complexes (RNPs), formed by two sgRNAs and Cas9 nuclease, were transfected to potato protoplasts. Up to 68% of regenerated plants contained mutations in at least one allele of the target gene, while 24% of edited lines carried mutations in all four alleles. No off-target mutations were identified in other analyzed StPPO genes. Mutations induced in the four alleles of StPPO2 gene, led to lines with a reduction of up to 69% in tuber PPO activity and a reduction of 73% in enzymatic browning, compared to the control. Our results demonstrate that the CRISPR/Cas9 system can be applied to develop potato varieties with reduced enzymatic browning in tubers, by the specific editing of a single member of the StPPO gene family.

Inhibition of plastid PPase and NTT leads to major changes in starch and tuber formation in potato.

The importance of a plastidial soluble inorganic pyrophosphatase (psPPase) and an ATP/ADP translocator (NTT) for starch composition and tuber formation in potato (Solanum tuberosum) was evaluated by individual and simultaneous down-regulation of the corresponding endogenous genes. Starch and amylose content of the transgenic lines were considerably lower, and granule size substantially smaller, with down-regulation of StpsPPase generating the most pronounced effects. Single-gene down-regulation of either StpsPPase or StNTT resulted in increased tuber numbers per plant and higher fresh weight yield. In contrast, when both genes were inhibited simultaneously, some lines developed only a few, small and distorted tubers. Analysis of metabolites revealed altered amounts of sugar intermediates, and a substantial increase in ADP-glucose content of the StpsPPase lines. Increased amounts of intermediates of vitamin C biosynthesis were also observed. This study suggests that hydrolysis of pyrophosphate (PPi) by action of a psPPase is vital for functional starch accumulation in potato tubers and that no additional mechanism for consuming, hydrolysing, or exporting PPi exists in the studied tissue. Additionally, it demonstrates that functional PPi hydrolysis in combination with efficient ATP import is essential for tuber formation and development.

Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery.

Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein-9 (CRISPR-Cas9) can be used as an efficient tool for genome editing in potato (Solanum tuberosum). From both a scientific and a regulatory perspective, it is beneficial if integration of DNA in the potato genome is avoided. We have implemented a DNA-free genome editing method, using delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to potato protoplasts, by targeting the gene encoding a granule bound starch synthase (GBSS, EC 2.4.1.242). The RNP method was directly implemented using previously developed protoplast isolation, transfection and regeneration protocols without further adjustments. Cas9 protein was preassembled with RNA produced either synthetically or by in vitro transcription. RNP with synthetically produced RNA (cr-RNP) induced mutations, i.e. indels, at a frequency of up to 9%, with all mutated lines being transgene-free. A mutagenesis frequency of 25% of all regenerated shoots was found when using RNP with in vitro transcriptionally produced RNA (IVT-RNP). However, more than 80% of the shoots with confirmed mutations had unintended inserts in the cut site, which was in the same range as when using DNA delivery. The inserts originated both from DNA template remnants from the in vitro transcription, and from chromosomal potato DNA. In 2-3% of the regenerated shoots from the RNP-experiments, mutations were induced in all four alleles resulting in a complete knockout of the GBSS enzyme function.

Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts.

KEY MESSAGE: Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts. Site-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines. Three different regions of the gene encoding granule-bound starch synthase (GBSS) were targeted under different experimental setups, resulting in mutations in at least one allele in 2-12 % of regenerated shoots, with multiple alleles mutated in up to 67 % of confirmed mutated lines. Most mutations resulted in small indels of 1-10 bp, but also vector DNA inserts of 34-236 bp were found in 10 % of analysed lines. No mutations were found in an allele diverging one bp from a used guide sequence, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential. To meet the challenge of screening large numbers of lines, a PCR-based high-resolution fragment analysis method (HRFA) was used, enabling identification of multiple mutated alleles with a resolution limit of 1 bp. Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. One remaining wild-type (WT) allele was shown sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.