An approach to improve nutritional properties of potato varieties by genome editing

Transcription

1 An approach to improve nutritional properties of potato varieties by genome editing Sadia Iqbal, Stephen Milroy, Stephen Wiley and Michael G.K. Jones Centre for Crop and Food Innovation/Potato Research WA, School of Veterinary and Life Sciences, WA State Agricultural Biotechnology Centre (SABC), Murdoch University, Perth (ARC LP ) Potato Research WA

2 Global potato production areas

3 Background Potato (Solanum tuberosum) is the world s 4 th largest food crop Its centre of origin is the high Andes of South America It is tetraploid, with 48 chromosomes Genome sequenced Haploid potato (n=12) 860Mbp (medium sized genome) It was introduced into Europe in the 1500s China (96 million tonnes) and India (46 million tones) are now the world s largest producers, followed by Russia, Ukraine, USA, Germany 2/3rds used for human consumption, remainder for cattle food or starch production

4 Potato improvement Potato varieties are vegetatively propagated heterozygous clones An established variety is lost if crossing is undertaken How can we improve deficiencies in current varieties? Only by GM technologies or mutagenic treatments Potato is therefore a prime target for genome editing technologies Our industry partner in the Netherlands does not want any possibility of the introduction of external DNA

5 A problem with potatoes High starch content Broadly classified as having a high glycemic index (GI) The GI is a ranking given to food to describe how quickly the carbohydrate in the food is broken down and glucose is absorbed into the bloodstream. High GI means a more rapid release of glucose into the blood stream, and this in turn can contribute to developing Type 2 diabetes and obesity The GI of potatoes depends on the variety and cooking method

6 The structure of starch

7 Synthesis of amylose and amylopectin Starch branching enzymes (SBEs) generate α-1,6 branches Starch synthases (SS) generate α-1,4 glucans High amylose starches are more resistant to α amylase digestion, leading to reduced digestibility, this can be achieved by suppression of SBEIIa and SBEIIb

8 Project aims To develop genome editing methods for potato which will not introduce any external DNA into an existing cultivar To reduce the ratio of amylopectin to amylose to improve dietary quality SSII SBEII SSIII SBEI Altered amylose to amylopectin ratio Amylose : Amylopectin = 20 : 80

9 CRISPR techniques - NHEJ

10 Project workflow Identify and sequence target genes Design and synthesis of sgrnas Determine the GI of edited potato tubers Delivery of Cas9 and guide RNAs to plant cells using different strategies DNA extraction and sequencing of target genes Regeneration of genome edited plants 6

11 Design of target sgrna For a polyploidlike potato, sequences targeting alleles of the same gene simultaneously should be chosen 20 nucleotides long with PAM (protospaceradjacent motif) at the end Preferably close to the 5 end of gene PAM: NGG sequence (depends on CRISPR type) No other targets in genome (off-target effects) Various softwares available (Zifit Targeter, E-CRISP, CRISPRdirect, CCTOP etc.) Check for off-target effects

12 Alignment of Starch Synthase (SS2) gene comparison of published sequences and three new potato varieties

13 Strategies to deliver genome editing in potato Plant transformation vectors coding for Cas9 and guide RNAs Ribonucleoprotein complex (Cas9 and guiderna complex)

14 Agrobacterium mediated transformation Leaf disc regeneration protocols have been optimised for two new potato varieties Innovator and Colombaand explants have been treated with Agrobacterium with plasmids encoding Cas 9 and guide RNAs Gene Target Variety transformed SS2 T1 Innovator and Colomba T2 Innovator and Colomba SS3 T1 Innovator and Colomba T2 Innovator and Colomba SBE1 T1 Innovator and Colomba T2 Innovator and Colomba SBE2 T1 Innovator and Colomba T2 Innovator and Colomba 14

15 Isolation and regeneration of plants from protoplasts the limiting process Potato protoplast after 1 week s culture Dividing protoplasts after 2 weeks culture 15

16 Protoplast culture and plant regeneration Protocol optimised to isolate large numbers of protoplasts from leaf mesophyll tissue, their transfection mediated by PEG and regeneration to develop into callus and shoots. A B C D (A) Cv Colomba protoplast-derived calli. (B) Optimising media for callus development. (C, D) Shoot initiation from protoplast-derived calli (from 0.5g leaves) after 3.5 months culture 16

17 PEG mediated transfection of potato protoplasts with 35S-gfp confirms uptake and expression (A) (B) (B) Protoplasts 48 hours after transfection (5 mins25% PEG) (A) Bright field and (B) GFP fluorescence (D) Innovator protoplasts, no PEG treatment after one week s culture Innovator protoplasts, PEG treatment, after one week s culture 17

18 Ribonucleoprotein transfections -in-vitro assays 1 2 M 3 4 M To test the DNA-free genome editing approach, the Cas9 protein and short guide RNA were combined in vitro to target the genes of interest (Cas 9 protein was produced by ProteoWA, SABC) Guide RNA for target DNA in lanes 1 directed complete digestion of the target DNA fragment at the specified site, cflane 3 control One guide RNA did not cause target cleavage (lanes 2 and 4) No difference between commercial and in house Cas9 activity Assay can be used to screen guide RNAs for their efficiency M = 100 bpdna ladder, lane 1 = Target 1 DNA incubated with Cas9 and guide RNA. Lane 2 = Target 2 DNA incubated with Cas9 and guide RNA. Lanes 3 and 4 = Control DNA amplicon for Gene 1 and Gene 2 to compare size differences. 18

19 Other targets under study in potato Using the protoplast-to-plant systems developed for current potato varieties Reduce bruising Reduce acrylamide levels after frying Reduce cold-sweetening on storage

20 Conclusions Because breeding new potato varieties and their market acceptance takes many years, a genome editing approach is ideal to modify the properties of existing cultivars without introducing external DNA Potato is amenable to genome editing There are a series of dietary properties of potato that could be modified by genome editing to improve the health of consumers Acceptance as non-gm is vital, and the current OGTR review of NBTs should exclude this type of SDN1 targeted mutagenesis as not being classified as GM

21 Acknowledgements Plant Biotechnology Research Group (SABC) Australian Research Council Potato Research WA PGAWA and Murdoch University HZPC Dr Sadia Iqbal Potato Research WA