Accelerated ex situ breeding of
            <i>GBSS</i>
            - and
            <i>PTST1</i>
            -edited cassava for modified starch

Bull, Simon E.; Seung, David; Chanez, Christelle; Mehta, Devang; Kuon, Joel‐Elias; Truernit, Elisabeth; Hochmuth, Anton; Zurkirchen, Irene; Zeeman, Samuel C.; Gruissem, Wilhelm; Vanderschuren, Hervé

doi:10.1126/sciadv.aat6086

Cited by 138 publications

(108 citation statements)

References 67 publications

(92 reference statements)

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“…Only recently, natural waxy and small granule starch cassava lines were identified from self-pollinated cassava lines in the CIAT cassava breeding program (Ceballos et al, 2007(Ceballos et al, , 2008(Ceballos et al, , 2017. Instead, successful approaches using transgenesis to obtain waxy cassava have been reported in the last decade by down-regulating expression of the GBSSI gene either constitutively or tissue-specifically (Raemakers et al, 2005;Zhao et al, 2011;Koehorst-van Putten et al, 2012), or by deletion mutating GBSSI and PTST1 via CRISPR/Cas9-based mutagenesis (Bull et al, 2018). Very-high-amylose cassava starch has not yet been identified through current traditional breeding programs.…”

Section: Discussionmentioning

confidence: 99%

“…Compared with other starches (e.g., from maize and potato), pure-white cassava starch contains low levels of fat (0.08-1.54%), proteins (0.03-0.6%), and phosphorus (0.75-4%), making it an excellent starch resource for various industrial applications (Richard et al, 1991;Moorthy, 2002;Balagopalan, 2002;Sharma et al, 2016). Modified starches of cassava have been broadly used in the food, textile, Wang et al, 2014;Bredeson et al, 2016) and development of cassava transformation technology (Liu et al, 2011;Zhang et al, 2017), we now have the capacity to breed novel cassava cultivars with desirable traits using state-of-the-art technologies including CRISPR/Cas9-based genome editing (Odipio et al, 2017;Sun et al, 2017;Bull et al, 2018;Gomez et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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Production of very-high-amylose cassava by post-transcriptional silencing of branching enzyme genes

Zhou

Zhao

et al. 2018

Preprint

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High amylose starch, a desired raw material in the starch industry, can be produced by plants deficient in the function of branching enzymes (BEs). Here we report the production of transgenic cassava plants with starches containing up to 50% amylose due to the constitutive expression of hair-pin dsRNAs targeting the BE1 or BE2 genes. A significant decrease in BE transcripts was confirmed in these transgenic plants by quantitative real-time RT-PCR. The absence of BE1 protein in the BE1-RNAi plant lines (BE1i) and a dramatically lower level of BE2 protein in the BE2-RNAi plant lines (BE2i) were further confirmed by Western blot assays. All transgenic plant lines were grown up in the field, but with reduced biomass production of the above-ground parts and storage roots compared to wild type (WT). Considerably high amylose content in the storage roots of BE2i plant lines was achieved, though not in BE1i plant lines. Storage starch granules of BE1i and BE2i plants had similar morphology as WT, however, the size of BE1i starch granules were bigger than that of WT. Comparisons of amylograms and thermograms of all three sources of storage starches revealed dramatic changes to the pasting properties and a higher melting temperature for BE2i starches. Glucan chain length distribution analysis showed a slight increase in chains of DP>36 in BE1i lines and a dramatic increase in glucan chains between DP 10-20 and DP>40 in BE2i lines, compared to that of WT starch. Furthermore, BE2i starches displayed a B-type X-ray diffraction pattern instead of the A-type pattern found in BE1i and WT starches. Therefore, cassava BE1 and BE2 function differently in storage root starch biosynthesis; silencing of cassava BE1 or BE2 caused various changes to starch physico-chemical properties and amylopectin structure. We also report that remarkably high amylose content in cassava starch has been first obtained in transgenic cassava by silencing of BE2 expression, thus showing a high potential for future industrial utilization.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Production of very-high-amylose cassava by post-transcriptional silencing of branching enzyme genes

Zhou

Zhao

et al. 2018

Preprint

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“…Amylose is a 36 nearly linear glucose polymer with α-1,4-linked residues, whereas amylopectin is made up of 37 α-1,4-linked chains with α-1,6-linkages at branch points, conferring crystallinity to the starch 38 granule. In higher plants, starch biosynthesis is mainly mediated by four classes of enzymes: Amylose determines many physicochemical properties of starch, namely its pasting 46 temperature and viscosity (Bull et al 2018;Park et al 2007). Therefore, modifying potato 47 starch composition by decreasing the amylose (or amylopectin) content may be useful for 48 industrial applications.…”

Section: Tubers 32mentioning

confidence: 99%

“…shown to be efficient in modifying amylose content in cassava (Bull et al 2018). With the 585 extraordinarily rapid development of genome editing tools in both animals and plants, 586…”

Section: Crispr-cas9 Cytidine Deaminase Fusion Precisely and Efficienmentioning

confidence: 99%

The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato

Veillet

Chauvin

Kermarrec

et al. 2019

Preprint

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The StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base editing strategies, leading to plants with impaired amylose biosynthesis. AbstractGenome editing has recently become a method of choice for basic research and functional 4 genomics, and holds great potential for molecular plant breeding applications. The powerful 5 CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to 6 generate knockout mutants. Recently, the development of base editors has broadened the 7 scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, 8we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum 9 tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knockout 10 the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective 11 screening strategy based on high-resolution melting analysis followed by direct Sanger 12 sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but 13 unwanted insertions of plasmid DNA were also observed. We successfully created tetra-14 allelic mutants with impaired amylose biosynthesis, confirming the loss-of-function of the 15 StGBSSI protein. The second main objective of this work was to demonstrate the proof of 16 concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding 17 catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently 18 and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete 19 variation in the amino acid sequence and generating a loss-of-function allele. The successful 20 application of base editing in the tetraploid potato opens up new avenues for genome 21 engineering in this species. 22 23 24 tuberosum. We identified single-and multi-allelic edited plants, and mutations in all four 113 alleles were observed, resulting in amylose biosynthesis impairment. To induce this 114 modification, we used protoplast transformation, which can produce non-transgenic plants. 115 Finally, we assessed the efficiency of the cytidine base editor (CBE) in two loci encoding 116 catalytic motifs of the StGBSSI enzyme, resulting in precise base conversion and amino-acid 117 substitution. Our results highlight that CRISPR-Cas9 gene and base editing can be efficiently 118 developed in a genetically complex and vegetatively propagated crop such as potato to 119 modify agronomic traits. 120 121 6 Materials and methods 122 Plant material 123 Potato cultivars Desiree (ZPC, the Netherlands) and Furia (Germicopa, France) were in vitro 124 propagated in 1X Murashige and Skoog (MS) medium (pH 5.8) including vitamins (Duchefa, 125 the Netherlands), 0.4 mg/L thiamine hydrochloride (Sigma-Aldrich, USA), 2.5% sucrose and 126 0.8% agar powder (VWR, USA). Plants were cultivated in a growth chamber at 19°C with a 127 16:8 h light:dark photoperiod. For the production of in vitro microtubers, plants w...

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“…FT is the key positive regulator of flowering in Arabidopsis, and at least some of the FT homologues in other plants species including medicago, rice, soybean and trees like poplar and pear also function as activator of flowering [25–28]. Due to its important role in flowering promotion, FT has been successfully used to reducing juvenile phase of many plants, therefore accelerated the process for plant breeding [15,27,29].…”

Section: Introductionmentioning

confidence: 99%

Integration of a FT expression cassette into CRISPR/Cas9 construct enables fast generation and easy identification of transgene-free mutants in Arabidopsis

Cheng

Zhang

Hussain

et al. 2019

Preprint

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21The CRISPR/Cas9 genome editing technique has been widely used to generate 22 transgene-free mutants in different plant species. Several different methods including 23 fluorescence marker-assisted visual screen of transgene-free mutants and programmed 24 self-elimination of CRISPR/Cas9 construct have been use to increase the efficiency of 25 genome edited transgene-free mutants isolation, but the overall time length required to 26 obtain transgene-free mutants has remained unchanged in these methods. We report 27 here a method for fast generation and easy identification of transgene-free mutants in 28 Arabidopsis. By generating and using a single FT expression cassette-containing 29 CRISPR/Cas9 construct, we targeted two sites of the AITR1 gene. We obtained many 30 early bolting plants in T1 generation, and found that about two thirds of these plants 31 have detectable mutations. We then analyzed T2 generations of two representative 32 lines of genome edited early bolting T1 plants, and identified plants without early 33 bolting phenotype, i.e., transgene-free plants, for both lines. Further more, 34 homologues aitr1 mutants were successful obtained for both lines from these 35 transgene-free plants. Taken together, these results suggest that the method described 36 here enables fast generation, and at the mean time, easy identification of 37 transgene-free mutants in plants. 38 39

show abstract

Accelerated ex situ breeding of GBSS - and PTST1 -edited cassava for modified starch

Abstract: The growing need for cassava, a food and fuel crop, has led to a new plant breeding technique designed to accelerate breeding of cassava with modified starch.

Cited by 138 publications

References 67 publications

Production of very-high-amylose cassava by post-transcriptional silencing of branching enzyme genes

Production of very-high-amylose cassava by post-transcriptional silencing of branching enzyme genes

The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato

Integration of a FT expression cassette into CRISPR/Cas9 construct enables fast generation and easy identification of transgene-free mutants in Arabidopsis

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