Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system

Wang, Shaohui; Zhang, Shuaibin; Wang, Wanxing; Xiong, Xingyao; Meng, Fanrong; Cui, Xia

doi:10.1007/s00299-015-1816-7

Cited by 236 publications

(140 citation statements)

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“…Recently, the first studies using TALEN and CRISPR-Cas9 to induce mutations in potato were published (Butler et al 2015, 2016; Clasen et al 2016; Nicolia et al 2015; Sawai et al 2014; Wang et al 2015). In a TALEN study, stable transformation was used for studying the disruption of a sterol side chain reductase 2 (StSSR2).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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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. AbstractSite-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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-016-2062-3) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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“…The complete knockout lines were free of foreign DNA and therefore indistinguishable from the variety except for small deletions in the targeted gene. In other cases, targeted mutations by CRISPR/Cas9 were only induced in reporter genes and/or studied in vitro at molecular level [52,53].…”

Section: Not All Gm Potatoes Are Equalmentioning

confidence: 99%

Effect of Organic Potato Farming on Human and Environmental Health and Benefits from New Plant Breeding Techniques. Is It Only a Matter of Public Acceptance?

Pacifico

Paris

2016

Sustainability

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Organic farming practices are commonly thought to reduce the environmental impact of agriculture and to preserve the naturalness of the products. Herein, we report the effect of crop management practices on nutritional and toxicological value of potato tubers. Comparative studies are often controversial and the results are dependent on genotype and methodological approach. Targeted analysis and "omics" strategies are discussed, pointing at the nutritional aspects and the corresponding biological and molecular processes involved. Organic farming supporters still do not accept the use of genetic modification to produce new varieties suited for organic agriculture and crop improvement by genetic engineering still sparks hot debate among various scientific and social factions whose major concern is the possible existence of unintended effects both on human and world health. In this context, the advent of "new plant breeding techniques" has reignited the discussion on genetic engineering and on the compatibility of the new technologies with an eco-friendly agriculture. Could cisgenic and genome-edited potatoes be new good options for organic agriculture? We discuss how these approaches can be used to address food security challenges and to overcome specific problems based on the biological characteristics of potato tubers, producing new varieties that can improve farmers' profit with a lower impact on public opinion. However, political, ethical, and social fears will probably persist much longer, mainly in Italy, historically a fiercely anti-GM country with a European leadership in organic food production and export.

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“…With the ability to introduce multiple targeted edits into plant genomes, CRISPR can also facilitate metabolic engineering, which usually requires gene manipulation of one or multiple pathways. CRISPR/Cas9-induced genome editing has been demonstrated in multiple plant species, including model species, major food crops and specialty crops Gao et al, 2015;Nekrasov et al, 2013;Shan et al, 2013;Jiang et al, 2013;Ron et al, 2014;Wang et al, 2015;Jia and Wang, 2014). However, the application of CRISPR in industrial crops has not been reported.…”

Section: Abbreviationsmentioning

confidence: 98%

CRISPR/Cas9 genome editing of rubber producing dandelion Taraxacum kok-saghyz using Agrobacterium rhizogenes without selection

Iaffaldano

Zhang

Cornish

2016

Industrial Crops and Products

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a b s t r a c t CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) is a highly accessible genome editing tool. Here we demonstrate its potential for use in Taraxacum species. Taraxacum kok-saghyz (TK, Rubber Dandelion) is notable for its ability to produce high molecular weight rubber in its roots and promise as an alternative source of natural rubber. In order to accelerate the domestication of TK, we have established a simple strategy to deploy CRISPR/Cas9 in this species. A critical gene encoding fructan:fructan 1-fructosyltransferase (1-FFT), implicated in inulin biosynthesis was selected as the target, as inulin is an expected antagonist of rubber production. TK plantlets were inoculated with Agrobacterium rhizogenes harboring a plasmid encoding a Cas9 and sgRNA (single guide RNA) targeting TK 1-FFT. We were able to rapidly induce hairy roots harboring knockout alleles without the selection of stable, herbicide or antibiotic resistant transformants. Mutagenesis was affirmed by observing a loss of restriction sites within 1-FFT, followed by sequencing. Of 11 hairy root samples tested, 10 showed the presence of genome editing, with mutation rates as high as 88.9%, suggesting a high efficiency mutagenesis induced by CRISPR/Cas9 via A. rhizogenes-mediated transformation. Whole TK plants were regenerated from hairy roots harboring knockout alleles. The regenerated plants contained knockout alleles, with mutation rates as high as 80.0%. TK plants with edited genomes were obtained within 10 weeks. By omitting a selection step, it was possible to generate edited TK plants lacking stably transformed CRISPR elements, which may potentially reduce off-target mutagenesis. Application of high efficiency CRISPR/Cas9 genome editing will facilitate the domestication and commercialization of TK as a rubber producing crop, and may accelerate basic research on the regulation of rubber biosynthesis.

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Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system

Cited by 236 publications

References 12 publications

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

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

Effect of Organic Potato Farming on Human and Environmental Health and Benefits from New Plant Breeding Techniques. Is It Only a Matter of Public Acceptance?

CRISPR/Cas9 genome editing of rubber producing dandelion Taraxacum kok-saghyz using Agrobacterium rhizogenes without selection

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