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

Abstract

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.

Crop genes modified using CRISPR/Cas system

“…CRISPR/Casнаправленный нокаут генов также пер спективен для улучшения признаков качества и техниче ских характеристик сырья. Например, у картофеля нокаут гена GBSS, кодирующего фермент биосинтеза крахмала, гранулсвязанную крахмалсинтазу, приводит к измене нию свойств крахмала за счет сдвига в нем соотношения количества линейных (амилоза) и разветвленных (амило пектин) полисахаридов в пользу последних (Andersson et al, 2017). Крахмал с высоким содержанием амилопектина дает гели повышенной оптической прозрачности, устой чивости при центрифугировании, а также демонстриру ет повышение максимальной и конечной температуры желатинизации и измененные реологические свойства (Хлесткин и др., 2017).…”

Use of CRISPR systems in plant genome editing: toward new opportunities in agriculture

“…As specific primers are used to detect the presence of transgenes encoding CRISPR/Cas components, scientists showed that transgene-free, T2 mutant lines could be obtained by genetic segregation: the targeted mutations were stably passed on in transgene-free plants [ Table 1, column 'Transgene-free plants studied (Yes/No)']. Other studies show how to generate transgene-free plants using alternative delivery methods [11,12]. These methods include ways of introducing the CRISPR components in a transient fashion such that integration is unlikely, for example using protoplast systems [12].…”

“…Other studies show how to generate transgene-free plants using alternative delivery methods [11,12]. These methods include ways of introducing the CRISPR components in a transient fashion such that integration is unlikely, for example using protoplast systems [12]. Alternatively, it is possible to avoid the presence of foreign DNA at any stage of the process, therefore avoiding the possibility of foreign DNA insertion.…”

Separate product from process: framing the debate that surrounds the potential uptake of new breeding technologies

“…Mariette Anderson has worked with an industrial partner, Lyckeby, to develop a potato variety that has reduced levels of amylose. This was achieved by successfully editing four copies of the GBSS gene (Box B; Anderson et al 2017). Attila Molnars' group used CRISPR-Cas9 to introduce a single base deletion into the Arabidopsis eLF(iso)4E gene, which subsequently confers viral resistance (Box C; Pyott et al 2016).…”