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Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts
Monday, 2017/01/30 | 01:56:12
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Mariette Andersson, Helle Turesson, Alessandro Nicolia, Ann-Sofie Fält, Mathias Samuelsson, Per Hofvander PLANT CELL REPORTS: January 2017, Volume 36, Issue 1, pp 117–128 AbstractKey messageAltered 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.
See http://link.springer.com/article/10.1007/s00299-016-2062-3
Fig. 1 Design of CRISPR-Cas9 constructs targeting the GBSS gene. a Illustration of Solanum tuberosum GBSS gene structure (GenBank accession no. A23741.1). Exons 1–13 are marked with blue arrows, outer boundaries of fragments amplified for sequence determination of allelic variation are marked with green arrows and CRISPR-Cas9 target regions GT1, GT2 and GT4 are marked with red triangles. b Target regions GT1, GT2 and GT4 are in red and PAM-site in purple. Allelic variation in GT1 marked in black bold text. c Constructs designed for CRISPR-Cas9-mediated induction of mutations in StGBSS; from left to right: terminator (poly-T), sgRNA scaffold, guide sequence (GT1, GT2 or GT4), U6 promoter of either Arabidopsis thaliana or Solanum tuberosum origin, 35S promoter of cauliflower mosaic virus origin (CaMV), nuclear localization sequence (NLS), plant codon-optimized Cas9 gene, NLS and nopaline synthase terminator (NOS) in vector pENTR™11. The elements shown in the illustration are not to scale in relation to each other |
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