title: Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts.
abstract: 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. Site-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.
authors.affiliation: Department of Plant Breeding, Swedish University of Agricultural Sciences, P.O. Box 101, SE-23053, Alnarp, Sweden.
authors.forename: Mariette
authors.initials: M
authors.lastname: Andersson
authors.affiliation: Department of Plant Breeding, Swedish University of Agricultural Sciences, P.O. Box 101, SE-23053, Alnarp, Sweden.
authors.forename: Helle
authors.initials: H
authors.lastname: Turesson
authors.affiliation: ENEA Research Centre Casaccia, SSPT-BIOAG-BIOTEC, Via Anguillarese, 301, 00123, Rome, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Nicolia
authors.affiliation: Department of Plant Breeding, Swedish University of Agricultural Sciences, P.O. Box 101, SE-23053, Alnarp, Sweden.
authors.forename: Ann-Sofie
authors.initials: AS
authors.lastname: Fält
authors.affiliation: Lyckeby Starch AB, Degebergavägen 60-20, SE-29191, Kristianstad, Sweden.
authors.forename: Mathias
authors.initials: M
authors.lastname: Samuelsson
authors.affiliation: Department of Plant Breeding, Swedish University of Agricultural Sciences, P.O. Box 101, SE-23053, Alnarp, Sweden. per.hofvander@slu.se.
authors.forename: Per
authors.identifier: http://orcid.org/0000-0003-1048-2608
authors.initials: P
authors.lastname: Hofvander
mesh_terms: D000483:Alleles; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D060005:Genotyping Techniques; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D011523:Protoplasts / Q000378:metabolism*; D012038:Regeneration; D015203:Reproducibility of Results; D011198:Solanum tuberosum / Q000235:genetics*; D013213:Starch / Q000378:metabolism; D057891:Tetraploidy; D014162:Transfection
chemical_list: D013213:Starch
keywords: Amylopectin; GBSS; Genome editing; New plant breeding technique; Starch