A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L.

作者：Umberto Salvagnin, Katharina Unkel, Thorben Sprink, Paul Bundock, Robert Sévenier, Milica Bogdanović, Slađana Todorović, Katarina Cankar, Johanna C. Hakkert, Elio Schijlen, Ronald Nieuwenhuis, Maria Hingsamer, Veronika Kulmer, Michael Kernitzkyi, Dirk Bosch, Stefan Martens, Mickaël Malnoy · 发表于：Frontiers in Plant Science · 年份：2023 · DOI：10.3389/fpls.2023.1111110 · 被引用次数：10 · 研究领域：Plant tissue culture and regeneration、CRISPR and Genetic Engineering

Root chicory ( Cichorium intybus L. var. sativum ) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associated costs, recently chicory variants with a lower sesquiterpene lactone content were created by inactivating the four copies of the germacrene A synthase gene ( CiGAS-S1, -S2, -S3, -L ) which encode the enzyme initiating bitter sesquiterpene lactone biosynthesis in chicory. In this study, different delivery methods for CRISPR/Cas9 reagents have been compared regarding their efficiency to induce mutations in the CiGAS genes, the frequency of off-target mutations as well as their environmental and economic impacts. CRISPR/Cas9 reagents were delivered by Agrobacterium-mediated stable transformation or transient delivery by plasmid or preassembled ribonucleic complexes (RNPs) using the same sgRNA. All methods used lead to a high number of INDEL mutations within the CiGAS -S1 and CiGAS -S2 genes, which match the used sgRNA perfectly; additionally, the CiGAS -S3 and CiGAS -L genes, which have a single mismatch with the sgRNA, were mutated but with a lower mutation efficiency. While using both RNPs and plasmids delivery resulted in biallelic, heterozygous or homozygous mutations, plasmid delivery resulted in 30% of unwanted integration of plasmid fragments in the genome. Plants transfo...