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

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 transformed via Agrobacteria often showed chimerism and a mixture of CiGAS genotypes. This genetic mosaic becomes more diverse when plants were grown over a prolonged period. While the genotype of the on-targets varied between the transient and stable delivery methods, no off-target activity in six identified potential off-targets with two to four mismatches was found. The environmental impacts (greenhouse gas (GHG) emissions and primary energy demand) of the methods are highly dependent on their individual electricity demand. From an economic view - like for most research and development activities - employment and value-added multiplier effects are high; particularly when compared to industrial or manufacturing processes. Considering all aspects, we conclude that using RNPs is the most suitable method for genome editing in chicory since it led to a high efficiency of editing, no off-target mutations, non-transgenic plants with no risk of unwanted integration of plasmid DNA and without needed segregation of transgenes.

Lactucin Synthase Inactivation Boosts the Accumulation of Anti-inflammatory 8-Deoxylactucin and Its Derivatives in Chicory (Cichorium intybus L.).

For several sesquiterpene lactones (STLs) found in Asteraceae plants, very interesting biomedical activities have been demonstrated. Chicory roots accumulate the guaianolide STLs 8-deoxylactucin, lactucin, and lactucopicrin predominantly in oxalated forms in the latex. In this work, a supercritical fluid extract fraction of chicory STLs containing 8-deoxylactucin and 11ß,13-dihydro-8-deoxylactucin was shown to have anti-inflammatory activity in an inflamed intestinal mucosa model. To increase the accumulation of these two compounds in chicory taproots, the lactucin synthase that takes 8-deoxylactucin as the substrate for the regiospecific hydroxylation to generate lactucin needs to be inactivated. Three candidate cytochrome P450 enzymes of the CYP71 clan were identified in chicory. Their targeted inactivation using the CRISPR/Cas9 approach identified CYP71DD33 to have lactucin synthase activity. The analysis of the terpene profile of the taproots of plants with edits in CYP71DD33 revealed a nearly complete elimination of the endogenous chicory STLs lactucin and lactucopicrin and their corresponding oxalates. Indeed, in the same lines, the interruption of biosynthesis resulted in a strong increase of 8-deoxylactucin and its derivatives. The enzyme activity of CYP71DD33 to convert 8-deoxylactucin to lactucin was additionally demonstrated in vitro using yeast microsome assays. The identified chicory lactucin synthase gene is predominantly expressed in the chicory latex, indicating that the late steps in the STL biosynthesis take place in the latex. This study contributes to further elucidation of the STL pathway in chicory and shows that root chicory can be positioned as a crop from which different health products can be extracted.

CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots.

Chicory taproots accumulate sesquiterpene lactones lactucin, lactucopicrin, and 8-deoxylactucin, predominantly in their oxalated forms. The biosynthetic pathway for chicory sesquiterpene lactones has only partly been elucidated; the enzymes that convert farnesyl pyrophosphate to costunolide have been described. The next biosynthetic step of the conversion of costunolide to the tricyclic structure, guaianolide kauniolide, has so far not been elucidated in chicory. In this work three putative kauniolide synthase genes were identified in chicory named CiKLS1, CiKLS2, and CiKLS3. Their activity to convert costunolide to kauniolide was demonstrated in vitro using yeast microsome assays. Next, introduction of CRISPR/Cas9 reagents into chicory protoplasts was used to inactivate multiple chicory KLS genes and several chicory lines were successfully regenerated. The inactivation of the kauniolide synthase genes in chicory by the CRISPR/Cas9 approach resulted in interruption of the sesquiterpene lactone biosynthesis in chicory leaves and taproots. In chicory taproots, but not in leaves, accumulation of costunolide and its conjugates was observed to high levels, namely 1.5 mg/g FW. These results confirmed that all three genes contribute to STL accumulation, albeit to different extent. These observations demonstrate that three genes oriented in tandem on the chicory genome encode kauniolide synthases that initiate the conversion of costunolide toward the sesquiterpene lactones in chicory.

Inactivation of the germacrene A synthase genes by CRISPR/Cas9 eliminates the biosynthesis of sesquiterpene lactones in Cichorium intybus L.

Chicory (Cichorium intybus var. sativum) is an industrial crop species cultivated for the production of a fructose polymer inulin, which is used as a low-calorie sweetener and prebiotic. Besides, inulin chicory taproots also accumulate sesquiterpene lactones (STLs). These are bitter tasting compounds, which need to be removed during inulin extraction, resulting in additional costs. In this work, we describe chicory lines where STL accumulation is almost completely eliminated. Genome editing using the CRISPR/Cas9 system was used to inactivate four genes that encode the enzyme that performs the first dedicated step in STL synthesis, germacrene A synthase (CiGAS). Chicory lines were obtained that carried null mutations in all four CiGAS genes. Lines lacking functional CiGAS alleles showed a normal phenotype upon greenhouse cultivation and show nearly complete elimination of the STL synthesis in the roots. It was shown that the reduction in STLs could be attributed to mutations in genetically linked copies of the CiGAS-short gene and not the CiGAS-long gene, which is relevant for breeding the trait into other cultivars. The inactivation of the STL biosynthesis pathway led to increase in phenolic compounds as well as accumulation of squalene in the chicory taproot, presumably due to increased availability of farnesyl pyrophosphate (FFP). These results demonstrate that STLs are not essential for chicory growth and that the inhibition of the STL biosynthesis pathway reduced the STL levels chicory which will facilitate inulin extraction.

Yeast (Saccharomyces cerevisiae).

The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic organisms. This species has enabled a detailed study of the (genetic) requirements for Agrobacterium-mediated DNA transformation. For instance research with this yeast has led to the recognition that the transforming DNA molecules integrate into the eukaryotic chromosomes either by homologous recombination, which is the preferred pathway in S. cerevisiae, or by nonhomologous end-joining. Based on the protocol for Agrobacterium-mediated transformation of S. cerevisiae methodology has been developed for the transformation of many other yeast and fungal species.

An Arabidopsis hAT-like transposase is essential for plant development.

A significant proportion of the genomes of higher plants and vertebrates consists of transposable elements and their derivatives. Autonomous DNA type transposons encode a transposase that enables them to mobilize to a new chromosomal position in the host genome by a cut-and-paste mechanism. As this is potentially mutagenic, the host limits transposition through epigenetic gene silencing and heterochromatin formation. Here we show that a transposase from Arabidopsis thaliana that we named DAYSLEEPER is essential for normal plant growth; it shares several characteristics with the hAT (hobo, Activator, Tam3) family of transposases. DAYSLEEPER was isolated as a factor binding to a motif (Kubox1) present in the upstream region of the Arabidopsis DNA repair gene Ku70. This motif is also present in the upstream regions of many other plant genes. Plants lacking DAYSLEEPER or strongly overexpressing this gene do not develop in a normal manner. Furthermore, DAYSLEEPER overexpression results in the altered expression of many genes. Our data indicate that transposase-like genes can be essential for plant development and can also regulate global gene expression. Thus, transposases can become domesticated by the host to fulfil important cellular functions.

The Arabidopsis AtLIG4 gene is required for the repair of DNA damage, but not for the integration of Agrobacterium T-DNA.

The joining of breaks in the chromosomal DNA backbone by ligases in processes of replication, recombination and repair plays a crucial role in the maintenance of genomic stability. Four ATP-dependent ligases, designated DNA ligases I-IV, have been identified in higher eukaryotes, and each one has distinct functions. In mammals and yeast, DNA ligase IV is exclusively involved in the repair of DNA double-strand breaks by non-homologous end joining. Recently, an Arabidopsis thaliana orthologue of the yeast and mammalian DNA ligase IV gene was found and termed AtLIG4. Here we describe the isolation and functional characterisation of a plant line with a T-DNA insertion in the AtLIG4 gene. Plants homozygous for the T-DNA insertion did not display any growth or developmental defects and were fertile. However, mutant seedlings were hypersensitive to the DNA-damaging agents methyl methanesulfonate and X-rays, demonstrating that AtLIG4 is required for the repair of DNA damage. Recently, we showed that a yeast lig4 mutant is deficient in Agrobacterium T-DNA integration. However, using tumorigenesis and germline transformation assays, we found that the plant AtLIG4 mutant is not impaired in T-DNA integration. Thus, in contrast to yeast, DNA ligase IV is not required for T-DNA integration in plants.

Severe developmental defects, hypersensitivity to DNA-damaging agents, and lengthened telomeres in Arabidopsis MRE11 mutants.

The Mre11 protein is essential for the long-term genetic stability of the cell and acts to ensure the efficient repair of DNA damage. Vertebrate cells lacking Mre11 function are not viable. However, we report here that this is not the case in the model plant Arabidopsis. We have isolated two different Arabidopsis lines containing a T-DNA copy integrated at a different point in the MRE11 gene (AtMRE11). Both mutant plant lines were hypersensitive to DNA-damaging treatments but exhibited strikingly different developmental phenotypes. Furthermore, we also observed lengthened telomeres in these plant lines, showing that AtMre11 is involved in telomere maintenance. Thus, the lines we have isolated are unique tools with which to study in detail the role of AtMre11 in the mature plant.

Increased telomere length and hypersensitivity to DNA damaging agents in an Arabidopsis KU70 mutant.

We have identified a putative homologue of the KU70 gene (AtKU70) from Arabidopsis thaliana. In order to study its function in plants we have isolated an A.thaliana line that contains a T-DNA inserted into AtKU70. Plants homozygous for this insertion appear normal and are fertile. In other organisms the KU70 gene has been shown to play a role in the repair of DNA damage induced by ionising radiation (IR) and by radiomimetic chemicals such as methylmethane sulfonate (MMS). We show that AtKU70(-/-) plants are hypersensitive to IR and MMS, and thus the AtKU70 gene plays a similar role in DNA repair in plants as in other organisms. The KU70 gene also plays a role in maintaining telomere length. Yeast and mammalian cells deficient for Ku70 have shortened telomeres. When we studied the telomeres in the AtKU70(-/-) plants we found unexpectedly that they were significantly longer (>30 kb) than was found in wild-type plants (2-4 kb). We propose several hypotheses to explain this telomere lengthening in the AtKU70(-/-) plants.

Insertional mutagenesis in yeasts using T-DNA from Agrobacterium tumefaciens.

Insertional mutagenesis is a powerful tool for the isolation of novel mutations. The gene delivery system of the bacterium Agrobacterium tumefaciens, which mediates transfer not only to plants but also to yeasts and fungi, could be exploited to generate collections of yeasts containing insertional mutations if there were no bias towards particular integration sites, as is the case in plants. To test this, we have analysed a small collection of Saccharomyces cerevisiae strains with T-DNA copies integrated in the S. cerevisiae genome. The position of 54 of these T-DNAs was determined. The T-DNA showed no clear preference for certain DNA sequences or genomic regions. We have isolated insertions in the coding regions of the genes YGR125w, YDR250c, YGR141w, YGR045c, YPL017c, YGR040w, YDL052c, YJL148w, YCL033c, YFL061w, YJR033c, YDR175c and YLR309c confirming that these genes are non-essential for S. cerevisiae haploid growth on minimal medium. Given the advantages of T-DNA, we propose its use as an ideal mobile DNA element for insertional mutagenesis in yeasts.