CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids.

Mahmoud M Zakaria,Brigitte Schemmerling,Dietrich Ober

doi:10.3390/molecules26061498

Mahmoud M Zakaria, Brigitte Schemmerling + Show 1 more

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https://doi.org/10.3390/molecules26061498

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Abstract

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.

Highlights

Several traditional medicinal plants contain, in addition to metabolites with various valuable nutritional and therapeutic properties, a structurally diverse group of compounds called the pyrrolizidine alkaloids (PAs) [1,2], many of which are regarded as being toxic and having the potential to cause human poisoning [3]
Intron positions of homospermidine synthase (HSS)- and deoxyhypusine synthase (DHS)-coding genes are known to be highly conserved with respect to position and phase, even when they are compared with the DHS of humans
1703 bp-long gene, nine exons of which exon 3/exon 4 and exon 7/exon 8 are not separated by introns in S. officinale (Figure 2a)

Summary

Introduction

Several traditional medicinal plants contain, in addition to metabolites with various valuable nutritional and therapeutic properties, a structurally diverse group of compounds called the pyrrolizidine alkaloids (PAs) [1,2], many of which are regarded as being toxic and having the potential to cause human poisoning [3]. Despite the beneficial characteristics of their valuable metabolites, the medicinal use of these herbs has been limited by several organizations such as BfArM, IPCS, FDA, and AHPA because of the reported toxicity of PAs [4,5,6,7]. The effect of such a restrictive policy is that, whenever a natural compound is shown to have some level of toxicity, as is the case for PAs, all medicinal herbs containing that compound are excluded from use. This limits our ability to make use of a natural health-care approach. New strategies might help to optimize the clinical and economic benefits of herbal medicine by minimizing its toxic and hazardous effects, e.g., by reducing or shutting down PA-production within in vitro systems

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