Botanisches Institut und Botanischer Garten, Christian-Albrechts-Universität zu Kiel, D-24098 Kiel, Germany.
Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University, 44519 Zagazig, Egypt.
Molecules. 2021 Mar 10;26(6):1498. doi: 10.3390/molecules26061498.
Comfrey () 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 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 alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated 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.
千里光（Comfrey）是一种具有抗炎、镇痛和增殖特性的药用植物。然而，其组织中同时存在有毒的吡咯里西啶生物碱（PAs），这限制了它在药物方面的应用。我们采用基于 CRISPR/Cas9 的方法，将有害突变引入编码同源亚精胺合酶（HSS）的 基因中，HSS 是 PA 生物合成途径中第一个特异性酶。对产生的毛状根（HR）系进行分析，以确定其表现出的基因编辑效应类型及其同源亚精胺和 PA 的含量。只有一个 等位基因失活会导致 HR 中同源亚精胺和 PAs 的水平显著降低，而两个 等位基因失活的 HR 中则检测不到生物碱。在缺乏 HSS 的 HR 系中添加同源亚精胺后，再次检测到 PAs，这证实了这些根无法产生 PAs 仅归因于失活的 HSS，而不是 CRISPR/Cas9 方法的任何未识别的脱靶效应。进一步的分析表明，无 PA 的 HR 系至少在痕量水平上仍具有可检测到的同源亚精胺含量，并且经过处理的 HR 系的 PA 模式与对照系不同。这些观察结果与在药用植物的体外系统中经济利用这种 CRISPR/Cas9 介导的方法以及进一步研究非模式植物中 PA 生物合成的潜在用途有关。
