Botanical Institute II, Karlsruhe Institute of Technology, Karlsruhe, Germany.
Plant Biotechnol J. 2021 Jul;19(7):1314-1324. doi: 10.1111/pbi.13546. Epub 2021 Jan 28.
Nicotiana tabacum is a non-food herb that has the potential to be utilized as bio-factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre-designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site-specific double-strand breaks to enhance homologous recombination-mediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas-based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature-insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium-mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T-DNA integration. Thus, we were not only able to obtain CRISPR/Cas-based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops.
烟草是一种非食用草本植物，具有作为生物工厂生产药物、疫苗或有价值的小代谢物的潜力。为了实现这些目标，改进用于预先设计的基因组修饰的遗传工具是必不可少的。CRISPR/Cas 核酸酶的发展允许诱导定点双链断裂，以增强同源重组介导的基因靶向 (GT)。然而，GT 的效率仍然是许多作物（包括烟草）面临的一个具有挑战性的障碍。最近，在几种植物物种中的研究表明，通过用其他 CRISPR/Cas 基核酸酶替代 SpCas9，GT 的效率可能会大大提高。因此，我们测试了 SaCas9 以及温度不敏感版本的 LbCas12a（ttLbCas12a）用于靶向烟草 SuRB 基因。同时，我们还优化了农杆菌介导的烟草转化和组织培养的方案。通过这种方式，当使用 ttLbCas12a 时，GT 的效率可以提高到接种子叶的三分之一，这明显优于 SaCas9。此外，我们可以证明 GT 反应的转换片段长度可以长达 606bp，并且在大多数情况下，它长于 250bp。我们获得了多个可遗传的 GT 事件，大多数是杂合的，但也有双等位 GT 事件和一些没有 T-DNA 整合。因此，我们不仅能够首次在异源四倍体烟草中获得基于 CRISPR/Cas 的可遗传 GT 事件，而且我们的结果还表明，ttLbCas12a 可能是基因编辑和 GT 的一种更好的替代物，不仅在烟草中，而且在其他作物中也是如此。
