Institute for Biology, Department of Plant Genetics, Martin Luther University Halle-Wittenberg, Weinbergweg 10, 06120, Halle (Saale), Germany.
Dipartimento di Biotecnologie, Università di Verona, Strada Le Grazie 15, 37134, Verona, Italy.
Funct Integr Genomics. 2020 Jan;20(1):151-162. doi: 10.1007/s10142-019-00665-4. Epub 2019 Feb 23.
Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T) generation.
拟南芥模式植物的遗传资源包括几乎在参考品系哥伦比亚中任何单个基因都有缺陷的突变系。然而，基因冗余和/或紧密连锁往往使得从分离群体中分离缺乏特定功能或一组基因的所需系变得极其费力甚至不可能。因此，我们在这里评估了基于 Cas9 的核酸酶和多重化来失活多个基因的策略和效率。在最初的尝试中，我们通过对众多个体进行基于 PCR 的筛选，成功地分离出了携带 70kb 缺失的突变系，该缺失在 T 代中的频率约为 1.6%。然而，我们未能分离出缺乏 Lhcb1 基因的系，该基因在拟南芥基因组的两个位点上以五个拷贝的形式存在。为了提高我们基于 Cas9 的核酸酶系统的效率，我们系统地比较了控制 Cas9 表达水平和时间的调控序列。事实上，与以前的泛素启动子相比，使用 DD45 和 RPS5a 启动子可将我们的基因组编辑系统的效率提高约 25-30 倍。使用优化的基因组编辑系统和 RPS5a 启动子驱动的 Cas9，缺乏可检测量 Lhcb1 蛋白的假定五倍体突变系代表了 T 转化体的约 30%。这些结果表明，改进的基因组编辑系统如何促进复杂突变等位基因的分离，这些等位基因以前被认为不可能在单个（T）代中以高频率产生。
