Sweet basil (Ocimum basilicum) is an economically important herb and its global production is threatened by basil downy mildew caused by the obligate biotrophic oomycete Peronospora belbahrii. Effective tools are required for functional understanding of its genes involved in synthesis of valuable secondary metabolites in essential oil and disease resistance, and breeding for varieties with improved traits. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene editing technology has revolutionized crop breeding and functional genomics. The applicability and efficacy of this genomic tool in the allotetraploid sweet basil were tested by editing a potential susceptibility (S) gene ObDMR1, the basil homolog of Arabidopsis DMR1 (Downy Mildew Resistant 1) whose mutations conferred nearly complete resistance against Arabidopsis downy mildew pathogen, Hyaloperonospora arabidopsidis. Two single guide RNAs targeting two different sites of the ObDMR1 coding sequence were designed. A total of 56 transgenic lines were obtained via Agrobacterium‐mediated stable transformation. Mutational analysis of 54 T0 transgenic lines identified 92.6% lines carrying mutations at target 1 site, while a very low mutation frequency was detected at target 2 site. Deep sequencing of six T0 lines revealed various mutations at target 1 site, with a complete knockout of all alleles in one line. ObDMR1 homozygous mutant plants with some being transgene free were identified from T1 segregating populations. T2 homozygous mutant plants with 1‐bp frameshift mutations exhibited a dwarf phenotype at young seedling stage. In summary, this study established a highly efficient CRISPR/Cas9‐mediated gene editing system for targeted mutagenesis in sweet basil. This system has the capacity to generate complete knockout mutants in this allotetraploid species at the first generation of transgenic plants and transgene‐free homozygous mutants in the second generation. The establishment of this system is expected to accelerate basil functional genomics and breeding.

中文翻译：

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利用 CRISPR/Cas9 对异源四倍体甜罗勒进行高效靶向诱变。


甜罗勒 ( Ocimum basilicum ) 是一种重要的经济药草，其全球生产受到专性生物营养卵菌Peronospora belbahrii引起的罗勒霜霉病的威胁。需要有效的工具来了解其基因的功能，这些基因涉及精油中有价值的次生代谢物的合成和抗病性，以及培育具有改良性状的品种。成簇规则间隔短回文重复序列 (CRISPR)/Cas9 基因编辑技术彻底改变了作物育种和功能基因组学。通过编辑潜在易感基因 ObDMR1，测试了该基因组工具在异源四倍体甜罗勒中的适用性和功效， ObDMR1是拟南芥 DMR1 （抗霜霉病 1 ）的罗勒同源物，其突变赋予拟南芥霜霉病病原体几乎完全的抗性，拟南芥霜霉属。设计了两个针对ObDMR1编码序列两个不同位点的单引导 RNA。通过农杆菌介导的稳定转化，总共获得了 56 个转基因株系。对54个T0转基因株系的突变分析发现，92.6%的株系在靶标1位点携带突变，而在靶标2位点检测到非常低的突变频率。对 6 个 T0 系的深度测序揭示了目标 1 位点的各种突变，并完全敲除了一个系中的所有等位基因。从 T1 分离群体中鉴定出ObDMR1纯合突变体植物，其中一些植物不含转基因。具有 1bp 移码突变的 T2 纯合突变体植物在幼苗阶段表现出矮化表型。 综上所述，本研究建立了一种高效的 CRISPR/Cas9 介导的基因编辑系统，用于甜罗勒的定向诱变。该系统有能力在第一代转基因植物中产生该异源四倍体物种的完全敲除突变体，并在第二代产生无转基因纯合突变体。该系统的建立有望加速罗勒功能基因组学和育种。