As the focus for CRISPR edited plants moves from proof-of-concept to real world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one gRNA per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in Populus tremula × P. alba INRA 717-1B4. Unexpected duplications of MYB186 and MYB138 resulted in a total of eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and PCR analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the non-glandular trichomes of poplar.

中文翻译：

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使用单个 gRNA 多重敲除杂种杨树中毛状体调节 MYB 重复

随着 CRISPR 编辑植物的重点从概念验证转向现实世界的应用，精确的基因操作将越来越需要对多基因性状进行并发多重编辑。跨多个位点编辑的常用方法是为每个目标设计一个 gRNA；然而，这使构建体组装复杂化并增加了脱靶突变的可能性。在这项研究中，我们利用一种 gRNA 靶向MYB186，一种已知的阳性毛状体调节剂，以及它的旁系同源物MYB138和MYB38，位于杨树× P. alba INRA 717-1B4突变的共有位点。MYB186和MYB138 的意外重复导致杂交杨树中三个靶基因的总共八个等位基因。深度测序和 PCR 分析证实了几乎所有产生的无毛突变体的所有八个目标的编辑，从小的插入缺失到大的基因组缺失，在四个潜在位点没有检测到脱靶活动。这突出了单个 gRNA 靶向保守外显子区域进行多重编辑的有效性。此外，表皮蜡和全叶分析表明，在无毛突变体中完全没有三萜，暗示了杨树非腺毛的先前未描述的作用。