Abstract

Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here we describe an efficient gene editing strategy for hybrid poplar, based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two sgRNAs were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target LBD12 and LBD4 genes simultaneously, using an independent construction. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype's characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in 3 (yuc4/plt1) and 2 (lbd12/lbd4) out of 4 transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site were the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple, and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.

中文翻译：

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简单、高效、开源的 CRISPR/Cas9 策略，用于山杨 × 白杨的多位点基因组编辑

摘要

尽管CRISPR/Cas9系统已成功用于农作物育种，但其在林木上的应用仍然有限。在这里，我们描述了一种基于金门 MoClo 克隆的高效杂交杨基因编辑策略。为了测试生成单基因突变体的系统效率，设计了两个 sgRNA，并将其与 Cas9 表达盒一起整合到 MoClo Tool Kit 2 级二元载体中，以突变短根( SHR ) 基因。此外，我们还通过表达一种针对YUC4基因单个位点的sgRNA和另一种针对PLT1基因的sgRNA来测试其在两个基因中同时引入突变的效率。为了对该方法进行稳健的评估，我们使用独立的构建重复了同时靶向LBD12和LBD4基因的策略。我们通过发根农杆菌介导的叶转化产生毛状根。测序结果证实了PtaSHR靶位点存在 CRISPR/Cas9 介导的突变。检测到双等位基因和纯合敲除突变。跨越靶位点的缺失和小插入/缺失是最常见的突变。在测序的 22 个 SHR 等位基因中，有 21 个发生突变。表型特征表明，具有SHR基因双等位基因突变的转基因根缺乏明确的内胚层单细胞层，这表明其保守的基因功能与其在拟南芥中的同源物相似。测序结果还揭示了该系统产生双突变体的高效性。在评估的 4 个转基因根中，有 3 个 ( yuc4 /plt1 ) 和 2 个 ( lbd12 /lbd4 )检测到yuc4/plt1和lbd12/lbd4根中两个基因的双等位基因突变。单个靶位点的小缺失或单个核苷酸插入是最常见的突变。这种 CRISPR/Cas9 策略作为一种快速、简单且标准化的基因编辑工具而出现，用于评估基因在基本发育程序（例如杨树根的径向细胞分化）中的作用。