Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana,The Plant Journal

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Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana
The Plant Journal ( IF 6.2 ) Pub Date : 2024-02-25 , DOI: 10.1111/tpj.16689
Lejon E M Kralemann ₁ , Niels van Tol ₁ , Paul J J Hooykaas ₁ , Marcel Tijsterman _{1,

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Affiliation

Precise genetic modification can be achieved via a sequence homology-mediated process known as gene targeting (GT). Whilst established for genome engineering purposes, the application of GT in plants still suffers from a low efficiency for which an explanation is currently lacking. Recently reported reduced rates of GT in A. thaliana deficient in polymerase theta (Polθ), a core component of theta-mediated end joining (TMEJ) of DNA breaks, have led to the suggestion of a direct involvement of this enzyme in the homology-directed process. Here, by monitoring homology-driven gene conversion in plants with CRISPR reagent and donor sequences pre-integrated at random sites in the genome (in planta GT), we demonstrate that Polθ action is not required for GT, but instead suppresses the process, likely by promoting the repair of the DNA break by end-joining. This finding indicates that lack of donor integration explains the previously established reduced GT rates seen upon transformation of Polθ-deficient plants. Our study additionally provides insight into ectopic gene targeting (EGT), recombination events between donor and target that do not map to the target locus. EGT, which occurs at similar frequencies as “true” GT during transformation, was rare in our in planta GT experiments arguing that EGT predominantly results from target locus recombination with nonintegrated T-DNA molecules. By describing mechanistic features of GT our study provides directions for the improvement of precise genetic modification of plants.

中文翻译：

聚合酶θ在拟南芥基因打靶中作用的分子分析

精确的基因修饰可以通过称为基因打靶（GT）的序列同源介导过程来实现。虽然 GT 是为基因组工程目的而建立的，但其在植物中的应用仍然效率低下，目前尚缺乏解释。最近报道的拟南芥中缺乏聚合酶theta（Polθ）的GT率降低，聚合酶theta是DNA断裂的θ介导的末端连接（TMEJ）的核心成分，这导致了这种酶直接参与同源性的建议。定向过程。在这里，通过使用 CRISPR 试剂和预整合在基因组随机位点（植物 GT 中）的供体序列监测植物中同源驱动的基因转换，我们证明 Polθ 作用不是 GT 所必需的，而是抑制了该过程，很可能通过末端连接促进 DNA 断裂的修复。这一发现表明，缺乏供体整合解释了先前确定的 Polθ 缺陷植物转化后 GT 率降低的原因。我们的研究还提供了对异位基因靶向（EGT）的深入了解，即供体和靶标之间未映射到靶基因座的重组事件。 EGT 在转化过程中与“真正的”GT 发生频率相似，在我们的植物GT 实验中很少见，我们认为 EGT 主要是由靶基因座与非整合 T-DNA 分子重组引起的。通过描述 GT 的机制特征，我们的研究为改进植物的精确遗传修饰提供了方向。

更新日期：2024-02-25

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