PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely unexplored. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal that Cas9 nuclease and base editors, when coupled with the same guide RNA (gRNA), prefer distinct gRNA-dependent off-target sites. De novo generated gRNAs by SpRY editors lead to additional, but insubstantial, off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. ABE8e’s preference for the TA motif is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scale and mechanisms of off-target and background mutations occurring during PAM-relaxed genome editing in plants.

中文翻译：

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对 PAM 松弛的 Cas9 基因组编辑器的全基因组分析揭示了 ABE8e 在水稻中的显着脱靶效应

PAM 松弛的 Cas9 核酸酶、胞嘧啶碱基编辑器和腺嘌呤碱基编辑器是在植物中进行精确基因组编辑的有前途的工具。然而，它们的全基因组脱靶效应在很大程度上尚未得到探索。在这里，我们对水稻中由 xCas9、Cas9-NGv1、Cas9-NG、SpRY、nCas9-NG-PmCDA1、nSpRY-PmCDA1 和 nSpRY-ABE8e 编辑的转基因植物进行全基因组测序 (WGS) 分析。我们的结果表明，Cas9 核酸酶和碱基编辑器与相同的引导 RNA (gRNA) 结合时，更喜欢不同的 gRNA 依赖性脱靶位点。从头SpRY 编辑器生成的 gRNA 会导致额外但非实质性的脱靶突变。引人注目的是，ABE8e 在每个转基因植物的 TA 基序位点导致约 500 个全基因组 A-to-G 脱靶突变。在目标位点也观察到 ABE8e 对 TA 基序的偏好。最后，我们研究了组织培养引起的体细胞克隆变异的时间线和机制，这主要导致了背景突变。本研究全面了解植物 PAM 松弛基因组编辑过程中发生的脱靶和背景突变的规模和机制。