Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding/Jiangsu Key Laboratory of Crop Genetics and Physiology, Agricultural College of Yangzhou University, Yangzhou, China.
Key Laboratory of Plant Functional Genomics of the Ministry of Education/Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, China.
Plant Biotechnol J. 2022 Sep;20(9):1670-1682. doi: 10.1111/pbi.13838. Epub 2022 May 23.
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.
PAM 放松的 Cas9 核酸酶、胞嘧啶碱基编辑器和腺嘌呤碱基编辑器是植物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，我们对经 xCas9、Cas9-NGv1、Cas9-NG、SpRY、nCas9-NG-PmCDA1、nSpRY-PmCDA1 和 nSpRY-ABE8e 编辑的转基因水稻进行了全基因组测序 (WGS) 分析。我们的结果表明，当 Cas9 核酸酶和碱基编辑器与相同的向导 RNA (gRNA) 结合时，它们优先选择不同的 gRNA 依赖性脱靶位点。SpRY 编辑器产生的新 gRNA 导致额外的、但不重要的脱靶突变。引人注目的是，ABE8e 导致每个转基因植物在 TA 基序位点处产生约 500 个全基因组 A 到 G 的脱靶突变。ABE8e 在靶位点也表现出对 TA 基序的偏好。最后，我们研究了由于组织培养而导致的体细胞变异的时间线和机制，这主要导致了背景突变。这项研究提供了对 PAM 放松的植物基因组编辑过程中发生的脱靶和背景突变的规模和机制的全面理解。
