SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition

文献类型： 外文期刊

第一作者： Xu, Ziyan

作者： Xu, Ziyan;Kuang, Yongjie;Ren, Bin;Yan, Daqi;Yan, Fang;Wang, Guirong;Zhou, Xueping;Zhou, Huanbin;Xu, Ziyan;Kuang, Yongjie;Ren, Bin;Zhou, Huanbin;Ren, Bin;Wang, Guirong;Yan, Daqi;Sun, Wenxian;Spetz, Carl;Sun, Wenxian;Zhou, Xueping

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关键词： CRISPR; SpG; SpRY; Genome editing; Base editing; Self-targeting; Oryza sativa L

期刊名称：GENOME BIOLOGY （ 影响因子：13.583； 五年影响因子：17.433 ）

ISSN： 1474-760X

年卷期： 2021 年 22 卷 1 期

页码：

收录情况： SCI

摘要： BackgroundPlant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome.ResultsIn this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Delta and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines.ConclusionsThe broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.

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