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

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

Posted 24 Sep 2020
bioRxiv DOI: 10.1101/2020.09.23.310839

Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, NNG, etc ., to initiate the target recognition, which notably restricts the editable range of the plant genome. In this study, we thoroughly investigated 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*Δ and adenosine deaminase TadA8e were generated, respectively. These constructs efficiently induced 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 were observed, whereas the self-editing of SpRY nickase-mediated base editor was quite low in transgenic rice lines. In conclusion, the broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering. ### Competing Interest Statement The authors have declared no competing interest.

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