Ziyan Xu1,2, Yongjie Kuang1,2, Bin Ren1,2,3, Daqi Yan1,4, Fang Yan1, Carl Spetz5, Wenxian Sun4,6, Guirong Wang1,3, Xueping Zhou7,8, Huanbin Zhou9,10. 1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China. 2. Scientific Observing and Experimental Station of Crop Pests in Guilin, Ministry of Agriculture and Rural Affairs, Guilin, 541399, China. 3. Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120, China. 4. College of Plant Protection, China Agricultural University, Beijing, 100193, China. 5. Norwegian Institute of Bioeconomy Research, 1432, Aas, Norway. 6. College of Plant Protection, Jilin Agricultural University, Changchun, 130118, China. 7. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China. zzhou@zju.edu.cn. 8. State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, 310058, China. zzhou@zju.edu.cn. 9. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China. hbzhou@ippcaas.cn. 10. Scientific Observing and Experimental Station of Crop Pests in Guilin, Ministry of Agriculture and Rural Affairs, Guilin, 541399, China. hbzhou@ippcaas.cn.
Abstract
BACKGROUND: Plant 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. RESULTS: In 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*Δ 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. CONCLUSIONS: The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.
BACKGROUND: Plant 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. RESULTS: In 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*Δ 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. CONCLUSIONS: The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.
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