Cas9-NG Greatly Expands the Targeting Scope of the Genome-Editing Toolkit by Recognizing NG and Other Atypical PAMs in Rice
作者机构：Ministry of Education Key Laboratory of Bio-Resource and Eco-Environment College of Life Sciences Sichuan University Chengdu 610065 China State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Beijing 100193 China State Key Laboratory of Plant Physiology and Biochemistry College of Biological Sciences China Agricultural University Beijing 100193 China State Key Laboratory of Rice Biology Institute of Biotechnology Zhejiang University Hangzhou 310058 China
出 版 物：《Molecular Plant》 (分子植物（英文版）)
年 卷 期：2019年第12卷第7期
页 面：1015-1026页
核心收录：
学科分类：0710[理学-生物学] 07[理学] 09[农学] 0901[农学-作物学] 0902[农学-园艺学]
基 金：National Natural Science Foundation of China (31871948) the National Key Research and Development Program of China (2017YFD0200900) the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences to H.Z
主 题：CRISPR Cas9-NG PAM gene editing base editing Oryza sativa L
摘 要：CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM)y such as NGG and TTN, for Cas protein recognition restricts the selection of targetable genomic loci in practical applications of CRISPR technologies. Recently Cas9-NG, which recognizes a minimal NG PAM, was reported to expand the targeting space of genome editing in human cells, but it remains unclear whether this Cas9 variant can be used in plants. In this study, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice. We found that Cas9-NG edits all NGG, NGA, NGT, and NGC sites with impaired activity, while the gene-edited plants were dominated by monoallelic mutations. Cas9-NG-engineered base editors were then developed and used to generate O s B Z R I gainof- function plants that can not be created by other available Cas9-engineered base editors. Moreover, we showed that a Cas9-NG-based transcriptional activator efficiently upregulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, and NCG apart from NG PAM. Together, these findings demonstrate that Cas9-NG can greatly expand the targeting scope of genome-editing tools, showing great potential for targeted genome editing, base editing, and genome regulation in plants.
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