Cas9-NG Greatly Expands the Targeting Scope of the Genome-Editing Toolkit by Recognizing NG and Other Atypical PAMs in Rice

文献类型： 外文期刊

第一作者： Zhang, Dawei

作者： Zhang, Dawei;Lin, Honghui;Ren, Bin;Liu, Lang;Kuang, Yongjie;Wang, Jingwen;Zhou, Xueping;Zhou, Huanbin;Li, Shaofang;Zhou, Xueping

作者机构：

关键词： CRISPR; Cas9-NG; PAM; gene editing; base editing; Oryza sativa L

期刊名称：MOLECULAR PLANT （ 影响因子：13.164； 五年影响因子：16.357 ）

ISSN： 1674-2052

年卷期： 2019 年 12 卷 7 期

页码：

收录情况： SCI

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), 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 OsBZR1 gain-of-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.

分类号：

  • 相关文献
作者其他论文 更多>>

微信小程序