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 for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Mol Plant. 2019 Jul 1;12(7):1015-1026. doi: 10.1016/j.molp.2019.03.010. Epub 2019 Mar 27.
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.
CRISPR 技术可实现精确的基因组操作，对于基因功能研究和分子作物育种具有重要价值。然而，Cas 蛋白识别需要一个原间隔序列邻近基序（PAM），如 NGG 和 TTN，这限制了 CRISPR 技术在实际应用中对靶基因组位点的选择。最近，报道称 Cas9-NG 可识别最小的 NG PAM，从而扩展了人类细胞中基因组编辑的靶向空间，但尚不清楚该 Cas9 变体是否可用于植物。在本研究中，我们通过靶向转基因水稻中的内源基因，评估了 Cas9-NG 对各种 NGN PAM 的核酸酶活性。我们发现 Cas9-NG 编辑所有 NGG、NGA、NGT 和 NGC 位点，但活性受损，而基因编辑植物主要表现为单等位基因突变。然后开发了 Cas9-NG 工程碱基编辑器，并用于生成其他可用的 Cas9 工程碱基编辑器无法创建的 OsBZR1 功能获得型植物。此外，我们表明 Cas9-NG 基转录激活因子可有效上调水稻内源靶基因的表达。此外，我们发现 Cas9-NG 除了识别 NG PAM 外，还可以识别 NAC、NTG、NTT 和 NCG。总之，这些发现表明 Cas9-NG 可以极大地扩展基因组编辑工具的靶向范围，在植物中的靶向基因组编辑、碱基编辑和基因组调控方面具有巨大的潜力。
