Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA.
Hubei Academy of Agricultural Sciences, Wuhan 430064, China.
Plant Commun. 2020 Jul 22;2(2):100101. doi: 10.1016/j.xplc.2020.100101. eCollection 2021 Mar 8.
The most popular CRISPR-SpCas9 system recognizes canonical NGG protospacer adjacent motifs (PAMs). Previously engineered SpCas9 variants, such as Cas9-NG, favor G-rich PAMs in genome editing. In this manuscript, we describe a new plant genome-editing system based on a hybrid iSpyMacCas9 platform that allows for targeted mutagenesis, C to T base editing, and A to G base editing at A-rich PAMs. This study fills a major technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants, which greatly expands the targeting scope of CRISPR-Cas9. Finally, our vector systems are fully compatible with Gateway cloning and will work with all existing single-guide RNA expression systems, facilitating easy adoption of the systems by others. We anticipate that more tools, such as prime editing, homology-directed repair, CRISPR interference, and CRISPR activation, will be further developed based on our promising iSpyMacCas9 platform.
最流行的 CRISPR-SpCas9 系统识别典型的 NGG 原间隔基序 (PAMs)。先前设计的 SpCas9 变体，如 Cas9-NG，在基因组编辑中偏爱富含 G 的 PAMs。在本手稿中，我们描述了一种基于杂交 iSpyMacCas9 平台的新型植物基因组编辑系统，该系统允许在富含 A 的 PAMs 上进行靶向诱变、C 到 T 的碱基编辑和 A 到 G 的碱基编辑。这项研究填补了 CRISPR-Cas9 系统在植物中编辑 NAAＲ PAMs 的重大技术空白，极大地扩展了 CRISPR-Cas9 的靶向范围。最后，我们的载体系统完全兼容 Gateway 克隆，并且可以与所有现有的单向导 RNA 表达系统兼容，便于其他研究人员采用这些系统。我们预计，将基于我们有前途的 iSpyMacCas9 平台进一步开发更多的工具，如 Prime 编辑、同源定向修复、CRISPR 干扰和 CRISPR 激活。
