通过靶向富含 A 的 PAM 序列的工程化 iSpyMacCas9 系统扩展植物基因组编辑范围。
Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences.
机构信息
Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA.
Hubei Academy of Agricultural Sciences, Wuhan 430064, China.
出版信息
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。
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