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 系统识别规范的 NGG protospacer 相邻基序 (PAM)。先前设计的 SpCas9 变体，例如 Cas9-NG，在基因组编辑中偏向于富含 G 的 PAM。在这份手稿中，我们描述了一种基于混合 iSpyMacCas9 平台的新植物基因组编辑系统，该系统允许在富含 A 的 PAM 上进行靶向诱变、C 到 T 碱基编辑和 A 到 G 碱基编辑。该研究填补了CRISPR-Cas9系统编辑植物NAAR PAMs的重大技术空白，极大扩展了CRISPR-Cas9的靶向范围。最后，我们的载体系统与 Gateway 克隆完全兼容，并将与所有现有的单向导 RNA 表达系统配合使用，便于其他人轻松采用这些系统。我们预计会有更多的工具，例如主要编辑、同源定向修复、CRISPR 干扰、