MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
Nucleic Acids Res. 2022 Apr 8;50(6):3565-3580. doi: 10.1093/nar/gkac166.
CRISPR/Cas-derived base editing tools empower efficient alteration of genomic cytosines or adenines associated with essential genetic traits in plants and animals. Diversified target sequences and customized editing products call for base editors with distinct features regarding the editing window and target scope. Here we developed a toolkit of plant base editors containing AID10, an engineered human AID cytosine deaminase. When fused to the N-terminus or C-terminus of the conventional Cas9 nickase (nSpCas9), AID10 exhibited a broad or narrow activity window at the protospacer adjacent motif (PAM)-distal and -proximal protospacer, respectively, while AID10 fused to both termini conferred an additive activity window. We further replaced nSpCas9 with orthogonal or PAM-relaxed Cas9 variants to widen target scopes. Moreover, we devised dual base editors with AID10 located adjacently or distally to the adenine deaminase ABE8e, leading to juxtaposed or spaced cytosine and adenine co-editing at the same target sequence in plant cells. Furthermore, we expanded the application of this toolkit in plants for tunable knockdown of protein-coding genes via creating upstream open reading frame and for loss-of-function analysis of non-coding genes, such as microRNA sponges. Collectively, this toolkit increases the functional diversity and versatility of base editors in basic and applied plant research.
CRISPR/Cas 衍生的碱基编辑工具可有效地改变植物和动物中与重要遗传特征相关的基因组胞嘧啶或腺嘌呤。多样化的靶序列和定制的编辑产物需要具有不同编辑窗口和靶标范围特征的碱基编辑器。在这里，我们开发了一套包含 AID10 的植物碱基编辑工具，AID10 是一种经过工程改造的人类 AID 胞嘧啶脱氨酶。当与传统 Cas9 切口酶 (nSpCas9) 的 N 端或 C 端融合时，AID10 在 PAM 远端和近端原间隔区分别表现出宽或窄的活性窗口，而 AID10 融合到两端则赋予了附加的活性窗口。我们进一步用正交或 PAM 放松的 Cas9 变体取代 nSpCas9，以扩大靶标范围。此外，我们设计了具有 AID10 位于腺嘌呤脱氨酶 ABE8e 附近或远端的双碱基编辑器，导致在同一靶序列中在植物细胞中进行毗邻或间隔的胞嘧啶和腺嘌呤共编辑。此外，我们通过创建上游开放阅读框，在植物中扩展了该工具包在可调控敲低编码蛋白基因和非编码基因（如 microRNA 海绵）功能丧失分析中的应用。总的来说，该工具包增加了碱基编辑器在基础和应用植物研究中的功能多样性和通用性。
