Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry Sciences, Shuguang Garden Middle Road No. 9, haidian district, Beijing, China.
BMC Plant Biol. 2019 Nov 21;19(1):511. doi: 10.1186/s12870-019-2131-1.
Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits.
We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture.
We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.
CRISPR/Cas9 系统或其衍生的碱基编辑器的应用使靶向基因组修饰成为可能，从而为利用基因功能和改良作物特性提供了一种可编程工具。
我们报告称，与 rAPOBEC1 融合的 PmCDA1 在将水稻中的胞嘧啶（C）转化为胸腺嘧啶（T）时，比 rAPOBEC1 更有效。三种高保真度 SpCas9 变体，eSpCas9(1.1)、SpCas9-HF2 和 HypaCas9，被设计为与 PmCDA1（pBEs）一起作为 C-to-T 碱基编辑器。这三种高保真度编辑器具有不同的多重基因组编辑效率。为了显著提高它们的碱基编辑效率，应用了串联排列的 tRNA 修饰的单指导 RNA（sgRNA）结构。eSpCas9(1.1)-pBE 的效率提高了 25.5 倍，而脱靶效应可接受。此外，在 tRNA 修饰 sgRNA 结构的指导下，这些高保真 Cas9s 的敲除突变频率提高了两到五倍。
我们已经为水稻中的高效和精确基因组工程设计了一个多样化的工具包，从而使植物研究和作物改良的基因组编辑更加灵活。
