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Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice

文献类型： 外文期刊

作者： Xu, Wen 1 ; Song, Wei 1 ; Yang, Yongxing 1 ; Wu, Ying 1 ; Lv, Xinxin 1 ; Yuan, Shuang 1 ; Liu, Ya 1 ; Yang, Jinxiao 1 ;

作者机构： 1.Beijing Acad Agr & Forestry Sci, Beijing Key Lab Maize DNA Fingerprinting & Mol Br, Shuguang Garden Middle Rd 9, Beijing, Peoples R China

关键词： CRISPR; Cas9; Base editing; High-fidelity Cas9 variants; tRNA-sgRNA; Off-target effect

期刊名称：BMC PLANT BIOLOGY （ 影响因子：4.215； 五年影响因子：4.96 ）

ISSN： 1471-2229

年卷期： 2019 年 19 卷 1 期

页码：

收录情况： SCI

摘要： Background 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. Results 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. Conclusions 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.

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