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  • Title: Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice.
    Author: Xu W, Song W, Yang Y, Wu Y, Lv X, Yuan S, Liu Y, Yang J.
    Journal: BMC Plant Biol; 2019 Nov 21; 19(1):511. PubMed ID: 31752697.
    Abstract:
    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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