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

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

doi:10.1186/s12870-019-2131-1

Wen Xu, Ying Wu + Show 6 more

Open Access

https://doi.org/10.1186/s12870-019-2131-1

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Abstract

BackgroundApplication 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.ResultsWe 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.ConclusionsWe 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.

Highlights

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
PmCDA1 cytidine deaminase and uracil glycosylase inhibitor (UGI) were successively fused to the C terminus of SpCas9 nickase (SpCas9n) to generate SpCas9-High-fidelity base editors (pBEs) (Fig. 1a)
We evaluated the base-editing and off-target efficiency of three highfidelity SpCas9 variants directed by tRNA-modified Single guide RNAs (sgRNAs)

Summary

Introduction

Application of the CRISPR/Cas system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits. The system induces double-strand breaks (DSBs) at a target locus, which triggers homologydirected repair (HDR) or non-homologous end joining (NHEJ) to enable site-directed genome modification [4]. This ability to engineer variation in gene regulatory regions or coding sequences can generate elite trait phenotypes in crops [5,6,7]. HeFSpCas9s and evoCas have been engineered and screened to maintain on-target editing efficiency while exceeding the fidelity of SpCas9 [27, 28] These refined high-fidelity Cas variants may be useful for a broad range of genome-editing applications requiring a high level of specificity

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