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Discriminated sgRNAs-Based SurroGate System Greatly Enhances the Screening Efficiency of Plant Base-Edited Cells

文献类型： 外文期刊

作者： Xu, Wen 1 ; Yang, Yongxing 1 ; Liu, Ya 1 ; Kang, Guiting 1 ; Wang, Feipeng 1 ; Li, Lu 1 ; Lv, Xinxin 1 ; Zhao, Si 1 ; Yuan, 1 ;

作者机构： 1.Beijing Acad Agr & Forestry Sci, Beijing Key Lab Maize DNA Fingerprinting & Mol Br, Beijing 100080, Peoples R China

关键词： cell enrichment; surrogate reporter; cytosine base editors; adenine base editors

期刊名称：MOLECULAR PLANT （ 影响因子：13.164； 五年影响因子：16.357 ）

ISSN： 1674-2052

年卷期： 2020 年 13 卷 1 期

页码：

收录情况： SCI

摘要： The development of CRISPR/Cas9-mediated base editing has made genomic modification more efficient. However, selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. In this study, an efficient surrogate reporter system based on a defective hygromycin resistance gene was established in rice to enrich base-edited cells. After step-by-step optimization, the Discriminated sgRNAs-based SurroGate system (DisSUGs) was established by artificially differentiating the editing abilities of a wild-type single guide RNA (sgRNA) targeting the surrogate reporter gene and an enhanced sgRNA targeting endogenous sites. The DisSUGs enhanced the efficiency of screening base-edited cells by 3- to 5-fold for a PmCDA1-based cytosine-to-tyrosine base editor (PCBE), and 2.5- to 6.5-fold for an adenine base editor (ABE) at endogenous targets. These targets showed editing efficiencies of <25% in the conventional systems. The DisSUGs greatly enhanced the frequency of homozygous substitutions and expanded the activity window slightly for both a PCBE and an ABE. Analyses of the total number of single-nucleotide variants from whole-genome sequencing revealed that, compared with the no-enrichment PCBE strategy, the DisSUGs did not alter the frequency of genome-wide sgRNA-independent off-target mutations, but slightly increased the frequency of target-dependent off-target mutations. Collectively, the DisSUGs developed in this study greatly enhances the efficiency of screening plant base-edited cells and will be a useful system in future applications.

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