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.

CRISPR/Cas9介导的碱基编辑技术的发展使得基因组修饰变得更加高效。然而，从数百万个经过处理的细胞，尤其是植物细胞中选择转基因细胞仍然具有挑战性。在这项研究中，在水稻中建立了基于有缺陷的潮霉素抗性基因的高效替代报告系统，以富集碱基编辑的细胞。经过逐步优化，通过人为区分针对替代报告基因的野生型单引导RNA（sgRNA）和针对内源位点的增强型sgRNA的编辑能力，建立了基于可区分sgRNA的SurroGate系统（DisSUGs）。 DisSUG 将基于 PmCDA1 的胞嘧啶至酪氨酸碱基编辑器 (PCBE) 的碱基编辑细胞筛选效率提高了 3 至 5 倍，将腺嘌呤碱基编辑器 (ABE) 的筛选效率提高了 2.5 至 6.5 倍。内生目标。这些目标在传统系统中显示出 <25% 的编辑效率。 DisSUG 大大提高了 PCBE 和 ABE 的纯合取代频率，并略微扩大了活性窗口。对全基因组测序的单核苷酸变异总数的分析表明，与非富集 PCBE 策略相比，DisSUG 并没有改变全基因组 sgRNA 独立的脱靶突变的频率，但略微增加了靶标依赖性脱靶突变的频率。总的来说，本研究中开发的 DisSUG 极大地提高了筛选植物基编辑细胞的效率，并将成为未来应用中的有用系统。