Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.

胞嘧啶碱基编辑器（CBE）在基因组靶位点中产生C-T核苷酸取代，而不会引起双链断裂。但是，诸如BE3之类的CBE可以通过不依赖sgRNA的DNA脱氨作用而引起全基因组脱靶变化。通过利用SaCas9切口酶产生的正交R环模拟更易受胞苷脱氨酶影响的主动转录的基因组位点，我们建立了高通量测定方法，用于评估水稻原生质体中CBE的不依赖sgRNA的脱靶效应。再生稻植物中10个碱基编辑者的全基因组测序（WGS）证实了该测定的可靠性。R环测定法用于筛选一系列合理设计的A3Bctd-BE3变体，以提高特异性。我们获得了2个有效的CBE变体A3Bctd-VHM-BE3和A3Bctd-KKR-BE3，WGS分析表明，这些新的CBE消除了水稻植物中不依赖sgRNA的DNA脱靶编辑。此外，这两个基本编辑器变体通过产生较少的多次C编辑，在其目标位置更加精确。