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. Display omitted •The nSaCas9-mediated R-loop assay enables specificity analysis of CBEs in plants•A3Bctd, a truncated APOBEC3B deaminase, is rationally designed to generate new CBEs•The 25 A3Bctd variants are screened with the R-loop assay•A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3 exhibit high specificity and precision in plants Jin et al. establish nSaCas9-mediated orthogonal R-loop assay, a rapid, high-throughput, and inexpensive method for assessing CBEs in plants. Using it, they assess the specificity of 25 rationally designed A3Bctd-BE3s, identifying A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3 with high specificity and precision.