Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-strand DNA binding protein domain

Cytidine base editors (CBEs) are powerful genetic tools which catalyze cytidine to thymidine conversion at specific genomic loci, but the efficiency and the editing range are required to be improved. We tested 10 non-sequence-specific single-strand DNA binding domains (ssDBDs) fused to the deaminase, and found that fusion of Rad51DBD to CBEs substantially increased CBE activity (up to 17-fold) and expanded A3A-BE4max editing window toward the PAM site in both cell lines and mouse embryos. Fusion of Rad51DBD to eA3A-BE4max (hyeA3A-BE4max) selectively catalyzed cytidine conversion in TC motifs with higher activity (up to 257-fold), broader editing range and similar accuracy compared with eA3A-BE4max, which was further confirmed in mouse embryos. Moreover, hyeA3A-BE4max selectively generated a C-to-T conversion without inducing a bystander mutation in the hemoglobin gamma (HBG) gene promoter to mimic a naturally occurring genetic variant for amelioration of beta-hemoglobinopathy, suggesting the therapeutic potential of the improved base editors.