Increasing the efficiency and targeting scope 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 DNA, and further improvement of the editing scope and efficiency is critical for their broader applications. Through insertion of asingle-strand DNA binding domain (ssDBD) from Rad51 protein between Cas9n and the deaminases, serial hyper CBEs (hyCBEs) were generated with substantially increased activity and an expanded editing window toward the protospacer adjacent motif (PAM) in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalyzed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations 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.