Increasing efficiency and targeting scope of 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. However, the current CBEs are not very efficient especially for the purpose of disease modeling in animals as well as gene therapy. To promote the activity of CBE, we screened 10 non-sequence specific single-strand DNA binding domains (ssDBDs) to fuse with the deaminase to increase its binding affinity with the substrate, and found that fusion of Rad51DBD to BE4max or A3A-BE4max substantially increased the activity (up to 17-fold) and expanded editing window toward PAM in both cell lines and mouse embryos. We also demonstrate that fusion Rad51DBD to eA3A-BE4max selectively catalyzes cytidines in TC motifs with higher activity (up to 250-fold) and broader editing scope comparing with eA3A-BE4max in cell lines.