RNA-DNA hybrid binding domain broadens the editing window of base editors

Adenine base editors (ABEs) and cytosine base editors (CBEs) are prominent tools for precise genome editing but are hindered by limited editing activity at positions proximal to the protospacer adjacent motif (PAM). This study investigates the potential of enhancing base editors editing activity by fusing them with RNA-DNA hybrid binding domains (RHBDs). Specifically, fusing ABE8e with the RHBD of Homo sapiens RNaseH1 (RHBD1) significantly increased A-to-G editing efficiency in the PAM-proximal region (A9-A15) by up to 3.5-fold, while reducing off-target cytosine editing. Additionally, RHBD1 is compatible with ABEmax, BE4max, and dual base editor (eA&C-BEmax), enhancing their editing activity at the PAM-proximal bases. Notably, RHBD1-fused BE4max led to a 3.1-fold improvement in C-to-T editing efficiency at PAM-proximal region (C9-C12). Furthermore, we demonstrated that RHBD1-fused ABE8e could effectively edit disease-related single nucleotide variations (SNVs) in human cells and validated its efficacy in adult mouse liver. These findings highlight the significance of the RHBD in expanding editing window and the applicability of base editors for gene therapy and disease modeling.