Increase base editing efficiency and product variety through fusion of Cas9n to cytosine and adenosine deaminases

Base editors, which are created through the fusion of Cas9 nickase to either cytidine or adenosine deaminase, efficiently catalyze site-specific nucleotide conversions with minimal indel rates1, 2. However, cytidine (CBE) or adenosine base editor (ABE) generates base conversion of only one nucleotide type. Here we developed a novel adenosine/cytidine-base editor (A&C-BE) through fusion of AID and TadA deaminases with Cas9n. A&C-BE is able to generate C-to-T and A-to-G conversions in the same allele to significantly increase the product multiplicity. Moreover, A&C-BE showed much higher editing activity of cytidine deaminase and greatly reduced RNA off-targeting effects comparing to ABE. We also demonstrate that A&C-BE induced efficient simultaneous A/C mutations in adjacent sites in a promoter region to strongly stimulate HBG expression for the therapy of β-hemoglobinopathies, indicating its therapeutic potential. It also exhibits a broad potential for investigation and modification of both coding and non-coding sequences at a single base resolution