Increase base editing efficiency and product variety through fusion of Cas9 nickase to cytosine and adenosine deaminases

Base editors, which are created through fusion of Cas9 nickase to either cytidine or adenine deaminase, efficiently catalyze site-specific nucleotide conversions with minimal indel rates. However, cytidine base editor (CBE) or adenine base editor (ABE) is able to only generate base conversion on single nucleotide type which limits the product multiplicity. Here we show that through fusion of cytidine and adenine deaminases with Cas9n, a novel cytidine-adenine base editor (ACBE) was developed. ACBE is able to generate C>T and A>G conversions in the same allele. Moreover, ACBE showed much higher (up to 14-fold) editing activity of cytidine deaminase with similar activity for adenine conversion compared to CBE and ABE respectively. We also demonstrate ACBE induced efficient simultaneous A>G and C>T mutations in adjacent sites to disrupt a inhibitory element and create an activation site on promoter region to strongly stimulate HBG expression, indicating potential clinical application for treatment of β-hemoglobinopathies.