Triple base editor catalyzes saturation mutation of adenine, cytidine, and guanine

Current base editors act on a maximum of two base substrates and generate limited base conversions or transversions, hindering their applicability for inducing DNA sequence diversity. Here, we engineered a triple base editor (named ACG-BEs) using a fusion of adenine base editor with high A/C catalytic activity and evolved N-methylpurine DNA glycosylase. ACG-BEs enables efficient, multiplexed saturation mutagenesis across adenine (A), cytosine (C), and guanine (G), achieving conversion efficiencies of up to 80.5% for A-to-G/C/T, 75.8% for C-to-T/G/A, and 63.4% for G-to-C/T/A in HEK293T cells. Leveraging ACG-BEs, we identify novel mutations in the HBG1/2 promoter region that confer efficient activation of γ-globin expression in HUDEP-2 cells-a promising advancement for therapeutic strategies targeting hemoglobinopathies. These findings highlight ACG-BEs as a cutting-edge platform for multiplexed saturation mutagenesis, offering broad applications in genetic screening and therapeutic base mutation introduction through enhanced DNA sequence diversity.