Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference

Emily Zhang, Monica E. Neugebauer, Nicholas A. Krasnow and David R. Liu ()
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Emily Zhang: Broad Institute of MIT and Harvard
Monica E. Neugebauer: Broad Institute of MIT and Harvard
Nicholas A. Krasnow: Broad Institute of MIT and Harvard
David R. Liu: Broad Institute of MIT and Harvard

Nature Communications, 2024, vol. 15, issue 1, 1-13

Abstract: Abstract TadA-derived cytosine base editors (TadCBEs) enable programmable C•G-to-T•A editing while retaining the small size, high on-target activity, and low off-target activity of TadA deaminases. Existing TadCBEs, however, exhibit residual A•T-to-G•C editing at certain positions and lower editing efficiencies at some sequence contexts and with non-SpCas9 targeting domains. To address these limitations, we use phage-assisted evolution to evolve CBE6s from a TadA-mediated dual cytosine and adenine base editor, discovering mutations at N46 and Y73 in TadA that prevent A•T-to-G•C editing and improve C•G-to-T•A editing with expanded sequence-context compatibility, respectively. In E. coli, CBE6 variants offer high C•G-to-T•A editing and no detected A•T-to-G•C editing in any sequence context. In human cells, CBE6 variants exhibit broad Cas domain compatibility and retain low off-target editing despite exceeding BE4max and previous TadCBEs in on-target editing efficiency. Finally, we show that the high selectivity of CBE6 variants is well-suited for therapeutically relevant stop codon installation without creating unwanted missense mutations from residual A•T-to-G•C editing.

Date: 2024
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DOI: 10.1038/s41467-024-45969-7

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