Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Nature biotechnology 2023 May

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Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing. © 2022. The Author(s).

Citation

Monica E Neugebauer, Alvin Hsu, Mandana Arbab, Nicholas A Krasnow, Amber N McElroy, Smriti Pandey, Jordan L Doman, Tony P Huang, Aditya Raguram, Samagya Banskota, Gregory A Newby, Jakub Tolar, Mark J Osborn, David R Liu. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nature biotechnology. 2023 May;41(5):673-685