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

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

doi:10.1038/s41587-022-01533-6

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

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

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9 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	673-685
Number of pages	13
Journal	Nature biotechnology
Volume	41
Issue number	5
DOIs	https://doi.org/10.1038/s41587-022-01533-6
State	Published - May 2023

Bibliographical note

Funding Information:
This work was supported by US National Institutes of Health (NIH) grants R01EB027793, R01EB031172, U01AI142756, R35GM118062, RM1HG009490 and R01AR063070-08; the Bill and Melinda Gates Foundation; and the Howard Hughes Medical Institute. We thank P. Chen, K. Everette, D. Nelson, A. Sousa and J. Queenan for materials, discussion and technical advice. M.E.N. was supported by a Ruth L. Kirschstein National Research Service Awards Postdoctoral Fellowship (GM143776-02). A.H. is a National Science Foundation (NSF) Graduate Research Fellow. M.A. is a recipient of the NIH Pathway to Independence Award (K99/R00NS119743). J.L.D. is supported by the Hertz Foundation and the NSF Graduate Research Fellowship Program. A.R. is an NSF Graduate Research Fellow and was supported by an NIH training grant (T32 GM095490). G.A.N. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation. M.J.O. receives funding from the Bill and Melinda Gates Foundation, the Saint Baldrick’s Foundation and the Kidz1stFund. J.T. receives funding from NIH R01 AR063070. For sourcing HSPCs, we acknowledge support from the National Institute of Diabetes and Digestive and Kidney Diseases under award U54DK106829: Fred Hutchinson Cancer Center Cooperative Center of Excellence in Hematology.

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© 2022, The Author(s).

PubMed: MeSH publication types

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, N.I.H., Extramural

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Neugebauer, M. E., Hsu, A., Arbab, M., Krasnow, N. A., McElroy, A. N., Pandey, S., Doman, J. L., Huang, T. P., Raguram, A., Banskota, S., Newby, G. A., Tolar, J., Osborn, M. J., & Liu, D. R. (2023). Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nature biotechnology, 41(5), 673-685. https://doi.org/10.1038/s41587-022-01533-6

Neugebauer, ME, Hsu, A, Arbab, M, Krasnow, NA, McElroy, AN, Pandey, S, Doman, JL, Huang, TP, Raguram, A, Banskota, S, Newby, GA, Tolar, J , Osborn, MJ & Liu, DR 2023, 'Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity', Nature biotechnology, vol. 41, no. 5, pp. 673-685. https://doi.org/10.1038/s41587-022-01533-6

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

abstract = "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.",

author = "Neugebauer, {Monica E.} and Alvin Hsu and Mandana Arbab and Krasnow, {Nicholas A.} and McElroy, {Amber N.} and Smriti Pandey and Doman, {Jordan L.} and Huang, {Tony P.} and Aditya Raguram and Samagya Banskota and Newby, {Gregory A.} and Jakub Tolar and Osborn, {Mark J.} and Liu, {David R.}",

note = "Funding Information: This work was supported by US National Institutes of Health (NIH) grants R01EB027793, R01EB031172, U01AI142756, R35GM118062, RM1HG009490 and R01AR063070-08; the Bill and Melinda Gates Foundation; and the Howard Hughes Medical Institute. We thank P. Chen, K. Everette, D. Nelson, A. Sousa and J. Queenan for materials, discussion and technical advice. M.E.N. was supported by a Ruth L. Kirschstein National Research Service Awards Postdoctoral Fellowship (GM143776-02). A.H. is a National Science Foundation (NSF) Graduate Research Fellow. M.A. is a recipient of the NIH Pathway to Independence Award (K99/R00NS119743). J.L.D. is supported by the Hertz Foundation and the NSF Graduate Research Fellowship Program. A.R. is an NSF Graduate Research Fellow and was supported by an NIH training grant (T32 GM095490). G.A.N. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation. M.J.O. receives funding from the Bill and Melinda Gates Foundation, the Saint Baldrick{\textquoteright}s Foundation and the Kidz1stFund. J.T. receives funding from NIH R01 AR063070. For sourcing HSPCs, we acknowledge support from the National Institute of Diabetes and Digestive and Kidney Diseases under award U54DK106829: Fred Hutchinson Cancer Center Cooperative Center of Excellence in Hematology. Publisher Copyright: {\textcopyright} 2022, The Author(s).",

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AU - Krasnow, Nicholas A.

AU - McElroy, Amber N.

AU - Pandey, Smriti

AU - Doman, Jordan L.

AU - Huang, Tony P.

AU - Raguram, Aditya

AU - Banskota, Samagya

AU - Newby, Gregory A.

AU - Tolar, Jakub

AU - Osborn, Mark J.

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N2 - 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.

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

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