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

Nat Biotechnol
Publication type
Journal Article
Authors
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.

Year of Publication
2022
Journal
Nat Biotechnol
Date Published
2022 Nov 10
ISSN
1546-1696
DOI
10.1038/s41587-022-01533-6
PubMed ID
36357719
Links
Grant list
Liu investigatorship / Howard Hughes Medical Institute (HHMI)
R01EB027793 / U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)
R01EB031172 / U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)
U01AI142756 / Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)
R35GM118062 / U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)
RM1HG009490 / U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)