Journal
NATURE BIOTECHNOLOGY
Volume 36, Issue 10, Pages 977-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.4199
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Funding
- National Institutes of Health [R35 GM118158, RM1 HG009490]
- Desmond and Ann Heathwood MGH Research Scholar Award
- St. Jude Children's Research Hospital Collaborative Research Consortium award
- National Science Foundation Graduate Research Fellowship Program
- NHLBI [DP2OD022716, P01HL032262]
- St. Jude Children's Research Hospital Collaborative Research Consortium
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [DP2HL137300, P01HL032262] Funding Source: NIH RePORTER
- NATIONAL HUMAN GENOME RESEARCH INSTITUTE [RM1HG009490] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R35GM118158] Funding Source: NIH RePORTER
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Base editor technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine DNA alterations(1-6). However, existing base editors create unwanted C-to-T alterations when more than one C is present in the enzyme's five-base-pair editing window. Here we describe a strategy for reducing bystander mutations using an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN hierarchy. In direct comparisons with the widely used base editor 3 (BE3) fusion in human cells, our eA3A-BE3 fusion exhibits similar activities on cytidines in TC motifs but greatly reduced editing on cytidines in other sequence contexts. eA3A-BE3 corrects a human beta-thalassemia promoter mutation with much higher (>40-fold) precision than BE3. We also demonstrate that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites.
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