Journal
NATURE BIOTECHNOLOGY
Volume 38, Issue 5, Pages 620-+Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41587-020-0414-6
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Funding
- US NIH [U01 AI142756, RM1 HG009490, R35 GM118062]
- HHMI
- Bill and Melinda Gates Foundation
- St. Jude Collaborative Research Consortium
- NIH [T32 GM095450]
- Hertz Foundation
- NSF GRFP fellowship
- NCI [P30-CA14051]
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Cytosine base editors (CBEs) enable targeted C center dot G-to-T center dot A conversions in genomic DNA. Recent studies report that BE3, the original CBE, induces a low frequency of genome-wide Cas9-independent off-target C center dot G-to-T center dot A mutation in mouse embryos and in rice. Here we develop multiple rapid, cost-effective methods to screen the propensity of different CBEs to induce Cas9-independent deamination in Escherichia coli and in human cells. We use these assays to identify CBEs with reduced Cas9-independent deamination and validate via whole-genome sequencing that YE1, a narrowed-window CBE variant, displays background levels of Cas9-independent off-target editing. We engineered YE1 variants that retain the substrate-targeting scope of high-activity CBEs while maintaining minimal Cas9-independent off-target editing. The suite of CBEs characterized and engineered in this study collectively offer similar to 10-100-fold lower average Cas9-independent off-target DNA editing while maintaining robust on-target editing at most positions targetable by canonical CBEs, and thus are especially promising for applications in which off-target editing must be minimized. Methods to efficiently detect Cas9-independent cytosine base editor off-target activity enable the identification and development of variants with minimal off-target editing and robust on-target editing.
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