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Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors

Abstract

Cytosine base editors (CBEs) enable targeted C•G-to-T•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•G-to-T•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 ~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.

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Fig. 1: On-target and Cas9-independent off-target DNA editing in E. coli.
Fig. 2: Cas9-independent deamination by CBEs in HEK293T cells.
Fig. 3: YE1 balances efficient on-target editing with greatly decreased Cas9-independent editing as confirmed by WGS.
Fig. 4: Expanding the utility of CBEs with decreased Cas9-independent off-targets through protein engineering.

Data availability

High-throughput sequencing and whole-genome sequencing data are deposited in the NCBI Sequence Read Archive (PRJNA553240). Plasmids used in this study are available from Addgene. Amino acid sequences of all base editors in this study are provided in the Supplementary Sequences.

Code availability

The script used to analyze the SNVs reported by Yang and coworkers12 is provided in Supplementary Note 1. The script and parameters used for running CRISPResso2 analyses are provided in Supplementary Note 2. The script used for calculating the number of pathogenic SNPs targetable by CBEs is provided in Supplementary Note 4.

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Acknowledgements

We thank B. Thuronyi for discussions; K. Zhao, T. Huang and S. Miller for providing guide RNA plasmids; and B. Mok for providing an sgRNA for in vitro experiments. M. Osborn, Aldevron and the Kidz1stFund provided the BE4 protein used for protein delivery experiments, and K. Tian and S. Pandey provided assistance with experiments. We thank D. Court for providing pSIM5, which was used for recombineering the HSV-TK gene onto the E. coli chromosome. We thank T. Mason and E. LaRoche for assistance with WGS library preparation and FAS Research Computing at Harvard University for computational resources. This work was supported by US NIH grant nos. U01 AI142756, RM1 HG009490, R35 GM118062 and HHMI; the Bill and Melinda Gates Foundation; and the St. Jude Collaborative Research Consortium. A.R. is an NSF Graduate Research Fellow and was supported by NIH Training Grant no. T32 GM095450. J.L.D. is supported by the Hertz Foundation and the NSF GRFP fellowship. G.A.N. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation. Flow cytometry was supported by Cancer Center Support (core) Grant no. P30-CA14051 from the NCI.

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J.L.D., A.R. and D.R.L. designed the research. A.R. and J.L.D. performed experiments. G.A.N. performed the BE4 protein delivery off-target experiment. All authors contributed to writing the manuscript.

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Correspondence to David R. Liu.

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D.R.L. is a consultant and cofounder of Editas Medicine, Pairwise Plants, Beam Therapeutics and Prime Medicine, companies that use genome editing. J.L.D., A.R. and D.R.L. through the Broad Institute have filed patent applications on aspects of this work.

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Supplementary Figs. 1–24, Tables 1–5, Notes 1–5 and Sequences.

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Doman, J.L., Raguram, A., Newby, G.A. et al. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat Biotechnol 38, 620–628 (2020). https://doi.org/10.1038/s41587-020-0414-6

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