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

Jordan L. Doman; Aditya Raguram; Gregory A. Newby; David R. Liu

doi:10.1038/s41587-020-0414-6

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

Jordan L. Doman, Aditya Raguram, Gregory A. Newby, David R. Liu

Research output: Contribution to journal › Article › peer-review

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.

Original language	English (US)
Pages (from-to)	620-628
Number of pages	9
Journal	Nature biotechnology
Volume	38
Issue number	5
DOIs	https://doi.org/10.1038/s41587-020-0414-6
State	Published - May 1 2020
Externally published	Yes

ASJC Scopus subject areas

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology
Molecular Medicine
Biomedical Engineering

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10.1038/s41587-020-0414-6

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title = "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.",

author = "Doman, {Jordan L.} and Aditya Raguram and Newby, {Gregory A.} and Liu, {David R.}",

note = "Funding Information: 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. Publisher Copyright: {\textcopyright} 2020, The Author(s), under exclusive licence to Springer Nature America, Inc.",

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doi = "10.1038/s41587-020-0414-6",

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AU - Doman, Jordan L.

AU - Raguram, Aditya

AU - Newby, Gregory A.

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N1 - Funding Information: 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. Publisher Copyright: © 2020, The Author(s), under exclusive licence to Springer Nature America, Inc.

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N2 - 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.

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

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