Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity

Shuai Jin; Hongyuan Fei; Zixu Zhu; Yingfeng Luo; Jinxing Liu; Shenghan Gao; Feng Zhang; Yu Hang Chen; Yanpeng Wang; Caixia Gao

doi:10.1016/j.molcel.2020.07.005

Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity

Shuai Jin, Hongyuan Fei, Zixu Zhu, Yingfeng Luo, Jinxing Liu, Shenghan Gao, Feng Zhang, Yu Hang Chen, Yanpeng Wang, Caixia Gao

Plant and Microbial Biology

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

59 Scopus citations

Abstract

Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.

Original language	English (US)
Pages (from-to)	728-740.e6
Journal	Molecular Cell
Volume	79
Issue number	5
DOIs	https://doi.org/10.1016/j.molcel.2020.07.005
State	Published - Sep 3 2020

Bibliographical note

Funding Information:
We thank Dr. Fei Lu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences [IGDB, CAS]) for advice in the statistics analysis. This work was supported by grants from the National Transgenic Science and Technology Program (2019ZX08010-003 and 2019ZX08010-001), the Strategic Priority Research Program of the CAS (Precision Seed Design and Breeding, XDA24020102), the National Natural Science Foundation of China (31788103), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020000003), and by a grant from the United Soybean Board (1920-152-0131-B) to F.Z. Y.W. and C.G. designed the experiments. S.J. H.F. and Z.Z. performed most of the experiments. Y.L. S.J. and S.G. performed the WGS analysis. J.L. performed the rice transformation experiments. Y.-H.C. analyzed the structure of the A3Bctd protein. Y.W. and C.G. supervised the project. S.J. H.F. Z.Z. Y.W. F.Z. and C.G. wrote the manuscript. The authors have submitted two patent applications based on the results reported in this article.

Funding Information:
We thank Dr. Fei Lu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences [IGDB, CAS]) for advice in the statistics analysis. This work was supported by grants from the National Transgenic Science and Technology Program ( 2019ZX08010-003 and 2019ZX08010-001 ), the Strategic Priority Research Program of the CAS (Precision Seed Design and Breeding, XDA24020102 ), the National Natural Science Foundation of China ( 31788103 ), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020000003), and by a grant from the United Soybean Board ( 1920-152-0131-B ) to F.Z.

Publisher Copyright:
© 2020 Elsevier Inc.

Keywords

A3Bctd deaminase
cytosine base editors
nSaCas9
off-target editing
rationally designing
specificity
whole-genome sequencing
Reproducibility of Results
Genes, Plant
Humans
Oryza/genetics
Minor Histocompatibility Antigens/genetics
Cytidine Deaminase/genetics
Gene Editing/methods
Cytosine/chemistry
RNA, Plant/chemistry
RNA, Guide/chemistry
Mutation

PubMed: MeSH publication types

Research Support, Non-U.S. Gov't
Journal Article
Validation Study

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10.1016/j.molcel.2020.07.005

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@article{5d79ba13ccc14c7cbfc01c74749ec506,

title = "Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity",

abstract = "Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.",

keywords = "A3Bctd deaminase, cytosine base editors, nSaCas9, off-target editing, rationally designing, specificity, whole-genome sequencing, Reproducibility of Results, Genes, Plant, Humans, Oryza/genetics, Minor Histocompatibility Antigens/genetics, Cytidine Deaminase/genetics, Gene Editing/methods, Cytosine/chemistry, RNA, Plant/chemistry, RNA, Guide/chemistry, Mutation",

author = "Shuai Jin and Hongyuan Fei and Zixu Zhu and Yingfeng Luo and Jinxing Liu and Shenghan Gao and Feng Zhang and Chen, {Yu Hang} and Yanpeng Wang and Caixia Gao",

note = "Funding Information: We thank Dr. Fei Lu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences [IGDB, CAS]) for advice in the statistics analysis. This work was supported by grants from the National Transgenic Science and Technology Program (2019ZX08010-003 and 2019ZX08010-001), the Strategic Priority Research Program of the CAS (Precision Seed Design and Breeding, XDA24020102), the National Natural Science Foundation of China (31788103), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020000003), and by a grant from the United Soybean Board (1920-152-0131-B) to F.Z. Y.W. and C.G. designed the experiments. S.J. H.F. and Z.Z. performed most of the experiments. Y.L. S.J. and S.G. performed the WGS analysis. J.L. performed the rice transformation experiments. Y.-H.C. analyzed the structure of the A3Bctd protein. Y.W. and C.G. supervised the project. S.J. H.F. Z.Z. Y.W. F.Z. and C.G. wrote the manuscript. The authors have submitted two patent applications based on the results reported in this article. Funding Information: We thank Dr. Fei Lu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences [IGDB, CAS]) for advice in the statistics analysis. This work was supported by grants from the National Transgenic Science and Technology Program ( 2019ZX08010-003 and 2019ZX08010-001 ), the Strategic Priority Research Program of the CAS (Precision Seed Design and Breeding, XDA24020102 ), the National Natural Science Foundation of China ( 31788103 ), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020000003), and by a grant from the United Soybean Board ( 1920-152-0131-B ) to F.Z. Publisher Copyright: {\textcopyright} 2020 Elsevier Inc.",

year = "2020",

month = sep,

day = "3",

doi = "10.1016/j.molcel.2020.07.005",

language = "English (US)",

volume = "79",

pages = "728--740.e6",

journal = "Molecular Cell",

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T1 - Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity

AU - Jin, Shuai

AU - Fei, Hongyuan

AU - Zhu, Zixu

AU - Luo, Yingfeng

AU - Liu, Jinxing

AU - Gao, Shenghan

AU - Zhang, Feng

AU - Chen, Yu Hang

AU - Wang, Yanpeng

AU - Gao, Caixia

N1 - Funding Information: We thank Dr. Fei Lu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences [IGDB, CAS]) for advice in the statistics analysis. This work was supported by grants from the National Transgenic Science and Technology Program (2019ZX08010-003 and 2019ZX08010-001), the Strategic Priority Research Program of the CAS (Precision Seed Design and Breeding, XDA24020102), the National Natural Science Foundation of China (31788103), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020000003), and by a grant from the United Soybean Board (1920-152-0131-B) to F.Z. Y.W. and C.G. designed the experiments. S.J. H.F. and Z.Z. performed most of the experiments. Y.L. S.J. and S.G. performed the WGS analysis. J.L. performed the rice transformation experiments. Y.-H.C. analyzed the structure of the A3Bctd protein. Y.W. and C.G. supervised the project. S.J. H.F. Z.Z. Y.W. F.Z. and C.G. wrote the manuscript. The authors have submitted two patent applications based on the results reported in this article. Funding Information: We thank Dr. Fei Lu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences [IGDB, CAS]) for advice in the statistics analysis. This work was supported by grants from the National Transgenic Science and Technology Program ( 2019ZX08010-003 and 2019ZX08010-001 ), the Strategic Priority Research Program of the CAS (Precision Seed Design and Breeding, XDA24020102 ), the National Natural Science Foundation of China ( 31788103 ), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020000003), and by a grant from the United Soybean Board ( 1920-152-0131-B ) to F.Z. Publisher Copyright: © 2020 Elsevier Inc.

PY - 2020/9/3

Y1 - 2020/9/3

N2 - Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.

AB - Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.

KW - A3Bctd deaminase

KW - cytosine base editors

KW - nSaCas9

KW - off-target editing

KW - rationally designing

KW - specificity

KW - whole-genome sequencing

KW - Reproducibility of Results

KW - Genes, Plant

KW - Humans

KW - Oryza/genetics

KW - Minor Histocompatibility Antigens/genetics

KW - Cytidine Deaminase/genetics

KW - Gene Editing/methods

KW - Cytosine/chemistry

KW - RNA, Plant/chemistry

KW - RNA, Guide/chemistry

KW - Mutation

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DO - 10.1016/j.molcel.2020.07.005

M3 - Article

C2 - 32721385

AN - SCOPUS:85089375155

SN - 1097-2765

VL - 79

SP - 728-740.e6

JO - Molecular Cell

JF - Molecular Cell

IS - 5

ER -

Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity

Abstract

Bibliographical note

Keywords

PubMed: MeSH publication types

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