Cited by 133CRISPR and Genetic EngineeringRNA regulation and diseaseGenetically Modified Organisms Research

Improved plant cytosine base editors with high editing activity, purity, and specificity

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Ren, Qiurong, Sretenovic, Simon, Liu, Guanqing, Zhong, Zhaohui, Wang, Jiaheng, Huang, Lan, Tang, Xu, Guo, Yachong +13 more·2021·Plant Biotechnology Journal

Abstract

Cytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C-to-T base editing in plants, we first compared seven cytidine deaminases in the BE3-like configuration in rice. We found A3A/Y130F-CBE_V01 resulted in the highest C-to-T base editing efficiency in both rice and Arabidopsis. Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9-mediated C-to-T base editing at A-rich PAMs. To showcase its applications, we first applied A3A/Y130F-CBE_V01 for multiplexed editing to generate microRNA-resistant mRNA transcripts as well as pre-mature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F-CBE_V01 for efficient artificial evolution of novel ALS and EPSPS alleles which conferred herbicide resistance in rice. To further improve C-to-T base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole-genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome-wide off-target effects in rice. Different levels of cytidine deaminase-dependent and sgRNA-independent off-target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome-wide sgRNA-dependent off-target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1-CBE_V04 and A3A/Y130F-CBE_V04 as next-generation plant CBEs with high editing efficiency, purity, and specificity.

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  • Base editing technologies have demonstrated high efficiency and specificity in various crops, including rice and Arabidopsis, allowing for the precise development of desirable traits compared to conventional breeding techniques.

    “Moreover, base editing technologies have demonstrated high efficiency and specificity in various crops, including rice and Arabidopsis, allowing for the precise development of desirable traits compared to conventional breeding techniques (Ren et al., 2021; Lyzenga et al., 2021).”

    From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Ren et al., 2021

    The Results report high editing efficiency in Arabidopsis protoplasts, and the Abstract identifies editors with high efficiency and specificity based on testing that includes rice. The Discussion describes rice trait-engineering examples. The paper does not establish high specificity in Arabidopsis or compare trait development with conventional breeding. Page numbers were not provided in our copy. (LLM, conf=0.88)

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Authors
Ren, Q., Sretenovic, S., Liu, G., Zhong, Z., Wang, J., Huang, L., Tang, X., Guo, Y., Liu, L., Wu, Y., Zhou, J., Zhao, Y., Yang, H., He, Y., Liu, S., Yin, D., Mayorga, R., Zheng, X., Zhang, T., Qi, Y., & Zhang, Y.
Affiliations
  • University of Electronic Science and Technology of China (CN)
  • University of Maryland, College Park (US)
  • Yangzhou University (CN)
  • Hubei Academy of Agricultural Sciences (CN)
  • Institute for Bioscience and Biotechnology Research (US)
Corresponding author
  • Zhang, Tao
  • Qi, Yiping
  • Zhang, Yong
Venue
Plant Biotechnology Journal
Publisher
Wiley
Published
2021-06-07
Volume
19
Issue
10
Pages
2052-2068
ISSN
  • 1467-7644 (ISSN-L)
  • 1467-7652
OpenAlex
W3164291647
Cited by
131
References
78
Citations by year
2026: 17 · 2025: 22 · 2024: 25 · 2023: 38 · 2022: 22 · 2021: 7
FWCI
7.46
Type
journal article
Language
en
Open access
Locations
  • Plant Biotechnology Journal (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • Division of Integrative Organismal Systems (IOS‐1758745, IOS‐2029889)
  • National Natural Science Foundation of China (31771486, 31960423, 32072045)
  • Fundamental Research Funds for the Central Universities (ZYGX2019J127)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Genetically Modified Organisms Research
Keywords
cytosine base editors, cytidine deaminase, off-target effects, single guide RNA, multiplexed genome editing, artificial evolution, herbicide resistance, rice protoplasts, Arabidopsis
MeSH terms
Gene Editing, Whole Genome Sequencing, RNA, Guide, CRISPR-Cas Systems, Cytosine, Mutation, RNA, Guide, Kinetoplastida, CRISPR-Cas Systems

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