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Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A

Nature Biotechnology volume 36, pages 950–953 (2018)Cite this article

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Abstract

Base editors (BEs) have been used to create C-to-T substitutions in various organisms. However, editing with rat APOBEC1-based BE3 is limited to a 5-nt sequence editing window and is inefficient in GC contexts. Here, we show that a base editor fusion protein composed of Cas9 nickase and human APOBEC3A (A3A-PBE) converts cytidine to thymidine efficiently in wheat, rice and potato with a 17-nucleotide editing window at all examined sites, independent of sequence context.

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Figure 1: Comparison of C-to-T base editing by A3A-PBE and PBE.
Figure 2: A3A-PBE is widely useful for C-to-T base editing.

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Acknowledgements

We thank M. Andersson for technical support on potato protoplast regeneration and Y. Huo for technical support on flow cytometry. This work was supported by grants from the National Key Research and Development Program of China (2016YFD0101804), the National Natural Science Foundation of China (31788103 and 31420103912), the National Transgenic Science and Technology Program (2016ZX08010002, 2018ZX0800102B-001 and 2018ZX0801002B-002) and the Chinese Academy of Sciences (QYZDY-SSW-SMC030 and GJHZ1602).

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Author notes

  1. Yuan Zong and Qianna Song: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Yuan Zong, Qianna Song, Chao Li, Shuai Jin, Dingbo Zhang, Yanpeng Wang & Caixia Gao

  2. University of Chinese Academy of Sciences, Beijing, China

    Yuan Zong, Qianna Song, Chao Li, Shuai Jin, Dingbo Zhang & Caixia Gao

  3. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Jin-Long Qiu

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  1. Yuan Zong
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Contributions

Y.Z. and C.G. designed the experiments; Y.Z., Q.S. and C.L. performed most of the experiments; D.Z. purified the protein. S.J. and Y.W. analyzed the results; C.G. supervised the project; Y.Z., J.-L.Q. and C.G. wrote the manuscript.

Corresponding author

Correspondence to Caixia Gao.

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Competing interests

The authors have submitted a patent application (application no. 201810816603.7) based on the results reported in this paper.

Integrated supplementary information

Supplementary Figure 1 Product purity of cytosine base editing for the wheat genomic loci tested.

Product distributions and indels frequencies at four representative wheat genomic DNA sites in wheat protoplasts treated with PBE, A3A-PBE and A3A-Gam are shown. A total of 19,000-140,000 sequencing reads were used at every position.

Supplementary Figure 2 Product purity of cytosine base editing for the rice genomic loci tested.

Product distributions and indels frequencies at six representative rice genomic DNA sites in rice protoplasts treated with PBE, A3A-PBE and A3A-Gam are shown. A total of 25,000-131,000 sequencing reads were used at every position.

Supplementary Figure 3 Frequencies of indels in the ten target sites on the wheat and rice genomes.

Indel frequencies induced by PBE, A3A-PBE, A3A-Gam and Cas9 were measured. Frequencies (mean ± s.e.m.) were calculated using the data from three biologically independent experiments (n=3).

Supplementary Figure 4 Comparison of C-to-T base editing efficiency between A3A-PBE and PBE base editors in potato protoplasts.

(a) Schematic representation of two cytosine base editors and one sgRNA vectors. (b) Sequence of the sgRNA targeting StALS and StGBSS. The C bases in the deamination window are highlighted in blue. The PAM sequences are shown in red. (c) Frequencies of indels in the ten target sites of potato. Indels frequencies induced by PBE, A3A-PBE and Cas9 with relative sgRNA. Frequencies (mean ± s.e.m.) were calculated using the data from three biologically independent experiments (n=3).

Supplementary Figure 5 Identification and analysis of the wheat plantlets with targeted C-to-T conversions by A3A-PBE.

(a) Sequence of the sgRNA targeting a conserved region of the exon of TaALS homoeologues. The C bases in the deamination window are highlighted in red. The protospacer-adjacent motif (PAM) sequence is highlighted in red bold and the EcoO109I restriction site is underlined. (b) PCR-RE analyses of 10 representative taals mutants. Lanes T0-1 to T0-10 show PCR fragments amplified from independent wheat plants that digested with EcoO109I. Lanes labelled WT/D and WT/U are PCR fragments amplified from WT plants with and without EcoO109I digestion, respectively. The bands marked by red arrowheads indicate positive base editing.

Supplementary Figure 6 Constructs used for base editing of TaALS and Ta MTL and detection of transgene integration in the resultant T0 mutants.

(a) Diagram of the A3A-PBE and pTaU6-sgRNA vectors used for base editing of TaALS and TaMTL. The positions of the five primer sets (F1/R1, F2/R2, F3/R3, F4/R4 and F5/R5) used for detecting transgene integration are shown. (b) Outcome of the tests for transgene integration using five primer sets for ten represented taals mutant plants and ten tamtl mutants. None of the five primer sets yielded the expected PCR amplicon in four mutants for TaALS (T0-3, T0-5, T0-6 and T0-7) and six mutants for TaMTL (T0-1, T0-2, T0-3, T0-5, T0-6 and T0-9), indicating that they were transgene free. The negative control for the tests was performed using the genomic DNA extracted from wild type wheat plants (cv Kenong 199). The positive control for the tests was conducted with the plasmid DNA of A3A-PBE or pTaU6-sgRNA. Data were from three biologically independent experiments (n=3).

Supplementary Figure 7 Purified A3A-PBE- ΔUGI protein analyzed by SDS–PAGE.

Three μg purified protein was separated on 10% SDS-PAGE, and was then visualized by Coomassie Blue staining.

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Supplementary Sequences

Supplementary Sequences (PDF 3433 kb)

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Zong, Y., Song, Q., Li, C. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat Biotechnol 36, 950–953 (2018). https://doi.org/10.1038/nbt.4261

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