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Strand-preferred base editing of organellar and nuclear genomes using CyDENT

Abstract

Transcription-activator-like effector (TALE)-based tools for base editing of nuclear and organellar DNA rely on double-stranded DNA deaminases, which edit substrate bases on both strands of DNA, reducing editing precision. Here, we present CyDENT base editing, a CRISPR-free, strand-selective, modular base editor. CyDENT comprises a pair of TALEs fused with a FokI nickase, a single-strand-specific cytidine deaminase and an exonuclease to generate a single-stranded DNA substrate for deamination. We demonstrate effective base editing in nuclear, mitochondrial and chloroplast genomes. At certain mitochondrial sites, we show editing efficiencies of 14% and strand specificity of 95%. Furthermore, by exchanging the CyDENT deaminase with one that prefers editing GC motifs, we demonstrate up to 20% mitochondrial base editing at sites that are otherwise inaccessible to editing by other methods. The modular nature of CyDENT enables a suite of bespoke base editors for various applications.

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Fig. 1: Strand-selective base editing using CyDENT in plant cells.
Fig. 2: Strand-selective mtDNA base editing using CyDENT in HEK293T cells.
Fig. 3: Optimization of CyDENT for mtDNA base editing.
Fig. 4: mtCyDENT enables efficient strand-selective base editing under GC contexts.
Fig. 5: Whole nuclear genome and mitochondrial genome-wide off-target analysis of mtCyDENT.

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Data availability

The deep amplicon sequencing data are deposited at the NCBI as Bioproject PRJNA957099 and PRJNA957096 ref. 54. All other data are available in the paper or Supplementary Information.

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Acknowledgements

We thank Y. Ai for kindly providing the HEK293T cell line. We also thank C. Yi for providing DdCBE vectors targeting ND1, ND4, ND5.1 and ND6. We are grateful to Y. Wang for the helpful discussions. We acknowledge Y. Li for assistance with figure drawing. We thank J. Huang at BGI Genomics for the whole-genome sequencing service. This work was supported by the Ministry of Agriculture and Rural Affairs of China, the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020102), the National Key Research and Development Program (2022YFF1002802) and the National Natural Science Foundation of China (32388201).

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J.H., K.T.Z. and C.G. conceived the project and designed the experiments; J.H. designed vectors and tested mtCyDENT in HEK293T cells; Y.S. and B.L. designed vectors and tested nuCyDENT and cpCyDENT in plants; J.H. and Y.S. collected and analyzed MiSeq data; J.H., Y.S. and G.L. prepared MiSeq samples; Z.L. and M.G. prepared plasmids and HEK293T cells; Z.W. and Q.G. wrote scripts and processed the MiSeq RAW data; Q.G. processed the whole-genome sequencing RAW data; J.H., Y.S. and B.L. prepared the figures; J.H., Y.S., B.L., K.T.Z. and C.G. wrote the manuscript with input from all authors; and C.G. and K.T.Z. supervised the study.

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Correspondence to Kevin Tianmeng Zhao or Caixia Gao.

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Competing interests

The authors have submitted a patent application based on the results reported in this paper. K.T.Z. is a founder and employee at Qi Biodesign. Z.L., Z.W., Q.G. and M.G. are employees of Qi Biodesign.

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Hu, J., Sun, Y., Li, B. et al. Strand-preferred base editing of organellar and nuclear genomes using CyDENT. Nat Biotechnol 42, 936–945 (2024). https://doi.org/10.1038/s41587-023-01910-9

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