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Split complementation of base editors to minimize off-target edits

Nature Plants volume 9, pages 1832–1847 (2023)Cite this article

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Abstract

Base editors (BEs) empower the efficient installation of beneficial or corrective point mutations in crop and human genomes. However, conventional BEs can induce unpredictable guide RNA (gRNA)-independent off-target edits in the genome and transcriptome due to spurious activities of BE-enclosing deaminases, and current improvements mostly rely on deaminase-specific mutagenesis or exogenous regulators. Here we developed a split deaminase for safe editing (SAFE) system applicable to BEs containing distinct cytidine or adenosine deaminases, with no need of external regulators. In SAFE, a BE was properly split at a deaminase domain embedded inside a Cas9 nickase, simultaneously fragmenting and deactivating both the deaminase and the Cas9 nickase. The gRNA-conditioned BE reassembly conferred robust on-target editing in plant, human and yeast cells, while minimizing both gRNA-independent and gRNA-dependent off-target DNA/RNA edits. SAFE also substantially increased product purity by eliminating indels. Altogether, SAFE provides a generalizable solution for BEs to suppress off-target editing and improve on-target performance.

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Fig. 1: Development of the split CBE complementation system.
Fig. 2: Split-AID10 exhibits improved on-target performance.
Fig. 3: Split-AID10 exhibits minimal off-target effects.
Fig. 4: Expansion of the SAFE system to CBEs with APOBEC deaminases.
Fig. 5: Expansion of the SAFE system to a TadA8e-based ABE.
Fig. 6: Split-ABE8e exhibits minimal gRNA-independent off-target effects.

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Data availability

The amplicon data, WGS data and RNA-seq data reported in this paper can be found at the CNGB Sequence Archive of the China National GeneBank DataBase with the accession number CNP0004043. Amino acid sequences of different SAFE BEs and target amplicon sequences for the gRNAs are provided in the Supplementary Information. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China grant no. 2019YFA0906202, the Guangdong Provincial Key Project for Basic and Applied Basic Research (Cornerstone grant) and the National Natural Science Foundation of China (NSFC) grant no. 32125004 to J.-F.L.; NSFC grant no. 32293191 to X.H.; NSFC grant no. 32200494 and the China Postdoctoral Science Foundation grant no. 2022M723662 to K.L.; and NSFC grant no. 32100485 to F.-N.X. We thank Azenta Life Sciences (Suzhou, China) for help in deep sequencing and Y. Chen for assistance in flow cytometry.

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  1. These authors contributed equally: Xiangyu Xiong, Kehui Liu.

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  1. State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    Xiangyu Xiong, Kehui Liu, Zhenxiang Li, Fan-Nv Xia, Xue-Ming Ruan, Xionglei He & Jian-Feng Li

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Contributions

J.-F.L. and K.L. conceived and designed the study. X.X., K.L., Z.L., F.-N.X. and X.-M.R. performed the experiments. K.L. and X.X. analysed the data. J.-F.L. and X.H. supervised the research. J.-F.L. wrote the manuscript. All authors approved the final version of the paper.

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Correspondence to Kehui Liu or Jian-Feng Li.

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X.X., K.L., Z.L., X.H. and J.-F.L. have been granted a China invention patent (ZL202210503831.5) based on some results reported in this paper. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 N- or C-terminal split fragment of PIGS-AID10 alone is catalytically inactive.

a, PIGS-AID10 exhibits higher editing efficiency than N-AID10. Comparison of C-to-T editing efficiencies between PIGS-AID10 and N-AID10 was conducted at indicated Arabidopsis endogenous genomic loci in protoplasts by deep sequencing of target amplicons. Data are shown as mean values of two biological replicates. Ctrl corresponds to cells transfected with the gRNA only. b, Amino acid sequence of AID10 with seven candidate split sites highlighted in red and the catalytic center shadowed in blue. c,d, None of the N-terminal (c) or C-terminal (d) split fragments of PIGS-AID10 alone can induce CBE reporter activity in the presence of gRNA. The gRNA only was used as a negative control (Ctrl). Relative luciferase (LUC) activity was calculated by setting the LUC activity of the unsplit CBE (PIGS-AID10) as 100%. Data are shown as mean values of two biological replicates.

Extended Data Fig. 2 Split-AID10 can edit the AtALS locus to confer herbicide resistance.

a, The AtALS target locus. Intended C·G to T·A (red) conversion by Split-AID10 would introduce an A-to-T amino acid substitution. The PAM is in green. b, Summary of Split-AID10 editing outcome in transgenic Arabidopsis T1 plants. The editing efficiency was calculated as the percentage of transgenic plants containing indicated mutation type at the target site. WT, Ho, He, Bi, and Chi denote wild type, homozygous, heterozygous, biallelic, and chimeric plants, respectively. c, Sanger sequencing validates homozygous C-to-T mutation introduced by Split-AID10. d, Homozygous C-to-T mutation at the AtALS locus confers the plant with imazethapyr herbicide resistance. Transgenic Arabidopsis T1 plants of 40-day-old were sprayed with 30 mg/l imazethapyr (IMZ) herbicide once and were photographed in one month. Unedited plants died of imazethapyr toxicity.

Extended Data Fig. 3 Split-AID10 enables robust on-target editing in human and yeast cells.

a,c, Split-AID10 exhibits sufficiently high on-target editing efficiency in human HEK293T cells (a) and yeast cells (c). Data are shown as mean values and standard deviation of three biological replicates for six target loci and the highest C-to-T editing efficiency among multiple Cs was used to represent the editing efficiency at a given target locus. b,d, Split-AID10 minimizes C-to-A/G and indel byproducts in human HEK293T cells (b) and yeast cells (d). The inlaid panel in d corresponds to a magnified view. Each dot in the violin plot represents the editing efficiency for the indicated editing product per target site, while the three lines mark quartile positions. e, Domain structures of Split-AID10N6, Split-AID10C6, and PIGS-AID10N6. Note that PIGS-AID10N6 contains a complete nCas9 and an intact deaminase catalytic center. f, None of Split-AID10N6, Split-AID10C6 and PIGS-AID10N6 is catalytically active in yeast cells. Data are shown as mean values and standard deviation of three biological replicates.

Extended Data Fig. 4 Rationale of visible selection of on-target editing in the EASY assay.

The intended nonsense mutation in ScAde1 or ScCan1 by converting a Trp or Gln codon (shadowed in yellow) to a stop codon via C-to-T editing (red) makes edited yeast clones visually distinguishable. The on-target C-to-T editing of ScAde1 generated red colonies in YPD agar plates, while that of ScCan1 produced white colonies in SC-Arg agar plates containing L-Canavanine.

Extended Data Fig. 5 Split-AID10 minimizes off-target DNA edits in human cells.

a,b, R-loop assay reveals that Split-AID10 eliminates unguided DNA edits relative to other tested CBEs in HEK293T cells. In a, data are shown as mean values and standard deviation of three biological replicates. In b, each dot in the violin plot represents the C-to-T editing efficiency per target site, while the three lines mark quartile positions. ns, not significant, two-sided Mann-Whitney U test. c, Split-AID10 induces substantially reduced edits at predicted off-target sites for the HEK-1 or HEK-5 targeting gRNA in HEK293T cells. Data are shown as mean values and standard deviation of three biological replicates.

Extended Data Fig. 6 Split-AID10 eliminates off-target RNA edits in human cells.

a,b, Gating strategies of flow cytometry for sorting base editor-expressing HEK293T cells and control cells. In a, cells expressing base editors were flow-sorted for the top 5% of gated cells (% parent) with the highest GFP (488 nm) and mScarlet (561 nm) signal. In b, the nCas9-UGI control was sorted for the top 5% of gated cells with the highest signal and for a population of cells with a mean fluorescence intensity matching the top 5% of BE3-transfected cells collected on the same day. c, On-target DNA editing validation for all base editor groups in HEK293T cell-based RNA off-target assay. Data are shown for three biological replicates (Rep.1-Rep.3). The lowercase number indicates the position of individual Cs in the protospacer (counting PAM as 21-23). d, Representative Manhattan plot (Rep.3) for transcriptome-wide off-target C-to-U edits in individual chromosomes in each group. n, total number of C-to-U edits. Dashed lines indicate a C-to-U editing level of 5%.

Extended Data Fig. 7 Split-BE3 eliminates off-target RNA edits in human cells.

Representative Manhattan plot (Rep.2) for transcriptome-wide off-target C-to-U edits in individual chromosomes in each group. n, total number of C-to-U edits. Dashed lines indicate a C-to-U editing level of 5%.

Extended Data Fig. 8 Split-ABE8e minimizes off-target RNA/DNA edits.

a,b, None of the N-terminal (a) or C-terminal (b) split fragments of PIGS-ABE8e alone can induce ABE reporter activity in protoplasts in the presence of gRNA. The gRNA only was used as a negative control (Ctrl). Relative luciferase (LUC) activity was calculated by setting the LUC activity of the unsplit ABE (PIGS-ABE8e) as 100%. Data are shown as mean values of two biological replicates. c, Representative Manhattan plot (Rep.3) for transcriptome-wide off-target A-to-I edits in individual chromosomes in each group in human HEK293T cell-based RNA off-target assay. n, total number of A-to-I edits. Dashed lines indicate a A-to-I editing level of 5%. d, Split-ABE8e induces substantially reduced edits at predicted off-target sites for the HEK-8 targeting gRNA in HEK293T cells. Data are shown as mean values and standard deviation of three biological replicates.

Extended Data Fig. 9 Split fragments of a BE are expressed at higher levels than the unsplit BE.

a, Split-AID10N6 and Split-AID10C6 are expressed at higher levels than PIGS-AID10 in both rice and Arabidopsis protoplasts. b, Split-ABE8eN6 and Split-ABE8eC6 are expressed at higher levels than PIGS-ABE8e in both rice and Arabidopsis protoplasts. In (a) and (b), 200 µL rice or Arabidopsis protoplasts were transfected with equal amounts of plasmid(s) encoding PIGS-BE alone or Split-BE-N plus Split-BE-C. Since Split-BE-N and Split-BE-C carry the same protein tag (2×HA tag for both PIGS-AID10 split fragments and 2×FLAG tag for both PIGS-ABE8e split fragments), Split-BE-N or Split-BE-C was also expressed alone to help distinguish individual products. A plasmid encoding GFP was co-transfected as an internal transfection control. The experiments were conducted twice with similar results. The white, orange, and blue arrowheads mark PIGS-BE, Split-BE-N, and Split-BE-C, respectively. Rubisco staining indicates equal protein loading.

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Xiong, X., Liu, K., Li, Z. et al. Split complementation of base editors to minimize off-target edits. Nat. Plants 9, 1832–1847 (2023). https://doi.org/10.1038/s41477-023-01540-8

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