Adenine base editors catalyze cytosine conversions in human cells

Abstract

Adenine base editors comprise an adenosine deaminase, evolved in vitro, and a Cas9 nickase. Here, we show that in addition to converting adenine to guanine, adenine base editors also convert cytosine to guanine or thymine in a narrow editing window (positions 5–7) and in a confined TC*N sequence context. Adenine base editor–induced cytosine substitutions occur independently of adenosine conversions with an efficiency of up to 11.2% and reduce the number of suitable targeting sites for high-specificity base editing.

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Fig. 1: Cytosine editing by ABEs.
Fig. 2: Sequence motif and editing window in cytosine editing by ABE7.10.

Data availability

High–throughput sequencing data have been deposited in the NCBI Sequence Read Archive database under accession number PRJNA525294.

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Acknowledgements

We thank G.-H. Hwang for his support on bioinformatics analysis; J.-S. Woo and S.-N. Lee for supplying purified ABE proteins. This research was supported by grants from IBS (no. IBS-R021-D1 to J.S.K.), the National Research Foundation of Korea (grant nos. 2017R1D1A1B03035094, 2017R1E1A1A01074529 and 2018M3A9H3021707 to J.K.H. and 2018M3A9H3022412 to S.B.) and the Next Generation BioGreen 21 Program (no. PJ01319301), Korea Healthcare Technology R&D Project (no. HI16C1012) and Technology Innovation Program (no. 20000158) to S.B.

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Contributions

H.S.K., J.S.K. and S.B. conceived this project. H.S.K. and Y.K.J. performed the experiments and bioinformatics analyses. J.K.H. gave critical comments. H.S.K. and S.B. wrote the manuscript with the approval of all other authors. J.S.K. and S.B. supervised the research.

Corresponding authors

Correspondence to Jin-Soo Kim or Sangsu Bae.

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Competing interests

S.B., J.-S.K., H.S.K. and Y.K.J. have filed a patent application based on this work.

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Integrated supplementary information

Supplementary Figure 1 Frequency of indel events induced by wild-type Cas9 at 22 target sites.

Frequency of indel events induced by wild-type Cas9 at 22 target sites. The efficiencies of 22 sgRNAs used for the ABE activity screen were confirmed using wild-type Cas9. (n = 22, mean ± S.D.)

Supplementary Figure 2 Defining the ABE cytosine deamination window for additional target sites in Figure 1b.

Defining the ABE cytosine deamination window for additional target sites in Fig. 1b. There are additional 4 target sites having a TC*N motif at positions 4, 7, 8, and 9 in the HPRT and EphA1 genes, considering up to position 10. The cytosines at positions 7 and 8 were converted at low frequencies (0.23% and 0.16%, respectively) and those at positions 4 and 9 showed no significant conversions.

Supplementary Figure 3 Sequence tables showing nucleotide frequencies at each position in the targeted sites in two genes (FANCF, RNF2).

Sequence tables showing nucleotide frequencies at each position in the targeted sites in two genes (FANCF, RNF2). The tables show the results from three independent replicates treated with ABE7.10 in the absence of sgRNA (Untreated) or with both ABE7.10 and sgRNA. Frequencies of expected nucleotides are highlighted in blue; frequencies of substituted nucleotides are highlighted in orange and PAM sequences in red.

Supplementary Figure 4 Sequence tables showing nucleotide frequencies at each position in the targeted site in the FANCF gene with or without UGI.

Sequence tables showing nucleotide frequencies at each position in the targeted site in the FANCF gene with or without UGI. The sites correspond to those used in experiments summarized in Fig. 1e. Each experiment was repeated three times. (UGI (-)) Results from cells expressing ABE 7.10 and guide RNAs without UGI. (UGI (+)) Results from cells expressing ABE 7.10, guide RNAs and UGI. Frequencies of expected nucleotides are highlighted in blue; frequencies of substituted nucleotides are highlighted in orange and PAM sequences in red.0.

Supplementary Figure 5 Cytosine conversion by ABE7.10 as usual in the absence of a neighboring adenine.

Cytosine conversion by ABE7.10 as usual in the absence of a neighboring adenine. a Analysis of base substitution efficiencies at each position in the targeted sites in two genes (BIRC7, ABHC12). Each experiment was repeated twice. Each bar indicates mean of the substitution efficiency. b Sequence tables showing nucleotide frequencies at each position in the targeted sites. (Untreated) Results in the absence of guide RNAs. (ABE7.10) Results from cells expressing ABE 7.10 and guide RNAs. Frequencies of expected nucleotides are highlighted in blue; frequencies of substituted nucleotides are highlighted in orange. PAM sequences are indicated in red.

Supplementary Figure 6 Cytosine editing efficiency of ABEmax in human HEK293T cells.

Cytosine editing efficiency of ABEmax in human HEK293T cells. Sequence tables show nucleotide frequencies at each position in the targeted sites in four genes (ABLIM3, CSRNP3, FANCF, RNF2) in human HEK293T cells. The sites correspond to those used in the experiments summarized in Fig. 2f. Each experiment was repeated three times and representative results are shown. (Untreated) Results in the absence of guide RNAs. (ABEmax) Results from cells expressing ABEmax and guide RNAs. Frequencies of expected nucleotides are highlighted in blue; frequencies of substituted nucleotides are highlighted in orange. PAM sequences are indicated in red.

Supplementary Figure 7 Cytosine editing efficiency of ABEmax in human HeLa cells.

Cytosine editing efficiency of ABEmax in human HeLa cells. Sequence tables show nucleotide frequencies at each position in the targeted sites in four genes (ABLIM3, CSRNP3, FANCF, RNF2) in human HeLa cells. The sites correspond to those used in the experiments summarized in Fig. 2f. Each experiment was repeated three times and representative results are shown. (Untreated) Results in the absence of guide RNAs. (ABEmax) Results from cells expressing ABEmax and guide RNAs. Frequencies of expected nucleotides are highlighted in blue; frequencies of substituted nucleotides are highlighted in orange. PAM sequences are indicated in red.

Supplementary Figure 8 Cytosine editing efficiency of ABEmax in human U-2 OS cells.

Cytosine editing efficiency of ABEmax in human U-2 OS cells. Sequence tables show nucleotide frequencies at each position in the targeted sites in four genes (ABLIM3, CSRNP3, FANCF, RNF2) in human U-2 OS cells. The sites correspond to those used in the experiments summarized in Fig. 2f. Each experiment was repeated three times and representative results are shown. (Untreated) Results in the absence of guide RNAs. (ABEmax) Results from cells expressing ABEmax and guide RNAs. Frequencies of expected nucleotides are highlighted in blue; frequencies of substituted nucleotides are highlighted in orange. PAM sequences are indicated in red.

Supplementary Figure 9 Cytosine editing efficiency of ABEmax in human K-562 cells.

Cytosine editing efficiency of ABEmax in human K-562 cells. Sequence tables show nucleotide frequencies at each position in the targeted sites in four genes (ABLIM3, CSRNP3, FANCF, RNF2) in human K-562 cells. The sites correspond to those used in the experiments summarized in Fig. 2f. Each experiment was repeated three times and representative results are shown. (Untreated) Results in the absence of guide RNAs. (ABEmax) Results from cells expressing ABEmax and guide RNAs. Frequencies of expected nucleotides are highlighted in blue; frequencies of substituted nucleotides are highlighted in orange. PAM sequences are indicated in red.

Supplementary Figure 10 Cytosine editing efficiency of ABEmax in human fibroblasts.

Cytosine editing efficiency of ABEmax in human fibroblasts. Sequence tables show nucleotide frequencies at each position in the targeted site in ABLIM3 gene in GM14867 cells derived from a patient with xeroderma pigmentosum. Each experiment was repeated twice. (Untreated) Results in the absence of guide RNAs. (ABEmax) Results from cells expressing ABEmax and guide RNAs. Frequencies of expected nucleotides are highlighted in blue; frequencies of substituted nucleotides are highlighted in orange. PAM sequences are indicated in red.

Supplementary Figure 11 In vitro ABE activity assay.

In vitro ABE activity assay. a Measurement of the frequency of cytosine substitutions mediated by ABE-sgRNA complexes using targeted deep sequencing. b Sequence tables showing nucleotide frequencies at each position in the targeted sites in three genes (BIRC7, ABHD12, ARAP2) after the in vitro ABE reaction. Each experiment was repeated two times and representative results are shown. (ABEmax) Results in the presence of the ABEmax protein-sgRNA complex. (Untreated) Results in the absence of sgRNAs (ABEmax protein only was used). Frequencies of expected nucleotides are highlighted in blue; frequencies of substituted nucleotides are highlighted in orange. PAM sequences are indicated in red and TC motifs are indicated in gray. c Frequencies of ABE-mediated cytosine conversions in vitro (left, n = 3) and frequencies of ABE-mediated cytosine conversions in human HEK293T cells (right, n = 27). The substitution frequencies of 27 target sites are summarized for HEK293T cells. The numbers indicate mean and S.D. Higher C>A and C>G conversion rates in HEK293T cells compared to the in vitro assay seem to be caused by the DNA repair mechanism, especially the uridine excision repair pathway through Uracil-DNA glycosylases (UDGs) in cells.

Supplementary Figure 12 Comparison of ABE-induced substitution rates at adenine versus cytosine.

Comparison of ABE-induced substitution rates at adenine versus cytosine. The adenines and cytosines were all located at position 4 or 6, respectively, downstream from the 5’ end of the target sites. Each experiment repeated twice. Each bar indicates mean of base substitution rates.

Supplementary Figure 13 Conserved motif in rAPOBEC1 of BE3 and TadA* of ABEs.

Conserved motif in rAPOBEC1 of BE3 and TadA* of ABEs. The rAPOBEC1 domain in BE3 and the TadA moiety in various versions of ABEs share a conserved motif, (C/H)XEXnPCXXC, which is highlighted in yellow. The probability that the conserved motif will be included in the TadA of ABEs by chance is calculated to be less than 0.000000625. (In case, over 100 base pairs encode enzyme).

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Kim, H.S., Jeong, Y.K., Hur, J.K. et al. Adenine base editors catalyze cytosine conversions in human cells. Nat Biotechnol 37, 1145–1148 (2019). https://doi.org/10.1038/s41587-019-0254-4

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