Letter | Published:

Genome-wide target specificity of CRISPR RNA-guided adenine base editors

Abstract

Adenine base editors1 enable efficient targeted adenine-to-guanine single nucleotide conversions to induce or correct point mutations in human cells, animals, and plants1,2,3,4. Here we present a modified version of Digenome-seq, an in vitro method for identifying CRISPR (clustered regularly interspaced short palindromic repeats)-induced double-strand breaks using whole-genome sequencing5,6,7,8, to assess genome-wide target specificity of adenine base editors. To produce double-strand breaks at sites containing inosines, the products of adenine deamination, we treat human genomic DNA with an adenine base editor 7.10 protein–guide RNA complex and either endonuclease V or a combination of human alkyladenine DNA glycosylase and endonuclease VIII in vitro. Digenome-seq detects adenine base editor off-target sites with a substitution frequency of 0.1% or more. We show that adenine base editor 7.10, the cytosine base editor BE3, and unmodified CRISPR-associated protein 9 (Cas9) often recognize different off-target sites, highlighting the need for independent assessments of their genome-wide specificities6. Using targeted sequencing, we also show that use of preassembled adenine base editor ribonucleoproteins, modified guide RNAs5,8,9,10,11, and Sniper/Cas9 (ref. 12) reduces adenine base editor off-target activity in human cells.

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Code availability

The source code used to generate the 2.0 version of Digenome can be accessed at https://github.com/chizksh/digenome-toolkit2. The source code used to calculate substitution and indel frequencies can be accessed at https://github.com/ibs-cge/maund.

Data availability

High-throughput sequencing data have been deposited in the NCBI Sequence Read Archive database with accession code PRJNA515331.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

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Acknowledgements

This research was supported by grants from the Institute for Basic Science (no. IBS-R021-D1) to J.-S.K. and ToolGen, Inc. (no. 0409-20160107) to Daesik Kim.

Author information

J.-S.K. supervised the research. Daesik Kim, Da-eun Kim, G.L., and S.-I.C. performed the experiments. Daesik Kim and Da-eun Kim carried out bioinformatics analyses. J.-S.K. and Daesik Kim wrote the manuscript.

Competing interests

Daesik Kim and J.-S.K. have filed a patent application based on this work. J.-S.K. is a cofounder of, and holds stock in, ToolGen, Inc.

Correspondence to Jin-Soo Kim.

Integrated supplementary information

Supplementary Figure 1 Mismatch tolerance of ABE7.10-induced base editing, Cas9-induced indel formation, and BE3-induced base editing in human cells.

sgRNAs targeting the HEK3 (a) or RNF2 (b) sites have 0 to 4 mismatches compared with the target sequence. The base editing and indel frequencies were measured using targeted deep sequencing. The red and blue characters depict the mismatched nucleotides and the PAM sequences, respectively. Asterisks indicate mismatched sgRNAs whose relative activity (mismatched sgRNA / matched sgRNA) with one enzyme is more than three times higher than that with the other two enzymes. Means ± s.e.m. were from three independent experiments.

Supplementary Figure 2 Digenome-seq workflow.

(a) Overview of the Digenome-seq workflow for ABE-mediated Digenome-seq (b) ABE7.10 mediates adenine-to-inosine conversion in one strand and produces a nick in the other strand. hAAG excises inosine to produce an AP site and Endo VIII (DNA glycosylase and AP-lyase) cleaves the AP site. Arrowheads indicate the sites cleaved by the ABE7.10 nickase and hAAG/Endo VIII.

Supplementary Figure 3 SDS–PAGE analysis of ABE7.10 protein purification using nickel affinity chromatography and heparin bead chromatography.

M: marker, I(-): cell lysate before IPTG induction, I(+): cell lysate after IPTG induction, S: soluble lysate fraction, IS: insoluble lysate fraction, FT: flow-through, W1, W2: waste after washing, Ni: Ni-NTA agarose beads after elution of bound protein, NE: protein fraction eluted from nickel beads, Hp: Heparin Sepharose 6 Fast Flow affinity resins after elution of bound protein, HE: protein fraction after purification using heparin beads. The red box represents the ABE7.10 protein band. We obtained similar results more than three times when the gel electrophoresis was repeated independently.

Supplementary Figure 4 ABE7.10-, hAAG- and Endo VIII–mediated Digenome-seq.

(a) A DSB occurs only when the PCR product is treated with ABE7.10, hAAG, and Endo VIII. We obtained similar results more than three times when the gel electrophoresis was repeated independently. (b) Sanger sequencing results showing an A-to-G conversion when genomic DNA was treated with ABE7.10 and then a G-to-A conversion when genomic DNA was treated with ABE7.10, hAAG, and Endo VIII. (c) qRT-PCR results showing digestion of genomic DNA by ABE7.10, hAAG, and Endo VIII. Means ± s.e.m. were from three independent experiments. (d) IGV images of the straight alignments of sequencing reads that are observed after treatment with ABE7.10, hAAG, and Endo VIII.

Supplementary Figure 5

In vitro DNA-cleavage scoring system for Digenome-seq analysis of ABE.

Supplementary Figure 6

IGV images showing the straight alignments of sequencing reads at the RNF2, TYRO3, WEE1, EphB4, HPRT-exon6 and HPRT-exon8 sites.

Supplementary Figure 7 On- and off-target activity measured using modified sgRNAs.

(a) Mean of base editing frequency when using modified sgRNAs targeted to HEK2, RNF2, EphB4, TYRO3, WEE1, HPRT-exon6, and HPRT-exon8. All data are the results of targeted deep sequencing. (b) Off-target activities measured using modified sgRNAs at the TYRO3 and HPRT-exon8 sites. The red and blue characters indicate the mismatched nucleotides and PAM sequences, respectively. The specificity ratio was calculated using the following formula: specificity of modified sgRNAs (on-target frequency/off-target frequency)/ specificity of conventional sgRNAs (on-target frequency/off-target frequency). Means ± s.e.m. were from three independent experiments.

Supplementary Figure 8 Specificity of ABE7.10 and Sniper ABE7.10.

On- and off-target activity and specificity ratio when using ABE7.10 and Sniper ABE7.10 at the HPRT-exon8, HEK2, EphB4 and TYRO3 sites. The red and blue characters indicate the mismatched nucleotides and PAM sequences, respectively. The specificity ratio was calculated using the following formula: specificity of Sniper ABE7.10 (on-target frequency/off-target frequency)/ specificity of conventional ABE7.10 (on-target frequency/off-target frequency). Means ± s.e.m. were from three independent experiments.

Supplementary Figure 9 Specificity of ABE7.10 depending on plasmid delivery or RNP delivery.

On- and off-target activity and specificity ratio when delivering plasmid or RNP at the HPRT-exon6, HEK2, EphB4 and TYRO3 sites. The red and blue characters indicate the mismatched nucleotides and PAM sequences, respectively. The specificity ratio was calculated using the following formula: specificity of RNP delivery (on-target frequency/off-target frequency)/ specificity of plasmid delivery (on-target frequency/off-target frequency). Means ± s.e.m. were from three independent experiments.

Supplementary Figure 10 Specificity of ABE7.10 by combining Sniper ABE7.10 with modified sgRNAs.

On- and off-target activity and specificity ratio using ABE7.10 and Sniper ABE7.10 with modified sgRNAs at the HPRT-exon6 and HPRT-exon8 sites. The red and blue characters indicate the mismatched nucleotides and PAM sequences, respectively. The specificity ratio was calculated using the following formula: specificity of Sniper ABE7.10 with modified sgRNAs (on-target frequency/off-target frequency)/specificity of ABE7.10 (on-target frequency/off-target frequency). Means ± s.e.m. were from three independent experiments.

Supplementary information

Supplementary Figures 1–10 and Supplementary Tables 1–6

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Further reading

Fig. 1: Tolerance of ABE7.10, BE3, and Cas9 for mismatched sgRNAs.
Fig. 2: Digenome-seq workflow for the identification of genome-wide ABE7.10 off-target sites.
Fig. 3: Genome-wide specificities of ABE7.10, BE3, and Cas9.
Fig. 4: Reducing ABE7.10 off-target effects with modified sgRNAs, Sniper ABE7.10, and RNP delivery.
Supplementary Figure 1: Mismatch tolerance of ABE7.10-induced base editing, Cas9-induced indel formation, and BE3-induced base editing in human cells.
Supplementary Figure 2: Digenome-seq workflow.
Supplementary Figure 3: SDS–PAGE analysis of ABE7.10 protein purification using nickel affinity chromatography and heparin bead chromatography.
Supplementary Figure 4: ABE7.10-, hAAG- and Endo VIII–mediated Digenome-seq.
Supplementary Figure 5
Supplementary Figure 6
Supplementary Figure 7: On- and off-target activity measured using modified sgRNAs.
Supplementary Figure 8: Specificity of ABE7.10 and Sniper ABE7.10.
Supplementary Figure 9: Specificity of ABE7.10 depending on plasmid delivery or RNP delivery.
Supplementary Figure 10: Specificity of ABE7.10 by combining Sniper ABE7.10 with modified sgRNAs.