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Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations

Abstract

The fusion of CRISPR–Cas9 with cytidine deaminases leads to base editors (BEs) capable of programmable C-to-T editing, which has potential in clinical applications but suffers from off-target (OT) mutations. Here, we used a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, the tBE remains inactive at OT sites with the fusion of a cleavable dCDI, therefore eliminating unintended mutations. When binding at on-target sites, the tBE is transformed to cleave off the dCDI domain and catalyses targeted deamination for precise base editing. After delivery into mice through a dual-adeno-associated virus (AAV) system, the tBE system created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9, resulting in a ~30–40% decrease in total cholesterol. The development of tBE establishes a highly specific base editing system and its in vivo efficacy has potential for therapeutic applications.

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Fig. 1: Identification of dCDI domains.
Fig. 2: Comparison of tBE with other BEs.
Fig. 3: Optimization of hsgRNA in the tBE system.
Fig. 4: The effect of split-TEV protease concentration on editing efficiency and sgRNA-independent OT mutation.
Fig. 5: tBE eliminates OT mutations genome-wide.
Fig. 6: tBE eliminates OT mutations transcriptome-wide.
Fig. 7: Dual-AAV delivery of tBE for in vivo base editing.
Fig. 8: Determining the in vivo OT effects of tBE-V5–mA3.

Data availability

Deep-sequencing data, whole-transcriptome sequencing data and whole-genome sequencing data can be accessed in Gene Expression Omnibus under the accession codes GSE164837 and GSE164477, at the NCBI BioProject under the accession code PRJNA692761 and in the National Omics Data Encyclopedia under the accession codes OEP001688, OEP001689 and OEP001690. All other data supporting the finding of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

The custom Perl and Shell scripts for calculating frequencies of base substitution and indels (CFBI) are available at GitHub (https://github.com/YangLab/CFBI). The computational pipeline of Base/Prime editor induced DNA off-target site identification unified toolkit (BEIDOU) to identify high-confidence base substitution in this paper is available at GitHub (https://github.com/YangLab/BEIDOU).

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Acknowledgements

We thank the staff at the Molecular and Cell Biology Core Facility, School of Life Science and Technology, ShanghaiTech University for providing experimental service. This work was supported by grants 2018YFA0801401 (to J.C. and H.Y.), 2019YFA0802804 (to L.Y. and B.Y.), 2018YFC1004602 (to J.C.), 2019YFA0802801 (to H.Y.) from MoST, 31925011 (to L.Y.), 91940306 (to L.Y.), 32070170 (to B.Y.), 32071442 (to H.Y.), 31871345 (to H.Y.), 31822016 (to J.C.), 81872305 (to J.C.), 31801073 (to W.X.) and 31972936 (to Y.Z.) from NSFC, Medical Science Advancement Program (to Basic Medical Sciences) of Wuhan University TFJC2018004 (to H.Y.), Applied Basic Frontier Program of Wuhan City (to H.Y.), the Youth Innovation Promotion Association from CAS (to W.X.), Fundamental Research Funds for the Central Universities (to H.Y. and Y.Z.), Hubei Health Commission Young Investigator Award (to H.Y.), National Postdoctoral Program for Innovative Talents (BX20190256 to H.Z.) and China Postdoctoral Science Foundation (2019M662704 to H.Z.).

Author information

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Authors

Contributions

J.C., L.Y., H.Y. and B.Y. conceived, designed and supervised the project. L.W., H.Z., R.G. and H.Q. performed most of the experiments with the help of L.Z., X.W., X.L., C.L., J. Wu and Q.C. on cell culture and plasmid construction. J.Wei prepared libraries for deep sequencing and W.X. and Y.-N.L. performed bioinformatics analyses, supervised by L.Y.; H.M., X.H., C.C and Y.Z. provided support with techniques. J.C., L.Y., H.Y. and B.Y. wrote the paper with input from the other authors. J.C. managed the project.

Corresponding authors

Correspondence to Bei Yang or Hao Yin or Li Yang or Jia Chen.

Ethics declarations

Competing interests

J.C., B.Y., L.Y., X.H. and L.W. have filed patent applications (PCT/CN2019/074577, PCT/CN2020/074218) relating to the published work through ShanghaiTech University.

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Peer review information Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

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

Extended data

Extended Data Fig. 1 Development and application of CESSCO to detect the sgRNA-independent mutations induced by BE3 at OTss sites.

a, Schematic diagrams illustrate the co-expressing S. aureus and S. pyogenes Cas9 orthologs (CESSCO) method, which expresses two Cas9 orthologs, to detect sgRNA-independent mutations induced by BEs at OTss sites. b, C-to-T mutation frequencies induced by dSpCas9, BE3, hA3A-BE3 and hA3A-BE3-Y130F at the indicated OTss sites triggered by Sa-sgRNA/dSaCas9 or Sa-sgRNA/nSaCas9 pairs. The data of C-to-T mutation frequencies induced by hA3A-BE3-Y130F at the indicated OTss sites triggered by Sa-sgRNA/nSaCas9 pairs are same as the ones shown in Fig. 2b. Data are presented as mean ± s.d. from three independent experiments. NT, non-transfected control. Numerical source data for b are provided.

Source data

Extended Data Fig. 2 Characterization of dCDI domains.

a, Schematic diagrams illustrate base editors constructed by fusing the indicated CDA domains to nSpCas9 and uracil DNA glycosylase inhibitor (UGI). The regulatory CDA domains are in grey shadow and the active CDA domains are in colors. NLS, nuclear localization sequence; XTEN and SGGS, linker peptides. b, C-to-T editing frequencies induced by the indicated BEs at six genomic loci. c, Schematic diagrams illustrate the fusion of different dCDI domains to the N-terminus of BE3 and hA3A-BE3. d, C-to-T editing frequencies induced by the indicated BEs at four genomic loci. Data in b and d are presented as mean ± s.d. from three independent experiments. Numerical source data for b and d are provided.

Source data

Extended Data Fig. 3 Versions of tBE and their performance in cells.

a, Schematic diagrams illustrate the construction and development of various versions of tBEs by using different strategies to cleave mA3dCDI off. b, The interaction of molecular components in different versions of tBEs. Due to free diffusion, the dCDI domain could be cleaved off from APOBEC through a two-component interaction of the TEV site and a free TEV protease (V2), a N22p-fused TEV protease (V3) or a TEV protease reconstituted by an sgRNA-boxB (V4) in cytosol/nucleosol, and then the resulted MCP-UGI-APOBEC fusion protein triggers mutations at sgRNA-independent OTss sites. In the version 5 (V5) of tBE, the dCDI is unlikely to be cleaved off from APOBEC in cytosol/nucleosol as the chance for the free diffusion induced three-component interaction of TEV site, TEVn and N22p-TEVc is very low. c-e, Comparison of the editing or mutation frequencies induced by different versions of tBEs at on-target sites (c), sgRNA-independent OTss sites (d) and sgRNA-dependent OT sites (e). Numerical source data for c-e are provided.

Source data

Extended Data Fig. 4 Comparing editing efficiencies of tBE-V5-mA3 with other BEs in 293FT cells.

a, Editing frequencies induced by indicated BEs at 22 genomic loci, including 6 on-target sites with natively high DNA methylation levels and 10 on-target sites containing GpC dinucleotides. Data are presented as mean ± s.d. from three independent experiments. b, Statistical analysis of normalized editing frequencies within the narrow window shown in a, Fig. 2a,d. n = 123 edited cytosines at 27 on-target sites from three independent experiments. c, Statistical analysis of normalized editing frequencies within the wide window excluding the narrow window shown in a, Figs. 2a,d. n = 96 edited cytosines at 22 on-target sites from three independent experiments. b-c, Setting the editing frequencies induced by the BE3 as 100%. P value, two-tailed Student’s t test. The median and interquartile range (IQR) are shown. Numerical source data for a-c are provided.

Source data

Extended Data Fig. 5 Comparing editing efficiencies of tBE-V5-mA3 with other BEs in U2OS cells.

a, Editing frequencies induced by indicated BEs at 23 genomic loci. Data are presented as mean ± s.d. from three independent experiments. b, Statistical analysis of normalized editing frequencies at all 23 on-target sites shown in a. n = 189 edited cytosines at 23 on-target sites from three independent experiments. c, Statistical analysis of normalized editing frequencies at 10 on-target sites containing GpC dinucleotides shown in a. n = 30 edited cytosines at 10 on-target sites from three independent experiments. b-c, Setting the editing frequencies induced by the BE3 as 100%. P value, two-tailed Student’s t test. The median and interquartile range (IQR) are shown. Numerical source data for a-c are provided.

Source data

Extended Data Fig. 6 Determining genome-wide OT mutations induced by various BEs.

a, Schematic diagrams illustrate the position of sgRNAs for the knockout of human APOBEC3 cluster and primers for PCR detection. b, The knockout of APOBEC3 cluster was confirmed by genomic DNA PCR. Agarose gel data are representative of three independent experiments. c, The numbers of genome-wide base substitutions on C or G in the single cell clones treated with Cas9 or indicated BEs. Data are presented as mean ± s.d. from n = 9 (Cas9), n = 6 (BE3, hA3A-BE3-Y130F), n = 5 (BE4max-YE1), n = 3 (tBE-V5-mA3) or n = 4 (tBE-V5-rA1) single cell colonies. d, Rare overlap was found for the genome-wide base substitutions among different clones treated with indicated BEs. Uncropped gels for b and numerical source data for c and are provided.

Source data

Extended Data Fig. 7 Determining transcriptome-wide OT mutations induced by various BEs.

a, Schematic diagrams illustrate the construction of mA3CDA1-nSpCas9-BE (mA3CDA1-BE3), mA3dCDI-nSpCas9-BE and mA3CDA1-mA3dCDI-nSpCas9-BE. b, Histograms show the numbers of all twelve types of RNA editing in different genome regions in the cells treated with mA3CDA1-nSpCas9-BE, mA3dCDI-nSpCas9-BE and mA3CDA1-mA3dCDI-nSpCas9-BE. Data are presented as mean from two independent experiments. c, Manhattan plot of RNA OT editing (C-to-U) frequency shown in b. d, Histograms show the numbers of all twelve types of RNA editing in different genomic regions in the cells treated with indicated BEs. Data are presented as mean ± s.d. from three (five for NT) independent experiments. e, Manhattan plot of RNA OT editing (C-to-U) frequency shown in d. Numerical source data for b and d are provided.

Source data

Extended Data Fig. 8 Enriching tBE system by the combination of different Cas9 orthologs and dCDIs.

a, Editing efficiencies induced by tBE-V5-mA3 and Pcsk9-targeting sgRNAs pairs at eight premature stop codon sites in six different exons of Pcsk9 in N2A cells. b, Comparison of editing efficiencies of indicated BEs before dual-AAV8 delivery in N2A cells. c, Schematic diagrams illustrate the dual-AAV system for intein-BEs delivery. d, Numbers of tBE correctable pathogenic mutations when combining with Cas9 or Cas9-NG. The gray portions show the number of preferentially correctable pathogenic mutations. A preferentially correctable pathogenic mutation is the only edited cytosine in the editing window of indicated tBE. e, The phylogenetic tree of mA3dCDI domain. Data in a and b are presented as mean ± s.d. from three independent experiments. Numerical source data for a,b,d are provided.

Source data

Supplementary information

Supplementary Information

BE sequences (amino acid sequences of plasmids).

Reporting Summary

Supplementary Tables 1 and 2

Oligos used for plasmid construction; gRNA target sequences and PCR primer sequences for genomic DNA amplification.

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Supplementary Protocol.

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Wang, L., Xue, W., Zhang, H. et al. Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations. Nat Cell Biol 23, 552–563 (2021). https://doi.org/10.1038/s41556-021-00671-4

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