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Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors

Abstract

Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA. Recent studies report that BE3, the original CBE, induces a low frequency of genome-wide Cas9-independent off-target C•G-to-T•A mutation in mouse embryos and in rice. Here we develop multiple rapid, cost-effective methods to screen the propensity of different CBEs to induce Cas9-independent deamination in Escherichia coli and in human cells. We use these assays to identify CBEs with reduced Cas9-independent deamination and validate via whole-genome sequencing that YE1, a narrowed-window CBE variant, displays background levels of Cas9-independent off-target editing. We engineered YE1 variants that retain the substrate-targeting scope of high-activity CBEs while maintaining minimal Cas9-independent off-target editing. The suite of CBEs characterized and engineered in this study collectively offer ~10–100-fold lower average Cas9-independent off-target DNA editing while maintaining robust on-target editing at most positions targetable by canonical CBEs, and thus are especially promising for applications in which off-target editing must be minimized.

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Fig. 1: On-target and Cas9-independent off-target DNA editing in E. coli.
Fig. 2: Cas9-independent deamination by CBEs in HEK293T cells.
Fig. 3: YE1 balances efficient on-target editing with greatly decreased Cas9-independent editing as confirmed by WGS.
Fig. 4: Expanding the utility of CBEs with decreased Cas9-independent off-targets through protein engineering.

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Data availability

High-throughput sequencing and whole-genome sequencing data are deposited in the NCBI Sequence Read Archive (PRJNA553240). Plasmids used in this study are available from Addgene. Amino acid sequences of all base editors in this study are provided in the Supplementary Sequences.

Code availability

The script used to analyze the SNVs reported by Yang and coworkers12 is provided in Supplementary Note 1. The script and parameters used for running CRISPResso2 analyses are provided in Supplementary Note 2. The script used for calculating the number of pathogenic SNPs targetable by CBEs is provided in Supplementary Note 4.

References

  1. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Komor, A. C., Badran, A. H. & Liu, D. R. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168, 20–36 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Paz Zafra, M. S. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–893 (2018).

    Article  CAS  Google Scholar 

  5. Kim, K. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35, 435–437 (2017).

    Article  CAS  PubMed  Google Scholar 

  6. Zhang, Y. et al. Programmable base editing of zebrafish genome using a modified CRISPR–Cas9 system. Nat. Commun. 8, 118 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9–cytidine deaminase fusion. Nat. Biotechnol. 35, 438–440 (2017).

    Article  CAS  PubMed  Google Scholar 

  8. Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kim, D. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat. Biotechnol. 35, 475–480 (2017).

    Article  CAS  PubMed  Google Scholar 

  10. Liang, P. et al. Genome-wide profiling of adenine base editor specificity by EndoV-seq. Nat. Commun. 10, 67 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat. Commun. 8, 15790 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zuo, E. S. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Jin, S. Z. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).

    Article  CAS  PubMed  Google Scholar 

  14. McGrath, E. et al. Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing. Nat. Commun. 10, 5353 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Badran, A. H. & Liu, D. R. Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Nat. Commun. 6, 8425 (2015).

    Article  CAS  PubMed  Google Scholar 

  16. Garibyan, L. Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome. DNA Repair 2, 593–608 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Harris, R. S., Petersen-Mahrt, S. K. & Neuberger, M. S. RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators. Mol. Cell 10, 1247–1253 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Kohli, R. M. et al. A portable hot spot recognition loop transfers sequence preferences from APOBEC family members to activation-induced cytidine deaminase. J. Biol. Chem. 284, 22898–22904 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lee, H., Popodi, E., Tang, H. & Foster, P. L. Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing. Proc. Natl Acad. Sci. USA 109, E2774–E2783 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Fukui, K. DNA mismatch repair in eukaryotes and bacteria. J. Nucleic Acids 2010, 260512 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Saraconi, G. S., Sala, C., Mattiuz, G. & Conticello, S. G. The RNA editing enzyme APOBEC1 induces somatic mutations and a compatible mutational signature is present in esophageal adenocarcinomas. Genome Biol. 15, 417 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729–aaf8729 (2016).

    Article  PubMed  CAS  Google Scholar 

  23. Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029–1035 (2016).

    Article  CAS  PubMed  Google Scholar 

  24. Hess, G. T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036–1042 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wang, X. et al. Efficient base editing in methylated regions with a human APOBEC3A–Cas9 fusion. Nat. Biotechnol. 36, 946–949 (2018).

    Article  CAS  PubMed  Google Scholar 

  26. Coelho, M. A. et al. BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B. BMC Biol. 16, 150 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. St Martin, A. et al. A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells. Nucleic Acids Res. 46, e84 (2018).

    Article  CAS  Google Scholar 

  28. Martin, A. S. et al. A panel of eGFP reporters for single base editing by APOBEC-Cas9 editosome complexes. Sci. Rep. 9, 497 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Liu, Z. et al. Highly precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions. bioRxiv https://www.biorxiv.org/content/10.1101/658351v1 (2019).

  30. Thuronyi, B. W. K. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol. 37, 1070–1079 (2019).

  31. Kim, Y. B. et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9–cytidine deaminase fusions. Nat. Biotechnol. 35, 371–376 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Grunewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Gehrke, J. M. et al. An APOBEC3A–Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tashiro, Y., Fukutomi, H., Terakubo, K., Saito, K. & Umeno, D. A nucleoside kinase as a dual selector for genetic switches and circuits. Nucleic Acids Res. 39, e12 (2011).

    Article  PubMed  CAS  Google Scholar 

  35. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Chan, K. et al. An APOBEC3A hypermutation signature is distinguishable from the signature of background mutagenesis by APOBEC3B in human cancers. Nat. Genet. 47, 1067–1072 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Eto, T., Kinoshita, K., Yoshiwaka, K., Muramatsu, M. & Honjo, T. RNA-editing cytidine deaminase Apobec-1 is unable to induce somatic hypermutation in mammalian cells. Proc. Natl Acad. Sci. USA 100, 12895–12898 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Carpenter, M. A. et al. Methylcytosine and normal cytosine deamination by the foreign DNA restriction enzyme APOBEC3A. J. Biol. Chem. 287, 34801–34808 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lei, L. et al. APOBEC3 induces mutations during repair of CRISPR–Cas9-generated DNA breaks. Nat. Struct. Mol. Biol. 25, 45–52 (2018).

    Article  CAS  PubMed  Google Scholar 

  40. Akre, M. K. et al. Mutation processes in 293-based clones overexpressing the DNA cytosine deaminase APOBEC3B. PLoS ONE 11, e0155391 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Nishimasu, H. S. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Huang, T. P. et al. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat. Biotechnol. 37, 626–631 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    Article  CAS  PubMed  Google Scholar 

  45. Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275–278 (2019).

    Article  CAS  PubMed  Google Scholar 

  46. Hazen, J. L. et al. The complete genome sequences, unique mutational spectra, and developmental potency of adult neurons revealed by cloning. Neuron 89, 1223–1236 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Milholland, B. et al. Differences between germline and somatic mutation rates in humans and mice. Nat. Commun. 8, 15183 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Dong, X. et al. Accurate identification of single-nucleotide variants in whole-genome-amplified single cells. Nat. Methods 14, 491–493 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lynch, M. Evolution of the mutation rate. Trends Genet. 26, 345–352 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Rahbari, R. et al. Timing, rates and spectra of human germline mutation. Nat. Genet. 48, 126–133 (2016).

    Article  CAS  PubMed  Google Scholar 

  51. Thomason, L. C. et al. Recombineering: genetic engineering in bacteria using homologous recombination. Curr. Protoc. Mol. Biol. 106, 1 16 11–39 (2014).

    Article  Google Scholar 

  52. Crooks, G. E. et al. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Clement, K. et al. CRISPResso2: accurate and rapid analysis of genome editing data from nucleases and base editors. Nat. Biotechnol. 37, 224–226 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Pedersen, B. S. & Quinlan, A. R. Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics 34, 867–868 (2018).

    Article  CAS  PubMed  Google Scholar 

  56. Van der Auwera, G. A. et al. From FastQ data to high-confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr. Protoc. Bioinformatics 43, 11–33 (2013).

    Article  PubMed  Google Scholar 

  57. Garrison, E. & Marth, G. Haplotype-based variant detection from short-read sequencing. Preprint at arXiv https://arxiv.org/abs/1207.3907 (2012).

  58. Koboldt, D. C. et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. 22, 568–576 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank B. Thuronyi for discussions; K. Zhao, T. Huang and S. Miller for providing guide RNA plasmids; and B. Mok for providing an sgRNA for in vitro experiments. M. Osborn, Aldevron and the Kidz1stFund provided the BE4 protein used for protein delivery experiments, and K. Tian and S. Pandey provided assistance with experiments. We thank D. Court for providing pSIM5, which was used for recombineering the HSV-TK gene onto the E. coli chromosome. We thank T. Mason and E. LaRoche for assistance with WGS library preparation and FAS Research Computing at Harvard University for computational resources. This work was supported by US NIH grant nos. U01 AI142756, RM1 HG009490, R35 GM118062 and HHMI; the Bill and Melinda Gates Foundation; and the St. Jude Collaborative Research Consortium. A.R. is an NSF Graduate Research Fellow and was supported by NIH Training Grant no. T32 GM095450. J.L.D. is supported by the Hertz Foundation and the NSF GRFP fellowship. G.A.N. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation. Flow cytometry was supported by Cancer Center Support (core) Grant no. P30-CA14051 from the NCI.

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J.L.D., A.R. and D.R.L. designed the research. A.R. and J.L.D. performed experiments. G.A.N. performed the BE4 protein delivery off-target experiment. All authors contributed to writing the manuscript.

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Correspondence to David R. Liu.

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D.R.L. is a consultant and cofounder of Editas Medicine, Pairwise Plants, Beam Therapeutics and Prime Medicine, companies that use genome editing. J.L.D., A.R. and D.R.L. through the Broad Institute have filed patent applications on aspects of this work.

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Supplementary Figs. 1–24, Tables 1–5, Notes 1–5 and Sequences.

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Doman, J.L., Raguram, A., Newby, G.A. et al. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat Biotechnol 38, 620–628 (2020). https://doi.org/10.1038/s41587-020-0414-6

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