Letter | Published:

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

Nature volume 533, pages 420424 (19 May 2016) | Download Citation

Abstract

Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction1,2. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks1,2. Here we report the development of ‘base editing’, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting ‘base editors’ convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15–75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Sequence Read Archive

Data deposits

High-throughput sequencing data have been deposited in the NCBI Sequence Read Archive database under accession code SRP072434. Plasmids encoding BE1, BE2, and BE3 are available from Addgene (plasmids 73018, 73019, 73020, 73021).

References

  1. 1.

    , & Therapeutic genome editing: prospects and challenges. Nature Med. 21, 121–131 (2015)

  2. 2.

    & Enabling functional genomics with genome engineering. Genome Res. 25, 1442–1455 (2015)

  3. 3.

    & CRISPR–Cas systems for editing, regulating and targeting genomes. Nature Biotechnol. 32, 347–355 (2014)

  4. 4.

    et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 44, D862–D868 (2015)

  5. 5.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)

  6. 6.

    et al. Genome engineering using the CRISPR–Cas9 system. Nature Protocols 8, 2281–2308 (2013)

  7. 7.

    et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012)

  8. 8.

    The AID/APOBEC family of nucleic acid mutators. Genome Biol. 9, 229 (2008)

  9. 9.

    , & RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators. Mol. Cell 10, 1247–1253 (2002)

  10. 10.

    et al. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nature Struct. Mol. Biol. 18, 529–536 (2011)

  11. 11.

    et al. Structures of a CRISPR–Cas9 R-loop complex primed for DNA cleavage. Science (2016)

  12. 12.

    et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nature Biotechnol. 32, 569–576 (2014)

  13. 13.

    et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nature Biotechnol. 27, 1186–1190 (2009)

  14. 14.

    , , , & The RNA editing enzyme APOBEC1 induces somatic mutations and a compatible mutational signature is present in esophageal adenocarcinomas. Genome Biology 15, 417 (2014)

  15. 15.

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

  16. 16.

    , & DNA repair in mammalian cells: mismatched repair: variations on a theme. Cell. Mol. Life Sci. 66, 1021–1038 (2009)

  17. 17.

    et al. Crystal structure of human uracil–DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA. Cell 82, 701–708 (1995)

  18. 18.

    , , & Mechanism and regulation of human non-homologous DNA end-joining. Nature Rev. Mol. Cell Biol. 4, 712–720 (2003)

  19. 19.

    & Replisome assembly and the direct restart of stalled replication forks. Nature Rev. Mol. Cell Biol. 7, 932–943 (2006)

  20. 20.

    et al. PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair. Proc. Natl Acad. Sci. USA 107, 16066–16071 (2010)

  21. 21.

    et al. Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo. J. Mol. Biol. 337, 585–596 (2004)

  22. 22.

    , , , & Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nature Biotechnol. 32, 677–683 (2014)

  23. 23.

    , & The role of apolipoprotein E in Alzheimer’s disease. Neuron 63, 287–303 (2009)

  24. 24.

    et al. The missing ApoE allele. Ann. Hum. Genet. 71, 496–500 (2007)

  25. 25.

    et al. The landscape of cancer genes and mutational processes in breast cancer. Nature 486, 400–404 (2012)

  26. 26.

    et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2015)

  27. 27.

    , , , & Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nature Chem. Biol. 11, 316–318 (2015)

  28. 28.

    et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016)

  29. 29.

    et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nature Biotechnol. 33, 73–80 (2015)

  30. 30.

    et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015)

  31. 31.

    et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnol. 31, 833–838 (2013)

  32. 32.

    et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnol. 31, 839–843 (2013)

  33. 33.

    & Near-infrared fluorescent proteins for multicolor in vivo imaging. Nature Methods 10, 751–754 (2013)

Download references

Acknowledgements

This work was supported by US National Institutes of Health (NIH) R01 EB022376 (formerly R01 GM065400), F-Prime Biomedical Research Initiative (A28161), and the Howard Hughes Medical Institute. A.C.K. is a Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellow (F32 GM 112366-2). Y.B.K. holds a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship (NSERC PGS-D). M.S.P. is an NSF Graduate Research Fellow and was supported by the Harvard Biophysics NIH training grant T32 GM008313. J.A.Z. was a Ruth L. Kirschstein National Research Service Award Postdoctoral Fellow (F32 GM 106601-2). We thank B. Hyman and E. Hudry for providing immortalized mouse astrocytes containing APOE4.

Author information

Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Alexis C. Komor
    • , Yongjoo B. Kim
    • , Michael S. Packer
    • , John A. Zuris
    •  & David R. Liu
  2. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA

    • Alexis C. Komor
    • , Yongjoo B. Kim
    • , Michael S. Packer
    • , John A. Zuris
    •  & David R. Liu

Authors

  1. Search for Alexis C. Komor in:

  2. Search for Yongjoo B. Kim in:

  3. Search for Michael S. Packer in:

  4. Search for John A. Zuris in:

  5. Search for David R. Liu in:

Contributions

A.C.K. and Y.B.K. designed the research, performed experiments, analysed data, and wrote the manuscript. M.S.P. assisted with the data analysis. J.A.Z. assisted with the preparation of materials and the design of experiments. D.R.L. designed and supervised the research and wrote the manuscript. All of the authors contributed to editing the manuscript.

Competing interests

A.C.K. and D.R.L. have filed a provisional patent application on this work. D.R.L. is a consultant and co-founder of Editas Medicine, a company that seeks to develop genome-editing therapeutics.

Corresponding author

Correspondence to David R. Liu.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains a Supplementary Discussion, Supplementary Notes, Supplementary Sequences, Supplementary Tables 1-9, Supplementary References and Supplementary Figure 1.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature17946

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.