CRISPR base editing enables the creation of targeted single-base conversions without generating double-stranded breaks. However, the efficiency of current base editors is very low in many cell types. We reengineered the sequences of BE3, BE4Gam, and xBE3 by codon optimization and incorporation of additional nuclear-localization sequences. Our collection of optimized constitutive and inducible base-editing vector systems dramatically improves the efficiency by which single-nucleotide variants can be created. The reengineered base editors enable target modification in a wide range of mouse and human cell lines, and intestinal organoids. We also show that the optimized base editors mediate efficient in vivo somatic editing in the liver in adult mice.

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  1. 1.

    , , , & Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

  2. 2.

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

  3. 3.

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

  4. 4.

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

  5. 5.

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

  6. 6.

    et al. RNA editing with CRISPR-Cas13. Science 358, 1019–1027 (2017).

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 10.

    , , & Rescue of high-specificity Cas9 variants using sgRNAs with matched 5′ nucleotides. Genome Biol. 18, 218 (2017).

  11. 11.

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

  12. 12.

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

  13. 13.

    et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat. Biotechnol. 33, 390–394 (2015).

  14. 14.

    et al. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat. Biotechnol. 35, 431–434 (2017).

  15. 15.

    et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci. Adv. 3, eaao4774 (2017).

  16. 16.

    et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614–620 (2009).

  17. 17.

    et al. Inhibiting Tankyrases sensitizes KRAS-mutant cancer cells to MEK inhibitors via FGFR2 feedback signaling. Cancer Res. 74, 3294–3305 (2014).

  18. 18.

    et al. mTOR signaling mediates resistance to tankyrase inhibitors in Wnt-driven colorectal cancer. Oncotarget 8, 47902–47915 (2017).

  19. 19.

    Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 487, 330–337 (2012).

  20. 20.

    et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015).

  21. 21.

    et al. New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism. Oncogene 21, 8293–8301 (2002).

  22. 22.

    et al. Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland. Genes Dev. 30, 1470–1480 (2016).

  23. 23.

    et al. Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum. Gene Ther. 26, 432–442 (2015).

  24. 24.

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

  25. 25.

    et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. (2018).

  26. 26.

    et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat. Biotechnol. 36, 536–539 (2018).

  27. 27.

    et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, 1179–1187 (2017).

  28. 28.

    et al. A pipeline for the generation of shRNA transgenic mice. Nat. Protoc. 7, 374–393 (2012).

  29. 29.

    et al. R-Spondin chromosome rearrangements drive Wnt-dependent tumour initiation and maintenance in the intestine. Nat. Commun. 8, 15945 (2017).

  30. 30.

    et al. Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer. Nat. Biotechnol. 35, 577–582 (2017).

  31. 31.

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

  32. 32.

    , & Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

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This work was supported by a project grant from the NIH/NCI (CA195787-01), a U54 grant from the NIH/NCI (U54OD020355), a project grant from the Starr Cancer Consortium (I10-0095), a Research Scholar Award from the American Cancer Society (RSG-17-202-01), and a Stand Up to Cancer Colorectal Cancer Dream Team Translational Research Grant (SU2C-AACR-DT22-17). Stand Up to Cancer is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, a scientific partner of SU2C. M.P.Z. is supported in part by National Cancer Institute (NCI) grant NIH T32 CA203702. E.M.S. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the NIH under award number T32GM07739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD–PhD Program and an F31 Award from the NCI/NIH under grant number 1 F31 CA224800-01. E.R.K. is supported by an F31 NRSA predoctoral fellowship from the NCI/NIH under award number F31CA192835. F.J.S.-R. was supported by the MSKCC TROT program (5T32CA160001) and is supported as an HHMI Hanna Gray Fellow. S.W.L. is supported as the Geoffrey Beene Chair of Cancer Biology and as an Investigator of the Howard Hughes Medical Institute. D.F.T. is supported by the Helmholtz Association (VH-NG-1114) and by the German Research Foundation (DFG) project B05, SFB/TR 209 'Liver Cancer'. L.E.D. was supported by a K22 Career Development Award from the NCI/NIH (CA 181280-01). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We thank H. Varmus (Weill Cornell Medicine) for providing cells.

Author information

Author notes

    • Maria Paz Zafra
    •  & Emma M Schatoff

    These authors contributed equally to this work.


  1. Sandra and Edward Meyer Cancer Center, Department of Medicine, Weill Cornell Medicine, New York, New York, USA.

    • Maria Paz Zafra
    • , Emma M Schatoff
    • , Alyna Katti
    • , Miguel Foronda
    • , Amber Simon
    • , Teng Han
    • , Sukanya Goswami
    • , Emma Montgomery
    •  & Lukas E Dow
  2. Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD–PhD program, New York, New York, USA.

    • Emma M Schatoff
  3. Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medicine, New York, New York, USA.

    • Alyna Katti
    • , Teng Han
    • , Jordana Thibado
    •  & Lukas E Dow
  4. Helmholtz-University Group 'Cell Plasticity and Epigenetic Remodeling', German Cancer Research Center (DKFZ) and Institute of Pathology, University Hospital, Heidelberg, Germany.

    • Marco Breinig
    • , Anabel Y Schweitzer
    •  & Darjus F Tschaharganeh
  5. Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Edward R Kastenhuber
    • , Francisco J Sánchez-Rivera
    •  & Scott W Lowe
  6. Gerstner Sloan Kettering Graduate School of Biomedical Sciences, New York, New York, USA.

    • Edward R Kastenhuber
  7. Cold Spring Harbor Laboratory, New York, New York, USA.

    • Junwei Shi
    •  & Christopher R Vakoc
  8. Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Junwei Shi
  9. Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Scott W Lowe
  10. Department of Biochemistry, Weill Cornell Medicine, New York, New York, USA.

    • Lukas E Dow


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M.P.Z. and E.M.S. performed experiments, analyzed data, and wrote the paper. A.K., M.F., A.S., S.G., E.M., T.H., J.T., and F.J.S.-R. performed experiments and analyzed data. M.B. and A.Y.S. performed and analyzed in vivo experiments. D.F.T. designed and supervised in vivo experiments. E.R.K. performed computational analysis of MSKCC IMPACT data. J.S., S.W.L., and C.R.V. supplied critical previously unreported reagents. L.E.D. performed and supervised experiments, analyzed data, and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Lukas E Dow.

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