Partial DNA-guided Cas9 enables genome editing with reduced off-target activity

  • Nature Chemical Biology volume 14, pages 311316 (2018)
  • doi:10.1038/nchembio.2559
  • Download Citation


CRISPR–Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9–sgRNA complex as a guide, the majority of the 3′ end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3′-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA–RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells.

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Change history

  • Corrected online 05 February 2018

    In the HTML version of this article initially published online, the received date was incorrectly stated as 18 May 2017. The date should be 5 October 2017. This error has been corrected in the HTML version of the article.


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We thank T. Jacks, P. Sharp, Z. Weng, C. Mello, and E. Sontheimer for discussions, and Y. Li for technical assistance. We thank K. Joung (Massachusetts General Hospital and Harvard Medical School) for sharing U2OS-GFP-PEST cells and J. Smith and A. Sheel for proofreading. This work is supported by grants from the National Institutes of Health (NIH), 5R00CA169512, DP2HL137167 and P01HL131471 (to W.X.). H.Y. is supported by 5-U54-CA151884-04 (NIH) and Skoltech Center. W.X. was supported by the Lung Cancer Research Foundation, Hyundai Hope on Wheels, and ALS Association. This work is supported in part by Cancer Center Support (core) grant P30-CA14051 from the NIH. We thank the Swanson Biotechnology Center at MIT for technical support.

Author information

Author notes

    • Hao Yin
    •  & Chun-Qing Song

    These authors contributed equally to this work.


  1. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Hao Yin
    • , Sneha Suresh
    • , Qiongqiong Wu
    • , Stephen Walsh
    • , Junmei Ding
    • , Roman L Bogorad
    • , Robert Langer
    •  & Daniel G Anderson
  2. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Chun-Qing Song
    • , Suet-Yan Kwan
    •  & Wen Xue
  3. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Lihua Julie Zhu
    • , Scot A Wolfe
    •  & Wen Xue
  4. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Lihua Julie Zhu
  5. Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Lihua Julie Zhu
    •  & Wen Xue
  6. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Scot A Wolfe
  7. Skolkovo Institute of Science and Technology, Skolkovo, Moscow Region, Russia.

    • Victor Koteliansky
  8. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer
    •  & Daniel G Anderson
  9. Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, USA.

    • Robert Langer
    •  & Daniel G Anderson
  10. Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer
    •  & Daniel G Anderson


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H.Y. conceived of and designed the study and directed the project. H.Y., C.-Q.S, S.S., S.W., Q.W., J.D., S.-Y.K., L.J.Z., and S.A.W. performed experiments and analyzed data. H.Y. made the figures with C.-Q.S. V.K., W.X., and R.L.B., and R.L. provided conceptual advice. H.Y. wrote the manuscript with comments from all authors. W.X., D.G.A. and R.L. supervised the project.

Competing interests

H.Y., C.-Q.S., W.X., D.G.A and R.L. have applied for patents related to this study. D.G.A. is a scientific co-founder of CRISPR Therapeutics.

Corresponding authors

Correspondence to Wen Xue or Robert Langer or Daniel G Anderson.

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Tables 1–2 and Supplementary Figures 1–6

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Data Set 1

    Details of GUIDE-seq analysis of native and 10 DNA crRNAs of three endogenous genes