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

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Abstract

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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Figure 1: Partial DNA replacement at the guide region of a GFP crRNA induces gene editing in human cells.
Figure 2: Partial DNA replacement at the guide region of crRNA or sgRNA induces efficient gene editing in human cells.
Figure 3: Partial DNA replacement at the guide region reduces off-target effects in human cells.
Figure 4: An optimized DNA–RNA chimeric crRNA enables efficient genome editing in human cells.

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  • 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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Acknowledgements

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.

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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.

Corresponding authors

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

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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.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–2 and Supplementary Figures 1–6 (PDF 1362 kb)

Life Sciences Reporting Summary (PDF 175 kb)

Supplementary Data Set 1

Details of GUIDE-seq analysis of native and 10 DNA crRNAs of three endogenous genes (XLSX 26 kb)

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Yin, H., Song, CQ., Suresh, S. et al. Partial DNA-guided Cas9 enables genome editing with reduced off-target activity. Nat Chem Biol 14, 311–316 (2018). https://doi.org/10.1038/nchembio.2559

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