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An efficient gene knock-in strategy using 5′-modified double-stranded DNA donors with short homology arms

A Publisher Correction to this article was published on 15 January 2020

This article has been updated

Abstract

Here, we report a rapid CRISPR–Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5′-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65/40% knock-in rates for 0.7/2.5 kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5′-end modification led to up to a fivefold increase in gene knock-in rates at various genomic loci in human cancer and stem cells.

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Fig. 1: Gene KI strategy using Cas9 RNP and end-modified dsDNA donors.
Fig. 2: End-modified dsDNA donors resulted in enhanced gene KI rate in various applications.

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The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

This work was supported by the US National Institutes of Health (grant nos. 1UM1HG009402 and 1U54DK107965) (H.Z.). We thank B. Pilas and B. Balhan (Flow Cytometry Facility, Biotechnology Center, University of Illinois at Urbana Champaign) for cell sorting experiments. We thank S. Long and K. Sobanska for help with ddPCR experiments. We thank W. Tang, T. Si, J. Lian, C. Field and S. Tsai for helpful suggestions for manuscript writing and experimental designs.

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Y.Y and H.Z. designed and conceived the study. Y.Y., Y.G. and Q.T. performed most of the experiments and analyzed the data. Y.L. and H.Y. contributed to construct generation, dsDNA donor preparation and fluorescence activated cell sorting analysis. M.Z. contributed to single-guide RNA preparation and gene KI experiments in HEK293T cell lines. I.T. contributed to gene KI experiments in the H1 and WTC G3 cell lines. S.J. contributed to gene KI experiments in the H1 cell line and off-target analysis. H.Z. supervised the research. Y.Y., Y.G., Q.T. and H.Z. wrote the manuscript.

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Correspondence to Huimin Zhao.

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The authors declare no competing interests.

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Supplementary Tables 1–5 and Figs. 1–6.

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Yu, Y., Guo, Y., Tian, Q. et al. An efficient gene knock-in strategy using 5′-modified double-stranded DNA donors with short homology arms. Nat Chem Biol 16, 387–390 (2020). https://doi.org/10.1038/s41589-019-0432-1

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