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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

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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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  1. Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  Google Scholar 

  2. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    Article  CAS  Google Scholar 

  3. Doudna, J. A. et al. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014).

    Article  Google Scholar 

  4. Leonetti, M. D. et al. A scalable strategy for high-throughput GFP tagging of endogenous human proteins. Proc. Natl Acad. Sci. USA 113, 3501–3508 (2016).

    Article  Google Scholar 

  5. Diao, Y. R. et al. A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat. Methods 14, 629–633 (2017).

    Article  CAS  Google Scholar 

  6. Stephens, C. J. et al. Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9. Gene Ther. 25, 139–156 (2018).

    Article  CAS  Google Scholar 

  7. Merkle, F. T. et al. Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell Rep. 11, 875–883 (2015).

    Article  CAS  Google Scholar 

  8. Renaud, J. B. et al. Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases. Cell Rep. 14, 2263–2272 (2016).

    Article  CAS  Google Scholar 

  9. He, X. et al. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res. 44, e85 (2016).

    Article  Google Scholar 

  10. Paix, A. et al. Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks. Proc. Natl Acad. Sci. USA 114, 10745–10754 (2017).

    Article  Google Scholar 

  11. Tasan, I. et al. CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci. Nucleic Acids Res. 46, e100 (2018).

    Article  Google Scholar 

  12. Brinkman, E. K. et al. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

    Article  Google Scholar 

  13. Gutierrez-Triana, J. A. et al. Efficient single-copy HDR by 5′ modified long dsDNA donors. eLife 7, e39468 (2018).

    Article  Google Scholar 

  14. DeWitt, M. A. et al. Genome editing via delivery of Cas9 ribonucleoprotein. Methods 121-122, 9–15 (2017).

    Article  CAS  Google Scholar 

  15. Hur, J. K. et al. Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat. Biotechnol. 34, 807–808 (2016).

    Article  CAS  Google Scholar 

  16. Strukov Y. G. et al. Development of mammalian cell lines with lac operator-tagged chromosomes. Cold Spring Harbor Protoc. (2008).

  17. Hindson, B. J. et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal. Chem. 83, 8604–8610 (2011).

    Article  CAS  Google Scholar 

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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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Authors and Affiliations



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).

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