Letter | Published:

Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA

Nature Biotechnology volume 34, pages 339344 (2016) | Download Citation

Abstract

Targeted genomic manipulation by Cas9 can efficiently generate knockout cells and organisms via error-prone nonhomologous end joining (NHEJ), but the efficiency of precise sequence replacement by homology-directed repair (HDR) is substantially lower1,2. Here we investigate the interaction of Cas9 with target DNA and use our findings to improve HDR efficiency. We show that dissociation of Cas9 from double-stranded DNA (dsDNA) substrates is slow (lifetime 6 h) but that, before complete dissociation, Cas9 asymmetrically releases the 3′ end of the cleaved DNA strand that is not complementary to the sgRNA (nontarget strand). By rationally designing single-stranded DNA (ssDNA) donors of the optimal length complementary to the strand that is released first, we increase the rate of HDR in human cells when using Cas9 or nickase variants to up to 60%. We also demonstrate HDR rates of up to 0.7% using a catalytically inactive Cas9 mutant (dCas9), which binds DNA without cleaving it.

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Acknowledgements

We thank J. Doudna, M. Botchan, and members of the Corn laboratory for critical reading of the manuscript. We thank the Doudna lab for the gift of wtCas9 expression plasmids and the Berkeley Macrolab for support with protein expression and purification. This work was supported by the Li Ka Shing Foundation. Reagents described in this work are available on Addgene (https://www.addgene.org/Jacob_Corn/), and detailed protocols are available on protocols.io (https://www.protocols.io/g/innovative-genomics-initiative).

Author information

Affiliations

  1. Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California, USA.

    • Christopher D Richardson
    • , Graham J Ray
    • , Mark A DeWitt
    • , Gemma L Curie
    •  & Jacob E Corn
  2. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.

    • Christopher D Richardson
    • , Graham J Ray
    • , Mark A DeWitt
    • , Gemma L Curie
    •  & Jacob E Corn

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Contributions

C.D.R. and J.E.C. designed experiments; C.D.R., G.J.R. and G.L.C. performed experiments; M.A.D. designed and constructed the BFP reporter cell line; C.D.R. and J.E.C. analyzed data; C.D.R. and J.E.C. wrote the manuscript with contributions from all authors.

Competing interests

C.D.R. and J.E.C. are inventors on US Patent Application No. 62/262,189 related to this work.

Corresponding author

Correspondence to Jacob E Corn.

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DOI

https://doi.org/10.1038/nbt.3481

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