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The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells

Alan S. Wang, Leo Chen, R. Alex Wu, Christopher D. Richardson, Benjamin G. Gowen, Katelynn R. Kazane, Jonathan T. Vu, Stacia K. Wyman, Jiyung Shin, Johannes C. Walter, View ORCID ProfileJacob E. Corn
doi: https://doi.org/10.1101/705657
Alan S. Wang
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
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Leo Chen
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
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R. Alex Wu
3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
4Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
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Christopher D. Richardson
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
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Benjamin G. Gowen
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
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Katelynn R. Kazane
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
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Jonathan T. Vu
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
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Stacia K. Wyman
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
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Jiyung Shin
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
5Department of Biology, ETH Zürich, 8093 Zürich, Switzerland
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Johannes C. Walter
3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
4Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
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Jacob E. Corn
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
5Department of Biology, ETH Zürich, 8093 Zürich, Switzerland
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  • ORCID record for Jacob E. Corn
  • For correspondence: jacob.corn@biol.ethz.ch
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Summary

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Using a proximity labeling system for unbiased detection of transient protein interactions in cell-free Xenopus laevis egg extract, we identified the dimeric histone chaperone FACT as an interactor of substrate-bound Cas9. Immunodepletion of FACT subunits from extract potently inhibits Cas9 unloading and converts Cas9’s activity from multi-turnover to single-turnover. In human cells, depletion of FACT delays genome editing and alters the balance between indel formation and homology directed repair. Depletion of FACT also increases epigenetic marking by dCas9-based transcriptional effectors with concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

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Posted July 23, 2019.
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The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells
Alan S. Wang, Leo Chen, R. Alex Wu, Christopher D. Richardson, Benjamin G. Gowen, Katelynn R. Kazane, Jonathan T. Vu, Stacia K. Wyman, Jiyung Shin, Johannes C. Walter, Jacob E. Corn
bioRxiv 705657; doi: https://doi.org/10.1101/705657
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The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells
Alan S. Wang, Leo Chen, R. Alex Wu, Christopher D. Richardson, Benjamin G. Gowen, Katelynn R. Kazane, Jonathan T. Vu, Stacia K. Wyman, Jiyung Shin, Johannes C. Walter, Jacob E. Corn
bioRxiv 705657; doi: https://doi.org/10.1101/705657

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