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Enabling large-scale genome editing by reducing DNA nicking

Cory J. Smith, Oscar Castanon, Khaled Said, Verena Volf, Parastoo Khoshakhlagh, Amanda Hornick, Raphael Ferreira, Chun-Ting Wu, Marc Güell, Shilpa Garg, Hannu Myllykallio, George M. Church
doi: https://doi.org/10.1101/574020
Cory J. Smith
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
2Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA.
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Oscar Castanon
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
2Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA.
3LOB, Ecole Polytechnique, CNRS, INSERM, Institut Polytechnique de Paris, 91128 Palaiseau, France.
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Khaled Said
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
2Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA.
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Verena Volf
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
2Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA.
4John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.
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Parastoo Khoshakhlagh
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
2Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA.
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Amanda Hornick
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
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Raphael Ferreira
5Department of Biology and Biological Engineering, Chalmers University of Technology, SE412 96 Gothenburg, Sweden.
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Chun-Ting Wu
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
2Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA.
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Marc Güell
6Pompeu Fabra University, Barcelona Biomedical Research Park, 08003-Barcelona, Spain.
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Shilpa Garg
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
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Hannu Myllykallio
3LOB, Ecole Polytechnique, CNRS, INSERM, Institut Polytechnique de Paris, 91128 Palaiseau, France.
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George M. Church
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
2Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA.
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  • For correspondence: gchurch@genetics.med.harvard.edu
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Abstract

To extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBEs) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements – ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.

One Sentence Summary Base editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.

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Posted March 15, 2019.
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Enabling large-scale genome editing by reducing DNA nicking
Cory J. Smith, Oscar Castanon, Khaled Said, Verena Volf, Parastoo Khoshakhlagh, Amanda Hornick, Raphael Ferreira, Chun-Ting Wu, Marc Güell, Shilpa Garg, Hannu Myllykallio, George M. Church
bioRxiv 574020; doi: https://doi.org/10.1101/574020
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Enabling large-scale genome editing by reducing DNA nicking
Cory J. Smith, Oscar Castanon, Khaled Said, Verena Volf, Parastoo Khoshakhlagh, Amanda Hornick, Raphael Ferreira, Chun-Ting Wu, Marc Güell, Shilpa Garg, Hannu Myllykallio, George M. Church
bioRxiv 574020; doi: https://doi.org/10.1101/574020

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