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Efficient Homology-directed Repair with Circular ssDNA Donors

View ORCID ProfileSukanya Iyer, View ORCID ProfileAamir Mir, Joel Vega-Badillo, Benjamin P. Roscoe, View ORCID ProfileRaed Ibraheim, View ORCID ProfileLihua Julie Zhu, View ORCID ProfileJooyoung Lee, View ORCID ProfilePengpeng Liu, Kevin Luk, Esther Mintzer, Josias Soares de Brito, Philip D. Zamore, View ORCID ProfileErik J. Sontheimer, View ORCID ProfileScot A. Wolfe
doi: https://doi.org/10.1101/864199
Sukanya Iyer
1Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Aamir Mir
2RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Joel Vega-Badillo
2RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Benjamin P. Roscoe
1Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Raed Ibraheim
2RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Lihua Julie Zhu
1Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
3Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Jooyoung Lee
2RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Pengpeng Liu
1Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Kevin Luk
1Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Esther Mintzer
1Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Josias Soares de Brito
1Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Philip D. Zamore
2RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA
4Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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Erik J. Sontheimer
2RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA
5Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA
6Li Weibo Institute for Rare Disease Research, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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  • For correspondence: erik.sontheimer@umassmed.edu scot.wolfe@umassmed.edu
Scot A. Wolfe
1Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
6Li Weibo Institute for Rare Disease Research, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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  • For correspondence: erik.sontheimer@umassmed.edu scot.wolfe@umassmed.edu
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Abstract

While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-Multi-Cas Variant 1 (MCV1)] that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knock-ins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knock-ins in mammalian cell lines.

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Posted December 05, 2019.
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Efficient Homology-directed Repair with Circular ssDNA Donors
Sukanya Iyer, Aamir Mir, Joel Vega-Badillo, Benjamin P. Roscoe, Raed Ibraheim, Lihua Julie Zhu, Jooyoung Lee, Pengpeng Liu, Kevin Luk, Esther Mintzer, Josias Soares de Brito, Philip D. Zamore, Erik J. Sontheimer, Scot A. Wolfe
bioRxiv 864199; doi: https://doi.org/10.1101/864199
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Efficient Homology-directed Repair with Circular ssDNA Donors
Sukanya Iyer, Aamir Mir, Joel Vega-Badillo, Benjamin P. Roscoe, Raed Ibraheim, Lihua Julie Zhu, Jooyoung Lee, Pengpeng Liu, Kevin Luk, Esther Mintzer, Josias Soares de Brito, Philip D. Zamore, Erik J. Sontheimer, Scot A. Wolfe
bioRxiv 864199; doi: https://doi.org/10.1101/864199

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