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Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

Jacob Lamberth, Laura Daley, Pachai Natarajan, Stanislav Khoruzhenko, Nurit Becker, Daniel Taglicht, Gregory D. Davis, Qingzhou Ji
doi: https://doi.org/10.1101/766493
Jacob Lamberth
1MilliporeSigma, 2909 Laclede Avenue, Saint Louis, MO 63103, USA. A Business of Merck KGaA, 64293 Darmstadt, Germany
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Laura Daley
1MilliporeSigma, 2909 Laclede Avenue, Saint Louis, MO 63103, USA. A Business of Merck KGaA, 64293 Darmstadt, Germany
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Pachai Natarajan
2MaxCyte, 22 Firstfield Road, Gaithersburg, MD 20878, USA
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Stanislav Khoruzhenko
2MaxCyte, 22 Firstfield Road, Gaithersburg, MD 20878, USA
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Nurit Becker
3Merck Israel, 13 Kiryat Hamada, Jerusalem, Israel
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Daniel Taglicht
3Merck Israel, 13 Kiryat Hamada, Jerusalem, Israel
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Gregory D. Davis
1MilliporeSigma, 2909 Laclede Avenue, Saint Louis, MO 63103, USA. A Business of Merck KGaA, 64293 Darmstadt, Germany
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Qingzhou Ji
1MilliporeSigma, 2909 Laclede Avenue, Saint Louis, MO 63103, USA. A Business of Merck KGaA, 64293 Darmstadt, Germany
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  • For correspondence: qingzhou.ji@milliporesigma.com
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ABSTRACT

CRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted September 20, 2019.
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Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins
Jacob Lamberth, Laura Daley, Pachai Natarajan, Stanislav Khoruzhenko, Nurit Becker, Daniel Taglicht, Gregory D. Davis, Qingzhou Ji
bioRxiv 766493; doi: https://doi.org/10.1101/766493
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Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins
Jacob Lamberth, Laura Daley, Pachai Natarajan, Stanislav Khoruzhenko, Nurit Becker, Daniel Taglicht, Gregory D. Davis, Qingzhou Ji
bioRxiv 766493; doi: https://doi.org/10.1101/766493

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