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Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array

Bernd Zetsche, Matthias Heidenreich, Prarthana Mohanraju, Iana Fedorova, Jeroen Kneppers, Ellen M. DeGennaro, Nerges Winblad, Sourav R. Choudhury, Omar O. Abudayyeh, Jonathan S. Gootenberg, Wen Y. Wu, David A. Scott, Konstantin Severinov, John van der Oost, Feng Zhang
doi: https://doi.org/10.1101/049122
Bernd Zetsche
1Broad Institute of MIT and Harvard, Cambridge, MA 02142
2McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139
3Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139
4Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
5Department of Developmental Pathology, Institute of Pathology, Bonn Medical School, Sigmund Freud Street 25, 53127 Bonn, Germany
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Matthias Heidenreich
1Broad Institute of MIT and Harvard, Cambridge, MA 02142
2McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139
3Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139
4Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
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Prarthana Mohanraju
6Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Stippeneng 4, 6708 WE Wageningen, Netherlands
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Iana Fedorova
1Broad Institute of MIT and Harvard, Cambridge, MA 02142
2McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139
3Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139
4Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
8Skolkovo Institute of Science and Technology, Skolkovo, 143025, Russia
9Peter the Great St.Petersburg Polytechinc University, St. Petersburg, 195251, Russia
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Jeroen Kneppers
1Broad Institute of MIT and Harvard, Cambridge, MA 02142
6Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Stippeneng 4, 6708 WE Wageningen, Netherlands
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Ellen M. DeGennaro
1Broad Institute of MIT and Harvard, Cambridge, MA 02142
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Nerges Winblad
1Broad Institute of MIT and Harvard, Cambridge, MA 02142
2McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139
3Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139
4Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
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Sourav R. Choudhury
1Broad Institute of MIT and Harvard, Cambridge, MA 02142
2McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139
3Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139
4Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
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Omar O. Abudayyeh
1Broad Institute of MIT and Harvard, Cambridge, MA 02142
2McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139
3Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139
4Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
7Department of Systems Biology, Harvard Medical School, Boston, MA 02115
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Jonathan S. Gootenberg
1Broad Institute of MIT and Harvard, Cambridge, MA 02142
2McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139
3Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139
4Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
7Department of Systems Biology, Harvard Medical School, Boston, MA 02115
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Wen Y. Wu
6Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Stippeneng 4, 6708 WE Wageningen, Netherlands
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David A. Scott
1Broad Institute of MIT and Harvard, Cambridge, MA 02142
2McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139
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Konstantin Severinov
8Skolkovo Institute of Science and Technology, Skolkovo, 143025, Russia
10Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
11Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182, Russia
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John van der Oost
6Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Stippeneng 4, 6708 WE Wageningen, Netherlands
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Feng Zhang
1Broad Institute of MIT and Harvard, Cambridge, MA 02142
2McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139
3Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139
4Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
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Abstract

Microbial CRISPR-Cas defense systems have been adapted as a platform for genome editing applications built around the RNA-guided effector nucleases, such as Cas9. We recently reported the characterization of Cpf1, the effector nuclease of a novel type V-A CRISPR system, and demonstrated that it can be adapted for genome editing in mammalian cells (Zetsche et al., 2015). Unlike Cas9, which utilizes a trans-activating crRNA (tracrRNA) as well as the endogenous RNaseIII for maturation of its dual crRNA:tracrRNA guides (Deltcheva et al., 2011), guide processing of the Cpf1 system proceeds in the absence of tracrRNA or other Cas (CRISPR associated) genes (Zetsche et al., 2015) (Figure 1a), suggesting that Cpf1 is sufficient for pre-crRNA maturation. This has important implications for genome editing, as it would provide a simple route to multiplex targeting. Here, we show for two Cpf1 orthologs that no other factors are required for array processing and demonstrate multiplex gene editing in mammalian cells as well as in the mouse brain by using a designed single CRISPR array.

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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 October 01, 2016.
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Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array
Bernd Zetsche, Matthias Heidenreich, Prarthana Mohanraju, Iana Fedorova, Jeroen Kneppers, Ellen M. DeGennaro, Nerges Winblad, Sourav R. Choudhury, Omar O. Abudayyeh, Jonathan S. Gootenberg, Wen Y. Wu, David A. Scott, Konstantin Severinov, John van der Oost, Feng Zhang
bioRxiv 049122; doi: https://doi.org/10.1101/049122
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Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array
Bernd Zetsche, Matthias Heidenreich, Prarthana Mohanraju, Iana Fedorova, Jeroen Kneppers, Ellen M. DeGennaro, Nerges Winblad, Sourav R. Choudhury, Omar O. Abudayyeh, Jonathan S. Gootenberg, Wen Y. Wu, David A. Scott, Konstantin Severinov, John van der Oost, Feng Zhang
bioRxiv 049122; doi: https://doi.org/10.1101/049122

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