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CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

View ORCID ProfileMiguel A. Moreno-Mateos, Juan P. Fernandez, Romain Rouet, Maura A. Lane, Charles E. Vejnar, Emily Mis, Mustafa K. Khokha, Jennifer A. Doudna, Antonio J. Giraldez
doi: https://doi.org/10.1101/156125
Miguel A. Moreno-Mateos
1 Department of Genetics, Yale University School of Medicine, New Haven, CT 06510
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  • ORCID record for Miguel A. Moreno-Mateos
  • For correspondence: antonio.giraldez@yale.edu miguel.moreno-mateos@yale.com
Juan P. Fernandez
1 Department of Genetics, Yale University School of Medicine, New Haven, CT 06510
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Romain Rouet
4 Department of Molecular and Cell Biology, University of California, Berkeley, USA
5 California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA
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Maura A. Lane
1 Department of Genetics, Yale University School of Medicine, New Haven, CT 06510
6 Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06520
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Charles E. Vejnar
1 Department of Genetics, Yale University School of Medicine, New Haven, CT 06510
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Emily Mis
1 Department of Genetics, Yale University School of Medicine, New Haven, CT 06510
6 Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06520
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Mustafa K. Khokha
1 Department of Genetics, Yale University School of Medicine, New Haven, CT 06510
6 Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06520
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Jennifer A. Doudna
4 Department of Molecular and Cell Biology, University of California, Berkeley, USA
5 California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA
7Department of Chemistry, University of California, Berkeley
8 Innovative Genomics Initiative, University of California, Berkeley
9 MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720
10 Howard Hughes Medical Institute, University of California, Berkeley, CA 94720
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Antonio J. Giraldez
1 Department of Genetics, Yale University School of Medicine, New Haven, CT 06510
2 Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT 06510
3 Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06510
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  • For correspondence: antonio.giraldez@yale.edu miguel.moreno-mateos@yale.com
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Abstract

Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.

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Posted June 26, 2017.
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CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing
Miguel A. Moreno-Mateos, Juan P. Fernandez, Romain Rouet, Maura A. Lane, Charles E. Vejnar, Emily Mis, Mustafa K. Khokha, Jennifer A. Doudna, Antonio J. Giraldez
bioRxiv 156125; doi: https://doi.org/10.1101/156125
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CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing
Miguel A. Moreno-Mateos, Juan P. Fernandez, Romain Rouet, Maura A. Lane, Charles E. Vejnar, Emily Mis, Mustafa K. Khokha, Jennifer A. Doudna, Antonio J. Giraldez
bioRxiv 156125; doi: https://doi.org/10.1101/156125

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