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.

Details

Title
CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing
Author
Moreno-Mateos, Miguel A 1 ; Fernandez, Juan P 1 ; Rouet, Romain 2 ; Vejnar, Charles E 1 ; Lane, Maura A 3 ; Mis, Emily 3 ; Khokha, Mustafa K 3 ; Doudna, Jennifer A 4   VIAFID ORCID Logo  ; Giraldez, Antonio J 5 

1  Department of Genetics, Yale University School of Medicine, New Haven, CT, USA 
2  Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA 
3  Department of Genetics, Yale University School of Medicine, New Haven, CT, USA; Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA 
4  Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA; Department of Chemistry, University of California, Berkeley, CA, USA; Innovative Genomics Initiative, University of California, Berkeley, CA, USA; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA; Howard Hughes Medical Institute, University of California, Berkeley, CA, USA 
5  Department of Genetics, Yale University School of Medicine, New Haven, CT, USA; Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA; Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA 
Pages
1-9
Publication year
2017
Publication date
Dec 2017
Publisher
Nature Publishing Group
e-ISSN
20411723
Source type
Scholarly Journal
Language of publication
English
DOI
https://doi.org/10.1038/s41467-017-01836-2
ProQuest document ID
1983413079
Copyright
© 2017. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.