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

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.

Authors:
 [1];  [1];  [2];  [1];  [1];  [1];  [1]; ORCiD logo [3];  [1]
+ Show Author Affiliations
  1. Yale Univ. School of Medicine, New Haven, CT (United States)
  2. Univ. of California, Berkeley, CA (United States)
  3. Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1560545
Grant/Contract Number:  
AC02-05CH11231; R21 HD073768; R01 HD074078; GM103789; GM102251; GM101108; GM081602
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Moreno-Mateos, Miguel A., Fernandez, Juan P., Rouet, Romain, Vejnar, Charles E., Lane, Maura A., Mis, Emily, Khokha, Mustafa K., Doudna, Jennifer A., and Giraldez, Antonio J. CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. United States: N. p., 2017. Web. doi:10.1038/s41467-017-01836-2.
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Moreno-Mateos, Miguel A., Fernandez, Juan P., Rouet, Romain, Vejnar, Charles E., Lane, Maura A., Mis, Emily, Khokha, Mustafa K., Doudna, Jennifer A., & Giraldez, Antonio J. CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. United States. doi:10.1038/s41467-017-01836-2.
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Moreno-Mateos, Miguel A., Fernandez, Juan P., Rouet, Romain, Vejnar, Charles E., Lane, Maura A., Mis, Emily, Khokha, Mustafa K., Doudna, Jennifer A., and Giraldez, Antonio J. Fri . "CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing". United States. doi:10.1038/s41467-017-01836-2. https://www.osti.gov/servlets/purl/1560545.
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@article{osti_1560545,
title = {CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing},
author = {Moreno-Mateos, Miguel A. and Fernandez, Juan P. and Rouet, Romain and Vejnar, Charles E. and Lane, Maura A. and Mis, Emily and Khokha, Mustafa K. and Doudna, Jennifer A. and Giraldez, Antonio J.},
abstractNote = {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.},
doi = {10.1038/s41467-017-01836-2},
journal = {Nature Communications},
number = 1,
volume = 8,
place = {United States},
year = {2017},
month = {12}
}
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Journal Article:
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Publisher's Version of Record
DOI:  10.1038/s41467-017-01836-2
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Cited by: 71 works
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Figures / Tables:

Fig. 1 Fig. 1: LbCpf1–crRNA RNP complexes are a robust genome editing system in zebrafish. a Diagram illustrating three crRNAs (orange) targeting slc45a2 and tyr exon 1 in zebrafish. b Schematic illustrating the experimental set-up to analyze CRISPR-Cpf1-mediated mutations in zebrafish. Three crRNAs (a) were either mixed with mRNA coding for AsCpf1more » or LbCpf1 or assembled into RNP complexes with their corresponding purified proteins and injected in one-cellstage embryos. c Phenotypic evaluation of crRNAs (30 pg/crRNA) and mRNA (100 pg) injections. Stacked barplots showing the percentage of mosaic (gray) and phenotypically wild-type (WT; black) embryos 48 h post fertilization (hpf) after injection. d Phenotypes obtained after the injection of the LbCpf1–crRNA RNP complexes targeting slc45a2 showing different levels of mosaicism compared to the WT. Lateral views (scale bar, 0.5 mm) and insets of the eyes (scale bar, 0.25 mm) of 48 hpf embryos are shown. e Phenotypic evaluation of Cpf1–crRNA RNP complexes injections targeting slc45a2 (albino). Stacked barplots showing the percentage of alb-like (white), mosaic (gray), and phenotypically WT (black) embryos 48 hpf after injection using different amounts (fmol) of RNP complexes. Number of embryos evaluated ($n$) is shown for each condition. f Phenotypes obtained after the injection of the LbCpf1–crRNA RNP complexes targeting tyr showing different levels of mosaicism compared to the WT. Lateral views (scale bar, 0.5 mm) and insets of the eyes (scale bar, 0.25 mm) of 48 hpf embryos are shown. g Phenotypic evaluation of Cpf1–crRNA RNP complexes targeting tyrosinase (tyr). Stacked barplots showing the percentage of tyr-like (white), severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection using different amounts (fmol) of RNP complexes. Number of embryos evaluated ( $n$) is shown for each condition« less
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