skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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]
  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.
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.
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.
@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}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 51 works
Citation information provided by
Web of Science

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

Save / Share:

Works referenced in this record:

Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System
journal, October 2015

  • Zetsche, Bernd; Gootenberg, Jonathan S.; Abudayyeh, Omar O.
  • Cell, Vol. 163, Issue 3, p. 759-771
  • DOI: 10.1016/j.cell.2015.09.038

Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development
journal, May 2016


A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
journal, June 2012


Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins
journal, June 2016

  • Hur, Junho K.; Kim, Kyoungmi; Been, Kyung Wook
  • Nature Biotechnology, Vol. 34, Issue 8
  • DOI: 10.1038/nbt.3596

CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo
journal, August 2015

  • Moreno-Mateos, Miguel A.; Vejnar, Charles E.; Beaudoin, Jean-Denis
  • Nature Methods, Vol. 12, Issue 10
  • DOI: 10.1038/nmeth.3543

Codon identity regulates mRNA stability and translation efficiency during the maternal‐to‐zygotic transition
journal, July 2016

  • Bazzini, Ariel A.; Viso, Florencia; Moreno‐Mateos, Miguel A.
  • The EMBO Journal, Vol. 35, Issue 19
  • DOI: 10.15252/embj.201694699

The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA
journal, April 2016

  • Fonfara, Ines; Richter, Hagen; Bratovič, Majda
  • Nature, Vol. 532, Issue 7600
  • DOI: 10.1038/nature17945

Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells
journal, June 2016

  • Kim, Daesik; Kim, Jungeun; Hur, Junho K.
  • Nature Biotechnology, Vol. 34, Issue 8
  • DOI: 10.1038/nbt.3609

Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus
journal, January 2017

  • Bothmer, Anne; Phadke, Tanushree; Barrera, Luis A.
  • Nature Communications, Vol. 8, Issue 1
  • DOI: 10.1038/ncomms13905

Method for isolation of PCR-ready genomic DNA from zebrafish tissues
journal, November 2007

  • Meeker, Nathan D.; Hutchinson, Sarah A.; Ho, Linh
  • BioTechniques, Vol. 43, Issue 5
  • DOI: 10.2144/000112619

Techniques and probes for the study ofXenopus tropicalis development
journal, November 2002

  • Khokha, Mustafa K.; Chung, Christina; Bustamante, Erika L.
  • Developmental Dynamics, Vol. 225, Issue 4
  • DOI: 10.1002/dvdy.10184

Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System
journal, July 2013


CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool
journal, April 2015


Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA
journal, January 2016

  • Richardson, Christopher D.; Ray, Graham J.; DeWitt, Mark A.
  • Nature Biotechnology, Vol. 34, Issue 3
  • DOI: 10.1038/nbt.3481

Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system
journal, August 2013

  • Jao, Li-En; Wente, Susan R.; Chen, Wenbiao
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 34
  • DOI: 10.1073/pnas.1308335110

GMAP: a genomic mapping and alignment program for mRNA and EST sequences
journal, February 2005


Multiplex Genome Engineering Using CRISPR/Cas Systems
journal, January 2013


Exon capture and bulk segregant analysis: rapid discovery of causative mutations using high-throughput sequencing
journal, January 2012


Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells
journal, June 2016

  • Kleinstiver, Benjamin P.; Tsai, Shengdar Q.; Prew, Michelle S.
  • Nature Biotechnology, Vol. 34, Issue 8
  • DOI: 10.1038/nbt.3620

Highly Efficient Cpf1-Mediated Gene Targeting in Mice Following High Concentration Pronuclear Injection
journal, December 2016

  • Watkins-Chow, Dawn E.; Varshney, Gaurav K.; Garrett, Lisa J.
  • G3: Genes|Genomes|Genetics, Vol. 7, Issue 2
  • DOI: 10.1534/g3.116.038091

LincSNP 2.0: an updated database for linking disease-associated SNPs to human long non-coding RNAs and their TFBSs
journal, October 2016

  • Ning, Shangwei; Yue, Ming; Wang, Peng
  • Nucleic Acids Research, Vol. 45, Issue D1
  • DOI: 10.1093/nar/gkw945

In vivo high-throughput profiling of CRISPR–Cpf1 activity
journal, December 2016

  • Kim, Hui K.; Song, Myungjae; Lee, Jinu
  • Nature Methods, Vol. 14, Issue 2
  • DOI: 10.1038/nmeth.4104

Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs
journal, September 2016

  • Port, Fillip; Bullock, Simon L.
  • Nature Methods, Vol. 13, Issue 10
  • DOI: 10.1038/nmeth.3972

Generation of knockout mice by Cpf1-mediated gene targeting
journal, June 2016

  • Kim, Yongsub; Cheong, Seung-A; Lee, Jong Geol
  • Nature Biotechnology, Vol. 34, Issue 8
  • DOI: 10.1038/nbt.3614

Targeted mutagenesis in rice using CRISPR-Cpf1 system
journal, January 2017


Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases
journal, November 2013


Optimized CRISPR–Cas9 System for Genome Editing in Zebrafish
journal, October 2016

  • Vejnar, Charles E.; Moreno-Mateos, Miguel A.; Cifuentes, Daniel
  • Cold Spring Harbor Protocols, Vol. 2016, Issue 10
  • DOI: 10.1101/pdb.prot086850

SSW Library: An SIMD Smith-Waterman C/C++ Library for Use in Genomic Applications
journal, December 2013


A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants
journal, February 2017


Effect of temperature on early embryological development of the zebra fish,Brachydanio rerio
journal, September 1968

  • Schirone, Robert C.; Gross, Leo
  • Journal of Experimental Zoology, Vol. 169, Issue 1
  • DOI: 10.1002/jez.1401690106

Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish
journal, November 2013

  • Hruscha, A.; Krawitz, P.; Rechenberg, A.
  • Development, Vol. 140, Issue 24
  • DOI: 10.1242/dev.099085

    Works referencing / citing this record:

    CRISPR–Cas ribonucleoprotein mediated homology-directed repair for efficient targeted genome editing in microalgae Nannochloropsis oceanica IMET1
    journal, March 2019

    • Naduthodi, Mihris Ibnu Saleem; Mohanraju, Prarthana; Südfeld, Christian
    • Biotechnology for Biofuels, Vol. 12, Issue 1
    • DOI: 10.1186/s13068-019-1401-3

      Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.