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Sci Rep. 2017 Aug 30;7(1):10061. doi: 10.1038/s41598-017-10347-5.

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes.

Author information

1
Department of Biology, University of Naples "Federico II", 80126, Napoli, Italy.
2
Institute of Biostructures and Bioimaging (IBB), CNR, 80134, Naples, Italy.
3
Department of Pharmacy, University of Naples "Federico II", 80134, Napoli, Italy.
4
Department of Physics "E. Pancini", University of Naples "Federico II", 80126, Napoli, Italy.
5
Stazione Zoologica Anton Dohrn, Center Villa Dohrn for Benthic Ecology, Punta San Pietro, 80077, Ischia, Italy.
6
Institute of Molecular Life Sciences, University of Zürich, Zürich, 8057, Switzerland.
7
SIB Swiss Institute of Bioinformatics, University of Zürich, Zürich, 8057, Switzerland.
8
Department of Biology, University of Naples "Federico II", 80126, Napoli, Italy. giuseppe.saccone@unina.it.

Abstract

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.

PMID:
28855635
PMCID:
PMC5577161
DOI:
10.1038/s41598-017-10347-5
[Indexed for MEDLINE]
Free PMC Article

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