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  A Versatile Two-Step CRISPR- and RMCE-Based Strategy for Efficient Genome Engineering in Drosophila

Zhang, X., Koolhaas, W. H., & Schnorrer, F. (2014). A Versatile Two-Step CRISPR- and RMCE-Based Strategy for Efficient Genome Engineering in Drosophila. G3-GENES GENOMES GENETICS, 4(12), 2409-2418. doi:10.1534/g3.114.013979.

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 Creators:
Zhang, Xu1, Author              
Koolhaas, Wouter H.1, Author              
Schnorrer, Frank1, Author              
Affiliations:
1Schnorrer, Frank / Muscle Dynamics, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565168              

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Free keywords: ZINC-FINGER NUCLEASES; HOMOLOGOUS RECOMBINATION; MELANOGASTER; CAS9; SYSTEM; GERMLINE; PROJECTIN; RESOURCE; PROTEIN; REPAIRDrosophila; CRISPR/Cas9; homologous recombination; RMCE; muscle;
 Abstract: The development of clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technologies promises a quantum leap in genome engineering of model organisms. However, CRISPR-mediated gene targeting reports in Drosophila melanogaster are still restricted to a few genes, use variable experimental conditions, and vary in efficiency, questioning the universal applicability of the method. Here, we developed an efficient two-step strategy to flexibly engineer the fly genome by combining CRISPR with recombinase-mediated cassette exchange (RMCE). In the first step, two sgRNAs, whose activity had been tested in cell culture, were co-injected together with a donor plasmid into transgenic Act5C-Cas9, Ligase4 mutant embryos and the homologous integration events were identified by eye fluorescence. In the second step, the eye marker was replaced with DNA sequences of choice using RMCE enabling flexible gene modification. We applied this strategy to engineer four different locations in the genome, including a gene on the fourth chromosome, at comparably high efficiencies. Our data suggest that any fly laboratory can engineer their favorite gene for a broad range of applications within approximately 3 months.

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Language(s): eng - English
 Dates: 2014
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: ISI: 000346341500011
DOI: 10.1534/g3.114.013979
 Degree: -

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Title: G3-GENES GENOMES GENETICS
Source Genre: Journal
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Publ. Info: 9650 ROCKVILLE AVE, BETHESDA, MD 20814 USA : GENETICS SOCIETY AMERICA
Pages: - Volume / Issue: 4 (12) Sequence Number: - Start / End Page: 2409 - 2418 Identifier: ISSN: 2160-1836