PT  - JOURNAL ARTICLE
AU  - Xu Zhang
AU  - Wouter Koolhaas
AU  - Frank Schnorrer
TI  - A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in <em>Drosophila</em>
AID  - 10.1101/007864
DP  - 2014 Jan 01
TA  - bioRxiv
PG  - 007864
4099  - http://biorxiv.org/content/early/2014/08/11/007864.short
4100  - http://biorxiv.org/content/early/2014/08/11/007864.full
AB  - The development of CRISPR/Cas9 technologies promises a quantum leap in genome-engineering of model organisms. However, CRISPR-mediated gene targeting reports in Drosophila 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 loci, including a gene on the fourth chromosome, at comparably high efficiencies, suggesting that any fly lab can engineer their favourite gene for a broad range of applications within about three months.