A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila.

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 id

SUBMITTER: Zhang X 

PROVIDER: S-EPMC4267936 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature