Removal of extra sequences with I-SceI in combination with CRISPR/Cas9 technique for precise gene editing in Drosophila

Abstract

The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies and has been adopted for many organisms, including Drosophila. Previously, an efficient two-step strategy was developed to engineer the fly genome by combining CRISPR/Cas9 with recom-binase-mediated cassette exchange (RMCE). This strategy allows the introduction of designed mutations into a gene of interest in vivo. However, the loxP or frt site remains in the edited locus. Here, we propose a modification of this approach for rapid and efficient seamless genome editing with CRISPR/Cas9 and site-specific recombinase-mediated integration (SSRMI) combined with recombination between homologous sequences induced by the rare-cutting endonuclease I-SceI. The induced homological recombination leads to the removal of the remaining extraneous sequences from the target locus. METHOD SUMMARY Modification of the method for Drosophila genome editing that allows different mutations to be easily and precisely introduced into a gene through three subsequent steps, namely, CRISPR/Cas9, SSRMI and I-SceI-mediated recombination techniques.