title: Accelerated homologous recombination and subsequent genome modification in Drosophila.
abstract: Gene targeting by 'ends-out' homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. These vectors allow the insertion of a variety of transcription reporters or cDNAs to express tagged or mutant isoforms at endogenous levels. In addition, they pave the way for difficult experiments such as tissue-specific allele switching and functional analysis in post-mitotic or polyploid cells. Therefore, our method retains the advantages of homologous recombination while capitalising on the mutagenic power of CRISPR.
authors.affiliation: MRC National Institute for Medical Research, London NW7 1AA, UK.
authors.forename: Luis Alberto
authors.initials: LA
authors.lastname: Baena-Lopez
authors.forename: Cyrille
authors.initials: C
authors.lastname: Alexandre
authors.forename: Alice
authors.initials: A
authors.lastname: Mitchell
authors.forename: Laurynas
authors.initials: L
authors.lastname: Pasakarnis
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Vincent
mesh_terms: D000818:Animals; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D004247:DNA / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D004331:Drosophila melanogaster / Q000235:genetics*; D018390:Gene Targeting; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics*; D059765:Homologous Recombination; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic; D017384:Sequence Deletion
chemical_list: D005819:Genetic Markers; D004247:DNA
keywords: Drosophila; Functional genomics; Gene targeting; Homologous recombination