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.

Keywords: Drosophila; Functional genomics; Gene targeting; Homologous recombination.

Fig. 1.

A targeting vector and protocol for accelerated gene targeting. (A) The targeting vector, pTV^Cherry, showing its key features, including cherry and mini-white. The cherry cDNA and the mini-white were flanked by non-canonical FRTs (FRT71) and this was followed by Gal4-encoding sequences with the aim of enabling easy conversion to a Gal4 driver although this turned out not to be functional. (B) Gene targeting by crossing scheme. P-element-mediated transformation was used to generate the donor strain, which carries the targeting vector pTV^Cherry[geneX] at a random genomic location. The donor strain was crossed to flies carrying hs-FLP and hs-I-SceI to mobilise and linearize the donor construct (first cross). Typically, 200 adult female progeny with mottled eye colour (an indication that they carry the targeting vector and the hs-FLP, hs-I-SceI chromosome) were then crossed in pools of 15 to ubiquitin-Gal4[3xP3-GFP] (2d cross) to eliminate unwanted events and parental flies. The progeny of this second cross were then screened for red eyes and screening was terminated when a PCR-confirmed (on both sides) homologous recombinant was obtained. All the strains used were mutant at the endogenous white locus, allowing the mini-white of the targeting construct to be followed throughout. (C) Gene targeting by direct injection in embryos. The injected mix contained pTV^Cherry[geneX] and vasa-FUS (expressing FLP and I-SceI). For CRISPR-aided gene targeting, the mix included, in addition, U6-target-gRNA and vasa-Cas9. (D) Top: The hedgehog locus. Coding exons are orange, the first unaffected exon is shown as a hatched box, untranslated sequences are in grey and the region deleted by homologous recombination is marked by a red box. Bottom: The resulting allele, highlighting the Cre-excisable region that includes cherry and mini-white. (E) Cherry expression in imaginal discs from a hedgehog[KO] larva.

Fig. 2.

A set of reintegration vectors (RIV): diagrams and features. (A) The key features of the reintegration vectors described in this paper. The restriction sites available for cloning (MCS) are listed below. Some of the vectors use mini-white and others use 3xP3-Cherry (pax-Cherry) as genetic markers. (B,C) Low-magnification fluorescence micrographs show the suitability of 3xP3-Cherry as a larval (B) and adult (C) markers. RIV^{FRT.Wg.FRT NRT-HA-Wg}/Cyo[white+] animals are shown.

Fig. 3.

Examples of reintegration vectors and their features. (A) The wild-type wingless locus before and after targeting by homologous recombination; Cre-mediated excision of the cherry and mini-white markers; and reintegration, via RIV^white, of a cDNA encoding HA-tagged Wingless. (B) Wild-type imaginal disc stained for anti-Wingless antibody. (C) Engineered allele expressing reintegrated HA-Wingless stained with anti-HA. (D) The rhogap102A locus before and after targeting by homologous recombination, Cre-mediated excision of the markers and reintegration of RIV^Gal4. Crossing such flies with UAS-GFP revealed that the rhogap102A locus is transcriptionally active in muscles. (E) The hedgehog locus before and after targeting by homologous recombination, Cre-mediated marker excision and reintegration of RIV^{FRT.HhOllas.FRTQF}. Larvae carrying the reintegrated allele hs-FLP and QUAS-Tomato were heat shocked to randomly excise the FRT cassette, thus triggering activation of QUAS-Tomato in a subset of the normal hedgehog expression domain. (F) Allele switching at the wingless locus. RIV^{FRT.Wg.FRT NRT-HA-Wg} was reintegrated so that excision of the FRT cassette makes the locus stop expressing wild-type Wingless and start expressing HA-tagged NRT-Wingless instead. Excision was induced in the posterior compartment with engrailed-Gal4-driven FLP. Hence, HA immunostaining from NRT-HA-Wg is detected only in the posterior subset of wingless-expressing cells. DAPI stains nuclei in blue (all panels). Coding exons are orange, the first unaffected exon is shown as a hatched box, untranslated sequences are in grey and the region deleted by homologous recombination is marked by a red box.