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.

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila

“…We next evaluated germ-line transmission of CRISPR/Casinduced mutations in wg and wls using nos-cas9. Nineteen randomly selected offspring of nos-cas9 U6:3-gRNA-wg flies were tested for wg mutations by genetic complementation with a previously characterized wg null allele (27). The wg null allele was not complemented in 12 of the 19 crosses, demonstrating efficient creation of nonfunctional wg mutations by CRISPR/Cas (Fig.…”

“…In Drosophila it has been possible for several years to create specific genome modifications by HDR, although targeting typically occurs with very low efficiency (28). Recent studies have suggested that zinc finger, transcriptional activator-like effector (TALE), and Cas9 nucleases can increase the efficiency of HDR in flies, implying that the generation of site-specific DSBs is rate limiting (11,12,27,29,30). Therefore we tested whether the highly effective creation of DSBs by our transgenic CRISPR/Cas system could be exploited to facilitate efficient HDR.…”

“…Individual flies of the genotype wg test /CyO then were crossed to wg TV-Cherry /CyO flies. wg TV-Cherry is a wg null allele in which the first exon of wg is replaced with a homologous recombination targeting cassette (27). The next generation of the crosses was screened for the presence or absence of flies without the CyO chromosome.…”

Methods to study Drosophila immunity

“…Extensive libraries of P-element-based transposon insertions are available through stock centers, along with deletion and duplication lines [78][79][80][81]. Finally, targeted gene knock-out using optimized targeting plasmids in combination with CRISPR will greatly accelerate full KO coverage of the fly genome [82].…”

Des insulines pour orchestrer la croissance

“…La validation fonctionnelle de cet élément a été réalisée d'une part in vitro, en démontrant une interaction physique directe, puis in vivo en suivant l'activation transcriptionnelle de dilp8 par Yki/ Sd. Dans le but de valider fonctionnellement le rôle de la voie Hippo dans le contrôle de dilp8, un mutant dilp8 a été généré par CRISPR/Cas9 [34,35] (➜). Les sites de fixation pour Sd/Yki dans le HRE endogène du promoteur dilp8 ont été mutés, créant ainsi un allèle du gène dilp8 insensible au contrôle transcriptionnel par Hippo/Yki.…”