期刊
DEVELOPMENT
卷 140, 期 23, 页码 4818-+出版社
COMPANY OF BIOLOGISTS LTD
DOI: 10.1242/dev.100933
关键词
Drosophila; Functional genomics; Gene targeting; Homologous recombination
资金
- UK Medical Research Council
- European Research Council grant [WNTEXPORT] from the European Union
- Sir Henry Wellcome post-doctoral fellowship [082694/Z/07/Z]
- Wellcome Trust [082694/Z/07/Z] Funding Source: Wellcome Trust
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.
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