Accelerated homologous recombination and subsequent genome modification in Drosophila

被引:142
作者
Baena-Lopez, Luis Alberto [1 ]
Alexandre, Cyrille [1 ]
Mitchell, Alice [1 ]
Pasakarnis, Laurynas [1 ]
Vincent, Jean-Paul [1 ]
机构
[1] Natl Inst Med Res, MRC, London NW7 1AA, England
来源
DEVELOPMENT | 2013年 / 140卷 / 23期
基金
英国医学研究理事会; 欧洲研究理事会; 英国惠康基金;
关键词
Drosophila; Functional genomics; Gene targeting; Homologous recombination; GUIDED CAS9 NUCLEASE; HUMAN-CELLS; ENDS-OUT; GENE; EFFICIENT; SYSTEM; MELANOGASTER; TALEN; TRANSGENESIS; EXPRESSION;
D O I
10.1242/dev.100933
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:4818 / +
页数:18
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