Parallel Genomic Engineering of Two Drosophila Genes Using Orthogonal attB/attP Sites

G3: Genes, Genomes, Genetics

Beatriz Blanco-Redondo^1¹ 

[1] Rudolf Schönheimer Institute of Biochemistry, Division of General Biochemistry, Medical Faculty, Leipzig University, 04103 Leipzig, Germany^1

关键词: phiC31;    transgenesis;    Drosophila melanogaster;    CRISPR;    orthogonal;   

DOI  :  10.1534/g3.118.200565

学科分类：生物科学（综合）

来源: Genetics Society of America

【 摘 要 】

Precise modification of sequences in the Drosophila melanogaster genome underlies the powerful capacity to study molecular structure-function relationships in this model species. The emergence of CRISPR/Cas9 tools in combination with recombinase systems such as the bacteriophage serine integrase ΦC31 has rendered Drosophila mutagenesis a straightforward enterprise for deleting, inserting and modifying genetic elements to study their functional relevance. However, while combined modifications of non-linked genetic elements can be easily constructed with these tools and classical genetics, the independent manipulation of linked genes through the established ΦC31-mediated transgenesis pipeline has not been feasible due to the limitation to one attB/attP site pair. Here we extend the repertoire of ΦC31 transgenesis by introducing a second pair of attB/attP targeting and transgenesis vectors that operate in parallel and independently of existing tools. We show that two syntenic orthologous genes, CG11318 and CG15556 , located within a 25 kb region can be genomically engineered to harbor attP^TT and attP^CC sites. These landing pads can then independently receive transgenes through ΦC31-assisted integration and facilitate the manipulation and analysis of either gene in the same animal. These results expand the repertoire of site-specific genomic engineering in Drosophila while retaining the well established advantages and utility of the ΦC31 transgenesis system.

【 授权许可】

CC BY