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

Abstract 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 inte...

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Bibliographic Details
Published in:G3 : genes - genomes - genetics 2018-09, Vol.8 (9), p.3109-3118
Main Authors: Blanco-Redondo, Beatriz, Langenhan, Tobias
Format: Article
Language:English
Subjects:
Animals
Attachment Sites, Microbiological
Bacteriophages - genetics
CRISPR
Drosophila melanogaster
Genetic Engineering - methods
Genetic Vectors - genetics
Investigations
orthogonal
phiC31
Transgenes
transgenesis
Virus Integration
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Summary:Abstract 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 attPTT and attPCC 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.
ISSN:2160-1836
2160-1836
DOI:10.1534/g3.118.200565