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

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 C31 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 C31-mediated transgenesis pipeline has not been feasible due to the limitation to one attB/attP site pair. Here we extend the repertoire of C31 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 harbour attP^TT and attP^CC sites. These landing pads can then independently receive transgenes through C31-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 whilst retaining the well established advantages and utility of the C31 transgenesis system.

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