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 Phi 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 nonlinked genetic elements can be easily constructed with these tools and classical genetics, the independent manipulation of linked genes through the established Phi C31-mediated transgenesis pipeline has not been feasible due to the limitation to one attB/attP site pair. Here we extend the repertoire of Phi 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 CG 15556, located within a 25 kb region can be genomically engineered to harbor att(PTT) and att(PCC) sites. These landing pads can then independently receive transgenes through Phi C31-assisted integration and facilitate the manipulation and analysis of either gene in the same animal. These results expand the repertoire of sitespecific genomic engineering in Drosophila while retaining the well established advantages and utility of the Phi C31 transgenesis system.