Comprehensive characterisation of the genomic insertion site of a transgene in highly repetitive, centromeric region of Anopheles mosquitoes

The availability of the genomic sequence of the malaria mosquito Anopheles gambiae has sparked in recent years the development of transgenic technologies with the potential to be used as novel tools for vector control. These technologies rely on genome editing that confers features able to affect vector capacity. This can be achieved by either reducing the mosquito population or by making mosquitoes refractory to the parasite infection. Although sophisticated molecular techniques such as those based on AttB/AttP site-specific recombination and CRISPR/Cas9 systems can lead to the integration of transgenes in specific sites of the genome, methods that allow semi-random integration are still in use due to their high efficiency; PiggyBac transposon-mediated integrations fall in this category. Characterization of the insertion site of transgenes in transgenic strains generated via PiggyBac integration can be hampered when the transgene is inserted in regions of the genome rich in repetitive sequences. Here we describe a number of techniques that were used to identify the genomic location of the transgene in a repetitive region in the Anopheles gambiae strain Ag(PMB)1 which was initially reported on Chromosome 3R 36D. Whilst Inverse PCR used in previous analysis was unable to distinguish between multiple genomic locations as potential insertion sites of the transgene, here we demonstrate that the use of FISH identifies clearly the integration of the transgene in a poorly annotated centromeric region of Chromosome 2R 19D. This study emphasises the need for accuracy in sequencing data for the genome of organisms of medical importance such as Anopheles mosquitoes. An effort to further improve reference genomes is of paramount importance to support and facilitate vector control interventions based on genome editing.