Fig. 1: Schematic representation of gene drive and anti-drive constructs.

Gene drive and anti-drive constructs respectively as inserted in the genome of  the gene drive lines previously generated (zpg:dsxF²; zpg:7280 and nos:7280, ref. ²⁶) and the newly generated anti-drive (vasa:A4) line. Specifically, the gene drive constructs tested in this work are inserted at target sites within the AGAP007280 or AgdsxF (AGAP004050-RB) gene-coding sequences and contain: the Streptococcus pyogenes Cas9 nuclease (Cas9), under the transcriptional control of the male and female germline-specific promoters zpg or nos; the gRNA, targeting the respective insertion site, transcribed by the RNA polymerase III responsive promoter (U6), and the DsRed fluorescent protein under the 3xP3 promoter (3xP3:DsRed) for the identification of larvae carrying the drive. The anti-drive construct carries the Listeria monocytogenes anti-CRISPR protein (AcrIIA4) expressed under the vasa male and female germline-specific promoter with the N-terminus addition of a nuclear localisation signal (NLS) and the eGFP fluorescent protein under the 3xP3 promoter (3xP3:eGFP) used for the screening of anti-drive positive larvae. The construct was inserted in a pre-existing docking line carrying the 3xP3:eCFP marker²⁵. As a result the AcrIIA4 protein is expected to interact and inhibit the Cas9–gRNA complex, when co-expressed in the mosquito germline cells.