Repair outcomes after germline homing endonuclease cleavage in Anopheles gambiae inform the design of synthetic gene drives

Homing endonuclease genes spread by cleaving homologous chromosomes that lack the endonuclease cassette, after which repair from the endonuclease-containing chromosome converts the cut allele into a copy of the drive allele. This mechanism has provided a conceptual foundation for synthetic gene drive systems, including CRISPR-based drives, that represent promising strategies for the genetic control of insect pests. However gene drive performance depends critically on the repair pathways available in the germline of the target organism. Here, we report a set of transgenic assays originally developed as part of an attempt to establish gene targeting in the malaria mosquito Anopheles gambiae using an in vivo-generated linear targeting molecule. Although the intended FLP-mediated excision step was not achieved in the mosquito germline, analysis of the component strains revealed efficient germline activity of the rare-cutting homing endonuclease I-SceI and a striking bias towards homology-based repair of I-SceI-induced double-strand breaks. Across reporter and donor configurations, cleavage outcomes were dominated by single-strand annealing, microhomology-mediated repair, synthesis-dependent strand annealing and gene conversion-like events, with comparatively limited evidence for classical non-homologous end joining. In reciprocal crosses designed to distinguish gene conversion from gamete loss, I-SceI cleavage also produced inheritance distortion consistent with both conversion of the cleaved allele and reduced recovery of gametes carrying extensively damaged donor alleles. These findings indicate that the An. gambiae germline can strongly favour homology-dependent repair following homing endonuclease cleavage and that cleavage can also generate meiotic drive-like distortion through selective loss of damaged gametes. The results have direct relevance for the design and interpretation of homing endonuclease and CRISPR-based gene drives in malaria mosquitoes, where the balance between homology-directed repair, end joining and gamete viability will determine drive efficiency, resistance formation and transmission bias. Author summaryGene drives depend on a simple but demanding principle: a nuclease cuts one chromosome, and the cell repairs the break using the homologous chromosome as a template, copying the drive element in the process. Before CRISPR, this type of system was explored using naturally occurring homing endonucleases such as I-SceI. We attempted to develop a gene targeting system in Anopheles gambiae based on the Rong and Golic strategy, in which FLP recombinase would excise a donor molecule and I-SceI would linearise it to stimulate recombination. The full knockout technology did not work because FLP-mediated excision was not detected in the mosquito germline. However, the component tests revealed something more broadly important: I-SceI-induced breaks were repaired predominantly through homology-based pathways rather than simple end joining. We also observed inheritance distortion consistent with both gene conversion and loss of damaged gametes. These results help explain why homing-based systems can work in mosquitoes, while also highlighting why repair pathway choice and gamete viability need to be measured directly in any new drive configuration.