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Targeting genes involved in sexual determinism for vector or pest control purpose, requires a better understanding of their polymorphism in natural populations in order to ensure a rapid spread of the construct. By using genomic data from An. gambiae s.l., we analyzed the genetic variation and the conservation score of the fru gene in 18 natural populations across Africa. A total of 34339 SNPs were identified including 3.11% non-synonymous segregating sites. Overall, the nucleotide diversity was low and the Tajima's D neutrality test was negative indicating an excess of low frequency SNPs in the fru gene. The allelic frequencies of the non-synonymous SNPs were low (freq < 0.26) except two SNPs identified at high frequencies (freq > 0.8) in the Zinc-finger A and B protein domains. The conservation score was variable throughout the fru gene with maximum values in the exonic compared to the intronic regions. These results showed a low genetic variation in overall the exonic regions especially the male sex-specific exon and the BTB-exon 1 of the fru gene. These findings are crucial for the development of a gene drive construct targeting the fru gene that can rapidly spread without encountering resistance in wild populations.

The analysis was conducted across 22 dipteran species and offered insight into the level of conservation at a genome-wide scale as well as at per base-pair resolution. The results of this analysis are gathered in data storage that allows for flexible extraction and bioinformatic manipulation.

Discovery and adaptation of CRISPR-Cas systems for genome editing have allowed us to gain an efficient and yet simple tool for genetic manipulation in various fields of molecular biology and biotechnology. One of the most promising applications is the use of CRISPR-Cas9 endonuclease for gene drive systems as a population control strategy for various insect pests of medical and agricultural importance. Use of CRISPR-Cas9 endonuclease in gene drive applications has shown great promise in the laboratory, particularly for the control of Anopheles gambiae, the major vector of malaria. However, the performance of such gene drives can be limited by the range of available target sequences and by a propensity of existing endonuclease formulations to generate resistant mutations that hinder the gene drive’s efficiency. To expand the flexibility of gene drive systems, computational analysis was performed to identify additional Cas9 orthologs and their specificities that could usefully augment the targeting range of endonuclease-based gene drives. Two alternative variants of CRISPR-Cas endonucleases found in the bacterial species Lactobacillus rhamnosus and Bacteroides fragilis were assessed for their potential to expand the targeting space in the genome Anopheles gambiae. In addition, a computational tool was developed that evaluates neighbouring sequences to the target site to measure both its likely functional constraint and its likely propensity for DNA repair that could generate in-frame alleles. Using this approach we were able to generate a prioritized list of Anopheles gambiae target sites for gene drive applications that are less likely to be compromised by resistant alleles. Open Access

Although of high priority for the development of genetic tools to control malaria-transmitting mosquitoes, only a few germline-specifc regulatory regions have been characterised to date and the\ud presence of global regulatory mechanisms, such as dosage compensation and meiotic sex chromosome inactivation (MSCI), are mostly assumed from transcriptomic analyses of reproductive tissues or whole gonads. In such studies, samples include a signifcant portion of somatic tissues inevitably complicating the reconstruction of a defned transcriptional map of gametogenesis. By exploiting recent advances in transgenic technologies and gene editing tools, combined with fuorescence-activated cell sorting and RNA sequencing, we have separated four distinct cell lineages from the Anopheles gambiae male gonads: premeiotic, meiotic (primary and secondary spermatocytes) and postmeiotic. By comparing\ud the overall expression levels of X-linked and autosomal genes across the four populations, we revealed\ud a striking transcriptional repression of the X chromosome coincident with the meiotic phase, classifable as MSCI, and highlighted genes that may evade silencing. In addition, chromosome-wide median\ud expression ratios of the premeiotic population confrmed the absence of dosage compensation in the male germline. Applying diferential expression analysis, we highlighted genes and transcript\ud isoforms enriched at specifc timepoints and reconstructed the expression dynamics of the main biological processes regulating the key stages of sperm development and maturation. We generated\ud the frst transcriptomic atlas of A. gambiae spermatogenesis that will expand the available toolbox for the genetic engineering of vector control technologies. We also describe an innovative and\ud multidimensional approach to isolate specifc cell lineages that can be used for the targeted analysis of other A. gambiae organs or transferred to other medically relevant species and model organisms.