**4. Spreading transgenes into mosquito populations**

Mobile genetic elements called transposons can spread rapidly through populations despite severe costs to the host [69–72]. Their ability to mobilize (excise and insert) led to their being developed as powerful systems for introducing exogenous DNA into several organisms. The adaptation of the P transposable element for transgenesis of the vinegar fly, *Drosophila melanogaster*, was followed 16 years later by the first reliable system for transforming mosquitoes using the *Hermes* elements in the yellow fever mosquito, *Aedes aegypti* [73, 74]. Shortly after this proof-of-principle in mosquitoes, additional systems based on *Hermes, piggyBac, Minos, and mariner Mos1* were demonstrated in both culicine and anopheline species [75–82]. Unfortunately, while transposons could mediate insertion into these genomes at experimentally-useful frequencies, they were not easily remobilized making them impractical as a basis of a gene-drive system to spread transgenes through a mosquito population [83–85].

Other tools and systems for introducing genes into mosquito genomes include site-specific recombinases. These require the presence of an endogenous nucleotide sequence in the genome that is identical to the recombinase target cleavage site, or a mechanism for introducing such a site (called a docking site; [86]) into that genome. This has been achieved using the previously-described transposons. Two recombinases have been used successfully to generate transgenic mosquitoes, the bacteriophage φC31 integrase and Cre/lox recombinase derived from yeast. Their dependence on a precise site for integration of the desired transgene limits their usefulness as the basis of gene-drive systems for spreading transgenes into populations [82, 87–90].

The application of zinc-finger nuclease (ZFNs) and the transcription activatorlike effector nucleases (TALENs) for engineering target-site recognition in mosquitoes introduced a major advance for genetic modification in mosquitoes. However, the high cost and low success rate limited their use [91–93]. The application of homing endonucleases nucleases genes (HEGs) for spreading genes into mosquito populations was proposed in 2003 [94] as useful basis for gene drives and in 2011 a successful HEG-based gene drive in *An. gambiae* was reported [95]. The latter required the fortuitous presence of a nuclease target site in the first chromosome (X) of this species.

A major breakthrough for mosquito transgenesis and gene-drive systems was achieved following the discovery and adaptation of the RNA-guided Cas9 nuclease from the bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) adaptive immune system [96]. This powerful tool simplified the highly-specific genome editing processes and made possible useful gene-drive systems. The Cas9 endonuclease is directed to its genomic target by a single 20 base-pair guide RNA (gRNA) complementary to its DNA target. This gRNA can be designed to target virtually any locus in a chromosome. CRISPR/Cas9 exploits the natural mechanism of cell repair to precisely insert a synthetic construct through homologydirected repair (HDR), a DNA repair system initiated by a double-strand break made at the site of a target location by the Cas9 nuclease [96]. CRISPR/Cas9 has been shown to be an excellent candidate technology for developing gene drive-based strategies to introduce beneficial genes into mosquito populations [28–30]. The properties of the system bias the inheritance of a desired trait, allowing them to quickly increase in frequency and spread through a mosquito population. CRISPR/Cas9 gene drives can efficiently convert pre-meiotic diploid germline cells in hemizygous mosquitoes (carrying one copy of the drive) into homozygotes carrying two copies [28–30]. Recently, the CRISPR/Cas9 technology has been adapted for the development of gene drives in anopheline mosquitoes and shows great promise for rapid introduction of anti-parasite genes into mosquito populations [28–30, 32].
