**5. CRISPR/Cas9 gene drives for population modification**

The recent adaptation of CRISPR/Cas9-based biology to generate gene drives has been proposed to provide a powerful, inexpensive, and easily-implemented solution for malaria control due to the rapid introduction of the antimalarial genes into mosquito populations [37]. To produce the desired epidemiological outcomes of reduced malaria transmission, the drive system and associated effector components must be introduced quickly and efficiently into wild populations. Rapid introduction requires population modification lines to have high rates of drive allele conversion in the germline so that maximally-biased inheritance is achieved. This will result in a remarkable increase in frequency of the gene-drive system in the following generations.

#### *Mosquito Population Modification for Malaria Control DOI: http://dx.doi.org/10.5772/intechopen.104907*

The first CRISPR/Cas9 gene drive for mosquito population modification was described in 2015 for the Indo-Pakistani vector, *An. stephensi* [28]. The AsMCRkh2 gene-drive synthetic cassette used targets the ommochrome biosynthesis pathway involved in development of mosquito eye-color, specifically the locus that encodes the kynurenine hydroxylase (kynurenine monooxygenase) enzyme (referred here as *kynurenine hydroxylase white* [*kh*w]). Mutations in the gene encoding this enzyme have a recessive white-eye phenotype. Drive efficiency in the AsMCRkh2 line was high with ~99% of progeny from both male and female hemizygous parents inheriting a copy of the drive allele [28]. Despite high initial efficiencies from both male and females, follow-up analyses of these lineages uncovered a return to near Mendelian inheritance in the progeny derived from female hemizygous parents. The diminished drive efficiency in female lineages was later attributed to the accumulation of indel alleles in these offspring (Section 6.2). Drive efficiency experiments in a second-generation *An. stephensi* population modification line, Reckh, resembled the observed efficiencies in the AsMCRkh2 line with ~99% of the progeny from hemizygous male parents inheriting a copy of the drive after two generations of outcrossing to wild-type mosquitoes and only ~56% of progeny from hemizygous female parents inheriting a copy of the drive after two generations of outcrosses [30].

A next-generation gene drive system for *An. gambiae* was developed [29]. The resulting strain, AgNosCd-1, targets the *An. gambiae cardinal* gene ortholog, encoding a protein downstream of the *kh* product in the ommochrome pathway. Mosquitoes with two loss-of function (LOF) alleles at this locus have a red-eye phenotype in subadult stages and newly-emerged adults (**Table 1**). AgNosCd-1 has a high drive efficiency in both male and female lineages (maternal/paternal daughters/sons and grand-daughters/grand-sons). AgNosCd-1 hemizygous males can pass the drive system with ~99% efficiency within their lineages and hemizygous females had only a slightly reduced ~95% drive efficiency within their lineages [29]. The individuals not inheriting a copy of the drive were found to have wild-type alleles as opposed to insertion or deletion (indel) alleles indicating that failures of the drive system were more likely due to Cas9/gRNA complexes not performing cleavage as opposed to cleavages that did not result in HDR [97]. Moreover, the AgNosCd-1 drive efficiency achieved a 98–100% inheritance bias in both males and females and full introduction within six to ten generations following single releases of gene drive males in small laboratory cage trials [29]. Drive efficiency experiments in a second-generation AgNosCd-1 population modification line, AgTP13 (AgNosCd-1 background linked to two antiparasite effector genes), resulted in similar rates of drive efficiency in hemizygous males and females suggesting no impact of the effector load on the ability of the drive system to facilitate accurate HDR in the germline [31].
