**6. CRISPR/Cas9 considerations**

#### **6.1 Fitness impacts**

The fitness load in population modification CRISPR/Cas9 drive lines have been assessed on male and female mosquitoes. An ideal CRISPR/Cas9 drive candidate for population modification would have little-to-no fitness effects resulting from the drive system and its corresponding locus, as it is predicted that the effector components are likely to have some effect on overall fitness [98, 99].

One notable example of a fitness cost was observed in the *An. stephensi* AsMCRkh2 gene drive line following disruption of both copies of the *kh*w gene. As described, the enzyme encoded by this gene is responsible for generating the precursors for the formation of eye-pigments, but interestingly it also plays an important role in tryptophan metabolism in adult females following a blood meal [100, 101]. AsMCRkh2 individuals containing two LOF alleles resulting from homozygous or heteroallelic combinations of gene-drive construct insertions or Non-Homologous End Joining (NHEJ) alleles produce a white-eye phenotype and show a high lethality and reduced fecundity following a blood meal. Follow up experiments showed that after achieving fixation in multi-generation cage trial experiments, the populations experienced extinction due to the significant fertility and fecundity load on homozygous AsMCRkh2 females [100]. The AsMCRkh2 prototype was later modified to include a re-coded version of the *kh* locus (Reckh) to reduce the previously observed fitness load on females by restoring the function of the *kh* gene and thereby reversing the eye phenotype to wild-type [30]. Homozygous Reckh female adults show no significant differences in fertility and fecundity in comparison to the hemizygous Reckh or wild-type females. The improvements of female fitness translated to the success of the drive system in multi-generational cage trials with >95% of individuals carrying a copy of the drive at the termination of the experiments [30].

In contrast, AgNosCd-1 individuals do not have reduced fitness in most of the fitness parameters evaluated (fertility, fecundity, longevity, larval and pupal development), but a mild reduction in male mating competitiveness was observed [29]. AgNosCd-1 males are slightly less likely to contribute to the next generation than wild-type males, ~2% less likely for hemizygote males and ~8% for homozygote males. Despite these observed reductions in fitness, the power of the drive system was sufficient to negate the effects in subsequent generations and the AgNosCd-1 line achieved fixation in all multi-generation cage trial experiments at different release ratios of homozygous AgNosCd-1 to wild type males [29]. However, the AgTP13 homozygous males were ~22% less likely to contribute to the next generation than wild-type males in competition experiments and have a significantly reduced median lifespan than the hemizygous AgTP13 or the wild-type males. Despite the increased fitness burden in AgTP13 males, there was no increased fitness load on AgTP13 females [31]. Theoretical modeling supports the conclusion that given an appropriate drive mechanism, a gene-drive system could have a significant fitness cost and still be driven through the population [102, 103].

Ideally, GEMs should have no or minimal fitness costs to avoid reducing the effectiveness of the genetic drive mechanism that is used to introduce the synthetic construct into field mosquito populations and to maximize the likelihood of successfully introducing refractory genes into a wild population [98]. Several factors can impact the fitness, including the possible negative effect of the transgene products, insertional position effects (chromatin rearrangement and/or new regulatory element interactions/pressure), inbreeding, and to "leaky (low level constitutive) promoter expression". GEMS can have different degrees of fitness cost and estimates of transgene fitness costs are essential for modeling and planning release strategies. However, it is clear that a robust drive system can compensate for reduced fitness.

#### **6.2 Maternal effects and resistant alleles**

The efficacy of population modification mosquito drive lines may be reduced by the presence of naturally-occurring cleavage-resistant allelic variants of the target site

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

in wild populations or by such alleles generated through NHEJ during the Cas9/gRNA targeting and DNA repair processes. The latter may result from double-stranded DNA breaks necessary for drive that are occasionally repaired through NHEJ resulting in insertions or deletions at the target site, making them refractory to the drive system. Both the naturally-occurring and induced allelic variants have been called resistance alleles [104–107]. The latter may arise in the germline and be passed on to subsequent generations or may be generated in somatic cells where they give rise to mosaic phenotypes [28–30]. Resistance alleles in the form of naturally-occurring mutations at the target site can be avoided by careful choice of the gene-drive target locus. Resistance alleles occurring because of NHEJ due to undesired Cas9 activity can be controlled by careful choice of the promoter used to induce Cas9 transcription.

Extensive analysis of suitable target loci must be performed prior to the creation of each proof-of-principle modification drive system. Loci must be chosen, in part, based on the minimization of naturally-present single nucleotide polymorphisms (SNPs) and overall conservation of the target site. Several SNPs in the AgNosCd-1 *cardinal* target site were identified after a screening effort of hundreds of diverse *Anopheles gambiae* s.l. sequences [29]. Interestingly, all these major variants still exhibited Cas9/gRNA-mediated cleavage in assays *in vitro*.

The pathways and frequency of resistance allele formation via undesired activity of the drive system was analyzed extensively for the AgNosCd-1 and AsMCRkh2 lines [29, 97]. Exceptional phenotype individuals (mosaics and LOF phenotypes) have been correlated to undesired Cas9 activity and possess indel mutations that would cause LOF in AgNosCd-1 and AsMCRkh2 lines. However, in contrast to the AgNosCd-1 drive system, the mosaic and LOF phenotypes made up the majority of the offspring (>99%) from AsMCRkh2 mothers [28]. The presence of mosaic and LOF phenotypes from female drive parents has been hypothesized to occur due to a maternal effect. The maternal effect is proposed to result from the accumulation of Cas9/gRNA complexes in the cytoplasm of embryos derived from mothers carrying the drive system, which perform cleavage on the paternally-donated allele during embryonic development. The differences in mosaic and LOF phenotypes observed in the progeny from AgNosCd-1 and AsMCRkh2 hemizygote females supports this hypothesis and this affect is higher in females with two copies (homozygous) of the drive system than those with one (hemizygous) [28, 29, 97]. In addition, the frequencies of such events are higher in the AsMCRkh2 line when compared to AgNosCd-1. These differences may result from the difference in the gene promoters used to express the Cas9 nuclease for each drive system, *vasa* for AsMCRkh2 and *nanos* for AgNosCd-1. Follow up studies showed that the transcripts expressed from the *nanos* promoter are more confined to germline cells than those expressed from the *vasa* promoter [108], which likely results in fewer Cas9/gRNA complexes in the cytoplasm of the former embryos.

As described previously, females homozygous for the drive system had a higher rate of resistance allele formation via maternal effect (~57% with mosaic phenotype and ~6% of progeny with LOF phenotype) than hemizygous females (~20% with mosaic phenotype and ~1% of progeny with LOF phenotype) but mosaic individuals were able to bias inheritance of the drive allele and had similar rates of drive efficiency when compared to AgNosCd-1 hemizygotes with wild-type eye phenotypes suggesting that the indels were primary somatic [29].

Suppression gene drive systems are much less flexible to drive-resistant alleles than population modification gene drive systems. Population modification mosquito lines can tolerate higher rates of drive-resistant alleles than population suppression

mosquitoes, however, the former are still susceptible to instability and inability to achieve fixation in a population due to resistance alleles, especially if the drive system and respective cargo are associated with a significant fitness load [109]. Recent work suggests that suppression drive systems that incur a 100% fitness cost (death of females) would require a very low frequency of drive resistant alleles <5 × 10−7 in order to provide a 4–5-year window of protection, as opposed to population modification systems, which would provide a 4–5-year window of protection at a resistance allele frequency of 1%, given that fitness costs of the population modification strain are below 15% [109].

Multiplexed gene drives using additional gRNA target sites are expected to substantially decrease the likelihood of gene-drive resistant allele formation [110]. Practical ways to multiplex Cas9-based gene drives have been demonstrated using post-transcriptional processing of several gRNAs expressed from a single promoter, but these have not yet been applied to mosquitoes [110–113].

#### **6.3 Off-targets**

The utility of CRISPR/Cas9 gene-drive systems may be affected by sequence similarity among gRNAs target and off-target sites in the mosquito genomes. Potential off-target sites can be predicted *in silico* by computational algorithms and then confirmed *in vivo* by deep-sequence screening of indels or SNPs by PCR-based assays. The possible impact of unwanted mutations linked to a drive system are higher since the arising mutations will have the potential to persist within the populations. Off-target mutations also can induce a potential fitness load. Efforts to detect Cas9 off-targets in *An. gambiae* gene drive mosquitoes found very few following sequencing of large number of samples containing putative target variants [29]. The detected indels neither increased in frequency nor were detected through multiple generations in long-term cage trials (indicating that they were not heritable) and did not significantly differ in number from variants observed in wild-type individuals [29]. New approaches to increase Cas9 specificity are being developed in other organisms and include the use of highly-specific Cas9 mutant enzymes together with the constant updating of computational algorithms to better predict the possible off-targets, but their applications for gene drive mosquitoes remain unclear [114–119].

#### **6.4 Deployment challenges**

The discovery, development, and deployment of CRISPR/Cas9 technologies is challenging due to the lack of an accepted pathway to move them from the laboratory to the field. The WHO released in 2014 the Guidance Framework for testing genetically modified (GM) mosquitoes (WHO Guidance Framework) describing a phased testing pathway and best practices to evaluate GEMs proposed as public health tools [120]. The Framework proposes a pathway to move from physically-confined studies in the laboratory/insectary (Phase 1) to a small-scale confined field-testing (Phase 2) that will lead to a staged open release trial (Phase 3). After successful completion of Phase 3, the national authorities in a malaria-endemic country will be responsible for determining if the tested GEMs can be included as part of their malaria control program and further deployment of the technology (Phase 4) [120]. However, pathways for moving gene-drive population modification mosquitoes to the field will be defined simultaneously with the laboratory work progress. As more CRISPR/Cas9 population modification gene-drive systems and strains are developed, new knowledge is being

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

generated about the impact of introduced anti-parasite genes on the mosquitoes that carry them. Insight into genetic loads and their effects on fitness, generation of driveresistant individuals as well as selection of resistant parasites and long-term stability of the system will emerge from these studies. The new empirical data generated is critical in the development of a phased pathway for further development and deployment. In 2018, James et al. published a series of recommendations that attempt to envision the development pathway for gene drive mosquitoes (from discovery to deployment) and to inform decision-making by regulators and policymakers [121]. They recognized that it is important to examine both the benefits and risks of this approach. Risk assessment will provide guidance on decision-making and information for the regulatory applications as well as for the development of mitigation plans, while cost-benefit analyses will compare the projected or estimated costs and benefits associated to the intervention. It also was recommended that these analyses be done by external third-party organizations or institutions with no interests in the success of the product and the outcomes of these analyses be made publicly available.

Any decision made to release gene-drive mosquitoes must be made on a case-bycase basis following a comprehensive environmental risk assessment [122], moreover, gene-drive population modification mosquitoes must meet the established Target Product Profile (TPP) criteria of safety and efficacy. A comprehensive draft TPP for gene-drive population modification mosquitoes was published providing the basis for evaluation of whether gene-drive mosquitoes should be made available for use [37]. Population modification TPPs will need to meet the efficacy and safety standards as well as the demands of different regulatory and social contexts. In addition, viable models for the inclusion of end-user and stakeholder involvement and control are absolutely needed before any such system can be brought to the field. We have favored the relationship-based model (RBM), which gives stakeholders and community key roles at the center of the decision-making processes [123]. It is important that open dialog and relationships with the scientists developing the technologies be established and that appropriate capacity-building take place to empower the communities affected by malaria to make informed decisions about the risk and use of the new technologies.

#### **7. Conclusions**

Population modification genetic control focuses on targeting the mosquito vector to interrupt the malaria transmission by introducing effector genes into the mosquito genome with the purpose of generating parasite-refractory mosquitoes.

Advances in gene-editing technologies using CRISPR/Cas9 gene drives have made available new possibilities for an efficient introduction of the desired genetic traits into mosquito populations. Gene drives represent a powerful tool to achieve genome editing in a species-specific targeted way with minimal infrastructure, are predicted to be self-sustaining and able to spread anti-parasite effectors to fixation.

Gene-drive systems for population modification of anopheline vector species to prevent transmission of parasites may play a future role in the malaria eradication agenda. Future steps will need to consider how to evaluate gene drives at large scale and evaluate their efficacy and robustness under more realistic ecological settings.

Challenges to such technologies are being addressed by scientists and regulators by development of pathways for their deployment and establishing acceptable efficacy and safety criteria. Importantly, the knowledge transfer process is being addressed

in new models for public engagement that will further development, testing and eventual deployment of gene drives for malaria control.
