Laboratory members at the University of California, Irvine Malaria Initiative, and staff have used a prototype Cas9 / gRNA-based gene propulsion system to modify the mosquito population to control malaria transmission by targeting the vectors.
A female Anopheles stephensi mosquito receiving a blood meal. Attribution to Jim Gathany, public domain, via Wikimedia Commons
Approximately 3.4 of the 7.8 billion people in the world are at risk of becoming infected with malaria parasites and developing diseases (WHO 2019). At the turn of the century, a renewed push to eradicate malaria began, and there was a significant decrease in the incidence and prevalence of disease in the first fifteen years of the initiative. Efforts in recent years to maintain this momentum have failed, however, and progress towards eradication has stalled. Vector control is an important low-cost part of the eradication agenda and 80% of the impact on malaria reduction since 2000 has been attributed to actions against vectors (see Figures 1 and 2). Consistent with these impacts, WHO recommended the research and development of a new generation of vector control tools to address the eradication challenge.
Our laboratory members and staff are among a number of scientists from the University of California, the Irvine Malaria Initiative (UCIMI), and the Tata Institute of Genetics and Society (TIGS) who wish to apply genetic engineering technologies to develop novel methods of controlling parasite transmission Mosquito vectors. Our preferred strategy, population modification, uses genetic tools to make vector mosquitoes incapable of transmitting parasites. We expect that the introduction of a gene into a mosquito population that confers parasite resistance should lead to a reduction in the transmission of the pathogen. We use synthetic approaches to create genes that disrupt the transmission of parasites and spread them through mosquito populations. After implementing such a strategy, we expect a measurable decrease in malaria transmission and disease.
We have had considerable success in the past, showing that we can use modern molecular biological and insect transgenesis tools to create genes that prevent mosquitoes from transmitting parasites (see Figures 1 and 2). We recently focused on laboratory experiments to find ways to transfer these genes into wild mosquito populations.
To this end, we and others have made significant efforts to leverage Cas9 / Guide RNA (Cas9 / gRNA) mediated gene propulsion systems.
Figure 1. An autonomous gene propulsion system (A) that is active in the germline comprises the gene that codes for the Cas9 nuclease (orange), a guide RNA-encoding DNA (yellow) that targets the gene of the Choice targets (blue) and the antimalarial molecules (green). . The system splits (scissors) the homologous chromosome (all blue) and copies itself from one chromosome to its homologue through homology-controlled repair (HDR). This leads to a distortion of the genome. All offspring receive a copy of the drive system from this germline cell. Occasionally, after cleavage, the system does not act as a template for DNA repair (B), and a non-homologous end link (EJ) can introduce insertions or deletions (red). These mutations can be resistant to further Cas9 / gRNA cleavage and manifest as drive-resistant alleles that result in 1: 1 inheritance patterns from this germline. Adapted from Adolfi 2020.
This clever and innovative genetic approach promotes the conversion of alternative forms of genes, alleles, from a heterozygous state to homozygous in diploid organisms. This deceptively simple technology can be used as a mechanism to create a gene propulsion system that will rapidly spread anti-parasite genes to the target mosquito population.
We previously demonstrated a prototype of a Cas9 / gRNA-based gene drive system for the population modification of the Indian-Pakistani malaria vector Anopheles stephensi. This system was autonomous, meaning it contained all the information necessary to drive on, and was able to move dual anti-Plasmodium falciparum genes (the deadliest human malaria parasite) in 99.5% of the offspring after outcrossing from gene drive lines Wild type mosquitoes. Small cage trials with this propulsion system revealed that a single release of 1: 1 gene propulsion: wild-type male could achieve 100% introduction within 6 to 12 generations (each mosquito carries at least one copy of the propulsion system and cargo) (Figure) 2A).
Figure 2. Comparisons of the dynamics of the gene drive system (AC) and population size (DF) in triplicate laboratory cage experiments. Propulsion propagation is measured as the change in the frequency of dominant fluorescent eye color phenotypes over several consecutive, non-overlapping generations (G0-G20 as indicated) following a 1: 1 release of gene propulsion systems containing wild-type males. It should be noted that two out of three cages in the cages of the prototype gene propulsion system have become extinct after reaching homozygosity (A, D :). The third cage did not achieve homozygosity and maintained population (A, D :). Second generation cages (recoded or alternative gRNA target genes B, E or C, F, drive systems achieved homozygosity and retained populations. Abbreviations: DsRed +, Discosoma sp, red fluorescent protein; GFP +, green fluorescent protein; CFP +, cyan fluorescent protein.
In addition, we observed that disruption of the target site for the insertion of the propulsion system, kynurenine hydroxylase (kh, also known as kynurenine monooxygenase, kmo, gene), a gene that encodes an enzyme important in the ocular pigment synthesis pathway, is significantly impaired female survival and offspring production after a blood meal (Figure 3).
Figure 3. Gene drive targets in the biosynthetic pathway of mosquito eye pigments (omommrom). The gene drive targeting of the An. stephensi kynurenine hydroxylase (Askh, dashed red line) or An. Gambiae cardinal genes (Agcd, dashed blue line) lead to eye color phenotypes visible in adults that are homozygous for corresponding mutated alleles (4). Abbreviations: TDO, tryptophan-2,3-dioxygenase; KAT, kynurenine aminotransferase; KH, kynurenine hydroxylase (KMO, kynurenine monooxygenase); HKT, 3-hydroxykynurenine transaminase. Adapted from Carballar-Lejarazú et al., 2020.
One approach involves a highly efficient version of the original 2nd generation gene propulsion system, called “recoded”, which provides the system with a functional copy of the target gene into which the engine will be inserted (Figure 4). In contrast to the original system, female mosquitoes of this strain show strong and consistent drive and negligible production of drive-resistant alleles in population cage experiments (2B, 2E). Mechanistically, non-functionally resistant alleles are eliminated via a maternal lethal / sterile effect in combination with a slower acting negative standard selection, and rare functionally resistant alleles do not prevent drive invasion. Small cage experiments showed that single releases from genetically engineered males robustly lead to efficient population modification, with ≥ 95% of mosquitoes carrying the engine within 5 to 11 generations over a range of initial release ratios. This strategy of inserting a gene drive into an essential gene (for viability or fertility) and then rescuing it with a functional gene offers a general solution to the emergence of drive resistance in women.
Figure 4. Eye color phenotypes of adult Anopheles gene mosquitoes. Above: wild type on. stephensi eyes (kh +), which have mutated to a phenotype with white eyes in mosquitoes that are homozygous for the gene drive system (kh-), show red fluorescent eyes (DsRed +). The same mosquitoes that were rescued by the Recoding System (khR) have wild-type colored eyes that are marked with green fluorescence (GFP +). Bottom: wild type on. Gambiae eyes (cd +) that have mutated to a red-eye phenotype in mosquitoes that are homozygous for the gene drive system (cd-) show cyan fluorescent eyes (CFP +). Abbreviations: kh +, wild-type kynurenine hydroxylase gene; kh, mutated kynurenine hydroxylase gene; khR, recoded kynurenine hydroxylase gene; cd + wild type cardinal gene; cd, mutated cardinal gene; DsRed +, Discosoma sp, red fluorescent protein; GFP +, green fluorescent protein; CFP +, cyan fluorescent protein. Photo credit: Kiona Parker, Rebeca Carballar-Lejarazú, and Anthony A. James, University of California, Irvine.
The second approach was to change the target for Cas / gRNA targeting to a gene without affecting fitness. This 2nd generation system, developed for the African malaria vector An, Gambiae, also targets a gene, cardinal (cd), which also encodes an enzyme involved in the synthesis of eye color pigments (Figures 3 and 4) ). The cd gene product acts “downstream” from the kh product and mutations caused by the insertion of driving elements are tolerated in homozygous women. The drive showed an average efficiency of ~ 97% in both sexes, minimal off-target effects in vitro, few adverse effects on the fitness of mosquitoes carrying a drive, and a low frequency of potentially resistant alleles that could counteract this. Importantly, tests in small cages in the laboratory showed that a single 1: 1 release from male instincts was sufficient to ensure that each mosquito carried at least one copy of the instinct construct within four to six generations, which is a period of 6 months and falls within a single annual cycle of malaria transmission (2C and 2F). This system can be a powerful means of delivering anti-parasite effector genes in mosquitoes and could help expedite the development of a shoot-bearing mosquito strain ready for field testing.
The use of Cas9 / gRNA systems has been used effectively by others (see the work of the Target Malaria team at Imperial College London). As part of a larger community involved in the development of this science, we adhere to strict recommendations and guidelines to ensure the safety of developing these systems. In addition, the entire work is made available via Open Access sources (e.g. PubMed) so that both specialists and interested stakeholders have free access to follow and evaluate the science in the further course. We assume that this transparency, combined with the leadership of the stakeholders, will lead to safe and scientifically relevant field tests with the technology.
Acknowledgments: The work discussed here was supported in part by the University of California, the Irvine Malaria Initiative, the Tata Institute of Genetics and Society, and the National Institutes of Health (NIH) (AI29746). AAJ is a Donald Bren Professor at the University of California at Irvine.