Combating Mosquito-Borne Diseases with CRISPR

Female mosquitoes are some of the deadliest organisms in the world due to their ability to spread infectious diseases through a simple bite. Mosquito-borne diseases such as yellow fever, Zika, Dengue fever, and malaria kill millions of humans every year, and there are limited therapeutics for their prevention and treatment.

While in college, Omar Akbari worked as a public service intern testing the local mosquito population for human pathogens and eradicating these insects with chemicals. During this experience, he felt dissatisfied with the insecticide-based method of controlling mosquito population and wanted to find a better way to tackle the problem of mosquito-borne disease spread. With a multidisciplinary team in his laboratory at the University of California, San Diego, he now develops tools through genetic engineering techniques such as CRISPR to solve the world’s insect control problems. 

How do you create new insect control strategies in the laboratory?

We ask basic developmental biology questions. Once we understand the basic biology and physiology of the insect, then we can manipulate it to develop alternative control technologies. For example, we manipulate a pathway to prevent mosquitoes from transmitting viral diseases. We also develop diagnostics so that people can detect in the field if an insect is infected with a pathogen. 

What makes CRISPR a useful tool for your research?

CRISPR is incredibly robust in terms of cutting DNA and targeting nucleic acids. It works really efficiently, we can program it, and it is easy to use. One of the first experiments I did with it was to genetically encode its components in flies and cross those flies together. When I targeted a gene for eye pigmentation or body pigmentation, close to 100 percent of the progeny had some type of mutation in those genes, and I could see the result. That efficiency was a big surprise. We have devised many strategies for utilizing CRISPR to control insect populations in a way that is safe enough to enable public acceptance and regulatory authorization in the near term. 

What are some CRISPR-based strategies for insect control that you have developed?

We recently developed a new technology that uses CRISPR to block viral transmission. There are CRISPR ribonucleases that can degrade RNA instead of DNA, and we engineered these to target viruses. We encoded the machinery in the mosquito so that they expressed CRISPR ribonucleases and designed guides that target different viruses. When the mosquito gets infected with a virus, the CRISPR machinery cuts the viral RNA sequences, resulting in collateral activity that reduces the mosquito’s fitness and ultimately kills the mosquito. Ideally, we want to multiplex the CRISPR machinery to target all mosquito-transmitted viruses that affect humans. 

Additionally, we used CRISPRa to overexpress insect developmental genes which, which results in complete lethality. We first did this as a proof of concept in flies, and we showed that this works in mosquitoes. CRISPRa uses a version of Cas9 that is inactive (dCas9). It cannot cut DNA, but it progressively binds to targets using guide RNAs, which recruits transcriptional machinery to the target promoter region and promotes gene expression. To rescue this overexpression, we mutated the promoters to prevent CRISPRa machinery binding. We created a creative genetic crossing scheme where we could maintain the engineered mosquito line in the laboratory by keeping CRISPRa inactive. When we cross these mosquitos with wild type insects that have the targets present, CRISPRa becomes active and causes lethality. This is complete postzygotic isolation, which is the definition of speciation. 

What do you see as the most promising technology for insect control?

The sterile insect technique is the most effective way of controlling insects in the wild, but it traditionally uses radiation for sterilization, which reduces insects’ fitness due to chromosomal damage. We developed a precision-guided sterile insect technique (pgSIT), which also uses CRISPR. This could be one way of combating mosquitoes that does not rely on using insecticides. By using CRISPR, we are not affecting the chromosomes, so the animal is more fit, which results in longer viability in the wild and a higher rate of population suppression. 

We make different insect lines homozygous for cas9 and a guide RNA. We design the guide RNAs to target genes important for female development and male fertility. When we cross lines with cas9 and these different guide RNAs together, all the progeny receive the cas9 gene and the guide RNAs through Mendelian segregation. CRISPR then targets those genes, and all the females die and all the males are sterilized. We want to kill the females because they bite and transmit disease. To control insect populations, one could deploy the progeny into the environment to hatch. When the sterile males try to mate with females, they would produce no progeny and the population would dwindle. 

What excites you about being a scientist?

Our laboratory is very applied, so what excites me is translating the technologies we develop. We are always trying to take on problems that are important in the world. For example, when COVID-19 hit, it was hard to diagnose, which we thought was very rate limiting. We got back into the lab—we wore masks and worked in isolated rooms—and we built a detection system called SENSR that uses CRISPR. We identified a new problem and came up with a solution for it. 

Right now, we are working on mosquitoes, but once we can deploy our technologies that solve some problems associated with mosquitos, we will move on to the next big problem. Ours is an engineering lab, so we design, build, learn, and iterate until we get something interesting. My background is molecular biology, and we have chemists, biochemists, geneticists, and engineers in the laboratory. With those fields combined, we can create really interesting products. 

This interview has been edited for length and clarity.