CRISPR/Cas9 is a modern genetic engineering tool that can be used to find specific gene sequences and delete, add to, or modify them in groundbreaking precision. One lab ventured to use CRISPR to test the effects of certain genetic factors on plasmodium susceptibility and fitness impacts on anopheles mosquitoes.

Whether or not plasmodium can survive and replicate inside of a mosquito depends on a variety of genetic factors, including the activity of agonists. Agonists control pathways that the development of plasmodium depends on, so to understand how specific agonists affect plasmodium infection, researchers investigated the effects of silencing the FREP1 (fibrinogen-related protein 1) gene on infection rates and fitness in Anopheles gambiae mosquitoes, a primary vector for malaria in humans.

Transgenic mosquitoes were created by putting synthesized gRNA (guide RNA) and Cas9 protein into mosquito embryos, along with a reporter gene. The reporter gene acts as an indicator for successful genome editing, as it caused GM mosquitoes to fluoresce blue under certain conditions. The gRNAs helped CRISPR/Cas9 locate the FREP1 gene, which was selected because of its effect on plasmodium growth in the mosquito midgut.

To test the impact of the FREP1 inactivation on susceptibility to plasmodium infection, researchers fed both the mutant and wild-type (wt) groups a culture with an unnaturally high concentration of plasmodium gametocytes. Eight days after infection, they measured the intensity of the infection with the amount of parasitic oocysts that developed. As a result, the wt mosquitoes developed a median of 91 oocysts, while the mutant population developed a median of just 17 oocysts. This test was then repeated with a tenfold lower concentration of gametocytes to reflect natural levels, with the median oocyst count at 2.0-2.5 in the wt groups and at 0.0 in the mutant groups.

Clearly, the silencing of the FREP1 gene was favorable for decreasing plasmodium susceptibility, but it had unintended side effects. The FREP1 gene not only regulates plasmodium infection rates, but also plays a role in blood feeding and digestion, so its complete inactivation had adverse effects on the transgenic mosquitoes’ fitness. In one test, both mutants and wt mosquitoes were prevented from feeding for 3-5 hours before being introduced to anesthetized mice, and while 90-95% of wt mosquitoes fed, only 68% of the mutants fed. Similarly, when the same trial was conducted with human blood-containing membrane feeders, the amount of the mosquitoes that took a blood meal was 73% for the wt group and just 28% for the mutant group. Other aspects of fitness were compromised in the mutant group, including the amount of eggs laid and percentage hatched and life expectancy after a blood meal was taken. The figure below was taken from the study.

One proposal mentioned in the study for the impact of genetic modification on fitness was that inactivating the FREP1 gene possibly “decreased nutritional uptake or in impaired midgut protection against stress,” or that unintended changes were made by CRISPR in non-target sites, because although this novel technology is incredible, it is not perfect, and has a tendency to make mistakes.

Though the compromised fitness of the transgenic mosquitoes is discouraging for this species of transgenic mosquito, this study proved the role of the FREP1 gene in plasmodium infection rates on anopheles mosquitoes, along with displaying how CRISPR/Cas9 can be used to identify and change parts of the genome to affect resistance to the parasite. This is certainly a valuable tool for gaining understanding about organisms’ genomes, which can be used in further studies on GM anopheles mosquitoes.

Perhaps this Synopsis should have been written and posted before the last one on mosquitoes, because these mutants did not fare nearly as well as the others. That being said, I did want to focus a bit on how CRISPR is being used in this field, because it shows a lot of promise, and I’ll hopefully be working with it in the future! I also liked how I learned a little bit more on how they changed the mosquitoes’ genome, which I didn’t get much of from the last episode.

These take me more time than I’d like to admit, which is not ideal considering how hectic my schedule is (three STEM exams within seven days is not for the weak), so I might sprinkle in some lighter articles in the future. I have to summarize ten scientific sources in my paper, but if I keep doing articles like this, it will be a million pages long, so maybe I should look at some simpler sources. Hope you’ve been enjoying the skeeters! Stay tuned to learn with me!

https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006898#ppat.1006898.s003