A team of scientists in the United States used Crispr Cas9 molecular scissors to cut and paste genes and alter the genome of the black Legged Tick, a large mite that is the cause of numerous dangerous infections for humans and animals, such as typhus and Lyme disease.
They were researchers from the universities of Penn State, Nevada in Reno and Maryland, who published their results in the latest issue of the journal iScience.
“In the United States alone, regarding 300,000 people are infected with Lyme disease each year by ticks, and if left untreated, the infection can spread to the joints, heart, and nervous system,” said co-author Jason Rasgon, Ph. of Entomology and Disease Epidemiology at Penn State. “Currently, there is no vaccine, and existing treatments are not always effective,” he stresses.
“Until now, no laboratory had shown that genome modification was possible in ticks. Some considered it too technically difficult,” says Nuss, “this is the first study to show that genetic transformation in ticks is possible not just by one, but by two different methods.”
Invasion of large areas due to climate change
Plus, Rasgon says, the problem is getting worse, as climate change is allowing ticks to rapidly invade new areas, putting even more people and animals at risk of infection.
In this sense, Monika Gulia-Nuss, co-author of the study and molecular biologist at the University of Nevada in Reno, points out that, “despite this ability to acquire and transmit debilitating pathogens, research on ticks has been lagging behind, compared to to other arthropod vectors, largely due to difficulties in applying the available genetic and molecular tools.”
The role of genetic scissors
This is where Crispr Cas9 comes in – the most exact molecular scissors, for which Jennifer Doudna and Emmanuelle Charpentier won the Nobel Prize in 2020 – which have revolutionized functional genetic research in many organisms.
However, he adds, “Crispr gene editing tools will unlock some of the secrets of the tick genome and determine how these unique animals survive in the environment, how they interact with pathogens, and how we might prevent them from transmitting disease to humans.” and cattle.”
Entomologist and co-author of the paper Andrew Nuss also acknowledges that knowledge regarding tick biology at the molecular level remains limited. Instead, he points out, “for insects like mosquitoes, numerous transgenic development and genome editing tools are already available. Advances in this field are fundamental to the progress of research in order to solve the growing problem of tick-borne diseases”, he stresses.
‘Armored’ embryos difficult to inject
The authors note that technical problems with injecting tick embryos to attempt gene editing have further slowed the progress of the research.
The embryos of this mite are very difficult to pierce due to the high pressure inside the eggs, a hard chorion (their outer shell), and a layer of wax that the females coat them with and that must be removed before injection.
Ticks use a specialized organ, called the organ of Gené, to make that hard layer of wax.
In this work, the team successfully developed a protocol for embryo injection and targeted gene disruption with CRISPR Cas9 using two methods: injection of embryos once outside the mother and cargo receptor-mediated ovarian transduction (ReMOT control). , a method of gene editing in arthropods that requires less work.
The researchers removed Gené’s organ to prevent wax deposition and then treated the eggs with benzalkonium chloride and sodium chloride to remove the chorion and decrease pressure inside the eggs.
Gulia-Nuss explains that they were able to carefully dissect gravid female ticks to surgically remove the organ responsible for coating the eggs with wax, but allowing the females to lay viable eggs.
“These eggs without wax allowed the tick embryos to be injected with the necessary materials for the modification of the genome”, adds the researcher.
Another important challenge they had to overcome was understanding the timing of the tick embryo’s development. “So little is known regarding its embryology, and we needed to determine the precise moment at which to introduce CRISPR Cas9 to ensure the highest probability of inducing genetic changes,” says Gulia-Nuss.
Embryo injection viability
The survival rate of the injected embryos was approximately 10%, comparable to that of well-established insect models. In the case of ReMOT Control, all injected ticks survived.
The data show the feasibility of embryo injection and genetic manipulation in ticks by both methods, the authors say.
More research is needed to fully understand the molecular mechanisms underlying efficient gene editing in ticks. Although these new tools will speed up genetic studies of these mites, it is necessary to improve the embryo injection protocol to increase the survival and hatching of larvae and the efficiency of gene editing.
Selective gene disruption in ticks, vectors of human pathogens, is a powerful method for uncovering the underlying biology of tick-pathogen-host interactions and thus developing new approaches and applications in tick-borne disease control, the researchers conclude.
New tools speed up genetic studies of mites, such as the black tick. Photo: Sync
