Gene editing is now possible in ticks

The researchers successfully used CRISPR-Cas9 to edit the genomes of the black-legged mite. To accomplish this feat, they developed an embryo injection protocol that overcame a serious barrier in this area. The work appears in the magazine on February 15th iScience.

“Despite their ability to extract and transmit many debilitating pathogens, studies of mites lag behind other arthropod vectors such as mosquitoes, largely due to problems with the use of available genetic and molecular tools“- says Monica Gulia-Nus, one of the lead authors of the study and a molecular biologist from the University of Nevada in Reno.

“Having genome editing tools will allow us to uncover some secrets tick genome and allow us to determine how these unique animals survive in the environment, how they interact with pathogenic microorganisms and how we can prevent ticks from spreading diseases to humans and animals, ”she says.

Despite the public importance of mites, which are carriers of a wide variety of pathogens for humans, wildlife and domestic animals, knowledge of the biology of mites at the molecular level is still limited. This is in contrast to insects such as mosquitoes, for which many tools are now available for transgenic development and genome editing. “Progress in this area is critical to advancing research to address the growing problem of tick-borne diseases,” says Andrew Nuss, second co-senior author of the study and entomologist at the University of Nevada at Reno.

CRISPR-Cas9 has revolutionized the study of functional genetics in many organisms. This method of gene editing has been applied to mosquitoes and other vectors of arthropod disease, but ticks have not yet reported successful gene editing. Technical problems with the introduction of ticks embryos attempts to edit genes have further slowed the progress of research. Embryos of ticks are very difficult to enter because of high pressure inside the eggs, the hard chorion (outer shell of the egg) and the wax layer outside the embryo that needs to be removed before injection. Female mites use a specialized organ called the Gin organ to cover their eggs with a solid layer of wax.

У iScience The researchers developed a successful mite-embryo injection protocol and targeted gene disruption using CRISPR-Cas9, using two methods: embryo injection and receptor-mediated ovarian cargo transduction (ReMOT Control), a less time-consuming method of editing. The researchers besieged Jenne’s body to prevent wax deposits and then treated the eggs with chemicals called benzalkonium chloride and sodium chloride to remove the chorion and lower the pressure in the eggs.

“We have managed to thoroughly dissect female female ticks to surgically remove the organ responsible for covering the eggs with wax, but still allowing the females to lay viable eggs. These are wax free eggs allowed the injection of mite embryos of the materials needed to modify the genome, “says Gulia-Nus.” Another major challenge was understanding the timing of the mite embryo’s development. So little is known about the embryology of ticks that we needed to determine the exact time when CRISPR-Cas9 was administered to ensure the highest probability of induction genetic changes».

The survival rate of the introduced embryos was approximately 10%, which is comparable to well-established insect models. For ReMOT Control all introduced mites survived. The data show the appropriateness of mite embryo injection and genetic manipulation ticked both methods that had comparative editing efficiency for Proboscipedia (ПрабП) a gene expressed in appendages.

“Previously, no laboratory has demonstrated that modification of the tick genome is possible. Some thought it was too technically difficult to do, ”Nuss said. “This is the first study that has shown that genetic transformation in ticks is possible with not just one but two different methods.”

Further research is needed to fully understand the molecular mechanisms underlying effective gene editing in ticks. Although these tools are accelerating genetic research on ticks, improvements in the embryo injection protocol are needed to increase survival and efficiency of larval hatching and gene editing.

“We expect that the tools we’ve developed here will open up new research pathways that will dramatically accelerate our understanding of the molecular biology of this and related mite species,” says Gulia-Nus. “Targeted gene disruption in human vectors of human pathogens is a powerful method of uncovering the underlying biology of tick-to-host interactions that can serve as a guide for developing and applying new approaches to controlling tick-borne diseases.”