Ticks are known to carry various pathogens that can be transmitted to humans and animals through bites, the most notorious being Lyme disease, which affects about 300,000 people per year in the United States. Previously, editing of the tick genome for research on tick-borne diseases has not been possible due to a number of challenges, such as the hard coating of the tick egg not being amenable to injection of CRISPR/Cas9 complexes. Now, researchers from Penn State, the University of Nevada, Reno and the University of Maryland have developed two methods enabling gene editing in ticks for the first time, providing access not only into eggs but directly into the ovaries of pregnant females.
One of the main barriers to accessing the tick genome for editing has been the hard wax coating that covers tick eggs. The delicate glass needles used to inject CRISPR/Cas9 complexes into embryos are unable to penetrate the coating and would shatter as a result, explained Jason Rasgon, professor of entomology and disease epidemiology at Penn State. The research team at the University of Nevada, Reno found a way to circumvent this problem; they removed the maternal organs that produce this wax from female ticks prior to egg laying. The resulting eggs lacked the coating and were able to be injected, and the team successfully made deletions from two different genes using this method.
The gene editing tool was then taken a step further using specialized technology developed at Penn State called Receptor-Mediated Ovary Transduction of Cargo, or ReMOT Control. This technique involves injection of CRISPR/Cas9 complexes directly into the ovaries of pregnant organisms, and has previously been successfully used in beetles, flies, whiteflies and mosquitos. ReMOT Control relies on the use of a small peptide that binds to receptors on the ovaries of most insect species, and the researchers found that the peptide was also functional in ticks, which are arachnids. By fusing the peptide to Cas9 and injecting it into pregnant adult female ticks, the researchers successfully delivered the complex to the ovaries where it was able to edit the genome of the developing offspring.
Applying both techniques to edit the ProbP gene in Ixodes scapularis black-legged ticks, the team observed a gene editing efficiency of 14% for egg/embryo injections and 11.7% using ReMOT control, suggesting that either method could be suitable for tick gene editing. According to Monika Gulia-Nuss, who led the University of Nevada, Reno team, the study is the first to demonstrate gene modification in ticks. This research was published in the journal iScience.
“The methods can be used to develop new control methods for diseases, such as Lyme disease, and also to further understand the biology of ticks,” said Rasgon.