Name: embryo injection technique for gene editing in the black-legged tick, ixodes scapularis
Uploaded: 2022-09-13T13:20:00
Description: Watch this Scientific Journal Video about Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis at JoVE.com

The authors acknowledge Channa Aluvihare and Yonus Gebermicale, ITF, UMD, for insight and support during the initial phase of protocol development. Tungsten needles were a generous gift from David O&#39;Brochta, ITF, UMD. We are thankful to Dr. Ladislav Simo for testing this protocol in I. ricinus and for insightful discussions. This project was funded by NIH-NIAID R21AI128393 and Plymouth Hill Foundation, NY to MG-N, startup funds from the University of Nevada to AN, the National Science Foundation Grant No. 2019609 to MG-N and AN, and a Peer-to-Peer Grant from IGTRCN to AS.

Ixodes scapularis adults were either purchased from Oklahoma State University (OSU) or reared at the University of Nevada, Reno (UNR) (IACUC protocol #21-001-1118).
1. Preparation of female ticks for embryo collection
NOTE: To collect eggs of appropriate age, it is important to synchronize egg-laying. Although egg-laying cues in ticks remain unclear, under the standard insectary conditions (27 &#176;C temperature and &#62;90% relative humidit...

<ol>
	<li>Hinckley, A. F. et al. Lyme disease testing by large commercial laboratories in the United States. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America. 59 (5), 676-681 (2014).</li>
	<li>Jongejan, F., Uilenberg, G. The global importance of ticks. Parasitology. 129 Suppl, S3-14 (2004).</li>
	<li>Eisen, R. J., Eisen, L. The blacklegged tick, Ixodes scapularis: An increasing public health concern. Trends in Parasitology. 34 (4), 295-309 (2018).</li>
	<li>Sharma, A. et al. Cas9-mediated gene editing in the black-legged tick, Ixodes scapularis, by embryo injection and ReMOT Control. iScience. 25 (3), 103781 (2022).</li>
	<li>Nuss, A., Sharma, A., Gulia-Nuss, M. Genetic manipulation of ticks: A paradigm shift in tick and tick-borne diseases research. Frontiers in Cellular and Infection Microbiology. 11, 678037 (2021).</li>
	<li>Heinze, S. D. et al. CRISPR-Cas9 targeted disruption of the yellow ortholog in the housefly identifies the brown body locus. Scientific Reports. 7 (1), 4582 (2017).</li>
	<li>Kistler, K. E., Vosshall, L. B., Matthews, B. J. Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti. Cell Reports. 11 (1), 51-60 (2015).</li>
	<li>Criscione, F., O&#39;Brochta, D. A., Reid, W. Genetic technologies for disease vectors. Current Opinion in Insect Science. 10, 90-97 (2015).</li>
	<li>Jasinskiene, N. et al. Stable transformation of the yellow fever mosquito, Aedes aegypti, with the Hermes element from the housefly. Proceedings of the National Academy of Sciences of the United States of America. 95 (7), 3743-3747 (1998).</li>
	<li>Dermauw, W. et al. Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae. Insect Biochemistry and Molecular Biology. 120, 103347 (2020).</li>
	<li>Santos, V. T. et al. The embryogenesis of the tick Rhipicephalus (Boophilus) microplus: the establishment of a new chelicerate model system. Genesis (New York, N.Y. 2000). 51 (12), 803-818 (2013).</li>
	<li>Ginsberg, H. S., Lee, C., Volson, B., Dyer, M. C., Lebrun, R. A. Relationships between maternal engorgement weight and the number, size, and fat content of larval Ixodes scapularis (Acari: Ixodidae). Journal of Medical Entomology. 54 (2), 275-280 (2017).</li>
	<li>Feldman-Muhsam, B., Havivi, Y. Accessory glands of Gene&#39;s organ in ticks. Nature. 187 (4741), 964 (1960).</li>
	<li>Kakuda, H., Koga, T., Mori, T., Shiraishi, S. Ultrastructure of the tubular accessory gland in Haemaphysalis longicornis (Acari: Ixodidae). Journal of Morphology. 221 (1), 65-74 (1994).</li>
	<li>Booth, T. F. Wax lipid secretion and ultrastructural development in the egg-waxing (Gen&#233;&#39;s) organ in ixodid ticks. Tissue &#38; Cell. 21 (1), 113-122 (1989).</li>
	<li>Arrieta, M. C., Leskiw, B. K., Kaufman, W. R. Antimicrobial activity in the egg wax of the African cattle tick Amblyomma hebraeum (Acari: Ixodidae). Experimental &#38; Applied Acarology. 39 (3-4), 297-313 (2006).</li>
	<li>Brady, J. A simple technique for making very fine, durable dissecting needles by sharpening tungsten wire electrolytically. Bulletin of the World Health Organization. 32 (1), 143-144 (1965).</li>
</ol>

This is the first protocol developed to inject early tick embryos successfully. A survival rate of ~4%-8% has been achieved, which is comparable to embryo injection in other well-established insect models5.
As this is the initial protocol, it is anticipated that this protocol will be further refined and specialized to individual tick species. In particular, injection timing will vary from species to species, dependent upon embryogenesis, especially the timing of cellula...

Ticks are vectors of medical and veterinary importance, capable of transmitting a variety of viral, bacterial, protozoan pathogens and nematodes1,2. In the eastern United States, the black-legged tick, Ixodes scapularis, is an important vector of the Lyme disease (LD) pathogen, the spirochete Borrelia burgdorferi. Over 400,000 cases of LD are reported each year in the United States, making it the top vector-borne infectious disease in the US1. In addition to B. burgdorferi, six other microorganisms are transmitted by I. scapularis- including four bacteria (Anaplasma phagocytophilum, B. mayonii, B. miyamotoi, and Ehrlichia muris eauclarensis), one protozoan parasite (Babesia microti), and one virus (Powassan virus), making this tick species a major public health concern3. While tick-borne diseases have become more prevalent in recent years, research on ticks has fallen behind other arthropod vectors, such as mosquitoes, due to the unique biology of ticks&#160;and challenges associated with applying genetic and functional genomic tools4,5.
Gene-editing techniques, particularly CRISPR/Cas9, have now made functional genomics studies feasible in non-model organisms. For creating heritable mutations in an organism, embryo injection remains the preferred method for delivering constructs for altering the germline6,7,8,9. However, until recently4, tick eggs were considered too difficult or even impossible to inject without killing the embryo10,11. A thick wax layer on eggs, hard chorion, and high intra-oval pressure were some of the main obstacles that prevented embryo injection in ticks. Adult, blood-fed I. scapularis deposit a single clutch of up to 2,000 eggs12 over 3-4 weeks (approximately 100 eggs/day). Eggs are laid singly, and each egg is coated with wax that is secreted by protrusions or &#34;horns&#34; of the glandular Gen&#233;&#39;s organ13,14,15&#160;of the mother. This wax protects the eggs from desiccation and contains antimicrobial compounds15. To successfully inject tick eggs, it is important to remove the wax layer, soften the chorion, and desiccate the eggs to decrease the intraoval pressure so that the injection does not irreversibly damage the egg. Understanding the critical importance of embryo injections for successful germline transformation, a protocol for I. scapularis is developed, which can be used to deliver a CRISPR/Cas9 construct and generate stable germline mutations4. In addition to its contribution to I. scapularis research, this protocol could also be optimized for other tick species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminum silicate capillaries, with filament</td>
			<td>Sutter instruments</td>
			<td>AF100-64-10</td>
			<td>Embryo injection</td>
		</tr>
		<tr>
			<td>Benzalkonium chloride 50% in water, 25 g</td>
			<td>TCI-America</td>
			<td>B0414</td>
			<td>Embryo treatment, 25 g is approximately 25 mL</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>1001-090</td>
			<td>Post-injection care</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Thomas Scientific</td>
			<td>300-101</td>
			<td>Gene`s organ manipulation</td>
		</tr>
		<tr>
			<td>Lab Wipes</td>
			<td>Genesee Scientific</td>
			<td>88-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td>Loading the pulled needles</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter instruments</td>
			<td>ROE-200</td>
			<td>Embryo injection</td>
		</tr>
		<tr>
			<td>Microscopic slides- plain, ground edges</td>
			<td>Genesee Scientific</td>
			<td>29-100</td>
			<td>Embryo alignment, ground edges are preferred, beveled edges could obscure the eggs from view</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Research Products International</td>
			<td>S23020-500.0</td>
			<td>Embryo treatment</td>
		</tr>
		<tr>
			<td>Needle Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Permanent Double sided tape</td>
			<td>Scotch</td>
			<td>34-8716-3417-5</td>
			<td>Embryo alignment</td>
		</tr>
		<tr>
			<td>Petri plates</td>
			<td>Genesee Scientific</td>
			<td>32-107G</td>
			<td>Post-injection care</td>
		</tr>
		<tr>
			<td>Tegaderm/ Transparent film dressing</td>
			<td>3M Healthcare</td>
			<td>1628</td>
			<td>Embryo alignment</td>
		</tr>
		<tr>
			<td>Tungsten needles</td>
			<td>Fine Science Tools</td>
			<td>10130-10</td>
			<td>Gene`s organ manipulation</td>
		</tr>
		<tr>
			<td>Tungsten Wire</td>
			<td>Amazon</td>
			<td>B08DNT7ZK3</td>
			<td>Gene`s organ manipulation</td>
		</tr>
		<tr>
			<td>XenoWorks Digital Microinjector</td>
			<td>Sutter instruments</td>
			<td>MPC-200</td>
			<td>Embryo injection</td>
		</tr>
	</tbody>
</table>

embryo injection technique for gene editing in the black-legged tick, ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some&#160;obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here.&#160;This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

A successful embryo injection protocol for I. scapularis is described in this article. Egg-laying females were kept at high humidity to avoid desiccation of partially-waxed eggs. The wax layer was removed to inject tick embryos by ablating the Gene&#39;s organ (wax gland) of the gravid female (Figure 1A-E). We used aluminosilicate glass needles with a shorter neck (Figure 2). This shape was ideal for tick egg injection ...

Watch this Scientific Journal Video about Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis at JoVE.com

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...

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