The authors thank&#160;Danny Reinberg&#39;s and Claude Desplan&#39;s labs at New York University and J&#252;rgen Liebig&#39;s lab at Arizona State University for their support on ant genetics. Hua Yan acknowledges support from the National Science Foundation I/UCRC, the Center for Arthropod Management Technologies under Grant No. IIP-1821914 and by industry partners. Maya Saar was supported by the United States - Israel Binational Agricultural Research and Development Fund, Vaadia-BARD Postdoctoral Fellowship No. FI-595-19.

1. Regular maintenance of Harpegnathos saltator colonies
<ol>
	<li>Maintain wild-type colonies of H. saltator in transparent plastic boxes in an ant rearing room at 22-25 &#176;C and a photoperiod of 12 hours light: 12 hours dark (12L:12D) lighting schedule.
	<ol>
		<li>Use small boxes (9.5 x 9.5 cm2) to rear individual workers or small colonies. Use medium boxes (19 x 13.5 cm2) or large boxes (27 x 19 cm2) to rear larger colonies (Figure 1).</li>
		<li>To create nest boxes, use plaster to make floors for the boxes. As the wet plaster is drying in the medium and the large boxes, press a foam block into the plaster a few centimeters deep and a few centimeters from the back of the box to designate a lower nest region. Once the plaster has dried, cover the designated nest region with a square piece of glass. 
		NOTE: In small boxes, there is no need to designate a lower nest region. If ants are neglecting to use the designated lower nest region, it may help to cover the glass with a square piece of red cellophane. This gives the impression of a dark underground space resembling nests that H. saltator use in the wild and may encourage them to move their brood to the designated region.</li>
	</ol>
	</li>
	<li>Feed colonies with live crickets twice per week. 
	NOTE: Colonies should be fed enough that they consume all crickets prior to their next feeding. Feed any isolated ants and mutant ant colonies with crickets pre-stung by workers of a regular colony 2-3 times every week.</li>
	<li>Apply water regularly to the plaster nest box flooring using a wash bottle. 
	NOTE: The plaster should be moist enough that it does not feel dusty to the touch, but it should be dry enough that all added water is absorbed by the plaster. It is important that the nests are not watered excessively. On average, nest boxes will need a small amount of water added once per week.</li>
	<li>Whenever feeding occurs, remove trash and dead individuals. Freeze all waste and dead ants overnight at -30 &#176;C before disposing of these materials as regular garbage.</li>
	<li>Add a pinch of dried sawdust to colonies periodically; this helps larvae as they undergo pupation and helps workers keep the nest box clean.</li>
</ol>
2. Preparation of quartz glass microinjection needles
<ol>
	<li>Use a micropipette puller to pull glass microinjection needles.</li>
	<li>Select the glass to be pulled. Ensure that the glass being used has been stored in a dust-free and clean environment. 
	NOTE: Here, thin-walled filamentous quartz glass with an outer diameter of 1.0 mm, inner diameter of 0.5 mm, and length of 7.5 cm has been used to produce microinjection needles. If injecting a soft-bodied embryo, borosilicate needles may also be applicable, but borosilicate needles are not able to penetrate hard chorion.</li>
	<li>Set the parameter settings of the puller. Use a two-step process to pull microinjection needles for H. saltator embryos: parameters for the first step include heat of 575, filament of 3, velocity of 35, delay of 145, and a pull of 75; parameters of the second step include heat of 425, filament of 0, velocity of 15, delay of 128, and a pull of 200. Following the second step, ensure that the resulting needle has a 2 mm taper and a tip of 0.5 &#181;m (Figure 2). 
	NOTE: This set of parameters, along with parameters for other needle types, can be found in the Operation Manual17. Manuals for pipette pullers often provide recommended parameters for a variety of techniques. Some trial and error may be required to determine which parameters generate the best needles for specific needs. A short taper is ideal for H. saltator injections, as it can penetrate the hard chorion of H. saltator embryos. If injecting a soft embryo, such as one that has been dechorionated (e.g., Drosophila), a longer taper around 10 mm may yield better results. Operation manuals for pipette pullers usually provide specific parameters for pulling needles suitable for different needs. It is important that gloves are worn while handling glass filaments and pulling needles. Oils from bare hands may transfer to the glass if gloves are not worn.</li>
	<li>Once parameters are set, use a micropipette puller to pull needles for microinjection. Ensure that needles are kept in a dust-free and clean environment until used. 
	&#8203;NOTE: It is recommended to use freshly pulled microinjection needles. If pre-pulled needles are used, they should be stored properly in a box to prevent damage of needle tips and potential contamination. Needles that have undergone long-term storage are not recommended for embryo injections.</li>
</ol>
3. Preparation of microinjector
<ol>
	<li>Use a microinjector to inject desired materials into ant embryos.</li>
	<li>Prepare the microinjection mixture of Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs)10. Keep the mixture on ice until it is time to load a microinjection needle. When not in use, store the microinjection mixture at -80 &#176;C. 
	NOTE: The concentrations vary in different species. High concentration may cause high mortality, whereas low concentration may reduce efficiency. We use 0.2 &#181;g/&#181;L Cas9 proteins and 0.2 &#181;g/&#181;L sgRNAs for H. saltator embryo injection. Design of our sgRNAs followed a previously established protocol18. Gene sequences were obtained from DNA databases. The genome sequence of H. saltator was also previously reported19,20.</li>
	<li>Adjust the injection parameters for H. saltator embryos: an injection pressure of 140 hectopascal (hPa), a constant pressure of 70 hPa, and a time of 0.4 seconds. Adjust constant pressure such that material only flows in one direction. Adjust the injection pressure and time only if no material is flowing from the needle into the embryo. 
	NOTE: If injecting a different type of embryo, the primary parameter that may change is constant pressure, which is responsible for ensuring that fluid from the embryo does not flow back into the needle. Volume is not controlled for in this protocol. Setting the parameters of the microinjector is sufficient for obtaining consistent injections.</li>
	<li>Load a microinjection needle with 2 &#181;L of the mixture using microloader pipette tips. Do this slowly to ensure that no bubbles are formed in the mixture. 
	NOTE: If bubbles form, it may be difficult to maintain consistent microinjections.</li>
	<li>Break just the tip of the needle by breaking along the edge of the tape such that a narrow taper is still maintained. Ensure that the needle is broken just enough that the tip is opened, but not so much that the taper is broken off. 
	NOTE: Importantly, if the opening of the needle is too wide after breaking, the user will see liquid run out of the needle when mounted to the pressurized microinjector prior to application of injection pressure. Some microinjection protocols advise breaking needles by cutting the tip with scissors. This method is not advised, as scissors may cause the tip of the needle to shatter. Injecting embryos with a shattered needle will be highly damaging.</li>
	<li>Mount the needle to the micromanipulator</li>
</ol>
4. Injection of embryos
<ol>
	<li>Select embryos for microinjection from the syncytial stage: the time during development in which nuclei divide without cytokinesis. 
	NOTE: This is the ideal time during development for genome editing by microinjection, as discovered previously in Drosophila21. H. saltator embryos pass the syncytial stage and reach cellularization around 36 h after egg deposition10. Higher efficiency is achieved if younger embryos are used for injections.</li>
	<li>Line embryos on a piece of double-sided tape stuck to a glass microscope slide. Ensure that embryos are secured well to the tape to prevent movement during injection. Lay the embryos in a vertical orientation, such that the lateral side of the embryo is at the edge of the tape (Figure 3). Place the slide and lined embryos onto the stage of the microscope at a designated microinjection workstation. 
	NOTE: It is recommended that the embryos are arranged such that successive injections can be performed by moving the slide on the stage instead of adjusting the needle position with every injection. This allows for successive injections to be performed more efficiently.</li>
	<li>Align the needle with the first embryo to be injected using the micromanipulator (Figure 4a).</li>
	<li>Laterally puncture the needle into the first embryo along its dorsal/ventral axis under a microscope.</li>
	<li>Inject the microinjection mixture. Look for slight movement of the embryo, indicating an increase in internal pressure due to the injected liquid. Additionally, watch for the formation of a small droplet containing visible trace of tissue and/or lipid on the outer membrane of the embryo (Figure 4b). 
	&#8203;NOTE: Presence of trace of tissue and/or lipid in the droplet indicates that the needle has successfully punctured both chorion and vitelline membrane of the embryo. If traces of these materials are not present, the injection was not performed successfully and should be repeated. After a few seconds, the droplet will be reabsorbed by the embryo and will no longer be visible.</li>
	<li>Gently remove the needle from the embryo immediately, and proceed to the next by adjusting the position of the microscope slide. Repeat until all the embryos have been injected.</li>
	<li>Once all embryos on the slide have been successfully injected, transfer the slide to a humid box for 1 hour to give the embryos time to recover from the injection process prior to being removed from the slide.</li>
</ol>
5. Rearing of injected embryos
<ol>
	<li>After 1 hour&#160;of incubation in a humid box, gently remove the injected embryos from the tape using featherweight forceps, and transfer them to a tube filled with a small amount of 70% ethanol. Invert the tube several times to transfer the embryos to the bottom of the tube. Repeat the ethanol wash once, followed by three washes with autoclaved water.</li>
	<li>Using a small and soft paintbrush, transfer all injected embryos to 1% agar plates with 2% Antibiotic-Antimycotic. Apply the Antibiotic-Antimycotic after poured agar plates have cooled by spreading over the surface of the plate using a cell spreader. Seal the agar plate with parafilm to prevent agar desiccation. 
	NOTE: Do not attempt to return injected embryos to a colony after injections as workers may destroy most injected embryos. Survival is therefore optimized by allowing embryos to develop on agar plates outside of a normal colony environment.</li>
	<li>Incubate the agar plates at 25 &#176;C for approximately 4 weeks. Check regularly for hatching.</li>
	<li>Once the first embryo has hatched into a larva, return all embryos and larvae to a nest box with a few young nurse workers to care for the hatchlings. Feed using crickets pre-stung by a larger wild-type colony, remove waste products, and add water following the same protocol discussed in Section 1. 
	NOTE: Small boxes (9.5 x 9.5 cm2) are ideal for such colonies.&#160;H. saltator reproduces well in captivity. Therefore, mutant embryos can be reared to adulthood. Isolation of mutant adult(s) induces transition to the reproductive gamergate stage. Controlled crosses are used to establish mutant colonies with heterozygous or homozygous individuals (Figure 5).</li>
</ol>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+genetic+differences+between+queens+and+workers+in+hybridizing+Pogonomyrmex+harvester+ants.">Extreme genetic differences between queens and workers in hybridizing Pogonomyrmex harvester ants.</a> Proceedings. Biological Sciences. 269 (1503), 1871-1877 (2002).</li><li>Volny, V. P., Gordon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+basis+for+queen-worker+dimorphism+in+a+social+insect.">Genetic basis for queen-worker dimorphism in a social insect.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (9), 6108-6111 (2002).</li><li>Yan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eusocial+insects+as+emerging+models+for+behavioural+epigenetics.">Eusocial insects as emerging models for behavioural epigenetics.</a> Nature Reviews Genetics. 15 (10), 677-688 (2014).</li><li>Liebig, J., H&#246;lldobler, B., Peeters, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+ant+workers+capable+of+colony+foundation.">Are ant workers capable of colony foundation.</a> Naturwissenschaften. 85 (3), 133-135 (1998).</li><li>Bonasio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+topics+in+epigenetics:+ants,+brains,+and+noncoding+RNAs.">Emerging topics in epigenetics: ants, brains, and noncoding RNAs.</a> Annals of the New York Academy of Sciences. 1260 (1), 14-23 (2012).</li><li>Peeters, C., Liebig, J., H&#246;lldobler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+reproduction+by+both+queens+and+workers+in+the+ponerine+ant+Harpegnathos+saltator.">Sexual reproduction by both queens and workers in the ponerine ant Harpegnathos saltator.</a> Insectes Sociaux. 47 (4), 325-332 (2000).</li><li>Trible, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=orco+mutagenesis+causes+loss+of+antennal+lobe+glomeruli+and+impaired+social+behavior+in+ants.">orco mutagenesis causes loss of antennal lobe glomeruli and impaired social behavior in ants.</a> Cell. 170 (4), 727-735 (2017).</li><li>Yan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+engineered+orco+mutation+produces+aberrant+social+behavior+and+defective+neural+development+in+ants.">An engineered orco mutation produces aberrant social behavior and defective neural development in ants.</a> Cell. 170 (4), 736-747 (2017).</li><li>Kohno, H., Suenami, S., Takeuchi, H., Sasaki, T., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+knockout+mutants+by+CRISPR/Cas9+in+the+European+honeybee,+Apis+mellifera+L.">Production of knockout mutants by CRISPR/Cas9 in the European honeybee, Apis mellifera L.</a> Zoological Science. 33 (5), 505-512 (2016).</li><li>Kohno, H., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mKast+is+dispensable+for+normal+development+and+sexual+maturation+of+the+male+European+honeybee.">mKast is dispensable for normal development and sexual maturation of the male European honeybee.</a> Scientific Reports. 8 (1), 1-10 (2018).</li><li>Schulte, C., Theilenberg, E., M&#252;ller-Borg, M., Gempe, T., Beye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+integration+and+expression+of+piggyBac-derived+cassettes+in+the+honeybee+(Apis+mellifera).">Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee (Apis mellifera).</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (24), 9003-9008 (2014).</li><li>Hu, X. 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The authors have nothing to disclose.

The evolution of eusociality amongst insects, including ants, bees, wasps, and termites, has resulted in the appearance of novel behavioral and morphological traits, many of which are understood to be influenced by a combination of environmental and genetic factors1,2,3,4. Unfortunately, the attractiveness and usefulness of eusocial insects as research models in the field of genetics has been h...

The evolution of eusociality in insects, namely those of the orders Hymenoptera and Blattodea (formerly Isoptera), has resulted in unique and often sophisticated behavioral traits that manifest on both the individual and the colony levels. Reproductive division of labor, a trait characterizing the most advanced groups of social insects, often involves caste systems composed of several behaviorally and often morphologically distinctive groups. Such behavioral and morphological diversity between castes is controlled not only by their genetic system, but also often by the environment1,2,3,4, making eusocial insects attractive subjects for genetic and epigenetic research.
The ability to manipulate the genetic system of eusocial insects has proven to be challenging as many species do not mate and reproduce in laboratory settings. Most eusocial insects also have very few reproductive individuals in a colony, limiting the number of offspring that can be produced and consequently, limiting the sample size for genetic manipulation5. Additionally, many eusocial insects have long generation times compared to insects commonly used for genetic studies (such as Drosophila), adding to the difficulty of establishing genetic lines5. Some eusocial species, however, can generate a large proportion of reproductively active individuals in a colony, which alleviates the challenges and provides opportunities to establish mutant or transgenic lines.
In the case of the ponerine ant species, Harpegnathos saltator, all female workers can become reproductively active upon the death of a queen or social isolation. These workers are referred to as &#34;gamergates&#34; and can be used to generate new colonies6. Furthermore, there may be more than one gamergate present in a colony, thus increasing offspring production5,7,8. Thus far, mutant and/or transgenic lines have been developed in the European honeybee, Apis mellifera, and in the ant species, H. saltator, Ooceraea biroi, and Solenopsis invicta9,10,11,12,13,14,15. Genetic analyses in social bees and ants have paved the way toward a better understanding of eusociality, providing an array of opportunities to study genes and their impacts on eusocial insect behavior and caste-specific physiology.
Here, we provide a protocol for genetic modification via the CRISPR/Cas9 system in H. saltator. Specifically, this technique was used to generate a germline mutation in orco, the gene encoding the obligate co-receptor of all odorant receptors (ORs)10. OR genes have been remarkably expanded in hymenopteran eusocial insects16, and orco plays an essential role in insect olfaction; in its absence, ORs do not assemble or function normally. Mutations of the orco gene therefore disrupt olfactory sensation, neural development, and associated social behaviors9,10.
In this protocol, Cas9 proteins and small guide RNAs (sgRNAs) are introduced into ant embryos using microinjection for the purpose of inducing mutagenesis of a target gene. Here, we will describe the microinjection procedure in detail along with directions regarding the care of colonies and injected embryos. These methods are appropriate for inducing mutagenesis in a variety of different genes in H. saltator ants and may be applied to a broader spectrum of hymenopteran insects.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>ThermoFisher</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Cas9 protein with NLS, high concentration</td>
			<td>PNA Bio</td>
			<td>CP02</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellophane Roll 20 inch X 5 feet</td>
			<td>Hypogloss Products</td>
			<td>B00254CNJA</td>
			<td>The product has many color variations. Purchase it in red for use in making ant nests.</td>
		</tr>
		<tr>
			<td>Eclipse Ci-S upright microscope&#160;</td>
			<td>Nikon</td>
			<td>Ci-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Featherweight forceps, narrow tip</td>
			<td>BioQuip</td>
			<td>4748</td>
			<td></td>
		</tr>
		<tr>
			<td>FemtoJet ll microinjector</td>
			<td>Eppendorf</td>
			<td>920010504</td>
			<td>This product is no longer sold or supported by Eppendorf. A comparable microinjector may be used instead.</td>
		</tr>
		<tr>
			<td>Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>NCBI database</td>
			<td>National Center for Biotechnology Information</td>
			<td>Gene ID: 105183395&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>P-2000 Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-2000/G</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic boxes (19 X 13.5 cm2)</td>
			<td>Pioneer Plastics</td>
			<td>079C&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic boxes (27 X 19 cm2)</td>
			<td>Pioneer Plastics</td>
			<td>195C</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic boxes (9.5 X 9.5 cm2)</td>
			<td>Pioneer Plastics</td>
			<td>028C&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz glass without filament</td>
			<td>Sutter Instruments</td>
			<td>Q100-50-7.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas scissors, 8.5 cm</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td></td>
		</tr>
		<tr>
			<td>Winsor &#38; Newton Cotman Water Colour Series 111 Short Handle Synthetic Brush - Round #000</td>
			<td>Winsor and Newton</td>
			<td>5301030</td>
			<td></td>
		</tr>
	</tbody>
</table>

embryo injections for crispr-mediated mutagenesis in the ant harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.

Using the protocol provided here, genome editing in Harpegnathos saltator embryos was performed successfully. These results were validated via polymerase chain reaction and pGEM cloning of DNA extracted from injected embryos followed by DNA sequencing. Efficiency of somatic mutagenesis using this protocol reached approximately 40%. F1 mutant males were mated to wild type females to produce heterozygous F2 females which, if not mated, produced F3 males. Mutant F3 males were mated ...

Watch this Scientific Journal Video about Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator at JoVE.com

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects.

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how ...

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Research

JoVE Journal

Genetics

2.5K Views.  University of Florida. Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects. 

Video: Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model organism. However, little is known about the sex-determination mechanisms found in other non-model hymenopteran taxa, such as ants. Because of the complex nature of the life cycles that have evolved in hymenopteran species, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory. Here, we describe the methods for conducting inbreeding crosses and for evaluating the success of those crosses in ant Vollenhovia emeryi. Inducing inbreeding in the laboratory using V. emeryi, is relatively simple because of the unique biology of the species. Specifically, this species produces androgenetic males, and female reproductives exhibit wing polymorphism, which simplifies identification of the phenotypes in genetic crosses. In addition, evaluating the success of inbreeding is straightforward as males can be produced continuously by inbreeding crosses, while normal males only appear during a well-defined reproductive season in the field. Our protocol allow for using V. emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ant species.

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

In this protocol, methods for conducting inbreeding crosses, and for evaluating the success of those crosses, are described for the ant Vollenhovia emeryi. These protocols are important for experiments aimed at understanding the genetic basis of sex determination systems in Hymenoptera.

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...