This work was supported by a grant from the Monsanto Corn Rootworm Knowledge Research Program, grant number AG/1005 (to MDL and YC) and start-up funds to MDL from NCSU. FC was supported by grants from Monsanto&#39;s Corn Rootworm Knowledge Research Program (AG/1005) and the National Science Foundation, grant number MCB-1244772 (to MDL). The authors declare no competing interests. FC and MDL conceived and designed the experiments; FC, PW and SP performed the experiments; FC and MDL analyzed the results; and FC, NG and MDL wrote the manuscript. We thank Teresa O&#39;Leary, William Klobasa, and Stephanie Gorski for their expert assistance in screening WCR. We also thank Dr. Wade French (USDA-ARS, North Central Agricultural Research Laboratory, Brookings, SD) for shipments of eggs to establish lab colonies and providing rearing protocols.

1. Colony-level Rearing of WCR Adults
<ol>
	<li>Obtain a sufficient quantity of WCR adults (500 - 1,000) from a reliable company or research laboratory (see Table of Materials for an example) and place in a 30 cm3 cage. 
	NOTE: Use of a non-diapausing strain is highly recommended.</li>
	<li>Prepare WCR artificial diet following manufacturer&#39;s protocol and pour a 1 cm-thick layer into a 38 oz container (see Table of Materials). After mixing, store unused diet at 4 &#176;C for up to 2 months.</li>
	<li>Put a Petri dish (100 x 15 mm) with adult diet (10&#160;- 15 g) into a 30 cm3 cage. Add more diet when the food is low or dry.</li>
	<li>Put a flask (300 mL) with water, covered with a cotton ball, which is used to hold in place a cotton roll (6&#34; x 3/8&#34;), to serve as a water source. Change the water when it runs low.</li>
	<li>Maintain WCR at 26 &#176;C with 60% humidity and a 14:10 light cycle in an insect rearing incubator.</li>
</ol>
2. Embryo Collection and Alignment
<ol>
	<li>Make an egg collection chamber using 1% agar in water in a sterile 100 x 15 mm2 Petri dish. Store at 4 &#176;C after the agar solidifies.</li>
	<li>To collect newly laid eggs, place a single layer of filter paper, followed by 4 layers of cheesecloth (each cut to size) on the surface of the agar (Figure 1A). 
	NOTE: Cheesecloth can be reused, after being washed in bleach and autoclaved, but it is not necessary to do this for the first use. Filter paper should not be reused and does not need to be sterilized.</li>
	<li>Place egg collection plate into WCR cage as late in the day as possible (end of work day) and cover with a tinfoil tent. Leave overnight. 
	NOTE: Having the lights on from 10 A.M. to 12 A.M. (14 h) delays egg laying, thus facilitating the collection of younger eggs for microinjection without requiring lab personnel to place collection chamber into cage late at night.</li>
	<li>Remove the egg collection chamber around 8 or 9 A.M.</li>
	<li>Use forceps to pick up 1 layer of cheesecloth at a time and place in a beaker (500 mL) of water. Wash eggs off of cheesecloth by gently stirring (Figure 1B and 1C).</li>
	<li>Use a bulb pipette to transfer eggs to another beaker (500 mL) of water. Repeat 2 - 3x to clean the eggs.</li>
	<li>Use the bulb pipette to transfer eggs onto filter paper (Figure 1D) while minimizing the amount of water carry-over.</li>
	<li>Cut black filter paper into strips as wide as a glass slide and tape firmly to the slide to make sure the paper lays flat (Figure 2A). 
	NOTE: Black filter paper helps improve visibility under the microscope by reducing the amount of reflected light.</li>
	<li>Apply non-toxic glue (see Table of Materials for an example) to the filter paper in fine lines (Figure 2B). Keep the glue lines thinner than the diameter of an egg to avoid getting the eggs coated in glue.</li>
	<li>Use a fine brush to gently move WCR eggs one by one from their filter paper to the glue line. Try to lay the eggs on the glue before it dries out and keep&#160;at least one egg&#39;s distance between each egg.</li>
	<li>Maximize injection efficiency by placing multiple lines of eggs on one filter paper slide (Figure 3).</li>
	<li>After all eggs are laid out on the filter paper, wait until all the glue dries before microinjection. 
	NOTE: For germline transformation and for CRISPR/Cas9-mediated genome editing, inject all embryos by noon (12 P.M.) so that the oldest embryo is no more than 19 h old. However, for RNAi, there is no time limitation because, in WCR, dsRNA can move between cells. In fact, for RNAi, aging embryos might result in better survival rates.</li>
</ol>
3. Preparation of Plasmid DNAs and Injection Needles
<ol>
	<li>Prepare high-quality supercoiled plasmid DNA using an endotoxin-free commercial kit (see Table of Materials for an example).</li>
	<li>To prepare the injection solution, check the concentration of each plasmid and then combine helper plasmid (250 ng/&#181;L final concentration), donor plasmid (750 ng/&#181;L final concentration) and phenol red buffer (20% final concentration). Vortex the solution briefly and centrifuge for 3 min at maximum speed to precipitate particles that could clog the needle.</li>
	<li>Pull borosilicate glass needles (O.D. 1 mm, I.D. 0.58 mm, 10 cm length) using a Flaming/Brown type micropipette puller. For storage, secure pulled needles on double-sided sticky tape in a Petri dish or other clear container. 
	NOTE: For the micropipette puller system, the settings used were: Heat = 335, FIL = 4, VEL = 40, DEL = 200, PUL = 100.</li>
	<li>Backfill the injection needle by using a micro-loader tip (see Table of Materials) to pipet 0.5 - 1.0 &#181;L down inside the needle, near the tapered end. Remove bubbles from the tip of the needle if any occur.</li>
	<li>Insert the injection needle into the needle holder.</li>
	<li>Open the tip of the needle by gently breaking with a fine pair of forceps, or gently touching/dragging the tip on the coverslip or glass slide. 
	NOTE: The goal is to have the smallest opening that still allows the injection mix to come out (smaller openings generally require higher injection pressure). Beveled needles don&#39;t require this step since they are already open.</li>
</ol>
4. Microinjection and Post-injection Care
<ol>
	<li>Using a fine paintbrush, carefully wet the surface of 1 - 3 eggs with sterile water, insert the needle, and inject the solution. Inject&#160;each egg before the surface dries. Look for the appearance a small amount of red color inside eggs during injection to indicate the successful microinjection. Crush or remove any eggs that look damaged, empty, or otherwise cannot be injected. 
	NOTE: Although injection pressure varies based on needle bore size, 10 - 13 psi is a good starting point.</li>
	<li>After all eggs are injected, remove the filter paper from the glass slide.</li>
	<li>Place the filter paper on the surface of a 1% agar dish (described above).</li>
	<li>Use plastic paraffin film to seal the dish, and put in a 26 &#176;C insect rearing incubator for 12 days. 
	NOTE: The paraffin film will help prevent cross-contamination of mold between dishes. However, any other treatment to prevent mold and bacterial growth could reduce the survival of the injected embryos.</li>
</ol>
5. Culturing and Hatching of Embryos
<ol>
	<li>Start checking embryos 12 days post-injection for newly-hatched larvae and continue daily for the next 2 weeks.</li>
	<li>Transfer larvae, using a fine brush, to a rearing box with sprouted corn and soil. 
	NOTE: Mold might grow in the agar dish, but larvae will still hatch in most cases. Look for larvae carefully within the mold to catch them all. Some larvae might move to the lid of the dish and might get stuck in the condensation.</li>
	<li>Rear larvae at 26 &#176;C following standard WCR rearing methods25.</li>
</ol>

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The authors have nothing to disclose.

Although microinjection of WCR with dsRNAs for the purpose of RNAi has been reported9, this is the first protocol to establish best practices for microinjecting precellular WCR embryos, a critical process for conducting germline transformation and/or CRISPR/Cas9 genome editing in this species. Successful microinjection of WCR embryos is dependent on many factors, as is transformation efficiency. Discussed below are some of the major issues impacting the outcome of using this protocol for germline ...

Western corn rootworm (WCR), Diabrotica virgifera virgifera, is an important pest of corn1. Interestingly, WCR appears to overcome control measures more rapidly than most agricultural pests since they not only adapt physiologically but also behaviorally2,3,4,5,6. Over the past decade, RNA interference (RNAi), a powerful functional genomic tool, has been investigated as a potential control method for WCR7,8, and has also been used as a means to study gene function9. However, while RNAi is frequently performed by microinjection of double-stranded RNA (dsRNA) in other species, injection-based RNAi is rare in WCR. In fact, there are only a few reports of RNAi via microinjection of dsRNA into WCR9. The reason is that WCR can attain high-levels of gene knockdown via ingestion of dsRNA7,10, even permitting the study of embryonic effects by feeding dsRNA to the mother11. While this method makes WCR an excellent subject for functional genomic analysis via RNAi, it has slowed progress on the development of methodologies for embryonic microinjection in this species.
Despite the power of RNAi, there are a few drawbacks. For example, not all genes respond equally to RNAi. This variability can make interpreting the results of a functional genomics assay more difficult. Also, RNAi is transient and does not generate heritable mutations. On the other hand, germline transformation, another functional genomic tool, can generate heritable mutations via insertion of a marked transposable element into the genome12,13. This makes germline transformation an excellent tool for creating mutant strains for use in long-term genetic studies. Transformation can also be used to rescue a mutation by delivering a functional copy of the gene to a mutant genome14. Moreover, germline transformation is the cornerstone of a wide variety of molecular genetic techniques. In addition to knocking out and/or rescuing gene function, germline transformation can be used for enhancer trapping15, gene trapping16, Gal4-based ectopic expression17, and genome-wide mutagenesis18.
More recently, tools that enable sophisticated genome editing&#160;have been developed. These tools include Transcription Activator-Like Effector Nucleases (TALEN) and the CRISPR/Cas9-nuclease system19,20. The advantage of these new tools is that they offer researchers the ability to induce a double-stranded DNA break at almost any location within almost any genome. Once induced, these breaks can be repaired through either non-homologous end joining, which can introduce deletions or insertions in the targeted gene, or through homology-directed repair, which in the presence of an engineered construct, can catalyze the replacement of entire genes or genetic regions21,22. However, these methods require microinjection of DNA, RNA and/or protein into very young embryos.
Importantly, unlike the dsRNAs used for RNAi, the nucleic acids and proteins used for germline transformation and genome editing cannot easily cross cell membranes. Therefore, microinjection of plasmid DNAs, mRNAs, and/or proteins must take place during the syncytial blastoderm stage (i.e. before the insect embryo cellularizes). This makes the timing of injections a critical factor. For example, in Drosophila melanogaster, embryos need to be injected within two hours after egg lay12. Therefore, development of a successful embryonic microinjection protocol for WCR must take into account the best conditions for female egg laying, as well as the best method for collecting sufficient quantities of precellular embryos.
An effort to bring a wide range of molecular genetic tools to bear on WCR biology requires developing methods for collecting and microinjecting precellular WCR embryos. Here we provide detailed instructions, along with tips and tricks, to help others use transformation-based techniques in WCR research. In addition to extending transgenic technologies to WCR for use in functional genomic studies, these techniques also enable powerful new pest control strategies such as gene drive23,24 to be tested in this economically important pest.

<table><tbody><tr><td>Qualitative Filter Paper (Black)</td><td>Ahlstrom</td><td>8613-0900</td><td>For egg microinjection</td></tr><tr><td>1 oz Containers&amp;nbsp;</td><td>Anny&#039;s Plastic Tableware</td><td>ASET101</td><td>Egg container</td></tr><tr><td>Drosophila Agar Type II</td><td>Apex</td><td>66-103</td><td>Substrate for WCR egg-laying dish</td></tr><tr><td>Featherweight Forceps&amp;nbsp;</td><td>Bioquip</td><td>4748</td><td>Handling larvae and pupae&amp;nbsp;</td></tr><tr><td>Clorox Regular-Bleach1</td><td>Clorox</td><td>44600-30770</td><td>Wash corn and dishes</td></tr><tr><td>Trucker&#039;s Favorite Yellow</td><td>Coor Farm Supply</td><td>502</td><td>Corn for feeding WCR</td></tr><tr><td>Microinjection System (Homemade)</td><td>NA</td><td>Parts &amp;amp; instructions available upon request</td><td>Controls injection pressure (0-20 psi)</td></tr><tr><td>Elmer&#039;s Non-toxic Glue</td><td>Elmer&#039;s Products, Inc.</td><td>E304</td><td>Glue eggs on filter paper</td></tr><tr><td>epTIPS Microloader Tips</td><td>Eppendorf</td><td>C2554691</td><td>Backfilling needle loading tips</td></tr><tr><td>Falcon Tissue Culture Dishes</td><td>Falcon</td><td>25383-103</td><td>Sprouting corn /150 x 25 mm</td></tr><tr><td>Fisherbrand&amp;nbsp;Quantitative-Grade Filter Paper Circles</td><td>Fisher Scientific</td><td>S47576C</td><td>Making agar dish for egg-lay/9cm</td></tr><tr><td>Sparkleen</td><td>Fisher Scientific</td><td>04-320-4</td><td>Wash dishes</td></tr><tr><td>Plain Microscope Slides</td><td>Fisher Scientific</td><td>12-549-3</td><td>Holding filter paper and eggs for microinjection</td></tr><tr><td>Western Corn Rootworm w/o Pollen Substitute</td><td>Frontier Agricultural Sciences&amp;nbsp;</td><td>F9766B</td><td>WCR adult artificial diet</td></tr><tr><td>Cotton Balls, Large</td><td>Genesee Scientific</td><td>51-101</td><td>Close the flask</td></tr><tr><td>Globe Scientific 3.0 mL Small Bulb Transfer Pipettes</td><td>Globe Scientific</td><td>137035</td><td>Collect and transfer eggs</td></tr><tr><td>Leica M165 FC Fluorescence Stereomicroscope&amp;nbsp;</td><td>Leica</td><td>M165 FC&amp;nbsp;</td><td>WCR screening</td></tr><tr><td>DsRed Filter Set for Fluorescence Stereomicroscope</td><td>Leica</td><td>DSR</td><td>Excitation filter: 510-560 nm, emission filter: 590-650 nm</td></tr><tr><td>EGFP filter</td><td>Leica</td><td>GFP2</td><td>Excitation filter: 440&amp;ndash;520 nm, emission filter: 510 nm</td></tr><tr><td>BugDorm</td><td>MegaView Science</td><td>DP1000_5P</td><td>Cage for adult colony</td></tr><tr><td>Miniature Paint Brush</td><td>MyArtscape</td><td>MAS-102-MINI</td><td>Liner 2/0&amp;nbsp;</td></tr><tr><td>Joystick Micromanipulator</td><td>Narishige</td><td>MN-151</td><td>Micromanipulator and needle holder</td></tr><tr><td>Microscope XY Stage</td><td>Olympus</td><td>265515</td><td>Microscope stage (adapted for use with stereoscope)</td></tr><tr><td>Grade 90 Cheesecloth</td><td>Online Fabric Store</td><td>CHEE90</td><td>For egg-lay</td></tr><tr><td>Plastic paraffin film&amp;nbsp;</td><td>Pechiney Plastic Packaging</td><td>PM-996</td><td>Seal agar dish after microinjection/Roll size 4 in. x 125 ft</td></tr><tr><td>Percival Incubator</td><td>Percival&amp;nbsp;</td><td>I41VLH3C8</td><td>WCR growing chamber (insect rearing incubator)</td></tr><tr><td>Narrow Mouth Erlenmeyer Flasks</td><td>VWR</td><td>4980-300-PK</td><td>Water container for adult colony&amp;nbsp;</td></tr><tr><td>Griffin Low Form Beakers</td><td>VWR</td><td>1000-600-PK</td><td>For egg wash</td></tr><tr><td>Braided Cotton Rolls</td><td>Richmond Dental Cotton Co.</td><td>605-3599</td><td>Use in water supply for adult colony&amp;nbsp;</td></tr><tr><td>Petri Dishes (35x10mm)</td><td>SIGMA</td><td>CLS430588-500EA</td><td>For diet plates</td></tr><tr><td>Phenol Red</td><td>Sigma</td><td>143-78-8</td><td>Microinjection buffer</td></tr><tr><td>Laser-based Micropipiette Puller</td><td>Sutter Instrument</td><td>P-2000/G</td><td>Needle puller/Heat = 335, FIL = 4, VEL = 40, DEL = 200, PUL = 100</td></tr><tr><td>Premium Topsoil</td><td>The Scotts Company</td><td>71130758</td><td>Soil for cron growing</td></tr><tr><td>Reloc Zippit 2 Mil Zipper Bags</td><td>United States Plastic Corporation</td><td>48342</td><td>Bag agar dish after microinjection/5x7</td></tr><tr><td>Petri Dishes (100x15m)</td><td>VWR</td><td>89038-968</td><td>Making agar dish for egg-lay/ 100 x 15 mm</td></tr><tr><td>6 oz Containers</td><td>Webstaurantstore</td><td>128E506</td><td>WCR single pair mating chamber&amp;nbsp;</td></tr><tr><td>38 oz Containers</td><td>Webstaurantstore</td><td>128NC888</td><td>Larvae rearing box/38 oz</td></tr><tr><td>ChoiceHD 16 oz. Microwavable Translucent Plastic Deli Container</td><td>Webstaurantstore</td><td>128HRD16</td><td>Larvae rearing box/16 oz</td></tr><tr><td>Microinjection Scope&amp;nbsp;</td><td>Wild Heerbrugg</td><td>WILD-M8</td><td>Microinjection scope outfited with an XY stage</td></tr><tr><td>Standard Glass Capillaries</td><td>World Precision Instruments</td><td>1B100F-4</td><td>Microinjection needles</td></tr><tr><td>Adult Western Corn Rootworms</td><td>North Centeral Aqricultural reasearch Laboratory</td><td>Non-diapase strain</td><td>Request from Dr. Bryan Wade French&#039;s lab</td></tr><tr><td>Plasmid DNA Midi Kit</td><td>Qiagen</td><td>12143</td><td>Purification of injection-ready plasmid DNAs</td></tr></tbody></table>

microinjection of western corn rootworm, diabrotica virgifera virgifera, embryos for germline transformation, or crispr/cas9 genome editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

An important consideration when developing this protocol was the stage of embryonic development at the time of microinjection. Specifically, germline transformation requires microinjection of DNAs prior to embryonic cellularization. This is because DNAs cannot easily cross cell membranes. Attempts to visualize the stages of WCR embryonic development using a nuclear stain were unsuccessful because dechorionation of WCR embryos essentially dissolves all of the protective membranes responsib...

Watch this Scientific Journal Video about Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing at JoVE.com

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

microinjection-western-corn-rootworm-diabrotica-virgifera-virgifera

Research

JoVE Journal

Bioengineering

7.5K Views.  North Carolina State University. Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

Video: Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

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. ...

Genetics

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

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 ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to ...

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

Behavior

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to their successful evolutionary radiation on the earth. The application of genetic tools such as genome editing is the key to understanding genetic changes that are responsible for evolutionary transitions in an Evo-Devo approach. In this article, we describe our current protocol for generating and maintaining mutant strains of T. domestica. We report a dry injection method, as an alternative to the reported wet injection method, that allows us to obtain stably high survival rates in injected embryos. We also report an optimized environment setting to mate adults and obtain subsequent generations with high efficiency. Our method underlines the importance of taking each species&#8217; unique biology into account for the successful application of genome editing methods to non-traditional model organisms. We predict that these genome editing protocols will help in implementing T. domestica as a laboratory model and to further accelerate the development and application of useful genetic tools in this species.

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

We provide a detailed protocol for rearing, microinjection of eggs and for efficient mating of the firebrat Thermobia domestica to generate and maintain mutant strains after genome editing.

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...

Developmental Biology

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Herein are protocols for collecting and microinjecting precellular corn planthopper embryos for the purpose of modifying their genome via CRISPR/Cas9-based genome editing or for the addition of marked transposable elements through germline transformation.

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Summary

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Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

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Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner)

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis