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

Summary

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.

Abstract

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'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.

Introduction

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 behaviorally^2,3,4,5,6. Over the past decade, RNA interference (RNAi), a powerful functional genomic tool, has been investigated as a potential control method for WCR^7,8, 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 dsRNA^7,10, 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 genome^12,13. 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 have been developed. These tools include Transcription Activator-Like Effector Nucleases (TALEN) and the CRISPR/Cas9-nuclease system^19,20. 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 regions^21,22. 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 drive^23,24 to be tested in this economically important pest.

Protocol

1. Colony-level Rearing of WCR Adults

1. 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.
2. Prepare WCR artificial diet following manufacturer'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 °C for up to 2 months.
3. Put a Petri dish (100 x 15 mm) with adult diet (10 - 15 g) into a 30 cm³ cage. Add more diet when the food is low or dry.
4. Put a flask (300 mL) with water, covered with a cotton ball, which is used to hold in place a cotton roll (6" x 3/8"), to serve as a water source. Change the water when it runs low.
5. Maintain WCR at 26 °C with 60% humidity and a 14:10 light cycle in an insect rearing incubator.

2. Embryo Collection and Alignment

1. Make an egg collection chamber using 1% agar in water in a sterile 100 x 15 mm² Petri dish. Store at 4 °C after the agar solidifies.
2. 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.
3. 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.
4. Remove the egg collection chamber around 8 or 9 A.M.
5. 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).
6. Use a bulb pipette to transfer eggs to another beaker (500 mL) of water. Repeat 2 - 3x to clean the eggs.
7. Use the bulb pipette to transfer eggs onto filter paper (Figure 1D) while minimizing the amount of water carry-over.
8. 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.
9. 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.
10. 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 at least one egg's distance between each egg.
11. Maximize injection efficiency by placing multiple lines of eggs on one filter paper slide (Figure 3).
12. 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.

3. Preparation of Plasmid DNAs and Injection Needles

1. Prepare high-quality supercoiled plasmid DNA using an endotoxin-free commercial kit (see Table of Materials for an example).
2. To prepare the injection solution, check the concentration of each plasmid and then combine helper plasmid (250 ng/µL final concentration), donor plasmid (750 ng/µL 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.
3. 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.
4. Backfill the injection needle by using a micro-loader tip (see Table of Materials) to pipet 0.5 - 1.0 µL down inside the needle, near the tapered end. Remove bubbles from the tip of the needle if any occur.
5. Insert the injection needle into the needle holder.
6. 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't require this step since they are already open.

4. Microinjection and Post-injection Care

1. Using a fine paintbrush, carefully wet the surface of 1 - 3 eggs with sterile water, insert the needle, and inject the solution. Inject 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.
2. After all eggs are injected, remove the filter paper from the glass slide.
3. Place the filter paper on the surface of a 1% agar dish (described above).
4. Use plastic paraffin film to seal the dish, and put in a 26 °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.

5. Culturing and Hatching of Embryos

1. Start checking embryos 12 days post-injection for newly-hatched larvae and continue daily for the next 2 weeks.
2. 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.
3. Rear larvae at 26 °C following standard WCR rearing methods²⁵.

Results

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...

Discussion

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 ...

Materials

List of materials used in this article

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