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 a....

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 ....

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